[Show abstract][Hide abstract] ABSTRACT: The rat is one of the most commonly used laboratory animals in biomedical research and the recent isolation of genuine pluripotent rat embryonic stem (ES) cell lines has provided new opportunities for applying contemporary genetic engineering techniques to the rat and enhancing the use of this rodent in scientific research. Technical refinements that improve the stability of the rat ES cell cultures will undoubtedly further strengthen and broaden the use of these stem cells in biomedical research. Here, we describe a relatively simple and robust protocol that supports the propagation of germ line competent rat ES cells, and outline how tuning stem cell signaling using small molecule inhibitors can be used to both stabilize self-renewal of rat ES cell cultures and aid evaluation of their differentiation potential in vitro.
[Show abstract][Hide abstract] ABSTRACT: Stabilisation of β-catenin, through inhibition of Glycogen synthase kinase 3 (GSK3) activity, in conjunction with inhibition of the enzyme MEK, promotes self-renewal of naïve-type mouse embryonic stem cells (ESC). In developmentally more advanced, primed-type, epiblast stem cells (EpiSC), however, β-catenin activity induces differentiation. We investigated the response of rat ESCs to β-catenin signalling and found that when maintained on feeder-support cells in the presence of a MEK inhibitor alone (1i culture), the derivation efficiency, growth, karyotypic stability, transcriptional profile, and differentiation potential of rat ESC cultures was similar to that of cell lines established using both MEK and GSK3 inhibitors (2i culture). Equivalent mouse ESCs, by comparison, differentiated in identical 1i conditions, consistent with insufficient β-catenin activity. This interspecies difference in reliance on GSK3 inhibition corresponded with higher overall levels of β-catenin activity in rat ESCs. Indeed, rat ESCs displayed widespread expression of the mesendoderm-associated β-catenin targets, Brachyury and Cdx2 in 2i medium, and overt differentiation upon further increases in β-catenin activity. In contrast, mouse ESC were resistant to differentiation at similarly elevated doses of GSK3 inhibitor. Interestingly, without feeder support, moderate levels of GSK3 inhibition were necessary to support effective growth of rat ESC, confirming the conserved role for β-catenin in ESC self-renewal. This work identifies β-catenin signalling as a molecular rheostat in rat ESC, regulating self-renewal in a dose-dependent manner, and highlights the potential importance of controlling flux in this signalling pathway to achieve effective stabilisation of naïve pluripotency.
[Show abstract][Hide abstract] ABSTRACT: Distinct signaling pathways are reported to maintain pluripotency in embryo-derived stem cells. Mouse embryonic stem cells (ESCs) respond to leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP)-mediated activity, whereas human ESCs depend upon Fibroblast growth factor (FGF) and activin signaling. In the majority of mammals investigated, however, the signals that support stem cell pluripotency are not well defined, as is evident by the persistent difficulties in maintaining authentic stable ESC lines. Induction of pluripotency by transcription factor-mediated reprogramming could provide an alternative way to produce ESC-like cells from nonpermissive species, and facilitate identification of core ESC signaling requirements. To evaluate the effectiveness of this approach in pigs, we transduced porcine foetal fibroblasts with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc, and maintained the resulting cultures in medium containing either LIF or FGF2. Alkaline phosphatase positive colonies with compact, mouse ESC-like morphology were preferentially recovered using serum-free medium supplemented with LIF. These cell lines expressed the endogenous stem cell transcription factors, OCT4, NANOG, and SOX2, and the cell surface marker SSEA-4, consistent with acquisition of an undifferentiated state. However, restricted differentiation potential, and persistent expression of retroviral transgenes indicated that reprogramming was incomplete. Interestingly, LIF activated both the transcription factor STAT3 and its target gene SOCS3, and stimulated cell growth, indicating functional coupling of the signaling pathway in these cells. This demonstration of LIF-dependence in reprogrammed pig cells supports the notion that the connection between LIF/STAT3 signaling and the core regulatory network of pluripotent stem cells is a conserved pathway in mammals.
[Show abstract][Hide abstract] ABSTRACT: Pluripotential stem cells from livestock offer an exciting prospect for the biotechnology industry. Applying strategies established for the derivation of murine induced pluripotential stem cells (iPSCs), we have isolated ovine iPSCs that can give rise to cells characteristic of all three germ cell layers both in vitro from embryoid bodies and in teratomas in vivo. Furthermore, although at a low level, these ovine iPS cells can contribute to live-born chimeric lambs. Colonies derived from ovine embryonic fibroblasts transfected with murine cMyc, Klf4, Oct4, and Sox2 displayed smooth domes with sharp edges when grown in human embryonic stem cell (ESC) medium but not in mouse ESC medium. These ovine iPSCs were alkaline phosphatase positive, expressed Nanog, and had a normal karyotype. These cells represent an important step in the understanding of mechanistic nature of pluripotency in ungulates.
[Show abstract][Hide abstract] ABSTRACT: The role of DNA sequence in determining chromatin state is incompletely understood. We have previously demonstrated that large chromosomal segments from human cells recapitulate their native chromatin state in mouse cells, but the relative contribution of local sequences versus their genomic context remains unknown. In this study, we compare orthologous chromosomal regions for which the human locus establishes prominent sites of Polycomb complex recruitment in pluripotent stem cells, whereas the corresponding mouse locus does not. Using recombination-mediated cassette exchange at the mouse locus, we establish the primacy of local sequences in the encoding of chromatin state. We show that the signal for chromatin bivalency is redundantly encoded across a bivalent domain and that this reflects competition between Polycomb complex recruitment and transcriptional activation. Furthermore, our results suggest that a high density of unmethylated CpG dinucleotides is sufficient for vertebrate Polycomb recruitment. This model is supported by analysis of DNA methyltransferase-deficient embryonic stem cells.
The EMBO Journal 11/2011; 31(2):317-29. · 9.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A major barrier to research on Parkinson's disease is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells from patients and differentiate them into neurons affected by disease. Triplication of SNCA, encoding α-synuclein, causes a fully penetrant, aggressive form of Parkinson's disease with dementia. α-Synuclein dysfunction is the critical pathogenic event in Parkinson's disease, multiple system atrophy and dementia with Lewy bodies. Here we produce multiple induced pluripotent stem cell lines from an SNCA triplication patient and an unaffected first-degree relative. When these cells are differentiated into midbrain dopaminergic neurons, those from the patient produce double the amount of α-synuclein protein as neurons from the unaffected relative, precisely recapitulating the cause of Parkinson's disease in these individuals. This model represents a new experimental system to identify compounds that reduce levels of α-synuclein, and to investigate the mechanistic basis of neurodegeneration caused by α-synuclein dysfunction.
[Show abstract][Hide abstract] ABSTRACT: The rat is the preferred experimental animal in many biological studies. With the recent derivation of authentic rat embryonic stem (ES) cells it is now feasible to apply state-of-the art genetic engineering in this species using homologous recombination. To establish whether rat ES cells are amenable to in vivo recombination, we tested targeted disruption of the hypoxanthine phosphoribosyltransferase (hprt) locus in ES cells derived from both inbred and outbred strains of rats. Targeting vectors that replace exons 7 and 8 of the hprt gene with neomycinR/thymidine kinase selection cassettes were electroporated into male Fisher F344 and Sprague Dawley rat ES cells. Approximately 2% of the G418 resistant colonies also tolerated selection with 6-thioguanine, indicating inactivation of the hprt gene. PCR and Southern blot analysis confirmed correct site-specific targeting of the hprt locus in these clones. Embryoid body and monolayer differentiation of targeted cell lines established that they retained differentiation potential following targeting and selection. This report demonstrates that gene modification via homologous recombination in rat ES cells is efficient, and should facilitate implementation of targeted, genetic manipulation in the rat.
PLoS ONE 01/2010; 5(12):e14225. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The POU domain transcription factor OCT4 is a key regulator of pluripotency in the early mammalian embryo and is highly expressed in the inner cell mass of the blastocyst. Consistent with its essential role in maintaining pluripotency, Oct4 expression is rapidly downregulated during formation of the trophoblast lineage. To enhance our understanding of the molecular basis of this differentiation event in humans, we used a functional genomics approach involving RNA interference-mediated suppression of OCT4 function in a human ESC line and analysis of the resulting transcriptional profiles to identify OCT4-dependent genes in human cells. We detected altered expression of >1,000 genes, including targets regulated directly by OCT4 either positively (NANOG, SOX2, REX1, LEFTB, LEFTA/EBAF DPPA4, THY1, and TDGF1) or negatively (CDX2, EOMES, BMP4, TBX18, Brachyury [T], DKK1, HLX1, GATA6, ID2, and DLX5), as well as targets for the OCT4-associated stem cell regulators SOX2 and NANOG. Our data set includes regulators of ACTIVIN, BMP, fibroblast growth factor, and WNT signaling. These pathways are implicated in regulating human ESC differentiation and therefore further validate the results of our analysis. In addition, we identified a number of differentially expressed genes that are involved in epigenetics, chromatin remodeling, apoptosis, and metabolism that may point to underlying molecular mechanisms that regulate pluripotency and trophoblast differentiation in humans. Significant concordance between this data set and previous comparisons between inner cell mass and trophectoderm in human embryos indicates that the study of human ESC differentiation in vitro represents a useful model of early embryonic differentiation in humans.
[Show abstract][Hide abstract] ABSTRACT: The transcription factor Oct-4 is a marker of pluripotency in mouse and human embryonic stem (ES) cells. Previous studies using a tetracycline-regulated Oct-4 transgene in the ZHBTc4 cell line demonstrated that downregulation of Oct-4 expression induced dedifferentiation into trophoblast, a lineage mouse ES cells do not normally generate. We found that transfection of Oct-4-specific short interfering RNA significantly reduced expression and functional activity of Oct-4 in mouse and human ES cells, enabling its role to be compared in both cell types. In mouse ES cells, Oct-4 knockdown produced a pattern of morphological differentiation and increase in expression of the trophoblast-associated transcription factor Cdx2, similar to that triggered by suppressing the Oct-4 transgene in the ZHBTc4 cell line. In addition, downregulation of Oct-4 was accompanied by increased expression of the endoderm-associated genes Gata6 and alpha-fetoprotein, and a gene trap associated with primitive liver/yolk sac differentiation. In human ES cells, Oct-4 knockdown also induced morphological differentiation coincident with the upregulation of Gata6. The induction of Cdx2 and other trophoblast-associated genes, however, was dependent on the culture conditions. These results establish the general requirement for Oct-4 in maintaining pluripotency in ES cells. Moreover, the upregulation of endoderm-associated markers in both mouse and human ES cells points to overlap between development of trophoblast and endoderm differentiation.
[Show abstract][Hide abstract] ABSTRACT: 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.
Trends in Cell Biology 10/2002; 12(9):432-8. · 11.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Genes & Development 08/1998; 12(13):2048-60. · 12.44 Impact Factor