Publications by year
In Press
Kinoshita M, Li MA, Barber M, Mansfield W, Dietmann S, Smith A (In Press). Disabling <i>de novo</i> DNA methylation in embryonic stem cells allows an illegitimate fate trajectory.
Abstract:
Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory
ABSTRACTGenome remethylation is essential for mammalian development but specific reasons are unclear. Here we examined embryonic stem (ES) cell fate in the absence of de novo DNA methyltransferases. We observed that ES cells deficient for both Dnmt3a and Dnmt3b are rapidly eliminated from chimaeras. On further investigation we found that in vivo and in vitro the formative pluripotency transition is derailed towards production of trophoblast. This aberrant trajectory is associated with failure to suppress activation of Ascl2. Ascl2 encodes a bHLH transcription factor expressed in placenta. Misexpression of Ascl2 in ES cells provokes transdifferentiation to trophoblast-like cells. Conversely, Ascl2 deletion rescues formative transition of Dnmt3a/b mutants and improves contribution to chimaeric epiblast. Thus, de novo DNA methylation safeguards against ectopic activation of Ascl2. However, Dnmt3a/b-deficient cells remain defective in ongoing embryogenesis. We surmise that multiple developmental transitions may be secured by DNA methylation silencing potentially disruptive genes.SIGNIFICANCE STATEMENTMammalian DNA is widely modified by methylation of cytosine residues. This modification is added to DNA during early development. If methylation is prevented, the embryo dies by mid-gestation with multiple abnormalities. In this study we found that stem cells lacking the DNA methylation enzymes do not differentiate efficiently into cell types of the embryo and are diverted into producing placental cells. This switch in cell fate is driven by a transcription factor, Ascl2, which should only be produced in placenta. In the absence of DNA methylation, the Ascl2 gene is mis-expressed. Removing Ascl2 redirects embryonic fate but not full differentiation potential, suggesting that methylation acts at multiple developmental transitions to restrict activation of disruptive genes.
Abstract.
Strawbridge SE, Blanchard GB, Smith A, Kugler H, Martello G (In Press). Embryonic stem cells commit to differentiation by symmetric divisions following a variable lag period.
Abstract:
Embryonic stem cells commit to differentiation by symmetric divisions following a variable lag period
ABSTRACTMouse embryonic stem (ES) cells are derived from the epiblast of the preimplantation embryo and retain the capacity to give rise to all embryo lineages. ES cells can be released into differentiation from a near-homogeneous maintenance condition. Exit from the ES cell state can be accurately monitored using the Rex1-GFPd2 transgenic reporter, providing a powerful system for examining a mammalian cell fate transition. Here, we performed live-cell imaging and tracking of ES cells during entry into differentiation for 48 hours in defined conditions. We observed a greater cell surface area and a modest shortening of the cell cycle prior to exit and subsequently a reduction in cell size and increase in motility. We did not see any instance of cells regaining ES cell identity, consistent with unidirectional developmental progression. Transition occurred asynchronously across the population but genealogical tracking revealed a high correlation in cell-cycle length and Rex1-GFPd2 expression between daughter cells. A population dynamics model was consistent with symmetric divisions during exit from naive pluripotency. Collapse of ES cell identity occurred acutely in individual cells but after a variable delay. The variation in lag period can extend up to three generations, creating marked population asynchrony.
Abstract.
Vallier L, Vitilio L, Durance C, Smith A (In Press). GMP-grade neural progenitor derivation and differentiation from clinical-grade human embryonic stem cells.
Stem Cell Research and TherapyAbstract:
GMP-grade neural progenitor derivation and differentiation from clinical-grade human embryonic stem cells
Background: a major challenge for the clinical use of human pluripotent stem cells is the development of safe, robust and controlled differentiation protocols. Adaptation of research protocols using reagents designated as research-only to those which are suitable for clinical use, often referred to as Good Manufacturing Practice (GMP) reagents, is a crucial and laborious step in the translational pipeline. However, published protocols to assist this process remain very limited. Methods: We adapted research-grade protocols for the derivation and differentiation of long-term neuroepithelial stem cell progenitors (lt-NES) to GMP-grade reagents and factors suitable for clinical applications. We screened the robustness of the protocol with six clinical-grade hESCs lines deposited in the UK Stem Cell Bank. Results: Here we present a new GMP-compliant protocol to derive lt-NES, which are multipotent, bankable, and karyotypically stable. This protocol resulted in robust and reproducible differentiation of several clinical-grade embryonic stem cells from which we derived lt-NES. Furthermore, GMP-derived lt-NES demonstrated a high neurogenic potential while retaining the ability to be redirected to several neuronal sub-types. Conclusions: Overall, we report the feasibility of derivation and differentiation of clinical grade embryonic stem cell lines into lt-NES under GMP-compliant conditions. Our protocols could be used as a flexible tool to speed up translation-to-clinic of pluripotent stem cells for a variety of neurological therapies or regenerative medicine studies.
Abstract.
Spindlow D, Stirparo GG, Smith A (In Press). Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species. Development
Dattani A, Huang T, Smith A, Guo G (In Press). Suppression of YAP Safeguards Human Naïve Pluripotency.
Abstract:
Suppression of YAP Safeguards Human Naïve Pluripotency
ABSTRACTPropagation of human naïve pluripotent stem cells (nPSCs) requires inhibition of MEK/ERK signalling. However, MEK/ERK inhibition also induces differentiation into trophectoderm (TE). Therefore, robust self-renewal requires active suppression of TE fate. Tankyrase inhibition using XAV939 has been shown to stabilise human nPSCs. Here we dissect the mechanism of this effect. Tankyrase inhibition blocks canonical Wnt/β-catenin signalling. However, nPSCs depleted of β-catenin remain dependent on XAV939. We show that XAV939 prevents TE induction by suppressing YAP activity independent of β-catenin. Tankyrase inhibition stabilises angiomotin, which reduces nuclear translocation of YAP1/TAZ. Upon deletion of Angiomotin-family members AMOT and AMOTL2, nuclear YAP increases and XAV939 fails to prevent TE induction. Conversely, nPSCs lacking YAP1 fail to undergo TE differentiation and sustain efficient self-renewal without XAV939. These findings explain the distinct requirement for tankyrase inhibition in human but not mouse naïve PSCs and highlight the pivotal role of YAP in human naïve pluripotency.
Abstract.
2023
Miroshnikova YA, Shahbazi MN, Negrete J, Chalut KJ, Smith A (2023). Cell state transitions: catch them if you can.
Development,
150(6).
Abstract:
Cell state transitions: catch them if you can.
The Company of Biologists' 2022 workshop on 'Cell State Transitions: Approaches, Experimental Systems and Models' brought together an international and interdisciplinary team of investigators spanning the fields of cell and developmental biology, stem cell biology, physics, mathematics and engineering to tackle the question of how cells precisely navigate between distinct identities and do so in a dynamic manner. This second edition of the workshop was organized after a successful virtual workshop on the same topic that took place in 2021.
Abstract.
Author URL.
Radley A, Corujo-Simon E, Nichols J, Smith A, Dunn SJ (2023). Entropy sorting of single-cell RNA sequencing data reveals the inner cell mass in the human pre-implantation embryo.
Stem Cell Reports,
18(1), 47-63.
Abstract:
Entropy sorting of single-cell RNA sequencing data reveals the inner cell mass in the human pre-implantation embryo
A major challenge in single-cell gene expression analysis is to discern meaningful cellular heterogeneity from technical or biological noise. To address this challenge, we present entropy sorting (ES), a mathematical framework that distinguishes genes indicative of cell identity. ES achieves this in an unsupervised manner by quantifying if observed correlations between features are more likely to have occurred due to random chance versus a dependent relationship, without the need for any user-defined significance threshold. On synthetic data, we demonstrate the removal of noisy signals to reveal a higher resolution of gene expression patterns than commonly used feature selection methods. We then apply ES to human pre-implantation embryo single-cell RNA sequencing (scRNA-seq) data. Previous studies failed to unambiguously identify early inner cell mass (ICM), suggesting that the human embryo may diverge from the mouse paradigm. In contrast, ES resolves the ICM and reveals sequential lineage bifurcations as in the classical model. ES thus provides a powerful approach for maximizing information extraction from high-dimensional datasets such as scRNA-seq data.
Abstract.
2022
Huth M, Santini L, Galimberti E, Ramesmayer J, Titz-Teixeira F, Sehlke R, Oberhuemer M, Stummer S, Herzog V, Garmhausen M, et al (2022). NMD is required for timely cell fate transitions by fine-tuning gene expression and regulating translation.
Genes and Development,
34(5), 348-367.
Abstract:
NMD is required for timely cell fate transitions by fine-tuning gene expression and regulating translation
Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expression levels of the translation initiation factor Eif4a2 and its premature termination codonencoding isoform (Eif4a2PTC). NMD deficiency leads to translation of the truncated eIF4A2PTC protein. eIF4A2PTC elicits increased mTORC1 activity and translation rates and causes differentiation delays. This establishes a previously unknown feedback loop between NMD and translation initiation. Furthermore, our results show a clear hierarchy in the severity of target deregulation and differentiation phenotypes betweenNMDeffector KOs (Smg5KO > Smg6 KO> Smg7 KO), which highlights heterodimer-independent functions for SMG5 and SMG7. Together, our findings expose an intricate link between mRNA homeostasis and mTORC1 activity that must be maintained for normal dynamics of cell state transitions.
Abstract.
Dattani A, Huang T, Liddle C, Smith A, Guo G (2022). Suppression of YAP safeguards human naïve pluripotency.
Development,
149(24).
Abstract:
Suppression of YAP safeguards human naïve pluripotency.
Propagation of human naïve pluripotent stem cells (nPSCs) relies on the inhibition of MEK/ERK signalling. However, MEK/ERK inhibition also promotes differentiation into trophectoderm (TE). Therefore, robust self-renewal requires suppression of TE fate. Tankyrase inhibition using XAV939 has been shown to stabilise human nPSCs and is implicated in TE suppression. Here, we dissect the mechanism of this effect. Tankyrase inhibition is known to block canonical Wnt/β-catenin signalling. However, we show that nPSCs depleted of β-catenin remain dependent on XAV939. Rather than inhibiting Wnt, we found that XAV939 prevents TE induction by reducing activation of YAP, a co-factor of TE-inducing TEAD transcription factors. Tankyrase inhibition stabilises angiomotin, which limits nuclear accumulation of YAP. Upon deletion of angiomotin-family members AMOT and AMOTL2, nuclear YAP increases and XAV939 fails to prevent TE induction. Expression of constitutively active YAP similarly precipitates TE differentiation. Conversely, nPSCs lacking YAP1 or its paralog TAZ (WWTR1) resist TE differentiation and self-renewal efficiently without XAV939. These findings explain the distinct requirement for tankyrase inhibition in human but not in mouse nPSCs and highlight the pivotal role of YAP activity in human naïve pluripotency and TE differentiation. This article has an associated 'The people behind the papers' interview.
Abstract.
Author URL.
2021
Stirparo GG, Smith A, Guo G (2021). Cancer-Related Mutations Are Not Enriched in Naive Human Pluripotent Stem Cells.
Cell Stem Cell,
28(1), 164-169.e2.
Abstract:
Cancer-Related Mutations Are Not Enriched in Naive Human Pluripotent Stem Cells.
Previous analysis of RNA sequencing (RNA-seq) data from human naive pluripotent stem cells reported multiple point "mutations" in cancer-related genes and implicated selective culture conditions. We observed, however, that those mutations were only present in co-cultures with mouse feeder cells. Inspection of reads containing the polymorphisms revealed complete identity to the mouse reference genome. After we filtered reads to remove sequences of mouse origin, the actual incidence of oncogenic polymorphisms arising in naive pluripotent stem cells is close to zero.
Abstract.
Author URL.
Kinoshita M, Barber M, Mansfield W, Cui Y, Spindlow D, Stirparo GG, Dietmann S, Nichols J, Smith A (2021). Capture of Mouse and Human Stem Cells with Features of Formative Pluripotency.
Cell Stem Cell,
28(3), 453-471.e8.
Abstract:
Capture of Mouse and Human Stem Cells with Features of Formative Pluripotency.
Pluripotent cells emerge as a naive founder population in the blastocyst, acquire capacity for germline and soma formation, and then undergo lineage priming. Mouse embryonic stem cells (ESCs) and epiblast-derived stem cells (EpiSCs) represent the initial naive and final primed phases of pluripotency, respectively. Here, we investigate the intermediate formative stage. Using minimal exposure to specification cues, we derive stem cells from formative mouse epiblast. Unlike ESCs or EpiSCs, formative stem (FS) cells respond directly to germ cell induction. They colonize somatic tissues and germline in chimeras. Whole-transcriptome analyses show similarity to pre-gastrulation formative epiblast. Signal responsiveness and chromatin accessibility features reflect lineage capacitation. Furthermore, FS cells show distinct transcription factor dependencies, relying critically on Otx2. Finally, FS cell culture conditions applied to human naive cells or embryos support expansion of similar stem cells, consistent with a conserved staging post on the trajectory of mammalian pluripotency.
Abstract.
Author URL.
Mulas C, Chaigne A, Smith A, Chalut KJ (2021). Cell state transitions: definitions and challenges.
Development,
148(20).
Abstract:
Cell state transitions: definitions and challenges
ABSTRACT
. A fundamental challenge when studying biological systems is the description of cell state dynamics. During transitions between cell states, a multitude of parameters may change – from the promoters that are active, to the RNAs and proteins that are expressed and modified. Cells can also adopt different shapes, alter their motility and change their reliance on cell-cell junctions or adhesion. These parameters are integral to how a cell behaves and collectively define the state a cell is in. Yet, technical challenges prevent us from measuring all of these parameters simultaneously and dynamically. How, then, can we comprehend cell state transitions using finite descriptions? the recent virtual workshop organised by the Company of Biologists entitled ‘Cell State Transitions: Approaches, Experimental Systems and Models’ attempted to address this question. Here, we summarise some of the main points that emerged during the workshop's themed discussions. We also present examples of cell state transitions and describe models and systems that are pushing forward our understanding of how cells rewire their state.
Abstract.
Lackner A, Sehlke R, Garmhausen M, Giuseppe Stirparo G, Huth M, Titz-Teixeira F, van der Lelij P, Ramesmayer J, Thomas HF, Ralser M, et al (2021). Cooperative genetic networks drive embryonic stem cell transition from naïve to formative pluripotency.
EMBO J,
40(8).
Abstract:
Cooperative genetic networks drive embryonic stem cell transition from naïve to formative pluripotency.
In the mammalian embryo, epiblast cells must exit the naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene networks involved in the exit from naïve pluripotency remains fragmentary. Here, we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large-scale transcriptomics and computational systems biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 ESC lines deficient for regulators of the exit from naïve pluripotency predominantly manifest delays on the trajectory from naïve to formative epiblast. We find that gene networks operative in ESCs are also active during transition from pre- to post-implantation epiblast in utero. We identified 496 naïve state-associated genes tightly connected to the in vivo epiblast state transition and largely conserved in primate embryos. Integrated analysis of mutant transcriptomes revealed funnelling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signalling pathways direct this pivotal mammalian cell state transition.
Abstract.
Author URL.
Kinoshita M, Li MA, Barber M, Mansfield W, Dietmann S, Smith A (2021). Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory.
Proceedings of the National Academy of Sciences,
118(38).
Abstract:
Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory
Significance
.
. Mammalian DNA is widely modified by methylation of cytosine residues. This modification is added to DNA during early development. If methylation is prevented, the embryo dies by midgestation with multiple abnormalities. In this study we found that stem cells lacking the DNA methylation enzymes do not differentiate efficiently into cell types of the embryo and are diverted into producing placental cells. This switch in cell fate is driven by a transcription factor, Ascl2, which should only be produced in the placenta. In the absence of DNA methylation, the
. Ascl2
. gene is misexpressed. Removing Ascl2 redirects embryonic fate but not full differentiation potential, suggesting that methylation acts at multiple developmental transitions to restrict activation of disruptive genes.
.
Abstract.
Kinoshita M, Barber M, Mansfield W, Cui Y, Spindlow D, Stirparo GG, Dietmann S, Nichols J, Smith A (2021). Erratum: Capture of Mouse and Human Stem Cells with Features of Formative Pluripotency (Cell Stem Cell (2021) 28(3) (453–471.e8), (S1934590920305439), (10.1016/j.stem.2020.11.005)).
Cell Stem Cell,
28(12).
Abstract:
Erratum: Capture of Mouse and Human Stem Cells with Features of Formative Pluripotency (Cell Stem Cell (2021) 28(3) (453–471.e8), (S1934590920305439), (10.1016/j.stem.2020.11.005))
(Cell Stem Cell 28, 1–19.e1–e8, March 4, 2021) the accession number for RNA-seq and ATAC-seq data listed in the Key Resources Table was incorrect due to a typographical error. The correct accession number is “GEO: GSE131556,” not GSE131566. This error has been corrected in the online version of the paper. The authors apologize for this error and any inconvenience caused.
Abstract.
Guo G, Stirparo GG, Strawbridge SE, Spindlow D, Yang J, Clarke J, Dattani A, Yanagida A, Li MA, Myers S, et al (2021). Human naive epiblast cells possess unrestricted lineage potential.
Cell Stem Cell,
28(6), 1040-1056.e6.
Abstract:
Human naive epiblast cells possess unrestricted lineage potential.
Classic embryological experiments have established that the early mouse embryo develops via sequential lineage bifurcations. The first segregated lineage is the trophectoderm, essential for blastocyst formation. Mouse naive epiblast and derivative embryonic stem cells are restricted accordingly from producing trophectoderm. Here we show, in contrast, that human naive embryonic stem cells readily make blastocyst trophectoderm and descendant trophoblast cell types. Trophectoderm was induced rapidly and efficiently by inhibition of ERK/mitogen-activated protein kinase (MAPK) and Nodal signaling. Transcriptome comparison with the human embryo substantiated direct formation of trophectoderm with subsequent differentiation into syncytiotrophoblast, cytotrophoblast, and downstream trophoblast stem cells. During pluripotency progression lineage potential switches from trophectoderm to amnion. Live-cell tracking revealed that epiblast cells in the human blastocyst are also able to produce trophectoderm. Thus, the paradigm of developmental specification coupled to lineage restriction does not apply to humans. Instead, epiblast plasticity and the potential for blastocyst regeneration are retained until implantation.
Abstract.
Author URL.
Yanagida A, Spindlow D, Nichols J, Dattani A, Smith A, Guo G (2021). Naive stem cell blastocyst model captures human embryo lineage segregation.
Cell Stem Cell,
28(6), 1016-1022.e4.
Abstract:
Naive stem cell blastocyst model captures human embryo lineage segregation.
Human naive pluripotent cells can differentiate into extraembryonic trophectoderm and hypoblast. Here we describe a human embryo model (blastoid) generated by self-organization. Brief induction of trophectoderm leads to formation of blastocyst-like structures within 3 days. Blastoids are composed of three tissue layers displaying exclusive lineage markers, mimicking the natural blastocyst. Single-cell transcriptome analyses confirm segregation of trophectoderm, hypoblast, and epiblast with high fidelity to the human embryo. This versatile and scalable system provides a robust experimental model for human embryo research.
Abstract.
Author URL.
Kinoshita M, Kobayashi T, Planells B, Klisch D, Spindlow D, Masaki H, Bornelöv S, Stirparo GG, Matsunari H, Uchikura A, et al (2021). Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species.
Development,
148(23).
Abstract:
Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species.
Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated 'The people behind the papers' interview.
Abstract.
Author URL.
Kinoshita M, Kobayashi T, Nagashima H, Alberio R, Smith A (2021). The people behind the papers - Masaki Kinoshita, Toshihiro Kobayashi, Hiroshi Nagashima, Ramiro Alberio and Austin Smith. Development (Cambridge), 148(23).
2020
Mulas C, Hodgson AC, Kohler TN, Agley CC, Humphreys P, Kleine-Brüggeney H, Hollfelder F, Smith A, Chalut KJ (2020). Microfluidic platform for 3D cell culture with live imaging and clone retrieval.
Lab Chip,
20, 2580-2591.
Abstract:
Microfluidic platform for 3D cell culture with live imaging and clone retrieval.
Combining live imaging with the ability to retrieve individual cells of interest remains a technical challenge. Combining imaging with precise cell retrieval is of particular interest when studying highly dynamic or transient, asynchronous, or heterogeneous cell biological and developmental processes. Here, we present a method to encapsulate live cells in a 3D hydrogel matrix, via hydrogel bead compartmentalisation. Using a small-scale screen, we optimised matrix conditions for the culture and multilineage differentiation of mouse embryonic stem cells. Moreover, we designed a custom microfluidic platform that is compatible with live imaging. With this platform we can long-term culture and subsequently extract individual cells-in-beads by media flow only, obviating the need for enzymatic cell removal from the platform. Specific beads may be extracted from the platform in isolation, without disrupting the adjacent beads. We show that we can differentiate mouse embryonic stem cells, monitor reporter expression by live imaging, and retrieve individual beads for functional assays, correlating reporter expression with functional response. Overall, we present a highly flexible 3D cell encapsulation and microfluidic platform that enables both monitoring of cellular dynamics and retrieval for molecular and functional assays.
Abstract.
Mayer D, Stadler MB, Rittirsch M, Hess D, Lukonin I, Winzi M, Smith A, Buchholz F, Betschinger J (2020). Zfp281 orchestrates interconversion of pluripotent states by engaging Ehmt1 and Zic2.
EMBO J,
39, e102591-e102591.
Abstract:
Zfp281 orchestrates interconversion of pluripotent states by engaging Ehmt1 and Zic2.
Developmental cell fate specification is a unidirectional process that can be reverted in response to injury or experimental reprogramming. Whether differentiation and de-differentiation trajectories intersect mechanistically is unclear. Here, we performed comparative screening in lineage-related mouse naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), and identified the constitutively expressed zinc finger transcription factor (TF) Zfp281 as a bidirectional regulator of cell state interconversion. We showed that subtle chromatin binding changes in differentiated cells translate into activation of the histone H3 lysine 9 (H3K9) methyltransferase Ehmt1 and stabilization of the zinc finger TF Zic2 at enhancers and promoters. Genetic gain-of-function and loss-of-function experiments confirmed a critical role of Ehmt1 and Zic2 downstream of Zfp281 both in driving exit from the ESC state and in restricting reprogramming of EpiSCs. Our study reveals that cell type-invariant chromatin association of Zfp281 provides an interaction platform for remodeling the cis-regulatory network underlying cellular plasticity.
Abstract.
2019
Dunn S-J, Li MA, Carbognin E, Smith A, Martello G (2019). A common molecular logic determines embryonic stem cell self-renewal and reprogramming.
EMBO J,
38Abstract:
A common molecular logic determines embryonic stem cell self-renewal and reprogramming.
During differentiation and reprogramming, new cell identities are generated by reconfiguration of gene regulatory networks. Here, we combined automated formal reasoning with experimentation to expose the logic of network activation during induction of naïve pluripotency. We find that a Boolean network architecture defined for maintenance of naïve state embryonic stem cells (ESC) also explains transcription factor behaviour and potency during resetting from primed pluripotency. Computationally identified gene activation trajectories were experimentally substantiated at single-cell resolution by RT-qPCR Contingency of factor availability explains the counterintuitive observation that Klf2, which is dispensable for ESC maintenance, is required during resetting. We tested 124 predictions formulated by the dynamic network, finding a predictive accuracy of 77.4%. Finally, we show that this network explains and predicts experimental observations of somatic cell reprogramming. We conclude that a common deterministic program of gene regulation is sufficient to govern maintenance and induction of naïve pluripotency. The tools exemplified here could be broadly applied to delineate dynamic networks underlying cell fate transitions.
Abstract.
Rostovskaya M, Stirparo GG, Smith A (2019). Capacitation of human naïve pluripotent stem cells for multi-lineage differentiation.
Development,
146Abstract:
Capacitation of human naïve pluripotent stem cells for multi-lineage differentiation.
Human naïve pluripotent stem cells (PSCs) share features with the pre-implantation epiblast. They therefore provide an unmatched opportunity for characterising the developmental programme of pluripotency in Homo sapiens Here, we confirm that naïve PSCs do not respond directly to germ layer induction, but must first acquire competence. Capacitation for multi-lineage differentiation occurs without exogenous growth factor stimulation and is facilitated by inhibition of Wnt signalling. Whole-transcriptome profiling during this formative transition highlights dynamic changes in gene expression, which affect many cellular properties including metabolism and epithelial features. Notably, naïve pluripotency factors are exchanged for postimplantation factors, but competent cells remain devoid of lineage-specific transcription. The gradual pace of transition for human naïve PSCs is consistent with the timespan of primate development from blastocyst to gastrulation. Transcriptome trajectory during in vitro capacitation of human naïve cells tracks the progression of the epiblast during embryogenesis in Macaca fascicularis, but shows greater divergence from mouse development. Thus, the formative transition of naïve PSCs in a simple culture system may recapitulate essential and specific features of pluripotency dynamics during an inaccessible period of human embryogenesis.
Abstract.
Kalkan T, Bornelöv S, Mulas C, Diamanti E, Lohoff T, Ralser M, Middelkamp S, Lombard P, Nichols J, Smith A, et al (2019). Complementary Activity of ETV5, RBPJ, and TCF3 Drives Formative Transition from Naive Pluripotency.
Cell Stem Cell,
24, 785-801.e7.
Abstract:
Complementary Activity of ETV5, RBPJ, and TCF3 Drives Formative Transition from Naive Pluripotency.
The gene regulatory network (GRN) of naive mouse embryonic stem cells (ESCs) must be reconfigured to enable lineage commitment. TCF3 sanctions rewiring by suppressing components of the ESC transcription factor circuitry. However, TCF3 depletion only delays and does not prevent transition to formative pluripotency. Here, we delineate additional contributions of the ETS-family transcription factor ETV5 and the repressor RBPJ. In response to ERK signaling, ETV5 switches activity from supporting self-renewal and undergoes genome relocation linked to commissioning of enhancers activated in formative epiblast. Independent upregulation of RBPJ prevents re-expression of potent naive factors, TBX3 and NANOG, to secure exit from the naive state. Triple deletion of Etv5, Rbpj, and Tcf3 disables ESCs, such that they remain largely undifferentiated and locked in self-renewal, even in the presence of differentiation stimuli. Thus, genetic elimination of three complementary drivers of network transition stalls developmental progression, emulating environmental insulation by small-molecule inhibitors.
Abstract.
Mulas C, Kalkan T, von Meyenn F, Leitch HG, Nichols J, Smith A (2019). Correction: Defined conditions for propagation and manipulation of mouse embryonic stem cells (doi:10.1242/dev.173146). Development, 146
Mulas C, Kalkan T, von Meyenn F, Leitch HG, Nichols J, Smith A (2019). Defined conditions for propagation and manipulation of mouse embryonic stem cells.
Development (Cambridge, England),
146(6).
Abstract:
Defined conditions for propagation and manipulation of mouse embryonic stem cells
The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation.
Abstract.
Huang M, Tailor J, Zhen Q, Gillmor AH, Miller ML, Weishaupt H, Chen J, Zheng T, Nash EK, McHenry LK, et al (2019). Engineering Genetic Predisposition in Human Neuroepithelial Stem Cells Recapitulates Medulloblastoma Tumorigenesis.
Cell Stem Cell,
25, 433-446.e7.
Abstract:
Engineering Genetic Predisposition in Human Neuroepithelial Stem Cells Recapitulates Medulloblastoma Tumorigenesis.
Human neural stem cell cultures provide progenitor cells that are potential cells of origin for brain cancers. However, the extent to which genetic predisposition to tumor formation can be faithfully captured in stem cell lines is uncertain. Here, we evaluated neuroepithelial stem (NES) cells, representative of cerebellar progenitors. We transduced NES cells with MYCN, observing medulloblastoma upon orthotopic implantation in mice. Significantly, transcriptomes and patterns of DNA methylation from xenograft tumors were globally more representative of human medulloblastoma compared to a MYCN-driven genetically engineered mouse model. Orthotopic transplantation of NES cells generated from Gorlin syndrome patients, who are predisposed to medulloblastoma due to germline-mutated PTCH1, also generated medulloblastoma. We engineered candidate cooperating mutations in Gorlin NES cells, with mutation of DDX3X or loss of GSE1 both accelerating tumorigenesis. These findings demonstrate that human NES cells provide a potent experimental resource for dissecting genetic causation in medulloblastoma.
Abstract.
Mulas C, Kalkan T, Von Meyenn F, Leitch HG, Nichols J, Smith A (2019). Erratum: Defined conditions for propagation and manipulation of mouse embryonic stem cells (Development (Cambridge) (2019) 146 (dev173146) DOI: 10.1242/dev.173146).
Development (Cambridge),
146(9).
Abstract:
Erratum: Defined conditions for propagation and manipulation of mouse embryonic stem cells (Development (Cambridge) (2019) 146 (dev173146) DOI: 10.1242/dev.173146)
There was an error in the supplementary data of Development (2019) 146, dev173146 (doi:10.1242/dev.173146). Part of Table S1 was omitted from the supplementary data. Table S1 has now been updated. The updated supplementary data are now available online at http://dev.biologists.org/content/146/6/dev173146.supplemental. The authors apologise to readers for this mistake.
Abstract.
Ostermann L, Ladewig J, Müller F-J, Kesavan J, Tailor J, Smith A, Brüstle O, Koch P (2019). In Vitro Recapitulation of Developmental Transitions in Human Neural Stem Cells.
Stem Cells,
37, 1429-1440.
Abstract:
In Vitro Recapitulation of Developmental Transitions in Human Neural Stem Cells.
During nervous system development, early neuroepithelial stem (NES) cells with a highly polarized morphology and responsiveness to regionalizing morphogens give rise to radial glia (RG) cells, which generate region-specific neurons. Recently, stable neural cell populations reminiscent of NES cells have been obtained from pluripotent stem cells and the fetal human hindbrain. Here, we explore whether these cell populations, similar to their in vivo counterparts, can give rise to neural stem (NS) cells with RG-like properties and whether region-specific NS cells can be generated from NES cells with different regional identities. In vivo RG cells are thought to form from NES cells with the onset of neurogenesis. Therefore, we cultured NES cells temporarily in differentiating conditions. Upon reinitiation of growth factor treatment, cells were found to enter a developmental stage reflecting major characteristics of RG-like NS cells. These NES cell-derived NS cells exhibited a very similar morphology and marker expression as primary NS cells generated from human fetal tissue, indicating that conversion of NES cells into NS cells recapitulates the developmental progression of early NES cells into RG cells observed in vivo. Importantly, NS cells generated from NES cells with different regional identities exhibited stable region-specific transcription factor expression and generated neurons appropriate for their positional identity. Stem Cells 2019;37:1429-1440.
Abstract.
Kleine-Brüggeney H, van Vliet LD, Mulas C, Gielen F, Agley CC, Silva JCR, Smith A, Chalut K, Hollfelder F (2019). Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation.
Small,
15(5).
Abstract:
Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation
Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (Ø: 80 µm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g. in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.
Abstract.
Bredenkamp N, Stirparo GG, Nichols J, Smith A, Guo G (2019). The Cell-Surface Marker Sushi Containing Domain 2 Facilitates Establishment of Human Naive Pluripotent Stem Cells.
Stem Cell Reports,
12(6), 1212-1222.
Abstract:
The Cell-Surface Marker Sushi Containing Domain 2 Facilitates Establishment of Human Naive Pluripotent Stem Cells.
Recently naive human pluripotent stem cells (hPSCs) have been described that relate to an earlier stage of development than conventional hPSCs. Naive hPSCs remain challenging to generate and authenticate, however. Here we report that Sushi Containing Domain 2 (SUSD2) is a robust cell-surface marker of naive hPSCs in the embryo and in vitro. SUSD2 transcripts are enriched in the pre-implantation epiblast of human blastocysts and immunostaining shows localization of SUSD2 to KLF17-positive epiblast cells. SUSD2 mRNA is strongly expressed in naive hPSCs but is negligible in other hPSCs. SUSD2 immunostaining of live or fixed cells provides unambiguous discrimination of naive versus conventional hPSCs. SUSD2 staining or flow cytometry enable monitoring of naive hPSCs in maintenance culture, and their isolation and quantification during resetting of conventional hPSCs or somatic cell reprogramming. Thus SUSD2 is a powerful non-invasive tool for reliable identification and purification of the naive hPSC phenotype.
Abstract.
Author URL.
Bredenkamp N, Yang J, Clarke J, Stirparo GG, von Meyenn F, Dietmann S, Baker D, Drummond R, Ren Y, Li D, et al (2019). Wnt Inhibition Facilitates RNA-Mediated Reprogramming of Human Somatic Cells to Naive Pluripotency.
Stem Cell Reports,
13(6), 1083-1098.
Abstract:
Wnt Inhibition Facilitates RNA-Mediated Reprogramming of Human Somatic Cells to Naive Pluripotency.
In contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs.
Abstract.
Author URL.
2018
Guo G, von Meyenn F, Rostovskaya M, Clarke J, Dietmann S, Baker D, Sahakyan A, Myers S, Bertone P, Reik W, et al (2018). Epigenetic resetting of human pluripotency (vol 144, pg 2748, 2017).
DEVELOPMENT,
145(8).
Author URL.
Zhang M, Leitch HG, Tang WWC, Festuccia N, Hall-Ponsele E, Nichols J, Surani MA, Smith A, Chambers I (2018). Esrrb Complementation Rescues Development of Nanog-Null Germ Cells.
Cell Rep,
22, 332-339.
Abstract:
Esrrb Complementation Rescues Development of Nanog-Null Germ Cells.
The transcription factors (TFs) Nanog and Esrrb play important roles in embryonic stem cells (ESCs) and during primordial germ-cell (PGC) development. Esrrb is a positively regulated direct target of NANOG in ESCs that can substitute qualitatively for Nanog function in ESCs. Whether this functional substitution extends to the germline is unknown. Here, we show that germline deletion of Nanog reduces PGC numbers 5-fold at midgestation. Despite this quantitative depletion, Nanog-null PGCs can complete germline development in contrast to previous findings. PGC-like cell (PGCLC) differentiation of Nanog-null ESCs is also impaired, with Nanog-null PGCLCs showing decreased proliferation and increased apoptosis. However, induced expression of Esrrb restores PGCLC numbers as efficiently as Nanog. These effects are recapitulated in vivo: knockin of Esrrb to Nanog restores PGC numbers to wild-type levels and results in fertile adult mice. These findings demonstrate that Esrrb can replace Nanog function in germ cells.
Abstract.
Stirparo GG, Boroviak T, Guo G, Nichols J, Smith A, Bertone P (2018). Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast.
Development (Cambridge),
145(3).
Abstract:
Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast
Single-cell profiling techniques create opportunities to delineate cell fate progression in mammalian development. Recent studies have provided transcriptome data from human pre-implantation embryos, in total comprising nearly 2000 individual cells. Interpretation of these data is confounded by biological factors, such as variable embryo staging and cell-type ambiguity, as well as technical challenges in the collective analysis of datasets produced with different sample preparation and sequencing protocols. Here, we address these issues to assemble a complete gene expression time course spanning human pre-implantation embryogenesis. We identify key transcriptional features over developmental time and elucidate lineage-specific regulatory networks. We resolve post-hoc cell-type assignment in the blastocyst, and define robust transcriptional prototypes that capture epiblast and primitive endoderm lineages. Examination of human pluripotent stem cell transcriptomes in this framework identifies culture conditions that sustain a naïve state pertaining to the inner cell mass. Our approach thus clarifies understanding both of lineage segregation in the early human embryo and of in vitro stem cell identity, and provides an analytical resource for comparative molecular embryology.
Abstract.
Stirparo GG, Boroviak T, Guo G, Nichols J, Smith A, Bertone P (2018). Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast (vol 145, dev158501, 2018).
DEVELOPMENT,
145(15).
Author URL.
Stirparo GG, Boroviak T, Guo G, Nichols J, Smith A, Bertone P (2018). Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human preimplantation epiblast.
DevelopmentAbstract:
Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human preimplantation epiblast
Single-cell profiling techniques create opportunities to delineate cell fate progression in mammalian development. Recent studies provide transcriptome data from human preimplantation embryos, in total comprising nearly 2000 individual cells. Interpretation of these data is confounded by biological factors such as variable embryo staging and cell-type ambiguity, as well as technical challenges in the collective analysis of datasets produced with different sample preparation and sequencing protocols. Here we address these issues to assemble a complete gene expression time course spanning human preimplantation embryogenesis. We identify key transcriptional features over developmental time and elucidate lineage-specific regulatory networks. We resolve post hoc cell-type assignment in the blastocyst, and define robust transcriptional prototypes that capture epiblast and primitive endoderm lineages. Examination of human pluripotent stem cell transcriptomes in this framework identifies culture conditions that sustain a naïve state pertaining to the inner cell mass. Our approach thus clarifies understanding both of lineage segregation in the early human embryo and of in vitro stem cell identity, and provides an analytical resource for comparative molecular embryology.
Abstract.
Dorn T, Kornherr J, Parrotta EI, Zawada D, Ayetey H, Santamaria G, Iop L, Mastantuono E, Sinnecker D, Goedel A, et al (2018). Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.
EMBO J,
37Abstract:
Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.
Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages.
Abstract.
Sutherland L, Ruhe M, Gattegno-Ho D, Mann K, Greaves J, Koscielniak M, Meek S, Lu Z, Waterfall M, Taylor R, et al (2018). LIF-dependent survival of embryonic stem cells is regulated by a novel palmitoylated Gab1 signalling protein.
J Cell Sci,
131(18).
Abstract:
LIF-dependent survival of embryonic stem cells is regulated by a novel palmitoylated Gab1 signalling protein.
The cytokine leukaemia inhibitory factor (LIF) promotes self-renewal of mouse embryonic stem cells (ESCs) through activation of the transcription factor Stat3. However, the contribution of other ancillary pathways stimulated by LIF in ESCs, such as the MAPK and PI3K pathways, is less well understood. We show here that naive-type mouse ESCs express high levels of a novel effector of the MAPK and PI3K pathways. This effector is an isoform of the Gab1 (Grb2-associated binder protein 1) adaptor protein that lacks the N-terminal pleckstrin homology (PH) membrane-binding domain. Although not essential for rapid unrestricted growth of ESCs under optimal conditions, the novel Gab1 variant (Gab1β) is required for LIF-mediated cell survival under conditions of limited nutrient availability. This enhanced survival is absolutely dependent upon a latent palmitoylation site that targets Gab1β directly to ESC membranes. These results show that constitutive association of Gab1 with membranes through a novel mechanism promotes LIF-dependent survival of murine ESCs in nutrient-poor conditions.
Abstract.
Author URL.
Nett IR, Mulas C, Gatto L, Lilley KS, Smith A (2018). Negative feedback via RSK modulates Erk-dependent progression from naïve pluripotency.
EMBO Rep,
19Abstract:
Negative feedback via RSK modulates Erk-dependent progression from naïve pluripotency.
Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signalling is implicated in initiation of embryonic stem (ES) cell differentiation. The pathway is subject to complex feedback regulation. Here, we examined the ERK-responsive phosphoproteome in ES cells and identified the negative regulator RSK1 as a prominent target. We used CRISPR/Cas9 to create combinatorial mutations in RSK family genes. Genotypes that included homozygous null mutations in Rps6ka1, encoding RSK1, resulted in elevated ERK phosphorylation. These RSK-depleted ES cells exhibit altered kinetics of transition into differentiation, with accelerated downregulation of naïve pluripotency factors, precocious expression of transitional epiblast markers and early onset of lineage specification. We further show that chemical inhibition of RSK increases ERK phosphorylation and expedites ES cell transition without compromising multilineage potential. These findings demonstrate that the ERK activation profile influences the dynamics of pluripotency progression and highlight the role of signalling feedback in temporal control of cell state transitions.
Abstract.
Kinoshita M, Smith A (2018). Pluripotency Deconstructed.
Dev Growth Differ,
60, 44-52.
Abstract:
Pluripotency Deconstructed.
Pluripotency denotes the flexible capacity of single cells to give rise to all somatic lineages and typically also the germline. Mouse ES cells and post-implantation epiblast-derived stem cells (EpiSC) are widely used pluripotent cell culture systems. These two in vitro stem cell types have divergent characteristics. They are considered as representative of distinct developmental stages, distinguished by using the terms "naïve" and "primed". A binary description is an over-simplification, however. Here, we discuss an intermediate stage of pluripotency that we term "formative". Formative pluripotency features a gene regulatory network switch from the naïve state and comprises capacitation of enhancers, signaling pathways and epigenetic machinery in order to install competence for lineage specification.
Abstract.
Boroviak T, Stirparo GG, Dietmann S, Hernando-Herraez I, Mohammed H, Reik W, Smith A, Sasaki E, Nichols J, Bertone P, et al (2018). Single cell transcriptome analysis of human, marmoset and mouse embryos reveals common and divergent features of preimplantation development.
Development,
145Abstract:
Single cell transcriptome analysis of human, marmoset and mouse embryos reveals common and divergent features of preimplantation development.
The mouse embryo is the canonical model for mammalian preimplantation development. Recent advances in single cell profiling allow detailed analysis of embryogenesis in other eutherian species, including human, to distinguish conserved from divergent regulatory programs and signalling pathways in the rodent paradigm. Here, we identify and compare transcriptional features of human, marmoset and mouse embryos by single cell RNA-seq. Zygotic genome activation correlates with the presence of polycomb repressive complexes in all three species, while ribosome biogenesis emerges as a predominant attribute in primate embryos, supporting prolonged translation of maternally deposited RNAs. We find that transposable element expression signatures are species, stage and lineage specific. The pluripotency network in the primate epiblast lacks certain regulators that are operative in mouse, but encompasses WNT components and genes associated with trophoblast specification. Sequential activation of GATA6, SOX17 and GATA4 markers of primitive endoderm identity is conserved in primates. Unexpectedly, OTX2 is also associated with primitive endoderm specification in human and non-human primate blastocysts. Our cross-species analysis demarcates both conserved and primate-specific features of preimplantation development, and underscores the molecular adaptability of early mammalian embryogenesis.
Abstract.
2017
Greaves RB, Dietmann S, Smith A, Stepney S, Halley JD (2017). A conceptual and computational framework for modelling and understanding the non-equilibrium gene regulatory networks of mouse embryonic stem cells.
PLoS Comput Biol,
13, e1005713-e1005713.
Abstract:
A conceptual and computational framework for modelling and understanding the non-equilibrium gene regulatory networks of mouse embryonic stem cells.
The capacity of pluripotent embryonic stem cells to differentiate into any cell type in the body makes them invaluable in the field of regenerative medicine. However, because of the complexity of both the core pluripotency network and the process of cell fate computation it is not yet possible to control the fate of stem cells. We present a theoretical model of stem cell fate computation that is based on Halley and Winkler’s Branching Process Theory (BPT) and on Greaves et al.’s agent-based computer simulation derived from that theoretical model. BPT abstracts the complex production and action of a Transcription Factor (TF) into a single critical branching process that may dissipate, maintain, or become supercritical. Here we take the single TF model and extend it to multiple interacting TFs, and build an agent-based simulation of multiple TFs to investigate the dynamics of such coupled systems. We have developed the simulation and the theoretical model together, in an iterative manner, with the aim of obtaining a deeper understanding of stem cell fate computation, in order to influence experimental efforts, which may in turn influence the outcome of cellular differentiation. The model used is an example of self-organization and could be more widely applicable to the modelling of other complex systems. The simulation based on this model, though currently limited in scope in terms of the biology it represents, supports the utility of the Halley and Winkler branching process model in describing the behaviour of stem cell gene regulatory networks. Our simulation demonstrates three key features: (i) the existence of a critical value of the branching process parameter, dependent on the details of the cistrome in question; (ii) the ability of an active cistrome to "ignite" an otherwise fully dissipated cistrome, and drive it to criticality; (iii) how coupling cistromes together can reduce their critical branching parameter values needed to drive them to criticality.
Abstract.
Li MA, Amaral PP, Cheung P, Bergmann JH, Kinoshita M, Kalkan T, Ralser M, Robson S, von Meyenn F, Paramor M, et al (2017). A lncRNA fine tunes the dynamics of a cell state transition involving Lin28, let-7 and de novo DNA methylation.
Elife,
6Abstract:
A lncRNA fine tunes the dynamics of a cell state transition involving Lin28, let-7 and de novo DNA methylation.
Execution of pluripotency requires progression from the naïve status represented by mouse embryonic stem cells (ESCs) to a state capacitated for lineage specification. This transition is coordinated at multiple levels. Non-coding RNAs may contribute to this regulatory orchestra. We identified a rodent-specific long non-coding RNA (lncRNA) linc1281, hereafter Ephemeron (Eprn), that modulates the dynamics of exit from naïve pluripotency. Eprn deletion delays the extinction of ESC identity, an effect associated with perduring Nanog expression. In the absence of Eprn, Lin28a expression is reduced which results in persistence of let-7 microRNAs, and the up-regulation of de novo methyltransferases Dnmt3a/b is delayed. Dnmt3a/b deletion retards ES cell transition, correlating with delayed Nanog promoter methylation and phenocopying loss of Eprn or Lin28a. The connection from lncRNA to miRNA and DNA methylation facilitates the acute extinction of naïve pluripotency, a pre-requisite for rapid progression from preimplantation epiblast to gastrulation in rodents. Eprn illustrates how lncRNAs may introduce species-specific network modulations.
Abstract.
Guo G, von Meyenn F, Rostovskaya M, Clarke J, Dietmann S, Baker D, Sahakyan A, Myers S, Bertone P, Reik W, et al (2017). Epigenetic resetting of human pluripotency.
Development (Cambridge),
144(15), 2748-2763.
Abstract:
Epigenetic resetting of human pluripotency
Much attention has focussed on the conversion of human pluripotent stem cells (PSCs) to a more naïve developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24 h. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from that of primed PSCs and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to a level equivalent to that in the ICM and is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of biallelic X-linked gene transcription indicates reactivation of the silenced X chromosome. On reconversion to primed status, XIST-induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells.
Abstract.
Guo G, von Meyenn F, Rostovskaya M, Clarke J, Dietmann S, Baker D, Sahakyan A, Myers S, Bertone P, Reik W, et al (2017). Epigenetic resetting of human pluripotency.
Smith A (2017). Formative pluripotency: the executive phase in a developmental continuum.
Development,
144, 365-373.
Abstract:
Formative pluripotency: the executive phase in a developmental continuum.
The regulative capability of single cells to give rise to all primary embryonic lineages is termed pluripotency. Observations of fluctuating gene expression and phenotypic heterogeneity in vitro have fostered a conception of pluripotency as an intrinsically metastable and precarious state. However, in the embryo and in defined culture environments the properties of pluripotent cells change in an orderly sequence. Two phases of pluripotency, called naïve and primed, have previously been described. In this Hypothesis article, a third phase, called formative pluripotency, is proposed to exist as part of a developmental continuum between the naïve and primed phases. The formative phase is hypothesised to be enabling for the execution of pluripotency, entailing remodelling of transcriptional, epigenetic, signalling and metabolic networks to constitute multi-lineage competence and responsiveness to specification cues.
Abstract.
Chen Y, Spitzer S, Agathou S, Karadottir RT, Smith A (2017). Gene Editing in Rat Embryonic Stem Cells to Produce in Vitro Models and in Vivo Reporters.
Stem Cell Reports,
9, 1262-1274.
Abstract:
Gene Editing in Rat Embryonic Stem Cells to Produce in Vitro Models and in Vivo Reporters.
Rat embryonic stem cells (ESCs) offer the potential for sophisticated genome engineering in this valuable biomedical model species. However, germline transmission has been rare following conventional homologous recombination and clonal selection. Here, we used the CRISPR/Cas9 system to target genomic mutations and insertions. We first evaluated utility for directed mutagenesis and recovered clones with biallelic deletions in Lef1. Mutant cells exhibited reduced sensitivity to glycogen synthase kinase 3 inhibition during self-renewal. We then generated a non-disruptive knockin of dsRed at the Sox10 locus. Two clones produced germline chimeras. Comparative expression of dsRed and SOX10 validated the fidelity of the reporter. To illustrate utility, live imaging of dsRed in neonatal brain slices was employed to visualize oligodendrocyte lineage cells for patch-clamp recording. Overall, these results show that CRISPR/Cas9 gene editing technology in germline-competent rat ESCs is enabling for in vitro studies and for generating genetically modified rats.
Abstract.
Mulas C, Kalkan T, Smith A (2017). NODAL Secures Pluripotency upon Embryonic Stem Cell Progression from the Ground State.
Stem Cell Reports,
9, 77-91.
Abstract:
NODAL Secures Pluripotency upon Embryonic Stem Cell Progression from the Ground State.
Naive mouse embryonic stem cells (ESCs) can develop multiple fates, but the cellular and molecular processes that enable lineage competence are poorly characterized. Here, we investigated progression from the ESC ground state in defined culture. We utilized downregulation of Rex1::GFPd2 to track the loss of ESC identity. We found that cells that have newly downregulated this reporter have acquired capacity for germline induction. They can also be efficiently specified for different somatic lineages, responding more rapidly than naive cells to inductive cues. Inhibition of autocrine NODAL signaling did not alter kinetics of exit from the ESC state but compromised both germline and somatic lineage specification. Transient inhibition prior to loss of ESC identity was sufficient for this effect. Genetic ablation of Nodal reduced viability during early differentiation, consistent with defective lineage specification. These results suggest that NODAL promotes acquisition of multi-lineage competence in cells departing naive pluripotency.
Abstract.
Ying Q-L, Smith A (2017). The Art of Capturing Pluripotency: Creating the Right Culture.
Stem Cell Reports,
8, 1457-1464.
Abstract:
The Art of Capturing Pluripotency: Creating the Right Culture.
Embryonic stem cells (ESCs) are a unique tool for genetic perturbation of mammalian cellular and organismal processes additionally in humans offer unprecedented opportunities for disease modeling and cell therapy. Furthermore, ESCs are a powerful system for exploring the fundamental biology of pluripotency. Indeed understanding the control of self-renewal and differentiation is key to realizing the potential of ESCs. Building on previous observations, we found that mouse ESCs can be derived and maintained with high efficiency through insulation from differentiation cues combined with consolidation of an innate cell proliferation program. This finding of a pluripotent ground state has led to conceptual and practical advances, including the establishment of germline-competent ESCs from recalcitrant mouse strains and for the first time from the rat. Here, we summarize historical and recent progress in defining the signaling environment that supports self-renewal. We compare the contrasting requirements of two types of pluripotent stem cell, naive ESCs and primed post-implantation epiblast stem cells (EpiSCs), and consider the outstanding challenge of generating naive pluripotent stem cells from different mammals.
Abstract.
Kalkan T, Olova N, Roode M, Mulas C, Lee HJ, Nett I, Marks H, Walker R, Stunnenberg HG, Lilley KS, et al (2017). Tracking the embryonic stem cell transition from ground state pluripotency.
Development,
144, 1221-1234.
Abstract:
Tracking the embryonic stem cell transition from ground state pluripotency.
Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naïve pluripotency. Here, we examine the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naïve status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.
Abstract.
2016
Yordanov B, Dunn S-J, Kugler H, Smith A, Martello G, Emmott S (2016). A Method to Identify and Analyze Biological Programs through Automated Reasoning.
NPJ Syst Biol Appl,
2Abstract:
A Method to Identify and Analyze Biological Programs through Automated Reasoning.
Predictive biology is elusive because rigorous, data-constrained, mechanistic models of complex biological systems are difficult to derive and validate. Current approaches tend to construct and examine static interaction network models, which are descriptively rich but often lack explanatory and predictive power, or dynamic models that can be simulated to reproduce known behavior. However, in such approaches implicit assumptions are introduced as typically only one mechanism is considered, and exhaustively investigating all scenarios is impractical using simulation. To address these limitations, we present a methodology based on automated formal reasoning, which permits the synthesis and analysis of the complete set of logical models consistent with experimental observations. We test hypotheses against all candidate models, and remove the need for simulation by characterizing and simultaneously analyzing all mechanistic explanations of observed behavior. Our methodology transforms knowledge of complex biological processes from sets of possible interactions and experimental observations to precise, predictive biological programs governing cell function.
Abstract.
Morrison G, Scognamiglio R, Trumpp A, Smith A (2016). Convergence of cMyc and β-catenin on Tcf7l1 enables endoderm specification.
EMBO J,
35, 356-368.
Abstract:
Convergence of cMyc and β-catenin on Tcf7l1 enables endoderm specification.
The molecular machinery that directs formation of definitive endoderm from pluripotent stem cells is not well understood. Wnt/β-catenin and Nodal signalling have been implicated, but the requirements for lineage specification remain incompletely defined. Here, we demonstrate a potent effect of inhibiting glycogen synthase kinase 3 (GSK3) on definitive endoderm production. We find that downstream of GSK3 inhibition, elevated cMyc and β-catenin act in parallel to reduce transcription and DNA binding, respectively, of the transcriptional repressor Tcf7l1. Tcf7l1 represses FoxA2, a pioneer factor for endoderm specification. Deletion of Tcf7l1 is sufficient to allow upregulation of FoxA2 in the presence of Activin. In wild-type cells, cMyc contributes by reducing Tcf7l1 mRNA, while β-catenin acts on Tcf7l1 protein. GSK3 inhibition is further required for consolidation of endodermal fate via upregulation of Sox17, highlighting sequential roles for Wnt signalling. The identification of a cMyc/β-catenin-Tcf7l1-FoxA2 axis reveals a de-repression mechanism underlying endoderm induction that may be recapitulated in other developmental and patho-logical contexts.
Abstract.
Marks H, Kerstens HHD, Barakat TS, Splinter E, Dirks RAM, van Mierlo G, Joshi O, Wang S-Y, Babak T, Albers CA, et al (2016). Erratum to: Dynamics of gene silencing during X inactivation using allele-specific RNA-seq. Genome Biol, 17, 22-22.
Scognamiglio R, Cabezas-Wallscheid N, Thier MC, Altamura S, Reyes A, Prendergast Á, Baumgärtner D, Carnevalli LS, Atzberger A, Haas S, et al (2016). Myc Depletion Induces a Pluripotent Dormant State Mimicking Diapause.
Cell,
164, 668-680.
Abstract:
Myc Depletion Induces a Pluripotent Dormant State Mimicking Diapause.
Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state.
Abstract.
Guo G, von Meyenn F, Santos F, Chen Y, Reik W, Bertone P, Smith A, Nichols J (2016). Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass.
STEM CELL REPORTS,
6(4), 437-446.
Author URL.
Mulas C, Kalkan T, Smith A (2016). Nodal secures pluripotency upon embryonic stem cell progression from the ground state.
Abstract:
Nodal secures pluripotency upon embryonic stem cell progression from the ground state
SUMMARY Naïve mouse embryonic stem (ES) cells can readily acquire specific fates, but the cellular and molecular processes that enable lineage specification are poorly characterised. Here we investigated progression from the ES cell ground state in adherent culture. We utilised down-regulation of Rex1::GFPd2 to track loss of ES cell identity. We found that cells that have newly down-regulated this reporter have acquired competence for germline induction. They can also be efficiently specified for different somatic lineages, responding more rapidly than naïve cells to inductive cues. Nodal is a candidate autocrine regulator of pluripotency. Abrogation of Nodal signalling did not substantially alter kinetics of exit from the ES cell state, but accelerated subsequent adoption of neural fate at the expense of other lineages. This effect was evident if Nodal was inhibited prior to extinction of ES cell identity. We suggest that Nodal is pivotal for non-neural competence in cells departing naïve pluripotency.
Abstract.
Carbognin E, Betto RM, Soriano ME, Smith AG, Martello G (2016). Stat3 promotes mitochondrial transcription and oxidative respiration during maintenance and induction of naive pluripotency.
EMBO J,
35, 618-634.
Abstract:
Stat3 promotes mitochondrial transcription and oxidative respiration during maintenance and induction of naive pluripotency.
Transcription factor Stat3 directs self-renewal of pluripotent mouse embryonic stem (ES) cells downstream of the cytokine leukemia inhibitory factor (LIF). Stat3 upregulates pivotal transcription factors in the ES cell gene regulatory network to sustain naïve identity. Stat3 also contributes to the rapid proliferation of ES cells. Here, we show that Stat3 increases the expression of mitochondrial-encoded transcripts and enhances oxidative metabolism. Chromatin immunoprecipitation reveals that Stat3 binds to the mitochondrial genome, consistent with direct transcriptional regulation. An engineered form of Stat3 that localizes predominantly to mitochondria is sufficient to support enhanced proliferation of ES cells, but not to maintain their undifferentiated phenotype. Furthermore, during reprogramming from primed to naïve states of pluripotency, Stat3 similarly upregulates mitochondrial transcripts and facilitates metabolic resetting. These findings suggest that the potent stimulation of naïve pluripotency by LIF/Stat3 is attributable to parallel and synergistic induction of both mitochondrial respiration and nuclear transcription factors.
Abstract.
2015
Marks H, Kerstens HHD, Barakat TS, Splinter E, Dirks RAM, van Mierlo G, Joshi O, Wang S-Y, Babak T, Albers CA, et al (2015). Dynamics of gene silencing during X inactivation using allele-specific RNA-seq.
Genome Biol,
16, 149-149.
Abstract:
Dynamics of gene silencing during X inactivation using allele-specific RNA-seq.
BACKGROUND: During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq. RESULTS: Induction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs. Also, the previously characterized gene clusters escaping XCI in human fibroblasts correlate with TADs. CONCLUSIONS: the gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of escape regions with TADs, in mouse and human, suggests that TADs are the primary targets during propagation of XCI over the X chromosome.
Abstract.
Boroviak T, Loos R, Lombard P, Okahara J, Behr R, Sasaki E, Nichols J, Smith A, Bertone P (2015). Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis.
Dev Cell,
35, 366-382.
Abstract:
Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis.
Naive pluripotency is manifest in the preimplantation mammalian embryo. Here we determine transcriptome dynamics of mouse development from the eight-cell stage to postimplantation using lineage-specific RNA sequencing. This method combines high sensitivity and reporter-based fate assignment to acquire the full spectrum of gene expression from discrete embryonic cell types. We define expression modules indicative of developmental state and temporal regulatory patterns marking the establishment and dissolution of naive pluripotency in vivo. Analysis of embryonic stem cells and diapaused embryos reveals near-complete conservation of the core transcriptional circuitry operative in the preimplantation epiblast. Comparison to inner cell masses of marmoset primate blastocysts identifies a similar complement of pluripotency factors but use of alternative signaling pathways. Embryo culture experiments further indicate that marmoset embryos utilize WNT signaling during early lineage segregation, unlike rodents. These findings support a conserved transcription factor foundation for naive pluripotency while revealing species-specific regulatory features of lineage segregation.
Abstract.
Pourquié O, Bruneau B, Keller G, Smith A (2015). Looking inwards: Opening a window onto human development. Development (Cambridge), 142, 1-2.
Pourquié O, Bruneau B, Keller G, Smith A (2015). Looking inwards: opening a window onto human development. Development, 142, 1-2.
Takashima Y, Guo G, Loos R, Nichols J, Ficz G, Krueger F, Oxley D, Santos F, Clarke J, Mansfield W, et al (2015). Resetting Transcription Factor Control Circuitry toward Ground-State Pluripotency in Human (vol 158, pg 1254, 2014).
CELL,
162(2), 452-453.
Author URL.
Rostovskaya M, Bredenkamp N, Smith A (2015). Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells.
Philos Trans R Soc Lond B Biol Sci,
370, 20140365-20140365.
Abstract:
Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells.
Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.
Abstract.
2014
Herberg M, Kalkan T, Glauche I, Smith A, Roeder I (2014). A model-based analysis of culture-dependent phenotypes of mESCs.
PLoS One,
9, e92496-e92496.
Abstract:
A model-based analysis of culture-dependent phenotypes of mESCs.
Mouse embryonic stem cells (mESCs) can be maintained in a proliferative and undifferentiated state over many passages (self-renewal) while retaining the potential to give rise to every cell type of the organism (pluripotency). Autocrine FGF4/Erk signalling has been identified as a major stimulus for fate decisions and lineage commitment in these cells. Recent findings on serum-free culture conditions with specific inhibitors (known as 2i) demonstrate that the inhibition of this pathway reduces transcription factor heterogeneity and is vital to maintain ground state pluripotency of mESCs. We suggest a novel mathematical model to explicitly integrate FGF4/Erk signalling into an interaction network of key pluripotency factors (namely Oct4, Sox2, Nanog and Rex1). The envisaged model allows to explore whether and how proposed mechanisms and feedback regulations can account for different expression patterns in mESC cultures. We demonstrate that an FGF4/Erk-mediated negative feedback is sufficient to induce molecular heterogeneity with respect to Nanog and Rex1 expression and thus critically regulates the propensity for differentiation and the loss of pluripotency. Furthermore, we compare simulation results on the transcription factor dynamics in different self-renewing states and during differentiation with experimental data on a Rex1GFPd2 reporter cell line using flow cytometry and qRT-PCR measurements. Concluding from our results we argue that interaction between FGF4/Erk signalling and Nanog expression qualifies as a key mechanism to manipulate mESC pluripotency. In particular, we infer that ground state pluripotency under 2i is achieved by shifting stable expression pattern of Nanog from a bistable into a monostable regulation impeding stochastic state transitions. Furthermore, we derive testable predictions on altering the degree of Nanog heterogeneity and on the frequency of state transitions in LIF/serum conditions to challenge our model assumptions.
Abstract.
Dunn S-J, Martello G, Yordanov B, Emmott S, Smith AG (2014). Defining an essential transcription factor program for naïve pluripotency.
Science,
344, 1156-1160.
Abstract:
Defining an essential transcription factor program for naïve pluripotency.
The gene regulatory circuitry through which pluripotent embryonic stem (ES) cells choose between self-renewal and differentiation appears vast and has yet to be distilled into an executive molecular program. We developed a data-constrained, computational approach to reduce complexity and to derive a set of functionally validated components and interaction combinations sufficient to explain observed ES cell behavior. This minimal set, the simplest version of which comprises only 16 interactions, 12 components, and three inputs, satisfies all prior specifications for self-renewal and furthermore predicts unknown and nonintuitive responses to compound genetic perturbations with an overall accuracy of 70%. We propose that propagation of ES cell identity is not determined by a vast interactome but rather can be explained by a relatively simple process of molecular computation.
Abstract.
Mohsen-Kanson T, Hafner A-L, Wdziekonski B, Takashima Y, Villageois P, Carrière A, Svensson M, Bagnis C, Chignon-Sicard B, Svensson P-A, et al (2014). Differentiation of human induced pluripotent stem cells into brown and white adipocytes: role of Pax3.
Stem Cells,
32, 1459-1467.
Abstract:
Differentiation of human induced pluripotent stem cells into brown and white adipocytes: role of Pax3.
Identification of molecular mechanisms involved in generation of different types of adipocytes is progressing substantially in mice. However, much less is known regarding characterization of brown (BAP) and white adipocyte progenitors (WAPs) in humans, highlighting the need for an in vitro model of human adipocyte development. Here, we report a procedure to selectively derive BAP and WAPs from human-induced pluripotent stem cells. Molecular characterization of APs of both phenotypes revealed that BMP4, Hox8, Hoxc9, and HoxA5 genes were specifically expressed in WAPs, whereas expression of PRDM16, Dio2, and Pax3 marked BAPs. We focused on Pax3 and we showed that expression of this transcription factor was enriched in human perirenal white adipose tissue samples expressing UCP1 and in human classical brown fat. Finally, functional experiments indicated that Pax3 was a critical player of human AP fate as its ectopic expression led to convert WAPs into brown-like APs. Together, these data support a model in which Pax3 is a new marker of human BAPs and a molecular mediator of their fate. The findings of this study could lead to new anti-obesity therapies based on the recruitment of APs and constitute a platform for investigating in vitro the developmental origins of human white and brown adipocytes.
Abstract.
Leeb M, Dietmann S, Paramor M, Niwa H, Smith A (2014). Genetic exploration of the exit from self-renewal using haploid embryonic stem cells.
Cell Stem Cell,
14, 385-393.
Abstract:
Genetic exploration of the exit from self-renewal using haploid embryonic stem cells.
Self-renewal circuitry in embryonic stem cells (ESCs) is increasingly defined. How the robust pluripotency program is dissolved to enable fate transition is less appreciated. Here we develop a forward genetic approach using haploid ESCs. We created libraries of transposon integrations and screened for persistent self-renewal in differentiation-permissive culture. This yielded multiple mutants in the Fgf/Erk and GSK3/Tcf3 modules known to drive differentiation and in epigenetic modifiers implicated in lineage commitment. We also identified and validated factors not previously considered. These include the conserved small zinc finger protein Zfp706 and the RNA binding protein Pum1. Pum1 targets several mRNAs for naive pluripotency transcription factors and accelerates their downregulation at the onset of differentiation. These findings indicate that the dismantling of pluripotent circuitry proceeds at multiple levels. More broadly they exemplify the power of haploid ESCs for genetic interrogation of developmental processes.
Abstract.
Kalkan T, Smith A (2014). Mapping the route from naive pluripotency to lineage specification.
Philos Trans R Soc Lond B Biol Sci,
369Abstract:
Mapping the route from naive pluripotency to lineage specification.
In the mouse blastocyst, epiblast cells are newly formed shortly before implantation. They possess a unique developmental plasticity, termed naive pluripotency. For development to proceed, this naive state must be subsumed by multi-lineage differentiation within 72 h following implantation. In vitro differentiation of naive embryonic stem cells (ESCs) cultured in controlled conditions provides a tractable system to dissect and understand the process of exit from naive pluripotency and entry into lineage specification. Exploitation of this system in recent large-scale RNAi and mutagenesis screens has uncovered multiple new factors and modules that drive or facilitate progression out of the naive state. Notably, these studies show that the transcription factor network that governs the naive state is rapidly dismantled prior to upregulation of lineage specification markers, creating an intermediate state that we term formative pluripotency. Here, we summarize these findings and propose a road map for state transitions in ESC differentiation that reflects the orderly dynamics of epiblast progression in the embryo.
Abstract.
Yang S-H, Kalkan T, Morissroe C, Marks H, Stunnenberg H, Smith A, Sharrocks AD (2014). Otx2 and Oct4 drive early enhancer activation during embryonic stem cell transition from naive pluripotency.
Cell Rep,
7, 1968-1981.
Abstract:
Otx2 and Oct4 drive early enhancer activation during embryonic stem cell transition from naive pluripotency.
Embryonic stem cells (ESCs) are unique in that they have the capacity to differentiate into all of the cell types in the body. We know a lot about the complex transcriptional control circuits that maintain the naive pluripotent state under self-renewing conditions but comparatively less about how cells exit from this state in response to differentiation stimuli. Here, we examined the role of Otx2 in this process in mouse ESCs and demonstrate that it plays a leading role in remodeling the gene regulatory networks as cells exit from ground state pluripotency. Otx2 drives enhancer activation through affecting chromatin marks and the activity of associated genes. Mechanistically, Oct4 is required for Otx2 expression, and reciprocally, Otx2 is required for efficient Oct4 recruitment to many enhancer regions. Therefore, the Oct4-Otx2 regulatory axis actively establishes a new regulatory chromatin landscape during the early events that accompany exit from ground state pluripotency.
Abstract.
Takashima Y, Guo G, Loos R, Nichols J, Ficz G, Krueger F, Oxley D, Santos F, Clarke J, Mansfield W, et al (2014). Resetting transcription factor control circuitry toward ground-state pluripotency in human.
Cell,
158(6), 1254-1269.
Abstract:
Resetting transcription factor control circuitry toward ground-state pluripotency in human.
Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells. Copyright © 2014 the Authors. Published by Elsevier Inc. All rights reserved.
Abstract.
Boroviak T, Loos R, Bertone P, Smith A, Nichols J (2014). The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification.
Nat Cell Biol,
16, 516-528.
Abstract:
The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification.
The precise relationship of embryonic stem cells (ESCs) to cells in the mouse embryo remains controversial. We present transcriptional and functional data to identify the embryonic counterpart of ESCs. Marker profiling shows that ESCs are distinct from early inner cell mass (ICM) and closely resemble pre-implantation epiblast. A characteristic feature of mouse ESCs is propagation without ERK signalling. Single-cell culture reveals that cell-autonomous capacity to thrive when the ERK pathway is inhibited arises late during blastocyst development and is lost after implantation. The frequency of deriving clonal ESC lines suggests that all E4.5 epiblast cells can become ESCs. We further show that ICM cells from early blastocysts can progress to ERK independence if provided with a specific laminin substrate. These findings suggest that formation of the epiblast coincides with competence for ERK-independent self-renewal in vitro and consequent propagation as ESC lines.
Abstract.
Martello G, Smith A (2014). The nature of embryonic stem cells.
Annu Rev Cell Dev Biol,
30, 647-675.
Abstract:
The nature of embryonic stem cells.
Mouse embryonic stem (ES) cells perpetuate in vitro the broad developmental potential of naïve founder cells in the preimplantation embryo. ES cells self-renew relentlessly in culture but can reenter embryonic development seamlessly, differentiating on schedule to form all elements of the fetus. Here we review the properties of these remarkable cells. Arising from the stability, homogeneity, and equipotency of ES cells, we consider the concept of a pluripotent ground state. We evaluate the authenticity of ES cells in relation to cells in the embryo and examine their utility for dissecting mechanisms that confer pluripotency and that execute fate choice. We summarize current knowledge of the transcription factor circuitry that governs the ES cell state and discuss the opportunity to expose molecular logic further through iterative computational modeling and experimentation. Finally, we present a perspective on unresolved questions, including the challenge of deriving ground state pluripotent stem cells from non-rodent species.
Abstract.
2013
Danovi D, Folarin A, Gogolok S, Ender C, Elbatsh AMO, Engström PG, Stricker SH, Gagrica S, Georgian A, Yu D, et al (2013). A high-content small molecule screen identifies sensitivity of glioblastoma stem cells to inhibition of polo-like kinase 1.
PLoS One,
8, e77053-e77053.
Abstract:
A high-content small molecule screen identifies sensitivity of glioblastoma stem cells to inhibition of polo-like kinase 1.
Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF(-/-), or p53(-/-)), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.
Abstract.
McLaren D, Gorba T, Marguerie de Rotrou A, Pillai G, Chappell C, Stacey A, Lingard S, Falk A, Smith A, Koch P, et al (2013). Automated large-scale culture and medium-throughput chemical screen for modulators of proliferation and viability of human induced pluripotent stem cell-derived neuroepithelial-like stem cells.
J Biomol Screen,
18, 258-268.
Abstract:
Automated large-scale culture and medium-throughput chemical screen for modulators of proliferation and viability of human induced pluripotent stem cell-derived neuroepithelial-like stem cells.
The aim of this study was to demonstrate proof-of-concept feasibility for the use of human neural stem cells (NSCs) for high-throughput screening (HTS) applications. For this study, an adherent human induced pluripotent stem (iPS) cell-derived long-term, self-renewing, neuroepithelial-like stem (lt-NES) cell line was selected as a representative NSC. Here, we describe the automated large-scale serum-free culture ("scale-up") of human lt-NES cells on the CompacT SelecT cell culture robotic platform, followed by their subsequent automated "scale-out" into a microwell plate format. We also report a medium-throughput screen of 1000 compounds to identify modulators of neural stem cell proliferation and/or survival. The screen was performed on two independent occasions using a cell viability assay with end-point reading resulting in the identification of 24 potential hit compounds, 5 of which were found to increase the proliferation and/or survival of human lt-NES on both occasions. Follow-up studies confirmed a dose-dependent effect of one of the hit compounds, which was a Cdk-2 modulator. This approach could be further developed as part of a strategy to screen compounds to either improve the procedures for the in vitro expansion of neural stem cells or to potentially modulate endogenous neural stem cell behavior in the diseased nervous system.
Abstract.
Martello G, Sugimoto T, Diamanti E, Joshi A, Hannah R, Ohtsuka S, Göttgens B, Niwa H, Smith A (2013). Erratum: Esrrb is a pivotal target of the Gsk3/Tcf3 axis regulating embryonic stem cell self-renewal (Cell Stem Cell (2012) 11 (491-504)). Cell Stem Cell, 12, 630-630.
Betschinger J, Nichols J, Dietmann S, Corrin PD, Paddison PJ, Smith A (2013). Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3.
Cell,
153, 335-347.
Abstract:
Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3.
Factors that sustain self-renewal of mouse embryonic stem cells (ESCs) are well described. In contrast, the machinery regulating exit from pluripotency is ill defined. In a large-scale small interfering RNA (siRNA) screen, we found that knockdown of the tumor suppressors Folliculin (Flcn) and Tsc2 prevent ESC commitment. Tsc2 lies upstream of mammalian target of rapamycin (mTOR), whereas Flcn acts downstream and in parallel. Flcn with its interaction partners Fnip1 and Fnip2 drives differentiation by restricting nuclear localization and activity of the bHLH transcription factor Tfe3. Conversely, enforced nuclear Tfe3 enables ESCs to withstand differentiation conditions. Genome-wide location and functional analyses showed that Tfe3 directly integrates into the pluripotency circuitry through transcriptional regulation of Esrrb. These findings identify a cell-intrinsic rheostat for destabilizing ground-state pluripotency to allow lineage commitment. Congruently, stage-specific subcellular relocalization of Tfe3 suggests that Flcn-Fnip1/2 contributes to developmental progression of the pluripotent epiblast in vivo.
Abstract.
Martello G, Bertone P, Smith A (2013). Identification of the missing pluripotency mediator downstream of leukaemia inhibitory factor.
EMBO J,
32, 2561-2574.
Abstract:
Identification of the missing pluripotency mediator downstream of leukaemia inhibitory factor.
Self-renewal of pluripotent mouse embryonic stem (ES) cells is sustained by the cytokine leukaemia inhibitory factor (LIF) acting through the transcription factor Stat3. Several targets of Stat3 have previously been identified, most notably the reprogramming factor Klf4. However, such factors are neither required nor sufficient for the potent effect of LIF. We took advantage of Stat3 null ES cells to confirm that Stat3 mediates the self-renewal response to LIF. Through comparative transcriptome analysis intersected with genome location data, we arrived at a set of candidate transcription factor effectors. Among these, Tfcp2l1 (also known as Crtr-1) was most abundant. Constitutive expression of Tfcp2l1 at levels similar to those induced by LIF effectively substituted for LIF or Stat3 in sustaining clonal self-renewal and pluripotency. Conversely, knockdown of Tfcp2l1 profoundly compromised responsiveness to LIF. We further found that Tfcp2l1 is both necessary and sufficient to direct molecular reprogramming of post-implantation epiblast stem cells to naïve pluripotency. These results establish Tfcp2l1 as the principal bridge between LIF/Stat3 input and the transcription factor core of naïve pluripotency.
Abstract.
Leitch HG, McEwen KR, Turp A, Encheva V, Carroll T, Grabole N, Mansfield W, Nashun B, Knezovich JG, Smith A, et al (2013). Naive pluripotency is associated with global DNA hypomethylation.
Nat Struct Mol Biol,
20, 311-316.
Abstract:
Naive pluripotency is associated with global DNA hypomethylation.
Naive pluripotent embryonic stem cells (ESCs) and embryonic germ cells (EGCs) are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs, in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however, we find that EGCs and ESCs exhibit equivalent global DNA methylation levels. Inhibition of MEK and Gsk3b by 2i conditions leads to pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i, and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instills a naive pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs.
Abstract.
Smith A (2013). Nanog heterogeneity: tilting at windmills?.
Cell Stem Cell,
13, 6-7.
Abstract:
Nanog heterogeneity: tilting at windmills?
Fluctuating expression of transcription factors in embryonic stem cells is an alluring observation, but, as outlined by two articles in this issue, appearances can be misleading.
Abstract.
Brilli E, Reitano E, Conti L, Conforti P, Gulino R, Consalez GG, Cesana E, Smith A, Rossi F, Cattaneo E, et al (2013). Neural stem cells engrafted in the adult brain fuse with endogenous neurons.
Stem Cells Dev,
22, 538-547.
Abstract:
Neural stem cells engrafted in the adult brain fuse with endogenous neurons.
Neural stem cells (NSCs) have become promising tools for basic research and regenerative medicine. Intracerebral transplantation studies have suggested that these cells may be able to adopt neuronal phenotypes typical of their engraftment site and to establish appropriate connections in the recipient circuitries. Here, we examined the in vivo neurogenic competence of well-characterized NSC lines subjected to in vitro priming and subsequent implantation into the adult intact mouse brain. Upon implantation into the hippocampus and, less frequently, in the striatum and in the cerebral cortex, numerous green fluorescent protein (GFP)-tagged cells acquired differentiated features indistinguishable from resident neurons. Upon closer examination, however, we found that this outcome resulted from fusion of donor cells with local neuronal elements generating long-term persistent GFP(+) neuronal hybrids. This fusogenic behavior of NSCs was unexpected and also observed in coculture with E18 hippocampal immature neural cells, but not with microglia or astrocytes. Similar findings were consistently obtained with different NSC lines, mouse recipients, and donor cell-labeling methods. The frequent and cell type-specific fusion of donor NSCs with host neurons highlights a previously underestimated biological property of the nervous tissue that might prove profitable for basic and therapeutically oriented studies.
Abstract.
Leitch HG, Nichols J, Humphreys P, Mulas C, Martello G, Lee C, Jones K, Surani MA, Smith A (2013). Rebuilding pluripotency from primordial germ cells.
Stem Cell Reports,
1, 66-78.
Abstract:
Rebuilding pluripotency from primordial germ cells.
Mammalian primordial germ cells (PGCs) are unipotent progenitors of the gametes. Nonetheless, they can give rise directly to pluripotent stem cells in vitro or during teratocarcinogenesis. This conversion is inconsistent, however, and has been difficult to study. Here, we delineate requirements for efficient resetting of pluripotency in culture. We demonstrate that in defined conditions, routinely 20% of PGCs become EG cells. Conversion can occur from the earliest specified PGCs. The entire process can be tracked from single cells. It is driven by leukemia inhibitory factor (LIF) and the downstream transcription factor STAT3. In contrast, LIF signaling is not required during germ cell ontogeny. We surmise that ectopic LIF/STAT3 stimulation reconstructs latent pluripotency and self-renewal. Notably, STAT3 targets are significantly upregulated in germ cell tumors, suggesting that dysregulation of this pathway may underlie teratocarcinogenesis. These findings demonstrate that EG cell formation is a robust experimental system for exploring mechanisms involved in reprogramming and cancer.
Abstract.
Bianco P, Barker R, Brüstle O, Cattaneo E, Clevers H, Daley GQ, De Luca M, Goldstein L, Lindvall O, Mummery C, et al (2013). Regulation of stem cell therapies under attack in Europe: for whom the bell tolls.
EMBO J,
32, 1489-1495.
Abstract:
Regulation of stem cell therapies under attack in Europe: for whom the bell tolls.
At the time of writing, the Italian Parliament is debating a new law that would make it legal to practice an unproven stem cell treatment in public hospitals. The treatment, offered by a private non-medical organization, may not be safe, lacks a rationale, and violates current national laws and European regulations. This case raises multiple concerns, most prominently the urgent need to protect patients who are severely ill, exposed to significant risks, and vulnerable to exploitation. The scientific community must consider the context-social, financial, medical, legal-in which stem cell science is currently situated and the need for stringent regulation. Additional concerns are emerging. These emanate from the novel climate, created within science itself, and stem cell science in particular, by the currently prevailing model of ’translational medicine’. Only rigorous science and rigorous regulation can ensure translation of science into effective therapies rather than into ineffective market products, and mark, at the same time, the sharp distinction between the striving for new therapies and the deceit of patients.
Abstract.
Chen Y, Blair K, Smith A (2013). Robust self-renewal of rat embryonic stem cells requires fine-tuning of glycogen synthase kinase-3 inhibition.
Stem Cell Reports,
1, 209-217.
Abstract:
Robust self-renewal of rat embryonic stem cells requires fine-tuning of glycogen synthase kinase-3 inhibition.
Germline-competent embryonic stem cells (ESCs) have been derived from mice and rats using culture conditions that include an inhibitor of glycogen synthase kinase 3 (GSK3). However, rat ESCs remain susceptible to sporadic differentiation. Here, we show that unsolicited differentiation is attributable to overinhibition of GSK3. The self-renewal effect of inhibiting GSK3 is mediated via β-catenin, which abrogates the repressive action of TCF3 on core pluripotency genes. In rat ESCs, however, GSK3 inhibition also leads to activation of differentiation-associated genes, notably lineage specification factors Cdx2 and T. Lowered GSK3 inhibition reduces differentiation and enhances clonogenicity and self-renewal. The differential sensitivity of rat ESCs to GSK3 inhibition is linked to elevated expression of the canonical Wnt pathway effector LEF1. These findings reveal that optimal GSK3 inhibition for ESC propagation is influenced by the balance of TCF/LEF factors and can vary between species.
Abstract.
Pourquié O, Bruneau B, Götz M, Keller G, Smith A (2013). Stem cells and regeneration: a special issue. Development, 140, 2445-2445.
Tailor J, Kittappa R, Leto K, Gates M, Borel M, Paulsen O, Spitzer S, Karadottir RT, Rossi F, Falk A, et al (2013). Stem cells expanded from the human embryonic hindbrain stably retain regional specification and high neurogenic potency.
J Neurosci,
33, 12407-12422.
Abstract:
Stem cells expanded from the human embryonic hindbrain stably retain regional specification and high neurogenic potency.
Stem cell lines that faithfully maintain the regional identity and developmental potency of progenitors in the human brain would create new opportunities in developmental neurobiology and provide a resource for generating specialized human neurons. However, to date, neural progenitor cultures derived from the human brain have either been short-lived or exhibit restricted, predominantly glial, differentiation capacity. Pluripotent stem cells are an alternative source, but to ascertain definitively the identity and fidelity of cell types generated solely in vitro is problematic. Here, we show that hindbrain neuroepithelial stem (hbNES) cells can be derived and massively expanded from early human embryos (week 5-7, Carnegie stage 15-17). These cell lines are propagated in adherent culture in the presence of EGF and FGF2 and retain progenitor characteristics, including SOX1 expression, formation of rosette-like structures, and high neurogenic capacity. They generate GABAergic, glutamatergic and, at lower frequency, serotonergic neurons. Importantly, hbNES cells stably maintain hindbrain specification and generate upper rhombic lip derivatives on exposure to bone morphogenetic protein (BMP). When grafted into neonatal rat brain, they show potential for integration into cerebellar development and produce cerebellar granule-like cells, albeit at low frequency. hbNES cells offer a new system to study human cerebellar specification and development and to model diseases of the hindbrain. They also provide a benchmark for the production of similar long-term neuroepithelial-like stem cells (lt-NES) from pluripotent cell lines. To our knowledge, hbNES cells are the first demonstration of highly expandable neuroepithelial stem cells derived from the human embryo without genetic immortalization.
Abstract.
Leitch HG, Smith A (2013). The mammalian germline as a pluripotency cycle.
Development,
140, 2495-2501.
Abstract:
The mammalian germline as a pluripotency cycle.
Naive pluripotency refers to the capacity of single cells in regulative embryos to engender all somatic and germline cell types. Only germ cells - conventionally considered to be unipotent - can naturally re-acquire pluripotency, by cycling through fertilisation. Furthermore, primordial germ cells express, and appear to be functionally dependent upon, transcription factors that characterise the pluripotent state. We hypothesise that germ cells require pluripotency factors to control a de-restricted epigenome. Consequently, they harbour latent potential, as manifested in teratocarcinogenesis or direct conversion into pluripotent stem cells in vitro. Thus, we suggest that there exists an unbroken cycle of pluripotency, naive in the early epiblast and latent in the germline, that is sustained by a shared transcription factor network.
Abstract.
Stricker SH, Feber A, Engström PG, Carén H, Kurian KM, Takashima Y, Watts C, Way M, Dirks P, Bertone P, et al (2013). Widespread resetting of DNA methylation in glioblastoma-initiating cells suppresses malignant cellular behavior in a lineage-dependent manner.
Genes Dev,
27, 654-669.
Abstract:
Widespread resetting of DNA methylation in glioblastoma-initiating cells suppresses malignant cellular behavior in a lineage-dependent manner.
Epigenetic changes are frequently observed in cancer. However, their role in establishing or sustaining the malignant state has been difficult to determine due to the lack of experimental tools that enable resetting of epigenetic abnormalities. To address this, we applied induced pluripotent stem cell (iPSC) reprogramming techniques to invoke widespread epigenetic resetting of glioblastoma (GBM)-derived neural stem (GNS) cells. GBM iPSCs (GiPSCs) were subsequently redifferentiated to the neural lineage to assess the impact of cancer-specific epigenetic abnormalities on tumorigenicity. GiPSCs and their differentiating derivatives display widespread resetting of common GBM-associated changes, such as DNA hypermethylation of promoter regions of the cell motility regulator TES (testis-derived transcript), the tumor suppressor cyclin-dependent kinase inhibitor 1C (CDKN1C; p57KIP2), and many polycomb-repressive complex 2 (PRC2) target genes (e.g. SFRP2). Surprisingly, despite such global epigenetic reconfiguration, GiPSC-derived neural progenitors remained highly malignant upon xenotransplantation. Only when GiPSCs were directed to nonneural cell types did we observe sustained expression of reactivated tumor suppressors and reduced infiltrative behavior. These data suggest that imposing an epigenome associated with an alternative developmental lineage can suppress malignant behavior. However, in the context of the neural lineage, widespread resetting of GBM-associated epigenetic abnormalities is not sufficient to override the cancer genome.
Abstract.
2012
Yang S-H, Kalkan T, Morrisroe C, Smith A, Sharrocks AD (2012). A genome-wide RNAi screen reveals MAP kinase phosphatases as key ERK pathway regulators during embryonic stem cell differentiation.
PLoS Genet,
8, e1003112-e1003112.
Abstract:
A genome-wide RNAi screen reveals MAP kinase phosphatases as key ERK pathway regulators during embryonic stem cell differentiation.
Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Complex interlinked transcriptional and signaling networks control the fate of these cells towards maintenance of pluripotency or differentiation. In this study we have focused on how mouse embryonic stem cells begin to differentiate and lose pluripotency and, in particular, the role that the ERK MAP kinase and GSK3 signaling pathways play in this process. Through a genome-wide siRNA screen we have identified more than 400 genes involved in loss of pluripotency and promoting the onset of differentiation. These genes were functionally associated with the ERK and/or GSK3 pathways, providing an important resource for studying the roles of these pathways in controlling escape from the pluripotent ground state. More detailed analysis identified MAP kinase phosphatases as a focal point of regulation and demonstrated an important role for these enzymes in controlling ERK activation kinetics and subsequently determining early embryonic stem cell fate decisions.
Abstract.
Smith AG, Dickneite G, Metzner H, Zettlmeissl G, Grundmann R, Lathe R, Li M (2012). A transgenic coagulation factor xiii defective animal and its use for testing wound healing and bleeding.
Abstract:
A transgenic coagulation factor xiii defective animal and its use for testing wound healing and bleeding
The invention provides a transgenic non-human mammalian animal which is heterozygous or homozygous for an at least partially defective coagulation factor XIII gene
Abstract.
Falk A, Koch P, Kesavan J, Takashima Y, Ladewig J, Alexander M, Wiskow O, Tailor J, Trotter M, Pollard S, et al (2012). Capture of neuroepithelial-like stem cells from pluripotent stem cells provides a versatile system for in vitro production of human neurons.
PLoS One,
7, e29597-e29597.
Abstract:
Capture of neuroepithelial-like stem cells from pluripotent stem cells provides a versatile system for in vitro production of human neurons.
Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) provide new prospects for studying human neurodevelopment and modeling neurological disease. In particular, iPSC-derived neural cells permit a direct comparison of disease-relevant molecular pathways in neurons and glia derived from patients and healthy individuals. A prerequisite for such comparative studies are robust protocols that efficiently yield standardized populations of neural cell types. Here we show that long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) derived from 3 hESC and 6 iPSC lines in two independent laboratories exhibit consistent characteristics including i) continuous expandability in the presence of FGF2 and EGF; ii) stable neuronal and glial differentiation competence; iii) characteristic transcription factor profile; iv) hindbrain specification amenable to regional patterning; v) capacity to generate functionally mature human neurons. We further show that lt-NES cells are developmentally distinct from fetal tissue-derived radial glia-like stem cells. We propose that lt-NES cells provide an interesting tool for studying human neurodevelopment and may serve as a standard system to facilitate comparative analyses of hESC and hiPSC-derived neural cells from control and diseased genetic backgrounds.
Abstract.
Blair K, Leitch HG, Mansfield W, Dumeau C-É, Humphreys P, Smith AG (2012). Culture parameters for stable expansion, genetic modification and germline transmission of rat pluripotent stem cells.
Biol Open,
1, 58-65.
Abstract:
Culture parameters for stable expansion, genetic modification and germline transmission of rat pluripotent stem cells.
The ability of cultured pluripotent cells to contribute to the germline of chimaeric animals is essential to their utility for genetic manipulation. In the three years since rat embryonic stem (ES) cells were first reported the anticipated proliferation of genetically modified rat models from this new resource has not been realised. Culture instability, karyotypic anomalies, and strain variation are postulated to contribute to poor germline colonisation capacity. The resolution of these issues is essential to bring pluripotent cell-based genetic manipulation technology in the rat to the level of efficiency achieved in the mouse. Recent reports have described various alternative methods to maintain rat ES cells that include provision of additional small molecules and selective passaging methods. In contrast, we report that euploid, germline competent rat ES and embryonic germ (EG) cell lines can be maintained by simple adherent culture methods in defined medium supplemented with the original two inhibitors (2i) of the mitogen-activated protein kinase (ERK1/2) cascade and of glycogen synthase kinase 3, in combination with the cytokine leukaemia inhibitory factor (LIF). We demonstrate genetic modification, clonal expansion and transmission through the germline of rat ES and EG cell lines. We also describe a marked preference for full-term chimaera contribution when SD strain blastocysts are used as recipients for either DA or SD pluripotent stem cells.
Abstract.
Martello G, Sugimoto T, Diamanti E, Joshi A, Hannah R, Ohtsuka S, Göttgens B, Niwa H, Smith A (2012). Esrrb is a pivotal target of the Gsk3/Tcf3 axis regulating embryonic stem cell self-renewal.
Cell Stem Cell,
11(4), 491-504.
Abstract:
Esrrb is a pivotal target of the Gsk3/Tcf3 axis regulating embryonic stem cell self-renewal
Inhibition of glycogen synthase kinase-3 (Gsk3) supports mouse embryonic stem cells (ESCs) by modulating Tcf3, but the critical targets downstream of Tcf3 are unclear. We analyzed the intersection between genome localization and transcriptome data sets to identify genes repressed by Tcf3. Among these, manipulations of Esrrb gave distinctive phenotypes in functional assays. Knockdown and knockout eliminated response to Gsk3 inhibition, causing extinction of pluripotency markers and loss of colony forming capability. Conversely, forced expression phenocopied Gsk3 inhibition or Tcf3 deletion by suppressing differentiation and sustaining self-renewal. Thus the nuclear receptor Esrrb is necessary and sufficient to mediate self-renewal downstream of Gsk3 inhibition. Leukaemia inhibitory factor (LIF) regulates ESCs through Stat3, independently of Gsk3 inhibition. Consistent with parallel operation, ESCs in LIF accommodated Esrrb deletion and remained pluripotent. These findings highlight a key role for Esrrb in regulating the naive pluripotent state and illustrate compensation among the core pluripotency factors. © 2012 Elsevier Inc.
Abstract.
Leeb M, Walker R, Mansfield B, Nichols J, Smith A, Wutz A (2012). Germline potential of parthenogenetic haploid mouse embryonic stem cells.
Development,
139, 3301-3305.
Abstract:
Germline potential of parthenogenetic haploid mouse embryonic stem cells.
Haploid embryonic stem cells (ESCs) have recently been derived from parthenogenetic mouse embryos and offer new possibilities for genetic screens. The ability of haploid ESCs to give rise to a wide range of differentiated cell types in the embryo and in vitro has been demonstrated. However, it has remained unclear whether haploid ESCs can contribute to the germline. Here, we show that parthenogenetic haploid ESCs at high passage have robust germline competence enabling the production of transgenic mouse strains from genetically modified haploid ESCs. We also show that differentiation of haploid ESCs in the embryo correlates with the gain of a diploid karyotype and that diploidisation is the result of endoreduplication and not cell fusion. By contrast, we find that a haploid karyotype is maintained when differentiation to an extra-embryonic fate is forced by induction of Gata6.
Abstract.
Roode M, Blair K, Snell P, Elder K, Marchant S, Smith A, Nichols J (2012). Human hypoblast formation is not dependent on FGF signalling.
Dev Biol,
361, 358-363.
Abstract:
Human hypoblast formation is not dependent on FGF signalling.
Mouse embryos segregate three different lineages during preimplantation development: trophoblast, epiblast and hypoblast. These differentiation processes are associated with restricted expression of key transcription factors (Cdx2, Oct4, Nanog and Gata6). The mechanisms of segregation have been extensively studied in the mouse, but are not as well characterised in other species. In the human embryo, hypoblast differentiation has not previously been characterised. Here we demonstrate co-exclusive immunolocalisation of Nanog and Gata4 in human blastocysts, implying segregation of epiblast and hypoblast, as in rodent embryos. However, the formation of hypoblast in the human is apparently not dependent upon FGF signalling, in contrast to rodent embryos. Nonetheless, the persistence of Nanog-positive cells in embryos following treatment with FGF inhibitors is suggestive of a transient naïve pluripotent population in the human blastocyst, which may be similar to rodent epiblast and ES cells but is not sustained during conventional human ES cell derivation protocols.
Abstract.
Scherf N, Herberg M, Thierbach K, Zerjatke T, Kalkan T, Humphreys P, Smith A, Glauche I, Roeder I (2012). Imaging, quantification and visualization of spatio-temporal patterning in mESC colonies under different culture conditions.
Bioinformatics,
28, i556-i561.
Abstract:
Imaging, quantification and visualization of spatio-temporal patterning in mESC colonies under different culture conditions.
MOTIVATION: Mouse embryonic stem cells (mESCs) have developed into a prime system to study the regulation of pluripotency in stable cell lines. It is well recognized that different, established protocols for the maintenance of mESC pluripotency support morphologically and functionally different cell cultures. However, it is unclear how characteristic properties of cell colonies develop over time and how they are re-established after cell passage depending on the culture conditions. Furthermore, it appears that cell colonies have an internal structure with respect to cell size, marker expression or biomechanical properties, which is not sufficiently understood. The analysis of these phenotypic properties is essential for a comprehensive understanding of mESC development and ultimately requires a bioinformatics approach to guarantee reproducibility and high-throughput data analysis. RESULTS: We developed an automated image analysis and colony tracking framework to obtain an objective and reproducible quantification of structural properties of cell colonies as they evolve in space and time. In particular, we established a method that quantifies changes in colony shape and (internal) motion using fluid image registration and image segmentation. The methodology also allows to robustly track motion, splitting and merging of colonies over a sequence of images. Our results provide a first quantitative assessment of temporal mESC colony formation and estimates of structural differences between colony growth under different culture conditions. Furthermore, we provide a stream-based visualization of structural features of individual colonies over time for the whole experiment, facilitating visual comprehension of differences between experimental conditions. Thus, the presented method establishes the basis for the model-based analysis of mESC colony development. It can be easily extended to integrate further functional information using fluorescence signals and differentiation markers. AVAILABILITY: the analysis tool is implemented C++ and Mathematica 8.0 (Wolfram Research Inc. Champaign, IL, USA). The tool is freely available from the authors. We will also provide the source code upon request. CONTACT: nico.scherf@tu-dresden.de.
Abstract.
Juliandi B, Abematsu M, Sanosaka T, Tsujimura K, Smith A, Nakashima K (2012). Induction of superficial cortical layer neurons from mouse embryonic stem cells by valproic acid.
Neurosci Res,
72, 23-31.
Abstract:
Induction of superficial cortical layer neurons from mouse embryonic stem cells by valproic acid.
Within the developing mammalian cortex, neural progenitors first generate deep-layer neurons and subsequently more superficial-layer neurons, in an inside-out manner. It has been reported recently that mouse embryonic stem cells (mESCs) can, to some extent, recapitulate cortical development in vitro, with the sequential appearance of neurogenesis markers resembling that in the developing cortex. However, mESCs can only recapitulate early corticogenesis; superficial-layer neurons, which are normally produced in later developmental periods in vivo, are under-represented. This failure of mESCs to reproduce later corticogenesis in vitro implies the existence of crucial factor(s) that are absent or uninduced in existing culture systems. Here we show that mESCs can give rise to superficial-layer neurons efficiently when treated with valproic acid (VPA), a histone deacetylase inhibitor. VPA treatment increased the production of Cux1-positive superficial-layer neurons, and decreased that of Ctip2-positive deep-layer neurons. These results shed new light on the mechanisms of later corticogenesis.
Abstract.
Wray J, Kalkan T, Gomez-Lopez S, Eckardt D, Cook A, Kemler R, Smith A (2012). Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation. Nat Cell Biol, 14, 555-555.
van Oosten AL, Costa Y, Smith A, Silva JCR (2012). JAK/STAT3 signalling is sufficient and. dominant over antagonistic cues for the establishment of naive pluripotency.
Nat Commun,
3, 817-817.
Abstract:
JAK/STAT3 signalling is sufficient and. dominant over antagonistic cues for the establishment of naive pluripotency.
Induced pluripotency depends on cooperativity between expression of defined factors. and the culture environment. The latter also determines the pluripotent cell state. that is, naïve or primed. LIF-JAK/STAT3. signalling was recently shown to be a limiting factor for reprogramming to. naïve pluripotency. Here we show that sufficient activation of. JAK/STAT3 overcomes the. reprogramming block of cell intermediates and enables somatic cell reprogramming in. absence of otherwise essential pluripotency medium requisites. Activation of FGF-ERK. signalling, which promotes exit of naïve pluripotent cells from. self-renewal, does not prevent JAK/STAT3 induced post-implantation epiblast-derived stem cell. conversion into naïve pluripotency. Moreover, even in the presence of FGF. plus Activin, which instructs and maintains the primed state, JAK/STAT3 enforces naïve pluripotency in. epiblast stem cells. We conclude that JAK/STAT3 signalling can be sufficient and dominant over. antagonistic cues to enable the induction of a naïve pluripotent state.
Abstract.
Nichols J, Smith A (2012). Pluripotency in the embryo and in culture.
Cold Spring Harb Perspect Biol,
4, a008128-a008128.
Abstract:
Pluripotency in the embryo and in culture.
Specific cells within the early mammalian embryo have the capacity to generate all somatic lineages plus the germline. This property of pluripotency is confined to the epiblast, a transient tissue that persists for only a few days. In vitro, however, pluripotency can be maintained indefinitely through derivation of stem cell lines. Pluripotent stem cells established from the newly formed epiblast are known as embryonic stem cells (ESCs), whereas those generated from later stages are called postimplantation epiblast stem cells (EpiSCs). These different classes of pluripotent stem cell have distinct culture requirements and gene expression programs, likely reflecting the dynamic development of the epiblast in the embryo. In this chapter we review current understanding of how the epiblast forms and relate this to the properties of derivative stem cells. We discuss whether ESCs and EpiSCs are true counterparts of different phases of epiblast development or are culture-generated phenomena. We also consider the proposition that early epiblast cells and ESCs may represent a naïve ground state without any prespecification of lineage choice, whereas later epiblasts and EpiSCs may be primed in favor of particular fates.
Abstract.
Halley JD, Smith-Miles K, Winkler DA, Kalkan T, Huang S, Smith A (2012). Self-organizing circuitry and emergent computation in mouse embryonic stem cells.
Stem Cell Res,
8, 324-333.
Abstract:
Self-organizing circuitry and emergent computation in mouse embryonic stem cells.
Pluripotency is a cellular state of multiple options. Here, we highlight the potential for self-organization to contribute to stem cell fate computation. A new way of considering regulatory circuitry is presented that describes the expression of each transcription factor (TF) as a branching process that propagates through time, interacting and competing with others. In a single cell, the interactions between multiple branching processes generate a collective process called ’critical-like self-organization’. We explain how this phenomenon provides a valid description of whole genome regulatory circuit dynamics. The hypothesis of exploratory stem cell decision-making proposes that critical-like self-organization (also called rapid self-organized criticality) provides the backbone for cell fate computation in regulative embryos and pluripotent stem cells. Unspecific amplification of TF expression is predicted to initiate this self-organizing circuitry, where cascades of gene expression propagate and may interact either synergistically or antagonistically. The emergent and highly dynamic circuitry is affected by various sources of selection pressure, such as the expression of TFs with disproportionate influence over other genes, and extrinsic biological and physical stimuli that differentially modulate particular gene expression cascades. Extrinsic conditions continuously trigger waves of transcription that ripple throughout regulatory networks on multiple spatiotemporal scales, providing the context within which circuitry self-organizes. In this framework, a distinction between instructive and selective mechanisms of fate determination is misleading because it is the ’interference pattern’, rather than any single instructing or selecting factor, that is ultimately responsible for computing and directing cell fate. Using this framework, we consider whether the idea of a naïve ground state of pluripotency and that of a fluctuating transcriptome are compatible, and whether a ground state like that captured in vitro could exist in vivo.
Abstract.
Camnasio S, Delli Carri A, Lombardo A, Grad I, Mariotti C, Castucci A, Rozell B, Lo Riso P, Castiglioni V, Zuccato C, et al (2012). The first reported generation of several induced pluripotent stem cell lines from homozygous and heterozygous Huntington’s disease patients demonstrates mutation related enhanced lysosomal activity.
Neurobiol Dis,
46, 41-51.
Abstract:
The first reported generation of several induced pluripotent stem cell lines from homozygous and heterozygous Huntington’s disease patients demonstrates mutation related enhanced lysosomal activity.
Neuronal disorders, like Huntington’s disease (HD), are difficult to study, due to limited cell accessibility, late onset manifestations, and low availability of material. The establishment of an in vitro model that recapitulates features of the disease may help understanding the cellular and molecular events that trigger disease manifestations. Here, we describe the generation and characterization of a series of induced pluripotent stem (iPS) cells derived from patients with HD, including two rare homozygous genotypes and one heterozygous genotype. We used lentiviral technology to transfer key genes for inducing reprogramming. To confirm pluripotency and differentiation of iPS cells, we used PCR amplification and immunocytochemistry to measure the expression of marker genes in embryoid bodies and neurons. We also analyzed teratomas that formed in iPS cell-injected mice. We found that the length of the pathological CAG repeat did not increase during reprogramming, after long term growth in vitro, and after differentiation into neurons. In addition, we observed no differences between normal and mutant genotypes in reprogramming, growth rate, caspase activation or neuronal differentiation. However, we observed a significant increase in lysosomal activity in HD-iPS cells compared to control iPS cells, both during self-renewal and in iPS-derived neurons. In conclusion, we have established stable HD-iPS cell lines that can be used for investigating disease mechanisms that underlie HD. The CAG stability and lysosomal activity represent novel observations in HD-iPS cells. In the future, these cells may provide the basis for a powerful platform for drug screening and target identification in HD.
Abstract.
Marks H, Kalkan T, Menafra R, Denissov S, Jones K, Hofemeister H, Nichols J, Kranz A, Stewart AF, Smith A, et al (2012). The transcriptional and epigenomic foundations of ground state pluripotency.
Cell,
149, 590-604.
Abstract:
The transcriptional and epigenomic foundations of ground state pluripotency.
Mouse embryonic stem (ES) cells grown in serum exhibit greater heterogeneity in morphology and expression of pluripotency factors than ES cells cultured in defined medium with inhibitors of two kinases (Mek and GSK3), a condition known as "2i" postulated to establish a naive ground state. We show that the transcriptome and epigenome profiles of serum- and 2i-grown ES cells are distinct. 2i-treated cells exhibit lower expression of lineage-affiliated genes, reduced prevalence at promoters of the repressive histone modification H3K27me3, and fewer bivalent domains, which are thought to mark genes poised for either up- or downregulation. Nonetheless, serum- and 2i-grown ES cells have similar differentiation potential. Precocious transcription of developmental genes in 2i is restrained by RNA polymerase II promoter-proximal pausing. These findings suggest that transcriptional potentiation and a permissive chromatin context characterize the ground state and that exit from it may not require a metastable intermediate or multilineage priming.
Abstract.
Fujimoto Y, Abematsu M, Falk A, Tsujimura K, Sanosaka T, Juliandi B, Semi K, Namihira M, Komiya S, Smith A, et al (2012). Treatment of a mouse model of spinal cord injury by transplantation of human induced pluripotent stem cell-derived long-term self-renewing neuroepithelial-like stem cells.
Stem Cells,
30, 1163-1173.
Abstract:
Treatment of a mouse model of spinal cord injury by transplantation of human induced pluripotent stem cell-derived long-term self-renewing neuroepithelial-like stem cells.
Because of their ability to self-renew, to differentiate into multiple lineages, and to migrate toward a damaged site, neural stem cells (NSCs), which can be derived from various sources such as fetal tissues and embryonic stem cells, are currently considered to be promising components of cell replacement strategies aimed at treating injuries of the central nervous system, including the spinal cord. Despite their efficiency in promoting functional recovery, these NSCs are not homogeneous and possess variable characteristics depending on their derivation protocols. The advent of induced pluripotent stem (iPS) cells has provided new prospects for regenerative medicine. We used a recently developed robust and stable protocol for the generation of long-term, self-renewing, neuroepithelial-like stem cells from human iPS cells (hiPS-lt-NES cells), which can provide a homogeneous and well-defined population of NSCs for standardized analysis. Here, we show that transplanted hiPS-lt-NES cells differentiate into neural lineages in the mouse model of spinal cord injury (SCI) and promote functional recovery of hind limb motor function. Furthermore, using two different neuronal tracers and ablation of the transplanted cells, we revealed that transplanted hiPS-lt-NES cell-derived neurons, together with the surviving endogenous neurons, contributed to restored motor function. Both types of neurons reconstructed the corticospinal tract by forming synaptic connections and integrating neuronal circuits. Our findings indicate that hiPS-lt-NES transplantation represents a promising avenue for effective cell-based treatment of SCI.
Abstract.
2011
Guo G, Huang Y, Humphreys P, Wang X, Smith A (2011). A PiggyBac-Based Recessive Screening Method to Identify Pluripotency Regulators.
PLOS ONE,
6(4).
Author URL.
Wray J, Kalkan T, Gomez-Lopez S, Eckardt D, Cook A, Kemler R, Smith A (2011). Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation.
Nat Cell Biol,
13, 838-845.
Abstract:
Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation.
Self-renewal of rodent embryonic stem cells is enhanced by partial inhibition of glycogen synthase kinase-3 (Gsk3; refs 1, 2). This effect has variously been attributed to stimulation of Wnt signalling by β-catenin, stabilization of Myc protein and global de-inhibition of anabolic processes. Here we demonstrate that β-catenin is not necessary for embryonic stem cell identity or expansion, but its absence eliminates the self-renewal response to Gsk3 inhibition. Responsiveness is fully restored by truncated β-catenin lacking the carboxy-terminal transactivation domain. However, requirement for Gsk3 inhibition is dictated by expression of T-cell factor 3 (Tcf3) and mediated by direct interaction with β-catenin. Tcf3 localizes to many pluripotency genes in embryonic stem cells. Our findings confirm that Tcf3 acts as a transcriptional repressor and reveal that β-catenin directly abrogates Tcf3 function. We conclude that Gsk3 inhibition stabilizes the embryonic stem cell state primarily by reducing repressive influence on the core pluripotency network.
Abstract.
Sun Y, Hu J, Zhou L, Pollard SM, Smith A (2011). Interplay between FGF2 and BMP controls the self-renewal, dormancy and differentiation of rat neural stem cells.
J Cell Sci,
124, 1867-1877.
Abstract:
Interplay between FGF2 and BMP controls the self-renewal, dormancy and differentiation of rat neural stem cells.
Mouse and human central nervous system progenitor cells can be propagated extensively ex vivo as stem cell lines. For the rat, however, in vitro expansion has proven to be problematic owing to proliferation arrest and differentiation. Here, we analyse the establishment, in adherent culture, of undifferentiated tripotent neural stem (NS) cell lines derived from rat foetal brain and spinal cord. Rat NS cells invariably undergo growth arrest and apparent differentiation after several passages; however, conditioned medium from proliferating cultures can overcome this block, enabling continuous propagation of undifferentiated rat NS cells. We found that dormancy is induced by autocrine production of bone morphogenetic proteins (BMPs). Accordingly, the BMP antagonist noggin can replace conditioned medium to sustain continuous self-renewal. Noggin can also induce dormant cells to re-enter the cell cycle, upon which they reacquire neurogenic potential. We further show that fibroblast growth factor 2 (FGF2) is required to suppress terminal astrocytic differentiation and maintain stem cell potency during dormancy. These findings highlight an extrinsic regulatory network, comprising BMPs, BMP antagonists and FGF2 signals, that governs the proliferation, dormancy and differentiation of rat NS cells and which can be manipulated to enable long-term clonogenic self-renewal.
Abstract.
Theunissen TW, van Oosten AL, Castelo-Branco G, Hall J, Smith A, Silva JCR (2011). Nanog overcomes reprogramming barriers and induces pluripotency in minimal conditions.
Curr Biol,
21, 65-71.
Abstract:
Nanog overcomes reprogramming barriers and induces pluripotency in minimal conditions.
Induced pluripotency requires the expression of defined factors and culture conditions that support the self-renewal of embryonic stem (ES) cells. Small molecule inhibition of MAP kinase (MEK) and glycogen synthase kinase 3 (GSK3) with LIF (2i/LIF) provides an optimal culture environment for mouse ES cells and promotes transition to naive pluripotency in partially reprogrammed (pre-iPS) cells. Here we show that 2i/LIF treatment in clonal lines of pre-iPS cells results in the activation of endogenous Nanog and rapid downregulation of retroviral Oct4 expression. Nanog enables somatic cell reprogramming in serum-free medium supplemented with LIF, a culture condition which does not support induced pluripotency or the self-renewal of ES cells, and is sufficient to reprogram epiblast-derived stem cells to naive pluripotency in serum-free medium alone. Nanog also enhances reprogramming in cooperation with kinase inhibition or 5-aza-cytidine, a small molecule inhibitor of DNA methylation. These results highlight the capacity of Nanog to overcome multiple barriers to reprogramming and reveal a synergy between Nanog and chemical inhibitors that promote reprogramming. We conclude that Nanog induces pluripotency in minimal conditions. This provides a strategy for imposing naive pluripotency in mammalian cells independently of species-specific culture requirements.
Abstract.
Gómez-López S, Wiskow O, Favaro R, Nicolis SK, Price DJ, Pollard SM, Smith A (2011). Sox2 and Pax6 maintain the proliferative and developmental potential of gliogenic neural stem cells in vitro.
Glia,
59, 1588-1599.
Abstract:
Sox2 and Pax6 maintain the proliferative and developmental potential of gliogenic neural stem cells in vitro.
Radial-glia-like neural stem (NS) cells may be derived from neural tissues or via differentiation of pluripotent embryonic stem (ES) cells. However, the mechanisms controlling NS cell propagation and differentiation are not yet fully understood. Here we investigated the roles of Sox2 and Pax6, transcription factors widely expressed in central nervous system (CNS) progenitors, in mouse NS cells. Conditional deletion of either Sox2 or Pax6 in forebrain-derived NS cells reduced their clonogenicity in a gene dosage-dependent manner. Cells heterozygous for either gene displayed moderate proliferative defects, which may relate to human pathologies attributed to SOX2 or PAX6 deficiencies. In the complete absence of Sox2, cells exited the cell cycle with concomitant downregulation of neural progenitor markers Nestin and Blbp. This occurred despite expression of the close relative Sox3. Ablation of Pax6 also caused major proliferative defects. However, a subpopulation of cells was able to expand continuously without Pax6. These Pax6-null cells retained progenitor markers but had altered morphology. They exhibited compromised differentiation into astrocytes and oligodendrocytes, highlighting that the role of Pax6 extends beyond neurogenic competence. Overall these findings indicate that Sox2 and Pax6 are both critical for self-renewal of differentiation-competent radial glia-like NS cells.
Abstract.
Blair K, Wray J, Smith A (2011). The liberation of embryonic stem cells.
PLoS Genet,
7, e1002019-e1002019.
Abstract:
The liberation of embryonic stem cells.
Mouse embryonic stem (ES) cells are defined by their capacity to self-renew and their ability to differentiate into all adult tissues including the germ line. Along with efficient clonal propagation, these properties have made them an unparalleled tool for manipulation of the mouse genome. Traditionally, mouse ES (mES) cells have been isolated and cultured in complex, poorly defined conditions that only permit efficient derivation from the 129 mouse strain; genuine ES cells have not been isolated from another species in these conditions. Recently, use of small molecule inhibitors of glycogen synthase kinase 3 (Gsk3) and the Fgf-MAPK signaling cascade has permitted efficient derivation of ES cells from all tested mouse strains. Subsequently, the first verified ES cells were established from a non-mouse species, Rattus norvegicus. Here, we summarize the advances in our understanding of the signaling pathways regulating mES cell self-renewal that led to the first derivation of rat ES cells and highlight the new opportunities presented for transgenic modeling on diverse genetic backgrounds. We also comment on the implications of this work for our understanding of pluripotent stem cells across mammalian species.
Abstract.
Nichols J, Smith A (2011). The origin and identity of embryonic stem cells.
Development,
138, 3-8.
Abstract:
The origin and identity of embryonic stem cells.
Embryonic stem (ES) cells are used extensively in biomedical research and as a model with which to study early mammalian development, but their exact origin has been subject to much debate. They are routinely derived from pre-implantation embryos, but it has been suggested that the cells that give rise to ES cells might arise from epiblast cells that are already predisposed to a primordial germ cell (PGC) fate, which then progress to ES cell status via the PGC lineage. Based on recent findings, we propose here that ES cells can be derived directly from early epiblast cells and that ES cells might arise via two different routes that are dictated by their culture conditions.
Abstract.
Smith A (2011). ’No’ to ban on stem-cell patents. Nature, 472, 418-418.
2010
Guo G, Smith A (2010). A genome-wide screen in EpiSCs identifies Nr5a nuclear receptors as potent inducers of ground state pluripotency.
DEVELOPMENT,
137(19), 3185-3192.
Author URL.
Leitch HG, Blair K, Mansfield W, Ayetey H, Humphreys P, Nichols J, Surani MA, Smith A (2010). Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state.
Development,
137, 2279-2287.
Abstract:
Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state.
Mouse and rat embryonic stem cells can be sustained in defined medium by dual inhibition (2i) of the mitogen-activated protein kinase (Erk1/2) cascade and of glycogen synthase kinase 3. The inhibitors suppress differentiation and enable self-renewal of pluripotent cells that are ex vivo counterparts of naïve epiblast cells in the mature blastocyst. Pluripotent stem cell lines can also be derived from unipotent primordial germ cells via a poorly understood process of epigenetic reprogramming. These are termed embryonic germ (EG) cells to denote their distinct origin. Here we investigate whether EG cell self-renewal and derivation are supported by 2i. We report that mouse EG cells can be established with high efficiency using 2i in combination with the cytokine leukaemia inhibitory factor (LIF). Furthermore, addition of fibroblast growth factor or stem cell factor is unnecessary using 2i-LIF. The derived EG cells contribute extensively to healthy chimaeric mice, including to the germline. Using the same conditions, we describe the first derivations of EG cells from the rat. Rat EG cells express a similar marker profile to rat and mouse ES cells. They have a diploid karyotype, can be clonally expanded and genetically manipulated, and are competent for multilineage colonisation of chimaeras. These findings lend support to the postulate of a conserved molecular ground state in pluripotent rodent cells. Future research will determine the extent to which this is maintained in other mammals and whether, in some species, primordial germ cells might be a more tractable source than epiblast for the capture of naïve pluripotent stem cells.
Abstract.
Danovi D, Falk A, Humphreys P, Vickers R, Tinsley J, Smith AG, Pollard SM (2010). Imaging-based chemical screens using normal and glioma-derived neural stem cells.
Biochem Soc Trans,
38, 1067-1071.
Abstract:
Imaging-based chemical screens using normal and glioma-derived neural stem cells.
The development of optimal culture methods for embryonic, tissue and cancer stem cells is a critical foundation for their application in drug screening. We previously described defined adherent culture conditions that enable expansion of human radial glia-like fetal NS (neural stem) cells as stable cell lines. Similar protocols proved effective in the establishment of tumour-initiating stem cell lines from the human brain tumour glioblastoma multiforme, which we termed GNS (glioma NS) cells. Others have also recently derived more primitive human NS cell lines with greater neuronal subtype differentiation potential than NS cells, which have similarities to the early neuroepithelium, named NES (neuroepithelial stem) cells. In the present paper, we discuss the utility of these cells for chemical screening, and describe methods for a simple high-content live-image-based platform. We report the effects of a panel of 160 kinase inhibitors (Inhibitor Select I and II; Calbiochem) on NES cells, identifying three inhibitors of ROCK (Rho-associated kinase) as promoting the expansion of NES cell cultures. For the GNS cells, we screened a panel of 1000 compounds and confirmed our previous finding of a cytotoxic effect of modulators of neurotransmitter signalling pathways. These studies provide a framework for future higher-throughput screens.
Abstract.
Kawaguchi J, Nichols J, Gierl MS, Faial T, Smith A (2010). Isolation and propagation of enteric neural crest progenitor cells from mouse embryonic stem cells and embryos.
Development,
137, 693-704.
Abstract:
Isolation and propagation of enteric neural crest progenitor cells from mouse embryonic stem cells and embryos.
Neural crest is a source of diverse cell types, including the peripheral nervous system. The transcription factor Sox10 is expressed throughout early neural crest. We exploited Sox10 reporter and selection markers created by homologous recombination to investigate the generation, maintenance and expansion of neural crest progenitors. Sox10-GFP-positive cells are produced transiently from mouse embryonic stem (ES) cells by treatment with retinoic acid in combination with Fgf8b and the cytokine leukaemia inhibitory factor (Lif). We found that expression of Sox10 can be maintained using noggin, Wnt3a, Lif and endothelin (NWLE). ES cell-derived Sox10-GFP-positive cells cultured in NWLE exhibit molecular markers of neural crest progenitors. They differentiate into peripheral neurons in vitro and are able to colonise the enteric network in organotypic gut cultures. Neural crest cells purified from embryos using the Sox10 reporter also survive in NWLE, but progressively succumb to differentiation. We therefore applied selection to eliminate differentiating cells. Sox10-selected cells could be clonally expanded, cryopreserved, and multiplied for over 50 days in adherent culture. They remained neurogenic in vitro and in foetal gut grafts. Generation of neural crest from mouse ES cells opens a new route to the identification and validation of determination factors. Furthermore, the ability to propagate undifferentiated progenitors creates an opportunity for experimental dissection of the stimuli and molecular circu that govern neural crest lineage progression. Finally, the demonstration of robust enteric neurogenesis provides a system for investigating and modelling cell therapeutic approaches to neurocristopathies such as Hirschsprung’s disease.
Abstract.
Moretti A, Bellin M, Jung CB, Thies T-M, Takashima Y, Bernshausen A, Schiemann M, Fischer S, Moosmang S, Smith AG, et al (2010). Mouse and human induced pluripotent stem cells as a source for multipotent Isl1+ cardiovascular progenitors.
FASEB J,
24, 700-711.
Abstract:
Mouse and human induced pluripotent stem cells as a source for multipotent Isl1+ cardiovascular progenitors.
Ectopic expression of defined sets of genetic factors can reprogram somatic cells to create induced pluripotent stem (iPS) cells. The capacity to direct human iPS cells to specific differentiated lineages and to their progenitor populations can be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. During mouse cardiogenesis, the major lineages of the mature heart, cardiomyocytes, smooth muscle cells, and endothelial cells arise from a common, multipotent cardiovascular progenitor expressing the transcription factors Isl1 and Nkx2.5. Here we show, using genetic fate-mapping, that Isl1(+) multipotent cardiovascular progenitors can be generated from mouse iPS cells and spontaneously differentiate in all 3 cardiovascular lineages in vivo without teratoma. Moreover, we report the identification of human iPS-derived ISL1(+) progenitors with similar developmental potential. These results support the possibility to use patient-specific iPS-generated cardiovascular progenitors as a model to elucidate the pathogenesis of congenital and acquired forms of heart diseases.-Moretti, A. Bellin, M. Jung, C. B. Thies, T.-M. Takashima, Y. Bernshausen, A. Schiemann, M. Fischer, S. Moosmang, S. Smith, A. G. Lam, J. T. Laugwitz, K.-L. Mouse and human induced pluripotent stem cells as a source for multipotent Isl1(+) cardiovascular progenitors.
Abstract.
Smith A (2010). Pluripotent stem cells: private obsession and public expectation. EMBO Mol Med, 2, 113-116.
Yang J, van Oosten AL, Theunissen TW, Guo G, Silva JCR, Smith A (2010). Stat3 Activation is Limiting for Reprogramming to Ground State Pluripotency.
CELL STEM CELL,
7(3), 319-328.
Author URL.
Wray J, Kalkan T, Smith AG (2010). The ground state of pluripotency.
Biochem Soc Trans,
38, 1027-1032.
Abstract:
The ground state of pluripotency.
Pluripotency is defined as the capacity of individual cells to initiate all lineages of the mature organism in response to signals from the embryo or cell culture environment. A pluripotent cell has no predetermined programme; it is a blank slate. This is the foundation of mammalian development and of ES (embryonic stem) cell biology. What are the design principles of this naïve cell state? How is pluripotency acquired and maintained? Suppressing activation of ERKs (extracellular-signal-regulated kinases) is critical to establishing and sustaining ES cells. Inhibition of GSK3 (glycogen synthase kinase 3) reinforces this effect. We review the effect of selective kinase inhibitors on pluripotent cells and consider how these effects are mediated. We propose that ES cells represent a ground state, meaning a basal proliferative state that is free of epigenetic restriction and has minimal requirements for extrinsic stimuli. The stability of this state is reflected in the homogeneity of ES cell populations cultured in the presence of small-molecule inhibitors of MEK (mitogen-activated protein kinase/ERK kinase) and GSK3.
Abstract.
2009
Pollard S, Clarke ID, Smith A, Dirks P (2009). Brain Cancer Stem Cells: a Level Playing Field. CELL STEM CELL, 5, 468-469.
Sun Y, Kong W, Falk A, Hu J, Zhou L, Pollard S, Smith A (2009). CD133 (Prominin) negative human neural stem cells are clonogenic and tripotent.
PLoS One,
4, e5498-e5498.
Abstract:
CD133 (Prominin) negative human neural stem cells are clonogenic and tripotent.
BACKGROUND: CD133 (Prominin) is widely used as a marker for the identification and isolation of neural precursor cells from normal brain or tumor tissue. However, the assumption that CD133 is expressed constitutively in neural precursor cells has not been examined. METHODOLOGY/PRINCIPAL FINDINGS: in this study, we demonstrate that CD133 and a second marker CD15 are expressed heterogeneously in uniformly undifferentiated human neural stem (NS) cell cultures. After fractionation by flow cytometry, clonogenic tripotent cells are found in populations negative or positive for either marker. We further show that CD133 is down-regulated at the mRNA level in cells lacking CD133 immunoreactivity. Cell cycle profiling reveals that CD133 negative cells largely reside in G1/G0, while CD133 positive cells are predominantly in S, G2, or M phase. A similar pattern is apparent in mouse NS cell lines. Compared to mouse NS cells, however, human NS cell cultures harbour an increased proportion of CD133 negative cells and display a longer doubling time. This may in part reflect a sub-population of slow- or non-cycling cells amongst human NS cells because we find that around 5% of cells do not take up BrdU over a 14-day labelling period. Non-proliferating NS cells remain undifferentiated and at least some of them are capable of re-entry into the cell cycle and subsequent continuous expansion. CONCLUSIONS: the finding that a significant fraction of clonogenic neural stem cells lack the established markers CD133 and CD15, and that some of these cells may be dormant or slow-cycling, has implications for approaches to identify and isolate neural stem cells and brain cancer stem cells. Our data also suggest the possibility that CD133 may be specifically down-regulated during G0/G1, and this should be considered when this marker is used to identify and isolate other tissue and cancer stem cells.
Abstract.
Smith A (2009). Design principles of pluripotency. EMBO Mol Med, 1, 251-254.
Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, Russell R, Bayani J, Head R, Lee M, Bernstein M, et al (2009). Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens.
Cell Stem Cell,
4, 568-580.
Abstract:
Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens.
Human brain tumors appear to have a hierarchical cellular organization suggestive of a stem cell foundation. In vitro expansion of the putative cancer stem cells as stable cell lines would provide a powerful model system to study their biology. Here, we demonstrate routine and efficient derivation of adherent cell lines from malignant glioma that display stem cell properties and initiate high-grade gliomas following xenotransplantation. Significantly, glioma neural stem (GNS) cell lines from different tumors exhibit divergent gene expression signatures and differentiation behavior that correlate with specific neural progenitor subtypes. The diversity of gliomas may, therefore, reflect distinct cancer stem cell phenotypes. The purity and stability of adherent GNS cell lines offer significant advantages compared to "sphere" cultures, enabling refined studies of cancer stem cell behavior. A proof-of-principle live cell imaging-based chemical screen (450 FDA-approved drugs) identifies both differential sensitivities of GNS cells and a common susceptibility to perturbation of serotonin signaling.
Abstract.
Da Silva JCR, Smith AG, Guo G (2009). IMPROVED REPROGRAMMING OF MAMMALIAN CELLS, AND THE CELLS OBTAINED.
Abstract:
IMPROVED REPROGRAMMING OF MAMMALIAN CELLS, AND THE CELLS OBTAINED
Expression of reprogramming factors such as Sox2, klf4, c-myc, Nanog, LIN28 and Oct4 followed by culture in a MEK inhibitor and a GSK3 inhibitor reprograms tissue cells. The invention provides new uses of these inhibitors, for example in inducing completion of the transcriptional resetting of so-called pre-pluripotent (pre-iPS) stem cells, for example as obtained from mammalian neural stem cells or epiblast stem cells treated with single or combinations of the reprogramming factors, expressed transiently or by integrative vectors. Also provided are systems for reprogramming an epiplast stem cells independently of the use of there inhibitors.
Abstract.
Author URL.
Moretti A, Jung C, Bellin M, Nhan TM, Takashima Y, Bernshausen A, Schiemann M, Frei EM, Moosmang S, Smith AG, et al (2009). Induced pluripotent stem cells as a source for multipotent ISL1+cardiovascular progenitors.
Guo G, Yang J, Nichols J, Hall JS, Eyres I, Mansfield W, Smith A (2009). Klf4 reverts developmentally programmed restriction of ground state pluripotency.
DEVELOPMENT,
136(7), 1063-1069.
Author URL.
Nichols J, Smith A (2009). Naive and primed pluripotent states.
Cell Stem Cell,
4, 487-492.
Abstract:
Naive and primed pluripotent states.
After maternal predetermination gives way to zygotic regulation, a ground state is established within the mammalian embryo. This tabula rasa for embryogenesis is present only transiently in the preimplantation epiblast. Here, we consider how unrestricted cells are first generated and then prepared for lineage commitment. We propose that two phases of pluripotency can be defined: naive and primed. This distinction extends to pluripotent stem cells derived from embryos or by molecular reprogramming ex vivo.
Abstract.
Silva J, Nichols J, Theunissen TW, Guo G, van Oosten AL, Barrandon O, Wray J, Yamanaka S, Chambers I, Smith A, et al (2009). Nanog is the Gateway to the Pluripotent Ground State.
CELL,
138(4), 722-737.
Author URL.
Hall J, Guo G, Wray J, Eyres I, Nichols J, Grotewold L, Morfopoulou S, Humphreys P, Mansfield W, Walker R, et al (2009). Oct4 and LIF/Stat3 Additively Induce Kruppel Factors to Sustain Embryonic Stem Cell Self-Renewal.
CELL STEM CELL,
5(6), 597-609.
Author URL.
Thornton K, Smith A, Merry CLR, Ulijn RV (2009). Phosphatase induced stiffness control in a self-assembled peptide hydrogel.
Nichols J, Silva J, Roode M, Smith A (2009). Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo.
Development,
136, 3215-3222.
Abstract:
Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo.
Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of small-molecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. We investigate the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. We find that blockade of Erk signalling from the 8-cell stage does not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, naïve epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. We probed directly the relationship between naïve epiblast and ES cells. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. We propose that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells.
Abstract.
Nichols J, Jones K, Phillips JM, Newland SA, Roode M, Mansfield W, Smith A, Cooke A (2009). Validated germline-competent embryonic stem cell lines from nonobese diabetic mice.
Nat Med,
15, 814-818.
Abstract:
Validated germline-competent embryonic stem cell lines from nonobese diabetic mice.
Nonobese diabetic (NOD) mice provide an excellent model of type 1 diabetes. The genetic contribution to this disease is complex, with more than 20 loci implicated in diabetes onset. One of the challenges for researchers using the NOD mouse model (and, indeed, other models of spontaneous autoimmune disease) has been the high density of sequence variation within candidate chromosomal segments. Furthermore, the scope for analyzing many putative disease loci via gene targeting has been hampered by the lack of NOD embryonic stem (ES) cells. We describe here the derivation of NOD ES cell lines capable of generating chimeric mice after stable genetic modification. These NOD ES cell lines also show efficient germline transmission, with offspring developing diabetes. The availability of these cells will not only enable the dissection of closely linked loci and the role they have in the onset of type 1 diabetes but also facilitate the generation of new transgenics.
Abstract.
2008
Buehr M, Meek S, Blair K, Yang J, Ure J, Silva J, McLay R, Hall J, Ying Q-L, Smith A, et al (2008). Capture of authentic embryonic stem cells from rat blastocysts.
Cell,
135, 1287-1298.
Abstract:
Capture of authentic embryonic stem cells from rat blastocysts.
Embryonic stem (ES) cells have been available from inbred mice since 1981 but have not been validated for other rodents. Failure to establish ES cells from a range of mammals challenges the identity of cultivated stem cells and our understanding of the pluripotent state. Here we investigated derivation of ES cells from the rat. We applied molecularly defined conditions designed to shield the ground state of authentic pluripotency from inductive differentiation stimuli. Undifferentiated cell lines developed that exhibited diagnostic features of ES cells including colonization of multiple tissues in viable chimeras. Definitive ES cell status was established by transmission of the cell line genome to offspring. Derivation of germline-competent ES cells from the rat paves the way to targeted genetic manipulation in this valuable biomedical model species. Rat ES cells will also provide a refined test-bed for functional evaluation of pluripotent stem cell-derived tissue repair and regeneration.
Abstract.
Silva J, Smith A (2008). Capturing pluripotency.
Cell,
132, 532-536.
Abstract:
Capturing pluripotency.
In this Essay, we argue that pluripotent epiblast founder cells in the embryo and embryonic stem (ES) cells in culture represent the ground state for a mammalian cell, signified by freedom from developmental specification or epigenetic restriction and capacity for autonomous self-replication. We speculate that cell-to-cell variation may be integral to the ES cell condition, safe-guarding self-renewal while continually presenting opportunities for lineage specification.
Abstract.
Pollard SM, Wallbank R, Tomlinson S, Grotewold L, Smith A (2008). Fibroblast growth factor induces a neural stem cell phenotype in foetal forebrain progenitors and during embryonic stem cell differentiation.
Mol Cell Neurosci,
38, 393-403.
Abstract:
Fibroblast growth factor induces a neural stem cell phenotype in foetal forebrain progenitors and during embryonic stem cell differentiation.
Neural stem (NS) cell lines may be derived via differentiation of pluripotent embryonic stem (ES) cells or from foetal forebrain. However, because NS cells arise in vitro from heterogeneous populations their immediate cellular origin remains unclear. We used microarray-based expression profiling to identify a set of markers expressed by mouse NS cells but not ES cells. One differentially expressed gene encodes the cell surface protein, CD44. CD44 expression is activated by FGF-2 in a subset of cells in both differentiating ES cells and foetal forebrain cultures. Following isolation by flow cytometry the CD44+ population was found to be highly enriched for NS cell founders. We found that other NS cell marker genes are also induced by FGF in culture, including: Adam12, Cadherin20, Cx3cl1, EGFR, Frizzled9, Kitl, Olig1, Olig2 and Vav3. We speculate that the self-renewing NS cell state may be generated in vitro following transcriptional resetting induced by FGF.
Abstract.
Sun Y, Pollard S, Conti L, Toselli M, Biella G, Parkin G, Willatt L, Falk A, Cattaneo E, Smith A, et al (2008). Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture.
Mol Cell Neurosci,
38, 245-258.
Abstract:
Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture.
Stem cell lines that provide a renewable and scaleable supply of central nervous system cell types would constitute an invaluable resource for basic and applied neurobiology. Here we describe the generation and long-term expansion of multiple human foetal neural stem (NS) cell lines in monolayer culture without genetic immortalization. Adherent human NS cells are propagated in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), under which conditions they stably express neural precursor markers and exhibit negligible differentiation into neurons or glia. However, they produce astrocytes, oligodendrocytes, and neurons upon exposure to appropriate differentiation factors. Single cell cloning demonstrates that human NS cells are tripotent. They retain a diploid karyotype and constant neurogenic capacity after over 100 generations. In contrast to human neurospheres, we observe no requirement for the cytokine leukaemia inhibitory factor (LIF) for continued expansion of adherent human NS cells. Human NS cells can be stably transfected to provide reporter lines and readily imaged in live monolayer cultures, creating the potential for high content genetic and chemical screens.
Abstract.
Batlle-Morera L, Smith A, Nichols J (2008). Parameters influencing derivation of embryonic stem cells from murine embryos.
Genesis,
46, 758-767.
Abstract:
Parameters influencing derivation of embryonic stem cells from murine embryos.
The derivation of ES cells is poorly understood and varies in efficiency between different strains of mice. We have investigated potential differences between embryos of permissive and recalcitrant strains during diapause and ES cell derivation. We found that in diapause embryos of the recalcitrant C57BL/6 and CBA strains, the epiblast failed to expand during the primary explant phase of ES cell derivation, whereas in the permissive 129 strain, it expanded dramatically. Epiblasts from the recalcitrant strains could be expanded by reducing Erk activation. Isolation of 129 epiblasts facilitated very efficient derivation of ES cell lines in serum- and feeder-free conditions, but reduction of Erk activity was required for derivation of ES cells from isolated C57BL/6 or CBA epiblasts. The results suggest that the discrepancy in ES cell derivation efficiency is not attributable merely to variable prodifferentiative effects of the extra-embryonic lineages but also to an intrinsic variability within the epiblast to maintain pluripotency.
Abstract.
Silva J, Barrandon O, Nichols J, Kawaguchi J, Theunissen TW, Smith A (2008). Promotion of reprogramming to ground state pluripotency by signal inhibition.
PLoS Biol,
6, e253-e253.
Abstract:
Promotion of reprogramming to ground state pluripotency by signal inhibition.
Induced pluripotent stem (iPS) cells are generated from somatic cells by genetic manipulation. Reprogramming entails multiple transgene integrations and occurs apparently stochastically in rare cells over many days. Tissue stem cells may be subject to less-stringent epigenetic restrictions than other cells and might therefore be more amenable to deprogramming. We report that brain-derived neural stem (NS) cells acquire undifferentiated morphology rapidly and at high frequency after a single round of transduction with reprogramming factors. However, critical attributes of true pluripotency–including stable expression of endogenous Oct4 and Nanog, epigenetic erasure of X chromosome silencing in female cells, and ability to colonise chimaeras–were not attained. We therefore applied molecularly defined conditions for the derivation and propagation of authentic pluripotent stem cells from embryos. We combined dual inhibition (2i) of mitogen-activated protein kinase signalling and glycogen synthase kinase-3 (GSK3) with the self-renewal cytokine leukaemia inhibitory factor (LIF). The 2i/LIF condition induced stable up-regulation of Oct4 and Nanog, reactivation of the X chromosome, transgene silencing, and competence for somatic and germline chimaerism. Using 2i /LIF, NS cell reprogramming required only 1-2 integrations of each transgene. Furthermore, transduction with Sox2 and c-Myc is dispensable, and Oct4 and Klf4 are sufficient to convert NS cells into chimaera-forming iPS cells. These findings demonstrate that somatic cell state influences requirements for reprogramming and delineate two phases in the process. The ability to capture pre-pluripotent cells that can advance to ground state pluripotency simply and with high efficiency opens a door to molecular dissection of this remarkable phenomenon.
Abstract.
Silva J, Smith AG (2008). Reprogramming and genetic modification of cells.
Abstract:
Reprogramming and genetic modification of cells
Methods for reprogramming and optional genetic modification of cells are provided. A pluripotent genome is obtained from a differentiated genome by fusing a pluripotent cell with a differentiated cell in the presence of Nanog or a MEK inhibitor. A cell is genetically modified by providing first and second cells, each containing chromosomes, fusing the first cell and the second cell, and culturing the fused cell so as to obtain a diploid cell containing at least one chromosome from the first cell and at least one chromosome form the second cell. A method of cell fusion comprises fusing a first cell and a second cell in the presence of Nanog or a MEK inhibitor. Cells obtained thereby and their uses are also described.
Abstract.
Ying Q-L, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A (2008). The ground state of embryonic stem cell self-renewal.
Nature,
453, 519-523.
Abstract:
The ground state of embryonic stem cell self-renewal.
In the three decades since pluripotent mouse embryonic stem (ES) cells were first described they have been derived and maintained by using various empirical combinations of feeder cells, conditioned media, cytokines, growth factors, hormones, fetal calf serum, and serum extracts. Consequently ES-cell self-renewal is generally considered to be dependent on multifactorial stimulation of dedicated transcriptional circuitries, pre-eminent among which is the activation of STAT3 by cytokines (ref. 8). Here we show, however, that extrinsic stimuli are dispensable for the derivation, propagation and pluripotency of ES cells. Self-renewal is enabled by the elimination of differentiation-inducing signalling from mitogen-activated protein kinase. Additional inhibition of glycogen synthase kinase 3 consolidates biosynthetic capacity and suppresses residual differentiation. Complete bypass of cytokine signalling is confirmed by isolating ES cells genetically devoid of STAT3. These findings reveal that ES cells have an innate programme for self-replication that does not require extrinsic instruction. This property may account for their latent tumorigenicity. The delineation of minimal requirements for self-renewal now provides a defined platform for the precise description and dissection of the pluripotent state.
Abstract.
2007
Smith AG, Ying QL (2007). Culture medium containing kinase inhibitors. and uses thereof including rat embryonic stem cells.
Abstract:
Culture medium containing kinase inhibitors. and uses thereof including rat embryonic stem cells
Pluripotent cells are maintained in a self-renewing state in serum-free culture medium comprising a MEK inhibitor, a GSK3 inhibitor and, optionally, an antagonist of an FGF receptor. Pluripotent cells are also maintained in a self-renewing state in serum-free culture medium comprising a MEK inhibitor and an antagonist of an FGF receptor.
Abstract.
Johnson CE, Crawford BE, Stavridis M, Ten Dam G, Wat AL, Rushton G, Ward CM, Wilson V, Van Kuppevelt TH, Esko JD, et al (2007). Erratum: Essential alterations of heparan sulfate during the differentiation of embryonic stem cells to sox1-enhanced green fluorescent protein-expressing neural progenitor cells (Stem Cells (2007) 25 (1913)-1923) DOI:10.1634/stemcells.2006-0445). Stem Cells, 25, 2389-2389.
Johnson CE, Crawford BE, Stavridis M, Ten Dam G, Wat AL, Rushton G, Ward CM, Wilson V, van Kuppevelt TH, Esko JD, et al (2007). Essential alterations of heparan sulfate during the differentiation of embryonic stem cells to Sox1-enhanced green fluorescent protein-expressing neural progenitor cells.
Stem Cells,
25, 1913-1923.
Abstract:
Essential alterations of heparan sulfate during the differentiation of embryonic stem cells to Sox1-enhanced green fluorescent protein-expressing neural progenitor cells.
Embryonic stem (ES) cells can be cultured in conditions that either maintain pluripotency or allow differentiation to the three embryonic germ layers. Heparan sulfate (HS), a highly polymorphic glycosaminoglycan, is a critical cell surface coreceptor in embryogenesis, and in this paper we describe its structural transition from an unusually low-sulfated variant in ES cells to a more highly sulfated form in fluorescence-activated cell sorting-purified neural progenitor cells. The characteristic domain structure of HS was retained during this transformation. However, qualitative variations in surface sulfation patterns between ES and differentiated cells were revealed using HS epitope-specific antibodies and the HS-binding growth factor fibroblast growth factor 2 (FGF-2). Expression profiles of the HS modification enzymes indicated that both "early" (N-sulfotransferases) and "late" (6O- and 3O-sulfotransferases) sulfotransferases contributed to the alterations in sulfation patterning. An HS-null ES line was used to demonstrate the necessity for HS in neural differentiation. HS is a coreceptor for many of the protein effectors implicated in pluripotency and differentiation (e.g. members of the FGF family, bone morphogenic proteins, and fibronectin). We suggest that the stage-specific activities of these proteins are finely regulated by dynamic changes in sulfation motifs in HS chains. Disclosure of potential conflicts of interest is found at the end of this article.
Abstract.
Kunath T, Saba-El-Leil MK, Almousailleakh M, Wray J, Meloche S, Smith A (2007). FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment.
Development,
134, 2895-2902.
Abstract:
FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment.
Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here, we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead, such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction, and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus, FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells, which fail to undergo either neural or mesodermal differentiation in adherent culture, and retain expression of pluripotency markers Oct4, Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for naïve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos.
Abstract.
Chambers I, Silva J, Colby D, Nichols J, Nijmeijer B, Robertson M, Vrana J, Jones K, Grotewold L, Smith A, et al (2007). Nanog safeguards pluripotency and mediates germline development.
Nature,
450, 1230-1234.
Abstract:
Nanog safeguards pluripotency and mediates germline development.
Nanog is a divergent homeodomain protein found in mammalian pluripotent cells and developing germ cells. Deletion of Nanog causes early embryonic lethality, whereas constitutive expression enables autonomous self-renewal of embryonic stem cells. Nanog is accordingly considered a core element of the pluripotent transcriptional network. However, here we report that Nanog fluctuates in mouse embryonic stem cells. Transient downregulation of Nanog appears to predispose cells towards differentiation but does not mark commitment. By genetic deletion we show that, although they are prone to differentiate, embryonic stem cells can self-renew indefinitely in the permanent absence of Nanog. Expanded Nanog null cells colonize embryonic germ layers and exhibit multilineage differentiation both in fetal and adult chimaeras. Although they are also recruited to the germ line, primordial germ cells lacking Nanog fail to mature on reaching the genital ridge. This defect is rescued by repair of the mutant allele. Thus Nanog is dispensible for expression of somatic pluripotency but is specifically required for formation of germ cells. Nanog therefore acts primarily in construction of inner cell mass and germ cell states rather than in the housekeeping machinery of pluripotency. We surmise that Nanog stabilizes embryonic stem cells in culture by resisting or reversing alternative gene expression states.
Abstract.
Takashima Y, Era T, Nakao K, Kondo S, Kasuga M, Smith AG, Nishikawa S-I (2007). Neuroepithelial cells supply an initial transient wave of MSC differentiation.
Cell,
129, 1377-1388.
Abstract:
Neuroepithelial cells supply an initial transient wave of MSC differentiation.
Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and are able to give rise to multiple mesenchymal lineages. Although MSCs are already used in regenerative medicine little is known about their in vivo behavior and developmental derivation. Here, we show that the earliest wave of MSC in the embryonic trunk is generated from Sox1+ neuroepithelium but not from mesoderm. Using lineage marking by direct gfp knock-in and Cre-recombinase mediated lineage tracing, we provide evidence that Sox1+ neuroepithelium gives rise to MSCs in part through a neural crest intermediate stage. This pathway can be distinguished from the pathway through which Sox1+ cells give rise to oligodendrocytes by expression of PDGFRbeta and A2B5. MSC recruitment from this pathway, however, is transient and is replaced by MSCs from unknown sources. We conclude that MSC can be defined as a definite in vivo entity recruited from multiple developmental origins.
Abstract.
Glaser T, Pollard SM, Smith A, Brüstle O (2007). Tripotential differentiation of adherently expandable neural stem (NS) cells.
PLoS One,
2, e298-e298.
Abstract:
Tripotential differentiation of adherently expandable neural stem (NS) cells.
BACKGROUND: a recent study has shown that pure neural stem cells can be derived from embryonic stem (ES) cells and primary brain tissue. In the presence of fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF), this population can be continuously expanded in adherent conditions. In analogy to continuously self-renewing ES cells, these cells were termed ’NS’ cells (Conti et al. PLoS Biol 3: e283, 2005). While NS cells have been shown to readily generate neurons and astrocytes, their differentiation into oligodendrocytes has remained enigmatic, raising concerns as to whether they truly represent tripotential neural stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Here we provide evidence that NS cells are indeed tripotent. Upon proliferation with FGF2, platelet-derived growth factor (PDGF) and forskolin, followed by differentiation in the presence of thyroid hormone (T3) and ascorbic acid NS cells efficiently generate oligodendrocytes ( approximately 20%) alongside astrocytes ( approximately 40%) and neurons ( approximately 10%). Mature oligodendroglial differentiation was confirmed by transplantation data showing that NS cell-derived oligodendrocytes ensheath host axons in the brain of myelin-deficient rats. CONCLUSIONS/SIGNIFICANCE: in addition to delineating NS cells as a potential donor source for myelin repair, our data strongly support the view that these adherently expandable cells represent bona fide tripotential neural stem cells.
Abstract.
2006
Smith A (2006). A glossary for stem-cell biology.
Nature,
441, 1060-1060.
Abstract:
A glossary for stem-cell biology
Stem-cell biology is in a phase of dynamic expansion and is forming connections with a broad range of basic and applied disciplines. The field is simultaneously exposed to public and political scrutiny. A common language in the stem-cell community is an important tool for coherent exposition to these diverse audiences, not least because certain terms in the stem-cell vocabulary are used differently in other fields. © 2006 Macmillan Publishers Ltd.
Abstract.
Pollard SM, Conti L, Sun Y, Goffred D, Smith A (2006). Adherent neural stem (NS) cells from fetal and adult forebrain. CEREBRAL CORTEX, 16, I112-I120.
Pollard SM, Conti L, Sun Y, Goffredo D, Smith A (2006). Adherent neural stem (NS) cells from fetal and adult forebrain.
Cereb Cortex,
16 Suppl 1, i112-i120.
Abstract:
Adherent neural stem (NS) cells from fetal and adult forebrain.
Stable in vitro propagation of central nervous system (CNS) stem cells would offer expanded opportunities to dissect basic molecular, cellular, and developmental processes and to model neurodegenerative disease. CNS stem cells could also provide a source of material for drug discovery assays and cell replacement therapies. We have recently reported the generation of adherent, symmetrically expandable, neural stem (NS) cell lines derived both from mouse and human embryonic stem cells and from fetal forebrain (Conti L, Pollard SM, Gorba T, Reitano E, Toselli M, Biella G, Sun Y, Sanzone S, Ying QL, Cattaneo E, Smith A. 2005. Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol 3(9):e283). These NS cells retain neuronal and glial differentiation potential after prolonged passaging and are transplantable. NS cells are likely to comprise the resident stem cell population within heterogeneous neurosphere cultures. Here we demonstrate that similar NS cell cultures can be established from the adult mouse brain. We also characterize the growth factor requirements for NS cell derivation and self-renewal. We discuss our current understanding of the relationship of NS cell lines to physiological progenitor cells of fetal and adult CNS.
Abstract.
Smith AG, Ying QL (2006). Culture medium containing kinase inhibitors, and uses thereof.
Abstract:
Culture medium containing kinase inhibitors, and uses thereof
Pluripotent cells are maintained in a self-renewing state in serum-free culture medium comprising a gpl30 agonist (LIF) and a GSK3 inhibitor.
Abstract.
Lowell S, Smith AG (2006). Directed neural differentiation.
Abstract:
Directed neural differentiation
Differentiation towards a neural fate, and away from a non-neural fate, is promoted by activation of Notch signalling in ES cells and then transferring the cells into neural differentiation protocols. Media for neural differentiation comprises a Notch activator, e.g. a notch ligand that can be clustered. Genetic manipulation is used as an alternative to media additives for Notch activation.
Abstract.
Smith AG (2006). Embryonic stem cells: potency and potential. EMBO Science & Society paper, Stem Cell Research - Status Prospectus Prerequisites
Pollard S, Conti L, Smith A (2006). Exploitation of adherent neural stem cells in basic and applied neurobiology.
Regen Med,
1, 111-118.
Abstract:
Exploitation of adherent neural stem cells in basic and applied neurobiology.
Evidence for neurogenesis within the adult brain has challenged traditional views that this tissue is devoid of stem cell activity. This raises the possibility of introducing new cells through cell transplantation or stimulating endogenous neurogenesis as routes to treat disease and injury. Fetal and adult neural stem/progenitor cells can be isolated and expanded in vitro and might provide a cell source for such transplantations. Embryonic stem (ES) cells, which can generate any adult tissues, offer an alternative unlimited supply of neural tissue. We recently showed that both mouse and human ES cells can be converted to adherent neural stem (NS) cell lines [1]. Here we discuss the benefits of working with NS cell lines and how they might be exploited for studies of fundamental cellular processes, such as neuronal specification and differentiation. NS cells also serve as versatile models of disease processes, either through genetic manipulations or direct isolation from disease carriers and can be exploited in pharmaceutical drug screening. Longer term, NS cells offer an opportunity to rigorously test the efficacy of cell-based therapies and develop strategies for tissue engineering.
Abstract.
Silva J, Chambers I, Pollard S, Smith A (2006). Nanog promotes transfer of pluripotency after cell fusion.
Nature,
441, 997-1001.
Abstract:
Nanog promotes transfer of pluripotency after cell fusion.
Through cell fusion, embryonic stem (ES) cells can erase the developmental programming of differentiated cell nuclei and impose pluripotency. Molecules that mediate this conversion should be identifiable in ES cells. One candidate is the variant homeodomain protein Nanog, which has the capacity to entrain undifferentiated ES cell propagation. Here we report that in fusions between ES cells and neural stem (NS) cells, increased levels of Nanog stimulate pluripotent gene activation from the somatic cell genome and enable an up to 200-fold increase in the recovery of hybrid colonies, all of which show ES cell characteristics. Nanog also improves hybrid yield when thymocytes or fibroblasts are fused to ES cells; however, fewer colonies are obtained than from ES x NS cell fusions, consistent with a hierarchical susceptibility to reprogramming among somatic cell types. Notably, for NS x ES cell fusions elevated Nanog enables primary hybrids to develop into ES cell colonies with identical frequency to homotypic ES x ES fusion products. This means that in hybrids, increased Nanog is sufficient for the NS cell epigenome to be reset completely to a state of pluripotency. We conclude that Nanog can orchestrate ES cell machinery to instate pluripotency with an efficiency of up to 100% depending on the differentiation status of the somatic cell.
Abstract.
Conti L, Pollard SM, Smith AG (2006). Neural Stem Cells.
Abstract:
Neural Stem Cells
A homogenous, symmetrically dividing population of adherent neural stem cells is obtained from ES cells or foetal or adult brain isolates, using an activator of a signalling pathway downstream of a receptor of the EGF receptor family, optionally in combination with an activator of a signalling pathway downstream of an FGF receptor. The neural stem cell population is highly pure and retains the ability to differentiate into neurons after in excess of 100 passages.
Abstract.
Lowell S, Benchoua A, Heavey B, Smith AG (2006). Notch promotes neural lineage entry by pluripotent embryonic stem cells.
PLoS Biol,
4, e121-e121.
Abstract:
Notch promotes neural lineage entry by pluripotent embryonic stem cells.
A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice.
Abstract.
Blelloch R, Wang Z, Meissner A, Pollard S, Smith A, Jaenisch R (2006). Reprogramming efficiency following somatic cell nuclear transfer is influenced by the differentiation and methylation state of the donor nucleus.
Stem Cells,
24, 2007-2013.
Abstract:
Reprogramming efficiency following somatic cell nuclear transfer is influenced by the differentiation and methylation state of the donor nucleus.
Reprogramming of a differentiated cell nucleus by somatic cell nuclear transplantation is an inefficient process. Following nuclear transfer, the donor nucleus often fails to express early embryonic genes and establish a normal embryonic pattern of chromatin modifications. These defects correlate with the low number of cloned embryos able to produce embryonic stem cells or develop into adult animals. Here, we show that the differentiation and methylation state of the donor cell influence the efficiency of genomic reprogramming. First, neural stem cells, when used as donors for nuclear transplantation, produce embryonic stem cells at a higher efficiency than blastocysts derived from terminally differentiated neuronal donor cells, demonstrating a correlation between the state of differentiation and cloning efficiency. Second, using a hypomorphic allele of DNA methyltransferase-1, we found that global hypomethylation of a differentiated cell genome improved cloning efficiency. Our results provide functional evidence that the differentiation and epigenetic state of the donor nucleus influences reprogramming efficiency.
Abstract.
2005
Fouladi-Nashta AA, Jones CJP, Nijjar N, Mohamet L, Smith A, Chambers I, Kimber SJ (2005). Characterization of the uterine phenotype during the peri-implantation period for LIF-null, MF1 strain mice.
Dev Biol,
281, 1-21.
Abstract:
Characterization of the uterine phenotype during the peri-implantation period for LIF-null, MF1 strain mice.
Leukemia inhibitory factor plays a major role in the uterus and in its absence embryos fail to implant. Our knowledge of the targets for LIF and the consequences of its absence is still very incomplete. In this study, we have examined the ultrastructure of the potential implantation site in LIF-null MF1 female mice compared to that of wild type animals. We also compared expression of proteins associated with implantation in luminal epithelium and stroma. Luminal epithelial cells (LE) of null animals failed to develop apical pinopods, had increased glycocalyx, and retained a columnar shape during the peri-implantation period. Stromal cells of LIF-null animals showed no evidence of decidual giant cell formation even by day 6 of pregnancy. A number of proteins normally expressed in decidualizing stroma did not increase in abundance in the LIF-null animals including desmin, tenascin, Cox-2, bone morphogenetic protein (BMP)-2 and -7, and Hoxa-10. In wild type animals, the IL-6 family member Oncostatin M (OSM) was found to be transiently expressed in the luminal epithelium on late day 4 and then in the stroma at the attachment site on days 5-6 of pregnancy, with a similar but not identical pattern to that of Cox-2. In the LIF-null animals, no OSM protein was detected in either LE or stroma adjacent to the embryo, indicating that expression requires uterine LIF in addition to a blastocyst signal. Fucosylated epitopes: the H-type-1 antigen and those recognized by lectins from Ulex europaeus-1 and Tetragonolobus purpureus were enhanced on apical LE on day 4 of pregnancy. H-type-1 antigen remained higher on day 5, and was not reduced even by day 6 in contrast to wild type uterus. These data point to a profound disturbance of normal luminal epithelial and stromal differentiation during early pregnancy in LIF-nulls. On this background, we also obtained less than a Mendelian ratio of null offspring suggesting developmental failure.
Abstract.
Nichols J, Smith AG, Ying QL (2005). Control of ES cell self renewal and lineage specification, and medium therefore including rat ES cells.
Abstract:
Control of ES cell self renewal and lineage specification, and medium therefore including rat ES cells
Self renewal of pluripotent cells in culture is promoted using a combination of an Id gene product and an activator of a gp130 downstream signalling pathway.
Abstract.
Wilmut I, West MD, Lanza RP, Gearhart JD, Smith A, Colman A, Trounson AO, Campbell KH (2005). Human embryonic stem cells. Science, 310, 1903-1903.
Conti L, Pollard SM, Gorba T, Reitano E, Toselli M, Biella G, Sun Y, Sanzone S, Ying Q-L, Cattaneo E, et al (2005). Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.
PLoS Biol,
3, e283-e283.
Abstract:
Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.
Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.
Abstract.
Kawaguchi J, Mee PJ, Smith AG (2005). Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors.
Bone,
36(5), 758-769.
Abstract:
Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors
Reliable in vitro conversion of pluripotent embryonic stem (ES) cells into bone and cartilage-forming cells would expand opportunities for experimental investigations of skeletogenesis and could also provide new cellular sources for pharmaceutical screening and for cell therapy applications. Here, we evaluate the generation of mesenchymal cell lineages from mouse ES cells following treatment of embryoid bodies with retinoic acid, previously reported to induce development of adipocyte precursors. We find that retinoic acid reduces mesodermal differentiation but enhances expression of markers of neural crest, an alternative origin of mesenchymal elements. Runx1 and Ptprv appear to provide early markers of mesenchymal potential. Subsequently, different mesenchymal fates are generated in response to particular growth factors. Substitution of the adipogenic factors insulin and triiodothyronine with bone morphogenetic protein (BMP-4) results in suppression of adipogenesis and development of a mature osteogenic phenotype. In contrast, treatment with transforming growth factor-β (TGF-β3) promotes chondrogenic differentiation. Thus, the use of appropriate growth factors and culture milieu steers differentiation of ES cell-derived precursors into distinct mesenchymal compartments. © 2005 Elsevier Inc. All rights reserved.
Abstract.
Kawaguchi J, Mee PJ, Smith AG (2005). Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors.
Bone,
36, 758-769.
Abstract:
Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors
Reliable in vitro conversion of pluripotent embryonic stem (ES) cells into bone and cartilage-forming cells would expand opportunities for experimental investigations of skeletogenesis and could also provide new cellular sources for pharmaceutical screening and for cell therapy applications. Here, we evaluate the generation of mesenchymal cell lineages from mouse ES cells following treatment of embryoid bodies with retinoic acid, previously reported to induce development of adipocyte precursors. We find that retinoic acid reduces mesodermal differentiation but enhances expression of markers of neural crest, an alternative origin of mesenchymal elements. Runx1 and Ptprv appear to provide early markers of mesenchymal potential. Subsequently, different mesenchymal fates are generated in response to particular growth factors. Substitution of the adipogenic factors insulin and triiodothyronine with bone morphogenetic protein (BMP-4) results in suppression of adipogenesis and development of a mature osteogenic phenotype. In contrast, treatment with transforming growth factor-β (TGF-β3) promotes chondrogenic differentiation. Thus, the use of appropriate growth factors and culture milieu steers differentiation of ES cell-derived precursors into distinct mesenchymal compartments. © 2005 Elsevier Inc. All rights reserved.
Abstract.
Wong KJ, Arvind DK, Sharwood-Smith N, Smith A (2005). Specknet-based responsive environments.
Abstract:
Specknet-based responsive environments
Abstract.
Smith A (2005). The battlefield of pluripotency.
Cell,
123, 757-760.
Abstract:
The battlefield of pluripotency.
How embryonic stem cells maintain the potential to differentiate into multiple cell lineages is still unclear. In this issue of Cell, Niwa et al. (2005) show that a duel between the transcription factors Oct3/4 and Cdx2 can restrict embryonic stem cells to either embryonic or placental fate. The vulnerability of lineage potential to transcriptional perturbation may reflect an essential feature of pluripotency.
Abstract.
Nichols J, Ying QL, Smith AG (2005). Understanding embryonic stem cells.
Matveeva NM, Pristyazhnyuk IE, Temirova SA, Menzorov AG, Vasilkova A, Shilov AG, Smith A, Serov OL (2005). Unequal segregation of parental chromosomes in embryonic stem cell hybrids.
Mol Reprod Dev,
71, 305-314.
Abstract:
Unequal segregation of parental chromosomes in embryonic stem cell hybrids.
Chromosome segregation was studied in 14 intra- and 20 inter-specific hybrid clones generated by fusion of Mus musculus embryonic stem (ES) cells with fibroblasts or splenocytes of DD/c mice or Mus caroli. As a control for in vitro evolution of tetraploid karyotype we used a set of hybrid clones obtained by fusion of ES cells (D3) with ES cells (TgTP6.3). Identification of the parental chromosomes in the clones was performed by microsatellite analysis and in situ hybridization with labeled species-specific probes. Both analyses have revealed three types of clones: (i) stable tetraploid, observed only for ES x ES cell hybrids; (ii) bilateral loss of chromosomes of both ES and somatic partners; (iii) unilateral segregation of chromosomes of the somatic partner. Observed unilateral segregation was extensive in ES-splenocyte cell hybrids, but lower in ES-fibroblast hybrid clones. Developmental state of the somatic partner is presumably responsible for directional chromosome loss. Nonrandom segregation implies that initial differences in the parental homologous chromosomes were not immediately equalized implying at least transient persistence of the differentiated epigenotype.
Abstract.
2004
Smith AG, Ying QL (2004). Control of ES cell self renewal and lineage specification, and medium therefor.
Abstract:
Control of ES cell self renewal and lineage specification, and medium therefor
Self renewal of pluripotent cells in culture is promoted using a combination of an activator of a signalling pathway downstream of a receptor of the TGF-ß superfamily and an activator of a gp130 downstream signalling pathway.
Abstract.
Smith A (2004). Converting ES cell into neurons. In (Ed) , 83-93.
Smith AG (2004). ES cells: Research progress in UK. Cell Technology (Japan), 23, 1268-1272.
Sherwin JRA, Freeman TC, Stephens RJ, Kimber S, Smith AG, Chambers I, Smith SK, Sharkey AM (2004). Identification of genes regulated by leukemia-inhibitory factor in the mouse uterus at the time of implantation.
Mol Endocrinol,
18, 2185-2195.
Abstract:
Identification of genes regulated by leukemia-inhibitory factor in the mouse uterus at the time of implantation.
The endometrium is prepared for implantation by the actions of estradiol (E2) and progesterone (P4). In mice the luminal epithelium (LE) only becomes fully receptive to the attaching blastocyst in response to the nidatory estrogen surge on d 4 of pregnancy. The cytokine leukemia-inhibitory factor (LIF) is rapidly induced by nidatory estrogen and has been shown to be the primary mediator of its action. Implantation fails in the absence of LIF, and injection of LIF on d 4 of pregnancy can substitute for the nidatory estrogen. In this study, we sought to identify genes regulated by LIF in the uterine epithelium. We used oligonucleotide microarrays to compare the transcript profiles of paired uterine horns from LIF-deficient MF1 mice after intraluminal injection of LIF or PBS on d 4 of pseudopregnancy. IGF-binding protein 3 was identified as a gene up-regulated by LIF; this was confirmed by RT-PCR. In situ hybridization showed that the primary site of IGF-binding protein 3 expression is the luminal epithelium (LE), the known site of LIF action in the uterus. We identified two other genes: amphiregulin and immune response gene-1, the expression of which were also up-regulated by LIF. Immune response gene 1 has recently been shown to be essential for implantation. Expression of all three of these genes in the LE is known to be regulated by P4. The expression of osteoblast-specific factor 2 and leukocyte 12/15 lipoxygenase, which are also expressed in LE under the control of P4, were not increased by LIF. This suggests that one of the actions of LIF on LE may be to enhance the expression of a subset of P4-regulated genes.
Abstract.
Dacquin R, Mee PJ, Kawaguchi J, Olmsted-Davis EA, Gallagher JA, Nichols J, Lee K, Karsenty G, Smith A (2004). Knock-in of nuclear localised beta-galactosidase reveals that the tyrosine phosphatase Ptprv is specifically expressed in cells of the bone collar.
Dev Dyn,
229, 826-834.
Abstract:
Knock-in of nuclear localised beta-galactosidase reveals that the tyrosine phosphatase Ptprv is specifically expressed in cells of the bone collar.
Ptprv is a member of the transmembrane tyrosine phosphatase gene family reported to be expressed in osteoblasts and gonads. To better define the developmental and tissue specificity of Ptprv expression, we generated knock-in mice expressing a nuclear localised beta-galactosidase reporter under the control of resident Ptprv regulatory elements. Histochemical staining of Ptprv-nLacZ mice revealed that Ptprv expression is readily detectable in the foetal gonadal ridge of both sexes and in adult gonads where it is localised to Sertoli cells of the testis and celomic epithelial cells of the ovaries. During early limb development, Ptprv expression is prominent in the apical ectodermal ridge of the limb bud. At latter stages of development, Ptprv is predominantly expressed in the perichondrial and periosteal region of long bones, known as the bone collar. In contrast to previous indications from in vitro studies, there is little if any expression in mature osteoblasts in vivo. Analysis of Ptprv mRNA localisation by in situ hybridization in parallel with molecular markers of chondrocytes and osteoblasts confirmed the specific expression of Ptprv in immature bone collar cells. The specificity of Ptprv expression in these cells may be a useful tool to elucidate their role in the transition of skeletal elements from cartilage template to bone.
Abstract.
Chuang TT, Griffiths D, Evans N, Smith A, Schroeder J (2004). Regulation of neural stem cells by beta amyloid in vitro.
Mohamet L, Fouladi-Nashta A, Nijjar N, Smith AG, Kimber SJ (2004). Regulation of uterine gene expression by leukaemia inhibitory factor.
Chambers I, Smith A (2004). Self-renewal of teratocarcinoma and embryonic stem cells.
Oncogene,
23, 7150-7160.
Abstract:
Self-renewal of teratocarcinoma and embryonic stem cells.
Pluripotent stem cells derived from preimplantation embryos, primordial germ cells or teratocarcinomas are currently unique in undergoing prolonged symmetrical self-renewal in culture. For mouse embryonic stem (ES) cells, self-renewal is dependent on signals from the cytokine leukaemia inhibitory factor (LIF) and from either serum or bone morphogenetic proteins (BMPs). In addition to the extrinsic regulation of gene expression, intrinsic transcriptional determinants are also required for maintenance of the undifferentiated state. These include Oct4, a member of the POU family of homeodomain proteins and a second recently identified homeodomain protein, Nanog. When overexpressed, Nanog allows ES cells to self-renew in the absence of the otherwise obligatory LIF and BMP signals. Although Nanog can act independent of the LIF signal, a contribution of both pathways provides maximal self-renewal efficiency. Nanog function also requires Oct4. Here, we review recent progress in ES cell self-renewal, relate this to the biology of teratocarcinomas and offer testable hypotheses to expose the mechanics of ES cell self-renewal.
Abstract.
Zhao S, Nichols J, Smith AG, Li M (2004). SoxB transcription factors specify neuroectodermal lineage choice in ES cells.
Mol Cell Neurosci,
27, 332-342.
Abstract:
SoxB transcription factors specify neuroectodermal lineage choice in ES cells.
Knowledge of lineage decision machinery in pluripotent embryonic stem (ES) cells may shed light on the process of germ layer segregation in the mammalian embryo and enable directed differentiation in vitro for biomedical applications. We have investigated the contribution of Class B1 Sox transcription factors to lineage choice during ES cell differentiation. We report that forced expression of Sox1 or Sox2 did not impair propagation of undifferentiated ES cells, but upon release from self-renewal promoted differentiation into neuroectoderm at the expense of mesoderm and endoderm. The efficient specification of a primary lineage by transcription factor manipulation provides a paradigm for instructing differentiation of ES cells for biopharmaceutical screening and cell therapy applications.
Abstract.
Johnson CE, Stavridis MP, Crawford BE, Wilson VA, Esko JD, Smith AG, Gallagher JT, Merry CLR (2004). The differentiation of ES cells into neuroectodermal precursors is associated with an increase in the levels and sulfation of heparan sulfate proteoglycans. INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, 85, A65-A66.
2003
Mee PJ, Shen MH, Smith AG, Brown WRA (2003). An unpaired mouse centromere passes consistently through male meiosis and does not significantly compromise spermatogenesis.
Chromosoma,
112, 183-189.
Abstract:
An unpaired mouse centromere passes consistently through male meiosis and does not significantly compromise spermatogenesis.
ST1 is an artificial mini-chromosome approximately 4.5 Mb in size containing mouse minor and major satellite DNA, human alphoid DNA and sequences derived from interval 5 of the human Y chromosome. Here we have measured the mitotic and meiotic transmission of ST1 and have used the mini-chromosome to define the ability of mice to monitor the presence of unpaired centromeres during meiosis. ST1 is mitotically stable, remaining intact and autonomous in mice for many generations. Female mice efficiently transmit ST1 to their offspring at a frequency approaching 50%. Male mice also reliably transmit the mini-chromosome, though to only 20% of their offspring. Presence of ST1 in males is not associated with any compromise in the output of the seminiferous epithelium nor with histological or immunocytochemical evidence of increased apoptosis, outcomes predicted for a synapsis checkpoint. These data indicate that the presence of an unpaired centromere is not sufficient to arrest male meiosis, implying that univalents are normally eliminated by a mechanism other than a tension-sensitive spindle checkpoint.
Abstract.
Ying QL, Nichols J, Chambers I, Smith A (2003). BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.
Cell,
115, 281-292.
Abstract:
BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.
The cytokine leukemia inhibitory factor (LIF) drives self-renewal of mouse embryonic stem (ES) cells by activating the transcription factor STAT3. In serum-free cultures, however, LIF is insufficient to block neural differentiation and maintain pluripotency. Here, we report that bone morphogenetic proteins (BMPs) act in combination with LIF to sustain self-renewal and preserve multilineage differentiation, chimera colonization, and germline transmission properties. ES cells can be propagated from single cells and derived de novo without serum or feeders using LIF plus BMP. The critical contribution of BMP is to induce expression of Id genes via the Smad pathway. Forced expression of Id liberates ES cells from BMP or serum dependence and allows self-renewal in LIF alone. Upon LIF withdrawal, Id-expressing ES cells differentiate but do not give rise to neural lineages. We conclude that blockade of lineage-specific transcription factors by Id proteins enables the self-renewal response to LIF/STAT3.
Abstract.
Ying Q-L, Stavridis M, Griffiths D, Li M, Smith A (2003). Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture.
Nat Biotechnol,
21, 183-186.
Abstract:
Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture.
Mouse embryonic stem (ES) cells are competent for production of all fetal and adult cell types. However, the utility of ES cells as a developmental model or as a source of defined cell populations for pharmaceutical screening or transplantation is compromised because their differentiation in vitro is poorly controlled. Specification of primary lineages is not understood and consequently differentiation protocols are empirical, yielding variable and heterogeneous outcomes. Here we report that neither multicellular aggregation nor coculture is necessary for ES cells to commit efficiently to a neural fate. In adherent monoculture, elimination of inductive signals for alternative fates is sufficient for ES cells to develop into neural precursors. This process is not a simple default pathway, however, but requires autocrine fibroblast growth factor (FGF). Using flow cytometry quantitation and recording of individual colonies, we establish that the bulk of ES cells undergo neural conversion. The neural precursors can be purified to homogeneity by fluorescence activated cell sorting (FACS) or drug selection. This system provides a platform for defining the molecular machinery of neural commitment and optimizing the efficiency of neuronal and glial cell production from pluripotent mammalian stem cells.
Abstract.
Ying Q-L, Smith AG (2003). Defined conditions for neural commitment and differentiation.
Methods Enzymol,
365, 327-341.
Abstract:
Defined conditions for neural commitment and differentiation.
The efficiency of monolayer differentiation establishes that commitment of ES cells to a neural fate needs neither multicellular aggregation nor extrinsic inducers. The entire process by which pluripotent ES cells acquire neural specification can be visualized and recorded at the level of individual colonies. Furthermore this simple culture system is amenable to cellular and molecular dissection, promising to yield new insights into the mechanism underlying neural determination in mammals and perhaps to deliver the goal of "directed" homogeneous differentiation of ES cells.
Abstract.
Surani A, Smith A (2003). Differentiation and gene regulation programming, reprogramming and regeneration - Editorial overview. CURR OPIN GENET DEV, 13, 445-447.
Testa G, Zhang Y, Vintersten K, Benes V, Pijnappel WWMP, Chambers I, Smith AJH, Smith AG, Stewart AF (2003). Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles.
Nat Biotechnol,
21, 443-447.
Abstract:
Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles.
The mouse is the leading vertebrate model because its genome can be altered by both random transgenesis and homologous recombination with targeting constructs. Both approaches have been hindered by the size and site limitations implicit in conventional Escherichia coli DNA-engineering methods. Homologous recombination in E. coli, or ’recombineering’, has overcome these limitations for bacterial artificial chromosome (BAC) transgenesis. Here we applied Red/ET recombineering (using the lambda Redalpha/Redbeta recombinase pair) to generate a 64 kilobase targeting construct that carried two selectable cassettes permitting the simultaneous mutation of the target gene, Mll, at sites 43 kb apart in one round of mouse embryonic stem (ES) cell targeting. The targeting frequency after dual selection was 6%. Because the two selectable cassettes were flanked by FRT or loxP sites, three more alleles can be generated by site-specific recombination. Our approach represents a simple way to introduce changes at two or more sites in a genetic locus, and thereafter generate allele combinations. The size of BAC templates offers new freedom for the design of targeting constructs. Combined with the use of two selectable cassettes placed far apart, BAC-based targeting constructs may be applicable to tasks such as regional exchanges, deletions, and insertions.
Abstract.
Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A (2003). Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells.
Cell,
113, 643-655.
Abstract:
Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells.
Embryonic stem (ES) cells undergo extended proliferation while remaining poised for multilineage differentiation. A unique network of transcription factors may characterize self-renewal and simultaneously suppress differentiation. We applied expression cloning in mouse ES cells to isolate a self-renewal determinant. Nanog is a divergent homeodomain protein that directs propagation of undifferentiated ES cells. Nanog mRNA is present in pluripotent mouse and human cell lines, and absent from differentiated cells. In preimplantation embryos, Nanog is restricted to founder cells from which ES cells can be derived. Endogenous Nanog acts in parallel with cytokine stimulation of Stat3 to drive ES cell self-renewal. Elevated Nanog expression from transgene constructs is sufficient for clonal expansion of ES cells, bypassing Stat3 and maintaining Oct4 levels. Cytokine dependence, multilineage differentiation, and embryo colonization capacity are fully restored upon transgene excision. These findings establish a central role for Nanog in the transcription factor hierarchy that defines ES cell identity.
Abstract.
Buehr M, Smith A (2003). Genesis of embryonic stem cells.
Philos Trans R Soc Lond B Biol Sci,
358, 1397-1402.
Abstract:
Genesis of embryonic stem cells.
Embryonic stem (ES) cells are permanent pluripotent stem cell lines established from pre-implantation mouse embryos. There is currently great interest in the potential therapeutic applications of analogous cells derived from human embryos. The isolation of ES cells is commonly presented as a straightforward transfer of cells in the early embryo into culture. In reality, however, continuous expansion of pluripotent cells does not occur in vivo, and in vitro is the exception rather than the norm. Both genetic and epigenetic factors influence the ability to derive ES cells. We have tracked the expression of a key marker and determinant of pluripotency, the transcription factor Oct-4, in primary cultures of mouse epiblasts and used this to assay the effect of experimental manipulations on the maintenance of a pluripotent cell compartment. We find that expression of Oct-4 is often lost prior to overt cytodifferentiation of the epiblast. The rate and extent of Oct-4 extinction varies with genetic background. We report that treatment with the MAP kinase/ERK kinase inhibitor PD98059, which suppresses activation of the mitogen-activated protein kinases Erk1 and Erk2, results in increased persistence of Oct-4-expressing cells. Oct-4 expression is also relatively sustained in cultures of diapause embryos and of isolated inner cell masses. Combination of all three conditions allowed the derivation of germline-competent ES cells from the normally refractory CBA mouse strain. These findings suggest that the genesis of an ES cell is a relatively complex process requiring epigenetic modulation of key gene expression over a brief time-window. Procedures that extend this time-window and/or directly regulate the critical genes should increase the efficiency of ES cell derivation.
Abstract.
Jones M, Rossant J, Robertson EJ, Smith A, Edwards RG (2003). Lineage allocation and asymmetries in the early mouse embryo - Discussion. PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON SERIES B-BIOLOGICAL SCIENCES, 358, 1349-1349.
Stavridis MP, Smith AG (2003). Neural differentiation of mouse embryonic stem cells.
Biochem Soc Trans,
31, 45-49.
Abstract:
Neural differentiation of mouse embryonic stem cells.
Pluripotent embryonic stem cells can give rise to neuroectodermal derivatives in culture. This potential could be harnessed to generate neurons and glia for cell-replacement therapies in the central nervous system and for use in drug discovery. However, current methods of neural differentiation are empirical and relatively inefficient. Here, we review these methodologies and present new tools for quantification, analysis and manipulation of embryonic stem cell neural determination.
Abstract.
Chambers I, Smith AG (2003). Pluripotency determining factors and uses thereof.
Abstract:
Pluripotency determining factors and uses thereof
Pluripotency determining factors are described which act intracellularly and maintain a pluripotent cell in a pluripotent state in the absence of gp130 activation, which maintain or confer pluripotency of a human stem cell, which maintain or confer pluripotency of a mouse ES cell, and which maintain or confer pluripotency of a stem cell from a non-permissive strain of mice. The factors and vectors encoding or activating the factors are used to maintain and derive pluripotent cells, especially of higher mammals, including humans.
Abstract.
Buehr M, Nichols J, Stenhouse F, Mountford P, Greenhalgh CJ, Kantachuvesiri S, Brooker G, Mullins J, Smith AG (2003). Rapid loss of Oct-4 and pluripotency in cultured rodent blastocysts and derivative cell lines.
Biol Reprod,
68, 222-229.
Abstract:
Rapid loss of Oct-4 and pluripotency in cultured rodent blastocysts and derivative cell lines.
The POU transcription factor Oct-4 is essential for the pluripotent character of the mouse inner cell mass (ICM) and derivative embryonic stem (ES) cells. We analyzed the expression of Oct-4 during culture and establishment of cell lines from mouse and rat preimplantation embryos. Oct-4 was rapidly lost in primary outgrowths of the majority of cultured embryos prior to any evidence of morphological differentiation. Oct-4 persisted in only a minority of strain 129 cultures, which can go on to give ES cells. We used transgenic rats in which the dual reporter/selection marker beta-geo is under control of Oct-4 regulatory elements to investigate the effect of direct selection for Oct-4 expressing cells. Ablation of all cells occurred, consistent with complete downregulation of Oct-4. Without selection, in contrast, continuous cultures of morphologically undifferentiated cells could be derived readily from rat blastocysts and ICMs. However, these cells did not express significant Oct-4 and, although capable of differentiating into extraembryonic cell types, appeared incapable of producing fetal germ layer derivatives. Downregulation of Oct-4 appears to be a limiting factor in attempts to derive pluripotent cell lines from preimplantation embryos.
Abstract.
Aubert J, Stavridis MP, Tweedie S, O’Reilly M, Vierlinger K, Li M, Ghazal P, Pratt T, Mason JO, Roy D, et al (2003). Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice.
Proc Natl Acad Sci U S A,
100 Suppl 1, 11836-11841.
Abstract:
Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice.
The transcription factor Sox1 is the earliest and most specific known marker for mammalian neural progenitors. During fetal development, Sox1 is expressed by proliferating progenitor cells throughout the central nervous system and in no tissue but the lens. We generated a reporter mouse line in which egfp is inserted into the Sox1 locus. Sox1GFP animals faithfully recapitulate the expression of the endogenous gene. We have used the GFP reporter to purify neuroepithelial cells by fluorescence-activated cell sorting from embryonic day 10.5 embryos. RNAs prepared from Sox1GFP+ and Sox1GFP- embryo cells were then used to perform a pilot screen of subtracted cDNAs prepared from differentiating embryonic stem cells and arrayed on a glass chip. Fifteen unique differentially expressed genes were identified, all previously associated with fetal or adult neural tissue. Whole mount in situ hybridization against two genes of previously unknown embryonic expression, Lrrn1 and Musashi2, confirmed the selectivity of this screen for early neuroectodermal markers.
Abstract.
Medvinsky A, Smith A (2003). Stem cells: Fusion brings down barriers. Nature, 422, 823-825.
Nichols J, Tzouanacou E, Wilson V, Smith A (2003). The role of Oct-4 in early post-implantation development.
Nichols J, Tzouanacou E, Wilson V, Smith A (2003). The role of Oct-4 in early post-implantation development.
2002
Ying Q-L, Nichols J, Evans EP, Smith AG (2002). Changing potency by spontaneous fusion.
Nature,
416, 545-548.
Abstract:
Changing potency by spontaneous fusion.
Recent reports have suggested that mammalian stem cells residing in one tissue may have the capacity to produce differentiated cell types for other tissues and organs 1-9. Here we define a mechanism by which progenitor cells of the central nervous system can give rise to non-neural derivatives. Cells taken from mouse brain were co-cultured with pluripotent embryonic stem cells. Following selection for a transgenic marker carried only by the brain cells, undifferentiated stem cells are recovered in which the brain cell genome has undergone epigenetic reprogramming. However, these cells also carry a transgenic marker and chromosomes derived from the embryonic stem cells. Therefore the altered phenotype does not arise by direct conversion of brain to embryonic stem cell but rather through spontaneous generation of hybrid cells. The tetraploid hybrids exhibit full pluripotent character, including multilineage contribution to chimaeras. We propose that transdetermination consequent to cell fusion 10 could underlie many observations otherwise attributed to an intrinsic plasticity of tissue stem cells 9.
Abstract.
Aubert J, Dunstan H, Chambers I, Smith A (2002). Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation.
Nat Biotechnol,
20, 1240-1245.
Abstract:
Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation.
The multilineage differentiation capacity of mouse embryonic stem (ES) cells offers a potential testing platform for gene products that mediate mammalian lineage determination and cellular specialization. Identification of such differentiation regulators is crucial to harnessing ES cells for pharmaceutical discovery and cell therapy. Here we describe the use of episomal expression technology for functional evaluation of cDNA clones during ES-cell differentiation in vitro. Several candidate cDNAs identified by subtractive cloning and expression profiling were introduced into ES cells in episomal expression constructs. Subsequent differentiation revealed that the Wnt antagonist Sfrp2 stimulates production of neural progenitors. The significance of this observation was substantiated by forced expression of Wnt-1 and treatment with lithium chloride, both of which inhibit neural differentiation. These findings reveal the importance of Wnt signaling in regulating ES-cell lineage diversification. More generally, this study establishes a path for rapid and direct validation of candidate genes in ES cells.
Abstract.
Andrews PW, Moore H, Smith A (2002). Human embryonic stem cells: prospects for human health - a 1-day international symposium held at the University of Sheffield. J Anat, 200, 221-223.
Billon N, Jolicoeur C, Ying QL, Smith A, Raff M (2002). Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells.
J Cell Sci,
115, 3657-3665.
Abstract:
Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells.
Oligodendrocytes are post-mitotic cells that myelinate axons in the vertebrate central nervous system (CNS). They develop from proliferating oligodendrocyte precursor cells (OPCs), which arise in germinal zones, migrate throughout the developing white matter and divide a limited number of times before they terminally differentiate. Thus far, it has been possible to purify OPCs only from the rat optic nerve, but the purified cells cannot be obtained in large enough numbers for conventional biochemical analyses. Moreover, the CNS stem cells that give rise to OPCs have not been purified, limiting one’s ability to study the earliest stages of commitment to the oligodendrocyte lineage. Pluripotent, mouse embryonic stem (ES) cells can be propagated indefinitely in culture and induced to differentiate into various cell types. We have genetically engineered ES cells both to positively select neuroepithelial stem cells and to eliminate undifferentiated ES cells. We have then used combinations of known signal molecules to promote the development of OPCs from selected, ES-cell-derived, neuroepithelial cells. We show that the earliest stages of oligodendrocyte development follow an ordered sequence that is remarkably similar to that observed in vivo, suggesting that the ES-cell-derived neuroepithelial cells follow a normal developmental pathway to produce oligodendrocytes. These engineered ES cells thus provide a powerful system to study both the mechanisms that direct CNS stem cells down the oligodendrocyte pathway and those that influence subsequent oligodendrocyte differentiation. This strategy may also be useful for producing human cells for therapy and drug screening.
Abstract.
Niwa H, Masui S, Chambers I, Smith AG, Miyazaki J-I (2002). Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells.
Mol Cell Biol,
22, 1526-1536.
Abstract:
Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells.
Transcription factors of the POU family govern cell fate through combinatorial interactions with coactivators and corepressors. The POU factor Oct-3/4 can define differentiation, dedifferentation, or self-renewal of pluripotent embryonic stem (ES) cells in a sensitive, dose-dependent manner (H. Niwa, J.-I. Miyazali, and A. G. Smith, Nat. Genet. 24:372-376, 2000). Here we have developed a complementation assay based on the ability of Oct-3/4 transgenes to rescue self-renewal in conditionally null ES cells and used this to define which domains of Oct-3/4 are required to sustain the undifferentiated stem cell phenotype. Surprisingly, we found that molecules lacking either the N-terminal or C-terminal transactivation domain, though not both, can effectively replace full-length Oct-3/4. Furthermore, a fusion of the heterologous transactivation domain of Oct-2 to the Oct-3/4 POU domain can also sustain self-renewal. Thus, the unique function of Oct-3/4 in ES cell propagation resides in combination of the specific POU domain with a generic proline-rich transactivation domain. Interestingly, however, Oct-3/4 target gene expression elicited by the N- and C-terminal transactivation domains is not identical, indicating that at least one class of genes activated by Oct-3/4 is not required for ES cell propagation.
Abstract.
Burdon T, Smith A, Savatier P (2002). Signalling, cell cycle and pluripotency in embryonic stem cells.
Trends Cell Biol,
12, 432-438.
Abstract:
Signalling, cell cycle and pluripotency in embryonic stem cells.
Pluripotent mouse embryonic stem (ES) cells can be expanded in large numbers in vitro owing to a process of symmetrical self-renewal. Self-renewal entails proliferation with a concomitant suppression of differentiation. Here we describe how the cytokine leukaemia inhibitory factor (LIF) sustains self-renewal through activation of the transcription factor STAT3, and how two other signals - extracellular-signal-related kinase (ERK) and phosphatidylinositol-3-OH kinase (PI3K) - can influence differentiation and propagation, respectively. We relate these observations to the unusual cell-cycle properties of ES cells and speculate on the role of the cell cycle in maintaining pluripotency.
Abstract.
Lauer P, Metzner HJ, Zettlmeissl G, Li M, Smith AG, Lathe R, Dickneite G (2002). Targeted inactivation of the mouse locus encoding coagulation factor XIII-A: hemostatic abnormalities in mutant mice and characterization of the coagulation deficit.
Thromb Haemost,
88, 967-974.
Abstract:
Targeted inactivation of the mouse locus encoding coagulation factor XIII-A: hemostatic abnormalities in mutant mice and characterization of the coagulation deficit.
Blood coagulation factor XIII (FXIII) promotes cross-linking of fibrin during blood coagulation; impaired clot stabilization in human genetic deficiency is associated with marked pathologies of major clinical impact, including bleeding symptoms and deficient wound healing. To investigate the role of FXIII we employed homologous recombination to generate a targeted deletion of the inferred exon 7 of the FXIII-A gene. FXIII transglutaminase activity in plasma was reduced to about 50% in mice heterozygous for the mutant allele, and was abolished in homozygous null mice. Plasma fibrin gamma-dimerization was also indetectable in the homozygous deficient animals, confirming the absence of activatable FXIII. Homozygous mutant mice were fertile, although reproduction was impaired. Bleeding episodes, hematothorax, hematoperitoneum and subcutaneous hemorrhage in mutant mice were associated with reduced survival. Arrest of tail-tip bleeding in FXIII-A deficient mice was markedly and significantly delayed; replacement of mutant mice with human plasma FXIII (Fibrogammin P) restored bleeding time to within the normal range. Thrombelastography (TEG) experiments demonstrated impaired clot stabilization in FXIII-A mutant mice, replacement with human FXIII led to dose-dependent TEG normalization. The mutant mice thus reiterate some key features of the human genetic disorder: they will be valuable in assessing the role of FXIII in other associated pathologies and the development of new therapies.
Abstract.
2001
Smith AG (2001). Embryo-derived stem cells: of mice and men.
Annu Rev Cell Dev Biol,
17, 435-462.
Abstract:
Embryo-derived stem cells: of mice and men.
Mouse embryonic stem cells are continuous cell lines derived directly from the fetal founder tissue of the preimplantation embryo. They can be expanded in culture while retaining the functional attributes of pluripotent early embryo cells. In particular, they can participate fully in fetal development when reintroduced into the embryo. The capacity for multilineage differentiation is reproduced in culture where embryonic stem cells can produce a wide range of well-defined cell types. This has stimulated interest in the isolation of analogous cells of human origin. Such human pluripotent stem cells could constitute a renewable source of more differentiated cells that could be employed to replace diseased or damaged tissue by cellular transplantation. In this review, the relationships between mouse embryonic stem cells, resident pluripotent cells in the embryo, and human embryo-derived cell lines are evaluated, and the prospects and challenges of embryo stem cell research are considered.
Abstract.
Smith AG (2001). Embryonic Stem Cells. In Marshak DR, Gardner RL, Gottlieb DI (Eds.)
Stem cell biology, Cold Spring Harbor Laboratory Pr, 205-230.
Abstract:
Embryonic Stem Cells
Abstract.
Li M, Price D, Smith A (2001). Lineage selection and isolation of neural precursors from embryonic stem cells. In (Ed) , 29-42.
Nichols J, Chambers I, Taga T, Smith A (2001). Physiological rationale for responsiveness of mouse embryonic stem cells to gp130 cytokines.
Development,
128, 2333-2339.
Abstract:
Physiological rationale for responsiveness of mouse embryonic stem cells to gp130 cytokines.
Embryonic stem cells are established directly from the pluripotent epiblast of the preimplantation mouse embryo. Their derivation and propagation are dependent upon cytokine-stimulated activation of gp130 signal transduction. Embryonic stem cells maintain a close resemblance to epiblast in developmental potency and gene expression profile. The presumption of equivalence between embryonic stem cells and epiblast is challenged, however, by the finding that early embryogenesis can proceed in the absence of gp130. To explore this issue further, we have examined the capacity of gp130 mutant embryos to accommodate perturbation of normal developmental progression. Mouse embryos arrest at the late blastocyst stage when implantation is prevented. This process of diapause occurs naturally in lactating females or can be induced experimentally by removal of the ovaries. We report that gp130(-/-) embryos survive unimplanted in the uterus after ovariectomy but, in contrast to wild-type or heterozygous embryos, are subsequently unable to resume development. Inner cell masses explanted from gp130(-/-) delayed blastocysts produce only parietal endoderm, a derivative of the hypoblast. Intact mutant embryos show an absence of epiblast cells, and Hoechst staining and TUNEL analysis reveal a preceding increased incidence of cell death. These findings establish that gp130 signalling is essential for the prolonged maintenance of epiblast in vivo, which is commonly required of mouse embryos in the wild. We propose that the responsiveness of embryonic stem cells to gp130 signalling has its origin in this adaptive physiological function.
Abstract.
Li M, Smith AG (2001). Selection of lineage specific cells after differentiation of progenitor cells.
Smith AG (2001). Self-renewal and differentiation of pluripotent embryonic stem cells. DEVELOPMENTAL BIOLOGY, 235, 289-290.
2000
Shen MH, Mee PJ, Nichols J, Yang J, Brook F, Gardner RL, Smith AG, Brown WR (2000). A structurally defined mini-chromosome vector for the mouse germ line.
Curr Biol,
10, 31-34.
Abstract:
A structurally defined mini-chromosome vector for the mouse germ line.
Yeast artificial mini-chromosomes have helped to define the features of chromosome architecture important for accurate segregation and replication and have been used to identify genes important for chromosome stability and as large-fragment cloning vectors. Artificial chromosomes have been developed in human cells but they do not have defined, experimentally predictable structures. Fragments of human chromosomes have also been introduced into mice and in one case passed through the germ line. In these experiments, however, the structure and sequence organization of the fragments was not defined. Structurally defined mammalian mini-chromosome vectors should allow large tracts of DNA to be introduced into the vertebrate germ line for biotechnological purposes and for investigations of features of chromosome structure that influence gene expression. Here, we have determined the structure and sequence organization of an engineered mammalian mini-chromosome, ST1, and shown that it is stably maintained in vertebrate somatic cells and that it can be transmitted through the mouse germ line.
Abstract.
Gillespie CS, Sherman DL, Fleetwood-Walker SM, Cottrell DF, Tait S, Garry EM, Wallace VC, Ure J, Griffiths IR, Smith A, et al (2000). Peripheral demyelination and neuropathic pain behavior in periaxin-deficient mice.
Neuron,
26, 523-531.
Abstract:
Peripheral demyelination and neuropathic pain behavior in periaxin-deficient mice.
The Prx gene in Schwann cells encodes L- and S-periaxin, two abundant PDZ domain proteins thought to have a role in the stabilization of myelin in the peripheral nervous system (PNS). Mice lacking a functional Prx gene assemble compact PNS myelin. However, the sheath is unstable, leading to demyelination and reflex behaviors that are associated with the painful conditions caused by peripheral nerve damage. Older Prx-/- animals display extensive peripheral demyelination and a severe clinical phenotype with mechanical allodynia and thermal hyperalgesia, which can be reversed by intrathecal administration of a selective NMDA receptor antagonist We conclude that the periaxins play an essential role in stabilizing the Schwann cell-axon unit and that the periaxin-deficient mouse will be an important model for studying neuropathic pain in late onset demyelinating disease.
Abstract.
Burdon T, Smith AG (2000). Propagation and/or derivation of embryonic stem cells.
Niwa H, Miyazaki J, Smith AG (2000). Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.
Nat Genet,
24, 372-376.
Abstract:
Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.
Cell fate during development is defined by transcription factors that act as molecular switches to activate or repress specific gene expression programmes. The POU transcription factor Oct-3/4 (encoded by Pou5f1) is a candidate regulator in pluripotent and germline cells and is essential for the initial formation of a pluripotent founder cell population in the mammalian embryo. Here we use conditional expression and repression in embryonic stem (ES) cells to determine requirements for Oct-3/4 in the maintenance of developmental potency. Although transcriptional determination has usually been considered as a binary on-off control system, we found that the precise level of Oct-3/4 governs three distinct fates of ES cells. A less than twofold increase in expression causes differentiation into primitive endoderm and mesoderm. In contrast, repression of Oct-3/4 induces loss of pluripotency and dedifferentiation to trophectoderm. Thus a critical amount of Oct-3/4 is required to sustain stem-cell self-renewal, and up- or downregulation induce divergent developmental programmes. Our findings establish a role for Oct-3/4 as a master regulator of pluripotency that controls lineage commitment and illustrate the sophistication of critical transcriptional regulators and the consequent importance of quantitative analyses.
Abstract.
1999
Wolf CR, Smith G, Smith AG, Brown K, Henderson CJ (1999). Adaptive responses to environmental chemicals.
Biochem Soc Symp,
64, 129-139.
Abstract:
Adaptive responses to environmental chemicals.
Adaption to chemical agents in the environment is a fundamental part of the evolutionary process and a large number of genes have evolved to specifically detoxify potentially harmful chemical agents. These genes can act at various levels within cells and determine circulating chemical or toxin concentrations, and uptake and efflux rates as well as intracellular detoxification enzymes, such as the cytochrome P-450-dependent mono-oxygenases and glutathione S-transferases. These multigene families of proteins play a central role in chemical and drug detoxification and their polymorphic expression may well be a factor in disease susceptibility.
Abstract.
Pagès G, Guérin S, Grall D, Bonino F, Smith A, Anjuere F, Auberger P, Pouysségur J (1999). Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice.
Science,
286, 1374-1377.
Abstract:
Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice.
The p42 and p44 mitogen-activated protein kinases (MAPKs), also called Erk2 and Erk1, respectively, have been implicated in proliferation as well as in differentiation programs. The specific role of the p44 MAPK isoform in the whole animal was evaluated by generation of p44 MAPK-deficient mice by homologous recombination in embryonic stem cells. The p44 MAPK-/- mice were viable, fertile, and of normal size. Thus, p44 MAPK is apparently dispensable and p42 MAPK (Erk2) may compensate for its loss. However, in p44 MAPK-/- mice, thymocyte maturation beyond the CD4+CD8+ stage was reduced by half, with a similar diminution in the thymocyte subpopulation expressing high levels of T cell receptor (CD3high). In p44 MAPK-/- thymocytes, proliferation in response to activation with a monoclonal antibody to the T cell receptor in the presence of phorbol myristate acetate was severely reduced even though activation of p42 MAPK was more sustained in these cells. The p44 MAPK apparently has a specific role in thymocyte development.
Abstract.
Aubert J, Dessolin S, Belmonte N, Li M, McKenzie FR, Staccini L, Villageois P, Barhanin B, Vernallis A, Smith AG, et al (1999). Leukemia inhibitory factor and its receptor promote adipocyte differentiation via the mitogen-activated protein kinase cascade.
J Biol Chem,
274, 24965-24972.
Abstract:
Leukemia inhibitory factor and its receptor promote adipocyte differentiation via the mitogen-activated protein kinase cascade.
Extracellular factors and intracellular signaling pathways involved in early events of adipocyte differentiation are poorly defined. It is shown herein that expression of leukemia inhibitory factor (LIF) and LIF receptor is developmentally regulated during adipocyte differentiation. Preadipocytes secrete bioactive LIF, and an antagonist of LIF receptor inhibits adipogenesis. Genetically modified embryonic stem (ES) cells combined with culture conditions to commit stem cells into the adipocyte lineage were used to examine the requirement of LIF receptor during in vitro development of adipose cells. The capacity of embryoid bodies derived from lifr(-/-) ES cells to undergo adipocyte differentiation is dramatically reduced. LIF addition stimulates adipocyte differentiation of Ob1771 and 3T3-F442A preadipocytes and that of peroxisome proliferator-activated receptor gamma2 ligand-treated mouse embryonic fibroblasts. Expression of the early adipogenic transcription factors C/EBPbeta and C/EBPdelta is rapidly stimulated following exposure of preadipose cells to LIF. The selective inhibitors of mitogen-activated protein kinase kinase, i.e. PD98059 and U0126, inhibit LIF-induced C/EBP gene expression and prevent adipocyte differentiation induced by LIF. These results are in favor of a model that implicates stimulation of LIF receptor in the commitment of preadipocytes to undergo terminal differentiation by controlling the early expression of C/EBPbeta and C/EBPdelta genes via the mitogen-activated protein kinase cascade.
Abstract.
Pevny L, Episkopou V, Smith AG, Lovell-Badge R (1999). Neuronal stem cell gene.
Svendsen CN, Smith AG (1999). New prospects for human stem-cell therapy in the nervous system.
Trends Neurosci,
22, 357-364.
Abstract:
New prospects for human stem-cell therapy in the nervous system.
It would be of enormous benefit if human neural tissue could be generated in vitro as this would allow screening for neuroactive compounds, and provide a source of tissue for testing cellular and gene therapies for CNS disorders. It is now well established that pluripotent embryonic stem cells (ES cells) from the mouse can be propagated in culture and differentiated into a range of tissues, including neuronal and glial cells. In other studies, more-restricted neural stem cells have been isolated from both the developing and adult rodent brain. Current reports now describe similar pluripotent and neural stem cells cultured from human embryos. While the exact nature of these cells continues to be explored, they can be grown for extended periods of time while retaining the capacity for neuronal and glial differentiation. In some cases, they have been shown to integrate into the developing or damaged adult brain. This article reviews their biology, with a focus on the possible links between ES-cell and neural stem-cell technologies, and the strategies used to isolate and expand defined cell populations.
Abstract.
Burdon T, Chambers I, Stracey C, Niwa H, Smith A (1999). Signaling mechanisms regulating self-renewal and differentiation of pluripotent embryonic stem cells.
Cells Tissues Organs,
165, 131-143.
Abstract:
Signaling mechanisms regulating self-renewal and differentiation of pluripotent embryonic stem cells.
An ability to propagate pluripotent embryonic cells in culture is the foundation both for defined germline modification in experimental rodents and for future possibilities for broad-based cellular transplantation therapies in humans. Yet, the molecular basis of the self-renewing pluripotent phenotype remains ill-defined. The relationship between factors that influence embryonic stem cell propagation in vitro and mechanisms of stem cell regulation operative in the embryo is also uncertain. In this article we discuss the role of intracellular signalling pathways in the maintenance of pluripotency and induction of differentiation in embryonic stem cell cultures and the mammalian embryo.
Abstract.
Perarnau B, Saron MF, San Martin BR, Bervas N, Ong H, Soloski MJ, Smith AG, Ure JM, Gairin JE, Lemonnier FA, et al (1999). Single H2K(b), H2D(b) and double H2K(b)D(b) knockout mice: peripheral CD8(+) T cell repertoire and anti-lymphocytic choriomeningitis virus cytolytic responses. EUROPEAN JOURNAL OF IMMUNOLOGY, 29, 1243-1252.
Pérarnau B, Saron MF, Reina San Martin B, Bervas N, Ong H, Soloski MJ, Smith AG, Ure JM, Gairin JE, Lemonnier FA, et al (1999). Single H2Kb, H2Db and double H2KbDb knockout mice: peripheral CD8+ T cell repertoire and anti-lymphocytic choriomeningitis virus cytolytic responses.
Eur J Immunol,
29, 1243-1252.
Abstract:
Single H2Kb, H2Db and double H2KbDb knockout mice: peripheral CD8+ T cell repertoire and anti-lymphocytic choriomeningitis virus cytolytic responses.
Single H2Kb, H2Db and double H2KbDb homozygous knockout (KO) mice were generated and their peripheral CD8+ T cell repertoires compared to that of C57BL/6 (B6) mice. Limited (10-20%, H2Db), substantial (30-50%, H2Kb) and profound (90%, H2KbDb) reduction of peripheral CD8+ T cells was observed in KO mice, without Vbeta diversity alteration. Classical class Ia molecules therefore ensure most but not all of the peripheral CD8+ T cell repertoire education. As expected, H2Kb but also H2Db KO mice developed choriomeningitis following intracranial infection by lymphocytic choriomeningitis virus with the same kinetics, lethality and CD8+ cell implication as wild-type B6 mice. By contrast, H2KbDb (class Ia-Ib+) KO mice survived. Choriomeningitis of H2Db KO mice was linked to the development of a subdominant (in normal B6 mice) H2Kb-restricted cytotoxic T lymphocyte response. Mice expressing a restricted set of histocompatibility class I molecules should represent useful tools to evaluate the immunological potentials of individual MHC class I molecules.
Abstract.
Burdon T, Stracey C, Chambers I, Nichols J, Smith A (1999). Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells.
Dev Biol,
210, 30-43.
Abstract:
Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells.
The propagation of pluripotent mouse embryonic stem (ES) cells depends on signals transduced through the cytokine receptor subunit gp130. Signalling molecules activated downstream of gp130 in ES cells include STAT3, the protein tyrosine phosphatase SHP-2, and the mitogen-activated protein kinases, ERK1 and ERK2. A chimaeric receptor in which tyrosine 118 in the gp130 cytoplasmic domain was mutated did not engage SHP-2 and failed to activate ERKs. However, this receptor did support ES cell self-renewal. In fact, stem cell colonies formed at 100-fold lower concentrations of cytokine than the unmodified receptor. Moreover, altered ES cell morphology and growth were observed at high cytokine concentrations. These indications of deregulated signalling in the absence of tyrosine 118 were substantiated by sustained activation of STAT3. Confirmation that ERK activation is not required for self-renewal was obtained by propagation of pluripotent ES cells in the presence of the MEK inhibitor PD098059. In fact, the growth of undifferentiated ES cells was enhanced by culture in PD098059. Thus activation of ERKs appears actively to impair self-renewal. These data imply that the self-renewal signal from gp130 is a finely tuned balance of positive and negative effectors.
Abstract.
1998
Smith A (1998). Cell therapy: in search of pluripotency.
Curr Biol,
8, R802-R804.
Abstract:
Cell therapy: in search of pluripotency.
Cell replacement as a route to treat cellular disease and injury is currently limited by the availability of suitable donor cell populations, but recent results with mouse embryonic stem cells suggest that isolated human pluripotent cells could provide a source of cells for transplantation and gene therapy.
Abstract.
Blackburn C, Chambers I, Medvinsky A, Niwa H, Smith AG (1998). DNA expression in transfected cells and assays carried out in transfected cells.
Loupart ML, Shen MH, Smith A (1998). Differential stability of a human mini-chromosome in mouse cell lines.
Chromosoma,
107, 255-259.
Abstract:
Differential stability of a human mini-chromosome in mouse cell lines.
A 4 Mb human mini-chromosome, DeltaDelta2, was transferred from Chinese hamster ovary (CHO) cells into a mouse L cell line. The mini-chromosome could be transferred intact into the L cells, with 112/119 clones maintaining a mini-chromosome of the same size as the original. Ten clones were grown for 30 days in continuous culture. The mini-chromosomes were maintained stably with or without selection at a copy number of 1-2 per cell and none experienced any size alterations, as determined by pulsed-field gel electrophoresis. Thus DeltaDelta2 is structurally and mitotically stable in L cells. This contrasts with results in embryonic stem cells, in which DeltaDelta2 is highly unstable. These findings indicate that established somatic cell lines, such as L cells and CHO cells, have less stringent controls over centromeric function than do normal embryonic cells.
Abstract.
Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Schöler H, Smith A (1998). Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.
Cell,
95, 379-391.
Abstract:
Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.
Oct4 is a mammalian POU transcription factor expressed by early embryo cells and germ cells. We report that the activity of Oct4 is essential for the identity of the pluripotential founder cell population in the mammalian embryo. Oct4-deficient embryos develop to the blastocyst stage, but the inner cell mass cells are not pluripotent. Instead, they are restricted to differentiation along the extraembryonic trophoblast lineage. Furthermore, in the absence of a true inner cell mass, trophoblast proliferation is not maintained in Oct4-/- embryos. Expansion of trophoblast precursors is restored, however, by an Oct4 target gene product, fibroblast growth factor-4. Therefore, Oct4 also determines paracrine growth factor signaling from stem cells to the trophectoderm.
Abstract.
Li M, Pevny L, Lovell-Badge R, Smith A (1998). Generation of purified neural precursors from embryonic stem cells by lineage selection.
Curr Biol,
8, 971-974.
Abstract:
Generation of purified neural precursors from embryonic stem cells by lineage selection.
Mouse embryonic stem (ES) cells are non-transformed cell lines derived directly from the pluripotent founder tissue in the mouse embryo, the epiblast [1-3]. Aggregation of ES cells triggers the generation of a diverse array of cell types, including neuronal cells [4-7]. This capacity for multilineage differentiation is retained during genetic manipulation and clonal expansion [8]. In principle, therefore, ES cells provide an attractive system for the molecular and genetic dissection of developmental pathways in vitro. They are also a potential source of cells for transplantation studies. These prospects have been frustrated, however, by the disorganised and heterogeneous nature of development in culture. We have therefore developed a strategy for genetic selection of lineage-restricted precursors from differentiating populations. Here, we report that application of such lineage selection enables efficient purification of neuroepithelial progenitor cells that subsequently differentiate efficiently into neuronal networks in the absence of other cell types.
Abstract.
Wastling JM, Knight P, Ure J, Wright S, Thornton EM, Scudamore CL, Mason J, Smith A, Miller HR (1998). Histochemical and ultrastructural modification of mucosal mast cell granules in parasitized mice lacking the beta-chymase, mouse mast cell protease-1.
Am J Pathol,
153, 491-504.
Abstract:
Histochemical and ultrastructural modification of mucosal mast cell granules in parasitized mice lacking the beta-chymase, mouse mast cell protease-1.
The soluble beta-chymases mouse mast cell protease-1 (mMCP-1) and rat mast cell protease-II are predominantly expressed by intestinal mucosal mast cells (IMMCs) and may promote mucosal epithelial permeability when released during intestinal allergic hypersensitivity responses. To study the function of these chymases, we generated mice with a homozygous null mutation of the mMCP-1 gene and investigated their response to infection with the intestinal nematode Nippostrongylus brasiliensis. Whereas mMCP-2, -4, and -5 were transcribed normally, there was no transcription of the mMCP-1 gene in null (-/-) mice, nor was mature mMCP-1 protein detected in (-/-) jejunal mucosa. In contrast, levels of mMCP-1 in wild-type (+/+) jejunal mucosa increased 200- to 350-fold from 0.66 microg mMCP-1/g wet weight in uninfected mice to 129 and 229 microg/g wet weight on days 8 and 10 of infection, respectively. The kinetics of IMMC recruitment differed in -/- mice compared with +/+ controls on days 8 (P < 0.05) and 10 (P < 0.03) of infection. The IMMCs in infected -/- mice stained poorly, if at all, for esterase with naphthol AS-D chloroacetate compared with the intense staining observed in +/+ controls. Ultrastructurally, the prominent crystal intragranular structures that are found in intraepithelial +/+ IMMCs were absent from -/- IMMCs. These data show that disruption of the mMCP-1 gene leads to profound histochemical and ultrastructural changes in IMMC granules.
Abstract.
Henderson CJ, Smith AG, Ure J, Brown K, Bacon EJ, Wolf CR (1998). Increased skin tumorigenesis in mice lacking pi class glutathione S-transferases.
Proc Natl Acad Sci U S A,
95, 5275-5280.
Abstract:
Increased skin tumorigenesis in mice lacking pi class glutathione S-transferases.
The activity of chemical carcinogens is a complex balance between metabolic activation by cytochrome P450 monooxygenases and detoxification by enzymes such as glutathione S-transferase (GST). Regulation of these proteins may have profound effects on carcinogenic activity, although it has proved impossible to ascribe the observed effects to the activity of a single protein. GstP appears to play a very important role in carcinogenesis, although the precise nature of its involvement is unclear. We have deleted the murine GstP gene cluster and established the effects on skin tumorigenesis induced by the polycyclic aromatic hydrocarbon 7, 12-dimethylbenz anthracene and the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate. After 20 weeks, a highly significant increase in the number of papillomas was found in the GstP1/P2 null mice [GstP1/P2(-/-) mice, 179 papillomas, mean 9.94 per animal vs. GstP1/P2(+/+) mice, 55 papillomas, mean 2.89 per animal, (P < 0.001)]. This difference in tumor incidence provides direct evidence that a single gene involved in drug metabolism can have a profound effect on tumorigenicity, and demonstrates that GstP may be an important determinant in cancer susceptibility, particularly in diseases where exposure to polycyclic aromatic hydrocarbons is involved, for instance in cigarette smoke-induced lung cancer.
Abstract.
Mountford P, Nichols J, Zevnik B, O’Brien C, Smith A (1998). Maintenance of pluripotential embryonic stem cells by stem cell selection.
Reprod Fertil Dev,
10, 527-533.
Abstract:
Maintenance of pluripotential embryonic stem cells by stem cell selection.
As gastrulation proceeds, pluripotential stem cells with the capacity to contribute to all primary germ layers disappear from the mammalian embryo. The extinction of pluripotency also occurs during the formation of embryoid bodies from embryonic stem (ES) cells. In this report we show that if the initial differentiated progeny are removed from ES cell aggregates, further differentiation does not proceed and the stem cell population persists and expands. Significantly, the presence of even minor populations of differentiated cells lead to the complete loss of stem cells from the cultures. This finding implies that the normal elimination of pluripotent cells is dictated by inductive signals provided by differentiated progeny. We have exploited this observation to develop a strategy for the isolation of pluripotential cells. This approach, termed stem cell selection, may have widespread applicability to the derivation and propagation of stem cells.
Abstract.
Dani C, Chambers I, Johnstone S, Robertson M, Ebrahimi B, Saito M, Taga T, Li M, Burdon T, Nichols J, et al (1998). Paracrine induction of stem cell renewal by LIF-deficient cells: a new ES cell regulatory pathway.
Dev Biol,
203, 149-162.
Abstract:
Paracrine induction of stem cell renewal by LIF-deficient cells: a new ES cell regulatory pathway.
The propagation of pluripotential mouse embryonic stem (ES) cells is sustained by leukemia inhibitory factor (LIF) or related cytokines that act through a common receptor complex comprising the LIF receptor subunit (LIF-R) and the signal transducer gp130. However, the findings that embryos lacking LIF-R or gp130 can develop beyond gastrulation argue for the existence of an alternative pathway(s) governing the maintenance of pluripotency in vivo. In order to define those factors that contribute to self-renewal in ES cell cultures, we have generated ES cells in which both copies of the lif gene are deleted. These cells showed a significantly reduced capacity for regeneration of stem cell colonies when induced to differentiate, confirming that LIF is the major endogenous regulatory cytokine in ES cell cultures. However, self-renewal was not abolished and undifferentiated ES cell colonies were still obtained in the complete absence of LIF. A differentiated, LIF-deficient, parietal endoderm-like cell line was derived and shown to support ES cell propagation via production of a soluble, macromolecular, trypsin-sensitive activity. This activity, which we name ES cell renewal factor (ESRF), is distinct from members of the IL-6/LIF family because (i) it is effective on ES cells lacking LIF-R; (ii) it is not blocked by anti-gp130 neutralizing antibodies; and (iii) it acts without activation of STAT3. ES cells propagated clonally using ESRF alone can contribute fully to chimaeras and engender germline transmission. These findings establish that ES cell pluripotency can be sustained via a LIF-R/gp130-independent, STAT-3 independent, signaling pathway. Operation of this pathway in vivo could play an important role in the regulation of pluripotency in the epiblast and account for the viability of lifr -/- and gp130 -/- embryos.
Abstract.
Nichols J, Smith A, Buehr M (1998). Rat and mouse epiblasts differ in their capacity to generate extraembryonic endoderm.
Reproduction, Fertility and Development,
10(7-8), 517-525.
Abstract:
Rat and mouse epiblasts differ in their capacity to generate extraembryonic endoderm
In this study we have compared the in vitro differentiation potential of epiblast tissue from mouse and rat embryos. Epiblasts were isolated from egg cylinder stage embryos by microdissection and placed in culture. Rat cultures were distinguished by the copious production of parietal endoderm cells. Mouse epiblasts, in contrast, did not produce parietal endoderm. This difference in capacity to regenerate extraembryonic endoderm marks a surprising distinction in development of the pluripotential lineage between these two closely related rodents.
Abstract.
Nichols J, Smith A, Buehr M (1998). Rat and mouse epiblasts differ in their capacity to generate extraembryonic endoderm. REPRODUCTION FERTILITY AND DEVELOPMENT, 10, 517-525.
Niwa H, Burdon T, Chambers I, Smith A (1998). Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.
Genes Dev,
12, 2048-2060.
Abstract:
Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.
The propagation of embryonic stem (ES) cells in an undifferentiated pluripotent state is dependent on leukemia inhibitory factor (LIF) or related cytokines. These factors act through receptor complexes containing the signal transducer gp130. The downstream mechanisms that lead to ES cell self-renewal have not been delineated, however. In this study, chimeric receptors were introduced into ES cells. Biochemical and functional studies of transfected cells demonstrated a requirement for engagement and activation of the latent trancription factor STAT3. Detailed mutational analyses unexpectedly revealed that the four STAT3 docking sites in gp130 are not functionally equivalent. The role of STAT3 was then investigated using the dominant interfering mutant, STAT3F. ES cells that expressed this molecule constitutively could not be isolated. An episomal supertransfection strategy was therefore used to enable the consequences of STAT3F expression to be examined. In addition, an inducible STAT3F transgene was generated. In both cases, expression of STAT3F in ES cells growing in the presence of LIF specifically abrogated self-renewal and promoted differentiation. These complementary approaches establish that STAT3 plays a central role in the maintenance of the pluripotential stem cell phenotype. This contrasts with the involvement of STAT3 in the induction of differentiation in somatic cell types. Cell type-specific interpretation of STAT3 activation thus appears to be pivotal to the diverse developmental effects of the LIF family of cytokines. Identification of STAT3 as a key transcriptional determinant of ES cell self-renewal represents a first step in the molecular characterization of pluripotency.
Abstract.
Wolf CR, Campbell SJ, Clark AJ, Smith A, Bishop JO, Henderson CJ (1998). The use of transgenic animals to assess the role of metabolism in target organ toxicity. In (Ed) , 443-453.
1997
Livesey FJ, O’Brien JA, Li M, Smith AG, Murphy LJ, Hunt SP (1997). A Schwann cell mitogen accompanying regeneration of motor neurons.
Nature,
390, 614-618.
Abstract:
A Schwann cell mitogen accompanying regeneration of motor neurons.
Motor neurons are the only adult mammalian neurons of the central nervous system to regenerate following injury. This ability is dependent on the environment of the peripheral nerve and an intrinsic capacity of motor neurons for regrowth. We report here the identification, using a technique known as messenger RNA differential display, of an extracellular signalling molecule, previously described as the pancreatic secreted protein Reg-2, that is expressed solely in regenerating and developing rat motor and sensory neurons. Axon-stimulated Schwann cell proliferation is necessary for successful regeneration, and we show that Reg-2 is a potent Schwann cell mitogen in vitro. In vivo, Reg-2 protein is transported along regrowing axons and inhibition of Reg-2 signalling significantly retards the regeneration of Reg-2-containing axons. During development, Reg-2 production by motor and sensory neurons is regulated by contact with peripheral targets. Strong candidates for peripheral factors regulating Reg-2 production are cytokines of the LIF/CNTF family, because Reg-2 is not expressed in developing motor or sensory neurons of mice carrying a targeted disruption of the LIF receptor gene, a common component of the receptor complexes for all of the LIF/CNTF family.
Abstract.
Dani C, Smith AG, Dessolin S, Leroy P, Staccini L, Villageois P, Darimont C, Ailhaud G (1997). Differentiation of embryonic stem cells into adipocytes in vitro.
J Cell Sci,
110 ( Pt 11), 1279-1285.
Abstract:
Differentiation of embryonic stem cells into adipocytes in vitro.
Embryonic stem cells, derived from the inner cell mass of murine blastocysts, can be maintained in a totipotent state in vitro. In appropriate conditions embryonic stem cells have been shown to differentiate in vitro into various derivatives of all three primary germ layers. We describe in this paper conditions to induce differentiation of embryonic stem cells reliably and at high efficiency into adipocytes. A prerequisite is to treat early developing embryonic stem cell-derived embryoid bodies with retinoic acid for a precise period of time. Retinoic acid could not be substituted by adipogenic hormones nor by potent activators of peroxisome proliferator-activated receptors. Treatment with retinoic acid resulted in the subsequent appearance of large clusters of mature adipocytes in embryoid body outgrowths. Lipogenic and lipolytic activities as well as high level expression of adipocyte specific genes could be detected in these cultures. Analysis of expression of potential adipogenic genes, such as peroxisome proliferator-activated receptors gamma and delta and CCAAT/enhancer binding protein beta, during differentiation of retinoic acid-treated embryoid bodies has been performed. The temporal pattern of expression of genes encoding these nuclear factors resembled that found during mouse embryogenesis. The differentiation of embryonic stem cells into adipocytes will provide an invaluable model for the characterisation of the role of genes expressed during the adipocyte development programme and for the identification of new adipogenic regulatory genes.
Abstract.
Lee K, Nichols J, Smith A (1997). Erratum: Identification of a developmentally regulated protein tyrosine phosphatase in embryonic stem cells that is a marker of pluripotential epiblast and early mesoderm (Mech. Dev. 59 (1996) 153-164). Mechanisms of Development, 61, 213-215.
Pascolo S, Bervas N, Ure JM, Smith AG, Lemonnier FA, Pérarnau B (1997). HLA-A2.1-restricted education and cytolytic activity of CD8(+) T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m double knockout mice.
J Exp Med,
185, 2043-2051.
Abstract:
HLA-A2.1-restricted education and cytolytic activity of CD8(+) T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m double knockout mice.
Three different HLA-A2.1 monochains were engineered in which either the human or mouse beta2-microglobulin (beta2m) is covalently linked to the NH2 terminus of the heavy chain by a 15- amino acid long peptide: HHH, entirely human, HHD, with the mouse H-2Db alpha3, transmembrane, and cytoplasmic domains, and MHD, homologous to HHD but linked to the mouse beta2mb. The cell surface expression and immunological capacities of the three monochains were compared with transfected cells, and the selected HHD construct was introduced by transgenesis in H-2Db-/- beta2m-/- double knockout mice. Expression of this monochain restores a sizable peripheral CD8(+) T cell repertoire essentially educated on the transgenic human molecule. Consequently, infected HHD, H-2Db-/- beta2m-/- mice generate only HLA-A2.1-restricted CD8(+) CTL responses against influenza a and vaccinia viruses. Interestingly, the CTL response to influenza a virus is mostly, if not exclusively, directed to the 58-66 matrix peptide which is the HLA-A2.1-restricted immunodominant epitope in humans. Such mice might constitute a versatile animal model for the study of HLA-A2.1-restricted CTL responses of vaccine interest.
Abstract.
Shen MH, Yang J, Loupart ML, Smith A, Brown W (1997). Human mini-chromosomes in mouse embryonal stem cells.
Hum Mol Genet,
6, 1375-1382.
Abstract:
Human mini-chromosomes in mouse embryonal stem cells.
We have introduced human mini-chromosomes of 4 Mb and approximately 15 Mb in size into mouse embryonal stem cells. Although these human mini-chromosomes are stable in hamster and chicken cells, they re-arrange or segregate aberrantly in the embryonal stem cells and are rapidly lost in the absence of selection. However, one of the mini-chromosomes re-arranged, acquired mouse centromeric sequences and was then stably maintained for at least 60 population doublings in culture. This mini-chromosome, which is 4 Mb in size, is a candidate for a mouse germ line chromosome vector.
Abstract.
Ramage AD, Clark AJ, Smith AG, Mountford PS, Burt DW (1997). Improved EBV-based shuttle vector system: dicistronic mRNA couples the synthesis of the Epstein-Barr nuclear antigen-1 protein to neomycin resistance.
Gene,
197, 83-89.
Abstract:
Improved EBV-based shuttle vector system: dicistronic mRNA couples the synthesis of the Epstein-Barr nuclear antigen-1 protein to neomycin resistance.
Use of EBV-based vector systems has been limited by the requirement to generate EBNA+ cells which are ’permissive’ for replication of an oriP-vector. In current constructs, selectable marker and EBNA-1 are not always co-expressed. This is a significant problem since the EBNA-1 gene product can be toxic in some cell types and may be selected against. In this paper, we describe a gene construct that overcomes this limitation. We have exploited the piconaviral internal ribosome entry site to allow the genes for Epstein-Barr nuclear antigen-1 and G-418 resistance to be transcribed as a dicistronic fusion mRNA under the control of the phosphoglucokinase promoter. This construct can be routinely integrated into human cell lines. The presence of EBNA-1 protein was reflected by a large increase in transfection frequencies (1000-fold) using an oriP-based vector which was shown to replicate stably in these cells with no apparent gross rearrangements detected after 8 weeks in culture. Using this system, G-418 resistance should directly reflect integration, as well as expression of the EBNA-1 gene, which, in turn, increases transfection frequencies and stability of EBV-based vector systems and should result in its increased use.
Abstract.
Schrank B, Götz R, Gunnersen JM, Ure JM, Toyka KV, Smith AG, Sendtner M (1997). Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos.
Proc Natl Acad Sci U S A,
94, 9920-9925.
Abstract:
Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos.
Proximal spinal muscular atrophy is an autosomal recessive human disease of spinal motor neurons leading to muscular weakness with onset predominantly in infancy and childhood. With an estimated heterozygote frequency of 1/40 it is the most common monogenic disorder lethal to infants; milder forms represent the second most common pediatric neuromuscular disorder. Two candidate genes-survival motor neuron (SMN) and neuronal apoptosis inhibitory protein have been identified on chromosome 5q13 by positional cloning. However, the functional impact of these genes and the mechanism leading to a degeneration of motor neurons remain to be defined. To analyze the role of the SMN gene product in vivo we generated SMN-deficient mice. In contrast to the human genome, which contains two copies, the mouse genome contains only one SMN gene. Mice with homozygous SMN disruption display massive cell death during early embryonic development, indicating that the SMN gene product is necessary for cellular survival and function.
Abstract.
Neophytou C, Vernallis AB, Smith A, Raff MC (1997). Müller-cell-derived leukaemia inhibitory factor arrests rod photoreceptor differentiation at a postmitotic pre-rod stage of development.
Development,
124, 2345-2354.
Abstract:
Müller-cell-derived leukaemia inhibitory factor arrests rod photoreceptor differentiation at a postmitotic pre-rod stage of development.
In the present study, we examine rod photoreceptor development in dissociated-cell cultures of neonatal mouse retina. We show that, although very few rhodopsin+ rods develop in the presence of 10% foetal calf serum (FCS), large numbers develop in the absence of serum, but only if the cell density in the cultures is high. The rods all develop from nondividing rhodopsin- cells, and new rods continue to develop from rhodopsin- cells for at least 6-8 days, indicating that there can be a long delay between when a precursor cell withdraws from the cell cycle and when it becomes a rhodopsin+ rod. We show that FCS arrests rod development in these cultures at a postmitotic, rhodopsin-, pre-rod stage. We present evidence that FCS acts indirectly by stimulating the proliferation of Müller cells, which arrest rod differentiation by releasing leukaemia inhibitory factor (LIF). These findings identify an inhibitory cell-cell interaction, which may help to explain the long delay that can occur both in vitro and in vivo between cell-cycle withdrawal and rhodopsin expression during rod development.
Abstract.
Chambers I, Cozens A, Broadbent J, Robertson M, Lee M, Li M, Smith A (1997). Structure of the mouse leukaemia inhibitory factor receptor gene: regulated expression of mRNA encoding a soluble receptor isoform from an alternative 5’ untranslated region.
Biochem J,
328 ( Pt 3), 879-888.
Abstract:
Structure of the mouse leukaemia inhibitory factor receptor gene: regulated expression of mRNA encoding a soluble receptor isoform from an alternative 5’ untranslated region.
The low-affinity leukaemia inhibitory factor receptor (LIF-R) is a component of cell-surface receptor complexes for the multifunctional cytokines leukaemia inhibitory factor, ciliary neurotrophic factor, oncostatin M and cardiotrophin-1. Both soluble and transmembrane forms of the protein have been described and several LIF-R mRNAs have been reported previously. In order to determine the coding potential of LIF-R mRNAs we have isolated and characterized the mouse LIF-R gene. mRNA encoding soluble LIF-R (sLIF-R) is formed by inclusion of an exon in which polyadenylation signals are provided by a B2 repeat. This exon is located centrally within the LIF-R gene but is excluded from the transmembrane LIF-R mRNA by alternative splicing. The transmembrane receptor is encoded by 19 exons distributed over 38 kb. Two distinct 5’ non-coding exons have been identified, indicating the existence of alternative promoters. One of these is G/C rich and possesses a consensus initiator sequence as well as potential Sp1 binding sites. Expression of exon 1 from this promoter occurs in a wide variety of tissues, whereas expression of the alternative 5’ untranslated region (exon 1a) is normally restricted to liver, the principal source of sLIF-R. During pregnancy expression of exon 1a becomes detectable also in the uterus. Expression of exon 1a increases dramatically during gestation and is accompanied by a similar quantitative rise in expression of sLIF-R mRNA. These findings establish that expression of LIF-R is under complex transcriptional control and indicate that regulated expression of the soluble cytokine receptor isoform may be due principally to an increase in the activity of a dedicated promoter.
Abstract.
1996
Nichols J, Davidson D, Taga T, Yoshida K, Chambers I, Smith A (1996). Complementary tissue-specific expression of LIF and LIF-receptor mRNAs in early mouse embryogenesis.
Mech Dev,
57, 123-131.
Abstract:
Complementary tissue-specific expression of LIF and LIF-receptor mRNAs in early mouse embryogenesis.
The maintenance of pluripotential embryonic stem (ES) cells is dependent on the cytokine LIF. This report documents the mRNA expression profiles of LIF and the two components of the LIF-receptor complex, LIF-R and gp130, during early mouse embryogenesis. These mRNAs were undetectable in 1- or 2-cell embryos, but all were present by the blastocyst stage. LIF transcripts were localised in the differentiated trophectoderm, and were absent from the pluripotential inner cell mass. In contrast, LIF-R mRNA was found in the inner cell mass but not in the trophectoderm. This complementary pattern of expression is suggestive of a paracrine coupling between stem cells and differentiated progeny at the earliest stage of mammalian development. After implantation, transcripts for all components were down-regulated in the embryo. High levels of LIF-R and gp130 mRNAs were observed in the deciduum, however. These dynamic, tissue-specific expression patterns are consistent with regulatory roles for LIF or related cytokines, both in the maintenance of pluripotency in the mouse embryo, and in development of the foeto-maternal interface.
Abstract.
Lee K, Nichols J, Smith A (1996). Identification of a developmentally regulated protein tyrosine phosphatase in embryonic stem cells that is a marker of pluripotential epiblast and early mesoderm.
Mech Dev,
59, 153-164.
Abstract:
Identification of a developmentally regulated protein tyrosine phosphatase in embryonic stem cells that is a marker of pluripotential epiblast and early mesoderm.
A sensitive RT-PCR display technique was used to examine the expression of protein tyrosine phosphatases (PTPs) during the differentiation of mouse embryonic stem (ES) cells. The majority of PTPs are expressed constitutively but one is present only in undifferentiated ES cells. This PTP was cloned and named ES cell phosphatase (ESP). ESP mRNA is detectable in oocytes and throughout early mouse embryo development. At early egg cylinder stages, transcripts are localised in the pluripotential epiblast. As gastrulation commences, however, epiblast expression is lost. Transcripts are present transiently in newly formed embryonic mesoderm. These data suggest that this transmembrane signaling molecule is associated with developmental lability in early embryogenesis.
Abstract.
1995
Wei XQ, Charles IG, Smith A, Ure J, Feng GJ, Huang FP, Xu D, Muller W, Moncada S, Liew FY, et al (1995). Altered immune responses in mice lacking inducible nitric oxide synthase.
Nature,
375, 408-411.
Abstract:
Altered immune responses in mice lacking inducible nitric oxide synthase.
Nitric oxide (NO) is important in many biological functions. It is generated from L-arginine by the enzyme NO synthase (NOS). The cytokine-inducible NOS (iNOS) is activated by several immunological stimuli, leading to the production of large quantities of NO which can be cytotoxic. To define the biological role of iNOS further, we generated iNOS mutant mice. These are viable, fertile and without evident histopathological abnormalities. However, in contrast to wild-type and heterozygous mice, which are highly resistant to the protozoa parasite Leishmania major infection, mutant mice are uniformly susceptible. The infected mutant mice developed a significantly stronger Th1 type of immune response than the wild-type or heterozygous mice. The mutant mice showed reduced nonspecific inflammatory response to carrageenin, and were resistant to lipopolysaccharide-induced mortality.
Abstract.
Li M, Sendtner M, Smith A (1995). Essential function of LIF receptor in motor neurons.
Nature,
378, 724-727.
Abstract:
Essential function of LIF receptor in motor neurons.
Development and maintenance of the mammalian nervous system is dependent upon neurotrophic cytokines. One class of neurotrophic factor acts through receptor complexes involving the low-affinity leukaemia inhibitory factor receptor subunit (LIF-R). Members of this family of cytokines, such as ciliary neurotrophic factor (CNTF) and leukaemia inhibitory factor (LIF), have profound effects on the survival and maintenance of motor neurons. Recently it was reported that mice lacking LIF-R die shortly after birth unlike mice lacking CNTF or LIF which are viable. Here we describe histopathological analyses of lifr mutants that reveal a loss > 35% of facial motor neurons, 40% of spinal motor neurons and 50% of neurons in the nucleus ambiguus. These findings point to the existence of a ligand for LIF-R that is required for the normal development of motor neurons in both brainstem nuclei and spinal cord.
Abstract.
Mountford PS, Smith AG (1995). Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis.
Trends Genet,
11, 179-184.
Abstract:
Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis.
Modification of the genetic content of cultured cells or of whole animals is now a key strategy in both basic biological research and applied biotechnology. Yet obtaining the desired level and specificity of expression of an introduced gene remains highly problematic. One solution could be to couple expression of a transgene to that of an appropriate intact genomic locus. The identification and functional characterization of RNA sequences known as internal ribosome entry sites now offer the possibility of achieving precise control of transgene expression through the generation of dicistronic fusion mRNAs.
Abstract.
1994
Nichols J, Chambers I, Smith A (1994). Derivation of germline competent embryonic stem cells with a combination of interleukin-6 and soluble interleukin-6 receptor.
Exp Cell Res,
215, 237-239.
Abstract:
Derivation of germline competent embryonic stem cells with a combination of interleukin-6 and soluble interleukin-6 receptor.
In this report we document the derivation of pluripotential embryonic stem (ES) cells in the absence of a feeder layer by supplementation of culture media with either ciliary neurotrophic factor or oncostatin M, or with a combination of interleukin-6 (IL-6) plus soluble interleukin-6 receptor (sIL-6R). These factors all activate gp130-associated signaling processes, as does the previously characterized ES cell maintenance factor Differentiation Inhibiting Activity (Leukemia Inhibitory Factor). In particular, the IL-6/sIL-6R complex is thought to act exclusively through gp130. All ES cell lines derived using IL-6/sIL-6R contributed extensively to chimeras and were transmitted through the germline at high frequency. These findings point to a pivotal role for gp130 in ES cell propagation and may be relevant to attempts to derive ES cells from species other than mouse.
Abstract.
Mountford P, Zevnik B, Düwel A, Nichols J, Li M, Dani C, Robertson M, Chambers I, Smith A (1994). Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression.
Proc Natl Acad Sci U S A,
91, 4303-4307.
Abstract:
Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression.
To investigate the activity of candidate regulatory molecules in mammalian embryogenesis, we have developed a general strategy for modifying and reporting resident chromosomal gene expression. The picornaviral internal ribosome-entry site was incorporated into gene targeting constructs to provide cap-independent translation of a selectable marker from fusion transcripts generated following homologous recombination. These promoterless constructs were highly efficient and have been used both to inactivate the stem-cell-specific transcription factor Oct-4 and to introduce a quantitative regulatory modification into the gene for a stem-cell maintenance factor, differentiation-inhibiting activity. In addition, the inclusion of a beta-galactosidase reporter gene in the constructs enabled accurate and sensitive detection of cellular sites of transcription. This has allowed visualization of putative "stem-cell niches" in which sources of elevated expression of differentiation-inhibiting activity were localized to the differentiated cells surrounding colonies of stem cells.
Abstract.
Smith AG, Hole N (1994). Embryonic Stem Cells and Haematopoiesis. In Freshney RI, Pragnell IB, Freshney MG (Eds.)
Culture of hematopoietic cells, Wiley-Blackwell, 235-250.
Abstract:
Embryonic Stem Cells and Haematopoiesis
Abstract.
Smith AG, Mountford P, Lathe R (1994). Expression of the heterologous genes according to a targeted expression profile.
Smith AG, Mountford P (1994). Isolation, selection and propagation of animal transgenic stem cell.
Yoshida K, Chambers I, Nichols J, Smith A, Saito M, Yasukawa K, Shoyab M, Taga T, Kishimoto T (1994). Maintenance of the pluripotential phenotype of embryonic stem cells through direct activation of gp130 signalling pathways.
Mech Dev,
45, 163-171.
Abstract:
Maintenance of the pluripotential phenotype of embryonic stem cells through direct activation of gp130 signalling pathways.
Propagation of the undifferentiated pluripotential phenotype of embryonic stem (ES) cells is dependent on the cytokine differentiation inhibiting activity/leukemia inhibitory factor (DIA/LIF). The DIA/LIF receptor complex is a heterodimer of DIA/LIF receptor (DIA/LIF-R) and gp130. The latter is also a component of the interleukin-6 (IL-6) receptor complex. We report that a combination of IL-6 and soluble IL-6 receptor (sIL-6R), which can induce homodimerisation of gp130 and activation of signalling processes, sustains self-renewal of pluripotential ES cells. Our findings indicate that the IL-6/sIL-6R complex acts on ES cells through gp130 alone, bypassing DIA/LIF-R, and therefore implicate gp130 as the key component in the signalling pathway responsible for stem cell renewal.
Abstract.
1993
Robertson M, Chambers I, Rathjen P, Nichols J, Smith A (1993). Expression of alternative forms of differentiation inhibiting activity (DIA/LIF) during murine embryogenesis and in neonatal and adult tissues.
Dev Genet,
14, 165-173.
Abstract:
Expression of alternative forms of differentiation inhibiting activity (DIA/LIF) during murine embryogenesis and in neonatal and adult tissues.
Differentiation inhibiting activity/leukaemia inhibitory factor (DIA/LIF) is a pleiotropic cytokine which has been implicated in a variety of developmental and physiological processes in mammals due to its broad range of biological activities in vitro. A role in very early development is suggested by the requirement for DIA/LIF to support the self-renewal of cultured embryonic stem (ES) cells. Other data point to potential roles in the establishment and maintenance of primordial germ cells, in osteogenesis and in haematopoiesis, and possibly in neuronal specification. DIA/LIF may also act as a mediator of the hepatic acute phase response. In the present study the expression of DIA/LIF transcripts during murine development and in adult mice has been determined using a highly sensitive ribonuclease protection analysis. In contrast to previous reports, it is apparent that DIA/LIF transcripts are present at low levels in many adult mouse tissues. Higher levels of expression are observed in skin, lung, intestine, and uterus. Elevated amounts of mRNA are also found in certain foetal tissue during late gestation and neonatally. In earlier embryogenesis, however, DIA/LIF mRNA is produced primarily in extraembryonic tissues. The alternative transcripts which produce either soluble or matrix-associated DIA/LIF exhibit overlapping but non-identical patterns of expression, consistent with the proposition that the two isoforms may have distinct biological functions. These findings are suggestive of widespread roles for DIA/LIF in vivo and are discussed in the light of available data on the phenotype of homozygous DIA/LIF-deficient mice.
Abstract.
Barnett MA, Buckle VJ, Evans EP, Porter AC, Rout D, Smith AG, Brown WR (1993). Telomere directed fragmentation of mammalian chromosomes.
Nucleic Acids Res,
21, 27-36.
Abstract:
Telomere directed fragmentation of mammalian chromosomes.
Cloned human telomeric DNA can integrate into mammalian chromosomes and seed the formation of new telomeres. This process occurs efficiently in three established human cell lines and in a mouse embryonic stem cell line. The newly seeded telomeres appear to be healed by telomerase. The seeding of new telomeres by cloned telomeric DNA is either undetectable or very inefficient in non-tumourigenic mouse or human somatic cell lines. The cytogenetic consequences of the seeding of new telomeres include large chromosome truncations but most of the telomere seeding events occur close to the pre-existing ends of natural chromosomes.
Abstract.
Whyatt LM, Düwel A, Smith AG, Rathjen PD (1993). The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes.
Mol Cell Biol,
13, 7971-7976.
Abstract:
The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes.
Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.
Abstract.
1992
Ure JM, Fiering S, Smith AG (1992). A rapid and efficient method for freezing and recovering clones of embryonic stem cells. Trends Genet, 8, 6-6.
Smith AG, Nichols J, Robertson M, Rathjen PD (1992). Differentiation inhibiting activity (DIA/LIF) and mouse development.
Dev Biol,
151, 339-351.
Abstract:
Differentiation inhibiting activity (DIA/LIF) and mouse development.
Analysis of the differentiation in culture of murine embryonic stem (ES) cells has resulted in the identification and characterization of the regulatory factor differentiation inhibiting activity (DIA). DIA specifically suppresses differentiation of the pluripotential ES cells without compromise of their developmental potential. DIA is identical to the pleiotropic cytokine leukaemia inhibitory factor (LIF) which has a broad range of biological activities in vitro and in vivo. It is produced in both diffusible and matrix-localised forms whose expression is differentially regulated. The compartmentalization of DIA/LIF and the modulation of its expression during stem cell differentiation and by other cytokines may be significant elements in the control of early embryo development. These features may also indicate general principles of the regulatory networks which govern stem cell renewal and differentiation in later development.
Abstract.
Smith AG (1992). Introduction: Mammalian stem cell systems. Seminars in Cell Biology, 3, 383-384.
Smith AG (1992). Mouse embryo stem cells: their identification, propagation and manipulation.
Semin Cell Biol,
3, 385-399.
Abstract:
Mouse embryo stem cells: their identification, propagation and manipulation.
The early mouse embryo contains a transient population of pluripotential stem cells which are responsible for generating both the foetal primordia and extraembryonic membranes. The characterisation of murine embryo stem cells and their isolation and propagation in culture provides the first instance in which pure populations of normal stem cells are directly accessible to the researcher. This marks a considerable advance in stem cell biology which may pave the way to the dissection of general stem cell control mechanisms and the identification of key regulatory factors. In addition, the genetic manipulation of embryo stem cells affords a unique avenue for experimental intervention in mammalian development and for controlled modification of the mouse germ line.
Abstract.
1991
Smith AG (1991). Culture and differentiation of embryonic stem cells.
Journal of Tissue Culture Methods,
13, 89-94.
Abstract:
Culture and differentiation of embryonic stem cells
Techniques are described for the culture of murine embryonic stem cells in the absence of heterologous feeder cells and for the induction of differentiation programs. The regulatory factor differentiation inhibiting activity/ leukaemia inhibitory factor (DIA/LIF) is produced at high concentration by transient expression in Cos cells and is used to suppress stem cell differentiation by addition to the culture medium. Differentiation is then induced in a controlled manner either by withdrawal of DIA/LIF or by exposure to the chemical inducers retinoic acid or 3-methoxybenzamide. © 1991 Tissue Culture Association.
Abstract.
Smith AG, Rathjen P (1991). Embryonic stem cells, differentiation inhibiting activity, and the mouse embryo. Seminars in Cell Biology, 317-327.
Heath JK, Smith AG, Rathjen P (1991). Leukaemia Inhibitory factor.
1990
Rathjen PD, Nichols J, Toth S, Edwards DR, Heath JK, Smith AG (1990). Developmentally programmed induction of differentiation inhibiting activity and the control of stem cell populations.
Genes Dev,
4, 2308-2318.
Abstract:
Developmentally programmed induction of differentiation inhibiting activity and the control of stem cell populations.
Differentiation inhibiting activity/leukemia inhibitory factor (DIA/LIF) is a glycoprotein that controls differentiation of pluripotential stem cells. Alternative transcription generates both diffusible and matrix-associated forms of DIA/LIF. Transcriptional analysis using a sensitive ribonuclease protection assay revealed that the two messages are expressed independently, consistent with the proposition that the two forms of DIA/LIF have distinct biological roles. DIA/LIF expression was found to be activated early during differentiation of embryonic stem (ES) cells, providing a mechanism for feedback regulation of stem cell renewal. Expression of DIA/LIF by mesenchymal cells was shown to be controlled in a paracrine manner by polypeptide regulatory factors. Specific expression of the two forms of DIA/LIF was also demonstrated in the egg cylinder-stage mouse embryo. The combination of cell type-specific and signal-specific regulation enables very precise control over DIA/LIF expression and may represent an important component of the regulatory networks that govern stem cell proliferation and differentiation during mammalian development.
Abstract.
Rathjen PD, Toth S, Willis A, Heath JK, Smith AG (1990). Differentiation inhibiting activity is produced in matrix-associated and diffusible forms that are generated by alternate promoter usage.
Cell,
62, 1105-1114.
Abstract:
Differentiation inhibiting activity is produced in matrix-associated and diffusible forms that are generated by alternate promoter usage.
The differentiation of embryonic stem (ES) cells is controlled by the regulatory factor differentiation inhibiting activity/leukemia inhibitory factor (DIA/LIF). Examination of feeder cell-mediated suppression of ES cell differentiation revealed that DIA/LIF is produced both as a diffusible protein and in an immobilized form associated with the extracellular matrix. This alternative localization arises from the expression of alternate transcripts that diverge throughout exon 1. The effect of alternate first exon usage is to change the amino terminus of the primary translation product and to direct incorporation of mature, biologically active DIA/LIF into the extracellular matrix. The production of a potent regulatory factor in both diffusible and immobilized forms may be an important element of developmental control mechanisms.
Abstract.
Nichols J, Evans EP, Smith AG (1990). Establishment of germ-line-competent embryonic stem (ES) cells using differentiation inhibiting activity.
Development,
110, 1341-1348.
Abstract:
Establishment of germ-line-competent embryonic stem (ES) cells using differentiation inhibiting activity.
The regulatory factor Differentiation Inhibiting Activity/Leukaemia Inhibitory Factor (DIA/LIF) suppresses the differentiation of cultured embryonic stem (ES) cells. In the present study, it is shown that ES cell lines can be derived and maintained in the absence of feeder layers using medium supplemented with purified DIA/LIF. These cells can differentiate normally in vitro and in vivo and they retain the capacity for germ-line transmission. DIA/LIF therefore fulfils the essential function of feeders in the isolation of pluripotential stem cells.
Abstract.
Heath JK, Smith AG, Hsu LW, Rathjen P (1990). Growth and Differentiation factors in embryonic stem cell function. Journal of Cell Science Suplement, 12, 75-85.
Heath JK, Smith AG, Hsu LW, Rathjen PD (1990). Growth and differentiation factors of pluripotential stem cells.
Abstract:
Growth and differentiation factors of pluripotential stem cells.
Abstract.
Smith AG, Leary AG, Wong GG, Clark SC, Ogawa M (1990). Leukemia inhibitory factor LIF/Differentiation-Inhibiting activity (DIA)/human interleukin for DA cells (HILDA) augments proliferation of human hematopoietic cells. Blood, 75, 1960-1964.
1989
Heath JK, Smith AG, Wills AJ, Edwards DR (1989). Growth and differentiation factors of embryonic stem cells. Cell to cell signalling in mammalian Development, 219-230.
Heath JK, Smith AG (1989). Growth factors in embryogenesis.
Br Med Bull,
45, 319-336.
Abstract:
Growth factors in embryogenesis.
The growing appreciation that embryonic material is a rich source of growth factors underlies increasing interest in the role of growth factors in early development. These developments have arisen from a desire to understand fundamental embryological processes; the control of differentiation and the generation of form. One expectation of this research is the discovery of new forms of regulatory agent with novel biological and therapeutic potential. Furthermore, there is a direct relationship between growth and differentiation processes in early development and pathological processes in the adult; for example, the behaviour of tumour cells has historically been compared to that of embryonic cells and regenerative processes in the adult such as angiogenesis, wound healing and fracture repair may be considered to recapitulate events that occur in the normal course of embryogenesis. An understanding of the mechanisms that underlie these fundamental processes in normal development may therefore lead to a concomitant understanding of related pathological events in the adult.
Abstract.
1988
Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, Rogers D (1988). Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides.
Nature,
336, 688-690.
Abstract:
Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides.
Murine embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo which can contribute differentiated progeny to all adult tissues, including the germ-cell lineage, after re-incorporation into the normal embryo. They provide both a cellular vector for the generation of transgenic animals and a useful system for the identification of polypeptide factors controlling differentiation processes in early development. In particular, medium conditioned by Buffalo rat liver cells contains a polypeptide factor, ES cell differentiation inhibitory activity (DIA), which specifically suppresses the spontaneous differentiation of ES cells in vitro, thereby permitting their growth as homogeneous stem cell populations in the absence of heterologous feeder cells. ES cell pluripotentiality, including the ability to give rise to functional gametes, is preserved after prolonged culture in Buffalo rat liver media as a source of DIA. Here, we report that purified DIA is related in structure and function to the recently identified hematopoietic regulatory factors human interleukin for DA cells and leukaemia inhibitory factor. DIA and human interleukin DA/leukaemia inhibitory factor have thus been identified as related multifunctional regulatory factors with distinct biological activities in both early embryonic and hematopoietic stem cell systems.
Abstract.
Heath JK, Smith AG (1988). Regulatory factors of embryonic stem cells.
J Cell Sci Suppl,
10, 257-266.
Abstract:
Regulatory factors of embryonic stem cells.
The analysis of factors which regulate cell proliferation and differentiation in early mammalian development has been facilitated by the existence of cell lines derived from pluripotential stem cells of the early embryo; embryonal carcinoma (EC) cells and embryonic stem (ES) cells. EC cells have proved to be a useful source of embryonic growth factors. A potent mitogen, ECDGF has been isolated from EC cell conditioned medium. ECDGF appears to be a novel member of the heparin binding growth factor family. A remarkable feature of heparin binding growth factors is their ability to induce mesodermal differences in Xenopus laevis animal pole explants. ES cell differentiation in vitro is controlled by exogenous factors, in particular a novel differentiation inhibitory factor DIA. Controlled bipotential differentiation of ES cells can be achieved by exposing ES cells to different combinations of regulatory signals.
Abstract.
1987
Smith AG, Hooper ML (1987). Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells.
Dev Biol,
121, 1-9.
Abstract:
Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells.
Many pluripotent embryonal carcinoma (EC) cell lines and all embryonic stem (ES) cell lines have hitherto been maintained in the undifferentiated state only by culture on feeder layers of mitomycin C-treated embryonic fibroblasts. We now demonstrate that medium conditioned by incubation with Buffalo rat liver (BRL) cells prevents the spontaneous differentiation of such cells which occurs when they are plated in the absence of feeders. This effect is not mediated via cell selection but represents a fully reversible inhibitory action ascribed to a differentiation-inhibiting activity (DIA). BRL-conditioned medium can therefore replace feeders in the propagation of homogeneous stem cell populations. Such medium also restricts differentiation in embryoid bodies formed via aggregation of EC cells and partially inhibits retinoic acid-induced differentiation. The PSA4 EC line gives rise only to extraembryonic endoderm-like cells when aggregated or exposed to retinoic acid in BRL-conditioned medium. This suggests that DIA may be lineage-specific. DIA is a dialysable, acid-stable entity of apparent molecular weight 20,000-35,000. Its actions are reproduced neither by insulin-like growth factor-II nor by transforming growth factor-beta. DIA thus appears to be a novel factor exerting a negative control over embryonic stem cell differentiation.
Abstract.
1986
Smith TA, Smith AG, Hooper ML (1986). Selection of a mouse embryonal carcinoma clone resistant to the inhibition of metabolic cooperation by retinoic acid.
Exp Cell Res,
165, 417-428.
Abstract:
Selection of a mouse embryonal carcinoma clone resistant to the inhibition of metabolic cooperation by retinoic acid.
A novel selection for cell lines resistant to the blocking of metabolic cooperation by chemical inhibitors is described. The selection has been used to isolate a pluripotent embryonal carcinoma clone which cooperates in retinoic acid (RA), an inhibitor of junctional communication in all other cell lines tested to date. The selected cells require higher levels of RA to induce differentiation, but show little or no increase in resistance to toxic effects of RA.
Abstract.
