Removal Of Leukemia Inhibitory Factor Research Articles

Changes in the Expression of Pyruvate Dehydrogenase Kinase Isoenzymes During Early Differentiation of Mouse Embryonic Stem Cells

The embryonic stem cells (ESCs) are pluripotent, self-renewing cells that able to differentiate into any of the germ layers involved in embryogenesis. However, the molecular mechanisms that control ESC pluripotency and differentiation remain poorly understood. The family of Pyruvate dehydrogenase kinase (PDK1-4), inactivates the mitochondrial pyruvate dehydrogenase complex via phosphorylation, plays a crucial role in the control of glucose homeostasis. In the current study, gene expression levels of PDK isoenzymes were analyzed on undifferentiated mouse embryonic stem cells (mESCs) and compared to mESCs induced to differentiate by removal of leukemia inhibitory factor (LIF) for 1, 3 and 5 days. Besides, we performed gene expression analysis of several genes related to pluripotency and differentiation. In addition, we also determined glucose uptake rates by a colorimetric assay kit in early differentiated and undifferentiated mESCs. Differently expression level of PDK isoenzymes in pluripotent and LIF-depleted mESCs suggest that they may have roles in differentiation and pluripotency of ESCs. Furthermore, this study lays the foundation for detailed investigation of molecular mechanisms underlying the roles of PDKs in the pluripotency and transition to differentiated state of ESCs.

Increased expression of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-3 is required for growth of mouse embryonic stem cells that are undergoing differentiation.

The unlimited proliferation capacity of embryonic stem cells (ESCs) coupled with their capability to differentiate into several cell types makes them an attractive candidate for studying the molecular mechanisms regulating self-renewal and transition from pluripotent state. Although the roles of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase family (PFKFB1-4) in cell survival, proliferation, and differentiation in tumor cells have been studied, their role in mouse ESC (mESC) biology is currently unkown. In the current study, Pfkfb isoenzyme expressions were analyzed in R1 and J1 mESCs that were cultured in the presence and absence of leukemia inhibitory factor (LIF). We report that expression of the Pfkfb3 isoenzyme was markedly increased when mESCs were promoted to differentiate upon LIF removal. We then demonstrated that Pfkfb3 silencing induced the differentiation marker Brachyury suggesting that Pfkfb3 may be required for the regulation of mesodermal differentiation of mESCs. Furthermore, we show that the increase in Pfkfb3 expression is required for the growth of early differentiated mESCs. Although these results provide important insights into the early differentiation of mESCs with regard to Pfkfb expressions, further mechanistic studies will be needed for understanding the pathways and mechanisms involved in regulation of proliferation and early differentiation of mESCs through Pfkfb3.

The Distinct Role of Tcfs and Lef1 in the Self-Renewal or Differentiation of Mouse Embryonic Stem Cells.

Background and ObjectivesTcfs and Lef1 are DNA-binding transcriptional factors in the canonical Wnt signaling pathway. In the absence of β-catenin, Tcfs and Lef1 generally act as transcriptional repressors with co-repressor proteins such as Groucho, CtBP, and HIC-5. However, Tcfs and Lef1 turn into transcriptional activators during the interaction with β-catenin. Therefore, the activity of Tcfs and Lef1 is regulated by β-catenin. However, the intrinsic role of Tcfs and Lef1 has yet to be examined. The purpose of this study was to determine whether Tcfs and Lef1 play differential roles in the regulation of self-renewal and differentiation of mouse ES cells.Methods and ResultsInterestingly, the expression of Tcfs and Lef1 was dynamically altered under various differentiation conditions, such as removal of LIF, EB formation and neuronal differentiation in N2B27 media, suggesting that the function of each Tcf and Lef1 may vary in ES cells. Ectopic expression of Tcf1 or the dominant negative form of Lef1 (Lef1-DN) contributes to ES cells to self-renew in the absence of leukemia inhibitory factor (LIF), whereas ectopic expression of Tcf3, Lef1 or Tcf1-DN did not support ES cells to self-renew. Ectopic expression of either Lef1 or Lef1-DN blocked neuronal differentiation, suggesting that the transient induction of Lef1 was necessary for the initiation and progress of differentiation. ChIP analysis shows that Tcf1 bound to Nanog promoter and ectopic expression of Tcf1 enhanced the transcription of Nanog.ConclusionsThe overall data suggest that Tcf1 plays a critical role in the maintenance of stemness whereas Lef1 is involved in the initiation of differentiation.

Bioenergetic Changes Underline Plasticity of Murine Embryonic Stem Cells.

Murine embryonic stem cells (mESCs) are endowed by a time-dependent window of plasticity during their early commitment steps. Indeed, while mESCs deprived of leukemia inhibitory factor (LIF) for 24 hours revert to their naive pluripotent state after subsequent LIF readdition, cells deprived of LIF for 48 hours are no longer efficient in reverting, upon LIF addition, and undergo irreversible differentiation. We investigated undisclosed bioenergetic profiles of early mESC-derived committed cells versus their undifferentiated states in order to reveal specific bioenergetic changes associated with mESC plasticity. Multiparametric bioenergetic analysis revealed that pluripotent (+LIF) and reversibly committed cells (-LIF24h) are energetically flexible, depending on both oxidative phosphorylation (OXPHOS) and glycolysis. They exhibit high mitochondrial respiration in the presence of the main energetic substrates and can also rely on glycolysis in the presence of OXPHOS inhibitor. Inhibition of the glycolysis or mitochondrial respiration does not change drastically the expression of pluripotency genes, which remain well expressed. In addition, cells treated with these inhibitors keep their capacity to differentiate efficiently upon embryoid bodies formation. Transition from metabolically active mESCs to irreversibly committed cells is associated with a clear change in mitochondrial network morphology, to an increase of adenosine triphosphate (ATP) produced from glycolysis and a decline of ATP turnover and of the mitochondrial activity without change in the mitochondrial mass. Our study pointed that plasticity window of mESCs is associated with the bivalent energetic metabolism and potency to shift to glycolysis or OXPHOS on demand. LIF removal provokes glycolytic metabolic orientation and consecutive loss of the LIF-dependent reversion of cells to the pluripotent state. Stem Cells 2019;37:463-475.

Biophysical subsets of embryonic stem cells display distinct phenotypic and morphological signatures.

The highly proliferative and pluripotent characteristics of embryonic stem cells engender great promise for tissue engineering and regenerative medicine, but the rapid identification and isolation of target cell phenotypes remains challenging. Therefore, the objectives of this study were to characterize cell mechanics as a function of differentiation and to employ differences in cell stiffness to select population subsets with distinct mechanical, morphological, and biological properties. Biomechanical analysis with atomic force microscopy revealed that embryonic stem cells stiffened within one day of differentiation induced by leukemia inhibitory factor removal, with a lagging but pronounced change from spherical to spindle-shaped cell morphology. A microfluidic device was then employed to sort a differentially labeled mixture of pluripotent and differentiating cells based on stiffness, resulting in pluripotent cell enrichment in the soft device outlet. Furthermore, sorting an unlabeled population of partially differentiated cells produced a subset of “soft” cells that was enriched for the pluripotent phenotype, as assessed by post-sort characterization of cell mechanics, morphology, and gene expression. The results of this study indicate that intrinsic cell mechanical properties might serve as a basis for efficient, high-throughput, and label-free isolation of pluripotent stem cells, which will facilitate a greater biological understanding of pluripotency and advance the potential of pluripotent stem cell differentiated progeny as cell sources for tissue engineering and regenerative medicine.

MiR-302b-3p Promotes Self-Renewal Properties in Leukemia Inhibitory Factor-Withdrawn Embryonic Stem Cells.

Objective Embryonic stem cells (ESCs) are regulated by a gene regulatory circuitry composed of transcription factors, signaling pathways, metabolic mediators, and non-coding RNAs (ncRNAs). MicroRNAs (miRNAs) are short ncRNAs which play crucial roles in ESCs. Here, we explored the impact of miR-302b-3p on ESC self-renewal in the absence of leukemia inhibitory factor (LIF). Materials and Methods In this experimental study, ESCs were cultured in the presence of 15% fetal bovine serum (FBS) and induced to differentiate by LIF removal. miR-302b-3p overexpression was performed by transient transfection of mature miRNA mimics. Cell cycle profiling was done using propidium iodide (PI) staining followed by flow cytometry. miRNA expression was quantified using a miR-302b-3p-specific TaqMan assay. Data were analyzed using t test, and a P<0.05 was considered statistically significant. Results We observed that miR-302b-3p promoted the viability of both wild-type and LIF-withdrawn ESCs. It also increased ESC clonogenicity and alkaline phosphatase (AP) activity. The defective cell cycling of LIF-deprived ESCs was completely rescued by miR-302b-3p delivery. Moreover, miR-302b-3p inhibited the increased cell death rate induced by LIF removal. ConclusionmiR-302b-3p, as a pluripotency-associated miRNA, promotes diverse features of ESC self-renewal in the absence of extrinsic LIF signals.

Leukemia inhibitory factor (LIF) withdrawal activates mTOR signaling pathway in mouse embryonic stem cells through the MEK/ERK/TSC2 pathway.

Leukemia inhibitory factor (LIF) is indispensable to maintain the pluripotent state of mouse embryonic stem cells (ESCs), but the mechanisms underlying the role of LIF/STAT3 pathway are yet poorly understood. Here we first showed that the LIF/STAT3-regulated signaling pathway contributes to the maintenance of self-renewal and pluripotency of mouse ESCs by suppressing mTOR (mammalian target of rapamycin), which is necessary for early differentiation. When LIF is withdrawn from culture medium, the mTOR activity rapidly increases as detected by phosphorylation of its targets – ribosomal protein S6 and translation factor 4EBP1. In turn, suppression of STAT3 phosphorylation on Tyr-705 by a specific small molecule WP1066 also activates phosphorylation of the mTOR target S6 ribosomal protein. LIF removal strongly activates ERK activity indicating that ERK can be involved in either direct phosphorylation of mTOR or phosphorylation of an upstream negative regulator of mTOR – TSC1/TSC2 proteins. According to western blotting data, LIF withdrawal leads to phosphorylation of TSC2 protein thereby relieving its negative effect on mTOR activity. mTOR activation is accompanied by a decrease of pluripotent gene expression Oct-4, Nanog, Sox2 and by an augmentation of fgf5 gene expression – a marker of post-implantation epiblast. Together, these data indicate that LIF-depleted mouse ESCs undergo a transition from the LIF/STAT3-supported pluripotent state to the FGFR/ERK-committed primed-like state with expression of early differentiation markers mediated through activation of mTOR signaling.

α6β1- and αV-integrins are required for long-term self-renewal of murine embryonic stem cells in the absence of LIF

BackgroundThe growth properties and self-renewal capacity of embryonic stem (ES) cells are regulated by their immediate microenvironment such as the extracellular matrix (ECM). Integrins, a central family of cellular ECM receptors, have been implicated in these processes but their specific role in ES cell self-renewal remains unclear.ResultsHere we have studied the effects of different ECM substrates and integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF) using short-term assays as well as long-term cultures. Removal of LIF from ES cell culture medium induced morphological differentiation of ES cells into polarized epistem cell-like cells. These cells maintained epithelial morphology and expression of key stemness markers for at least 10 passages in the absence of LIF when cultured on laminin, fibronectin or collagen IV substrates. The specific functional roles of α6-, αV- and β1-integrin subunits were dissected using stable lentivirus-mediated RNAi methodology. β1-integrins were required for ES cell survival in long-term cultures and for the maintenance of stem cell marker expression. Inhibition of α6-integrin expression compromised self-renewal on collagen while αV-integrins were required for robust ES cell adhesion on laminin. Analysis of the stemness marker expression revealed subtle differences between α6- and αV-depleted ES cells but the expression of both was required for optimal self-renewal in long-term ES cell cultures.ConclusionsIn the absence of LIF, long-term ES cell cultures adapt an epistem cell-like epithelial phenotype and retain the expression of multiple stem cell markers. Long-term maintenance of such self-renewing cultures depends on the expression of β1-, α6- and αV-integrins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-015-0051-y) contains supplementary material, which is available to authorized users.

Upregulated acetylcholine synthesis during early differentiation in the embryonic stem cell line CGR8

Stem cells are used to generate differentiated somatic cells including neuronal cells. Synthesis and release of acetylcholine, a neurotransmitter and widely expressed signaling molecule, were investigated in the murine embryonic stem cell line CGR8 during early differentiation, i.e. in the presence of leukemia inhibitory factor (LIF) to maintain pluripotency and in the absence of LIF to induce early differentiation. CGR8 cells express choline acetyltransferase (ChAT) as demonstrated by measurement of enzyme activity and substantial inhibition by bromoacetylcholine. Pluripotent CGR8 cells showed a ChAT activity of 250pmol acetylcholine/mg/h, contained 1.1pmol acetylcholine/106cells and released about 12.00pmol acetylcholine/1x106cells/6h. Removal of LIF induced early differentiation as evidenced by reduced transcription factors Oct-4 and Nanog and a substantial slowing of the proliferation rate. Under this condition acetylcholine synthesis increased to 1640pmol/mg/h; related to the pluripotent state the content of acetylcholine increased 10-fold and the release to about 32pmol acetylcholine/1x106cells/6h. Enzyme kinetic analysis showed a significant increase of the Km for the precursor acetyl-CoA and of Vmax without a change of the Km for the precursor choline. In conclusion, early differentiation of the stem cell line CGR8 is associated with a substantial increase in ChAT activity and acetylcholine release.

Reduced Oct4 Expression Directs a Robust Pluripotent State with Distinct Signaling Activity and Increased Enhancer Occupancy by Oct4 and Nanog

SummaryEmbryonic stem cell (ESC) pluripotency is governed by a gene regulatory network centered on the transcription factors Oct4 and Nanog. To date, robust self-renewing ESC states have only been obtained through the chemical inhibition of signaling pathways or enforced transgene expression. Here, we show that ESCs with reduced Oct4 expression resulting from heterozygosity also exhibit a stabilized pluripotent state. Despite having reduced Oct4 expression, Oct4+/− ESCs show increased genome-wide binding of Oct4, particularly at pluripotency-associated enhancers, homogeneous expression of pluripotency transcription factors, enhanced self-renewal efficiency, and delayed differentiation kinetics. Cells also exhibit increased Wnt expression, enhanced leukemia inhibitory factor (LIF) sensitivity, and reduced responsiveness to fibroblast growth factor. Although they are able to maintain pluripotency in the absence of bone morphogenetic protein, removal of LIF destabilizes pluripotency. Our findings suggest that cells with a reduced Oct4 concentration range are maintained in a robust pluripotent state and that the wild-type Oct4 concentration range enables effective differentiation.

Histone H1 Depletion Impairs Embryonic Stem Cell Differentiation

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.

Induction of Differentiation into Endoderm, Insulin-Secreting Cells from Mouse Embryonic Stem Cells using Activin A and Retinoic Acid

Abstract: Embryonic stem (ES) cells have been known to differentiate into various progenitor cells. In this study, we investigated the differentiation capacity of mouse ES cells into pancreatic hormone-secreting cells, and insulin-secreting cells. ES cells were cultured in Dulbecco's modified Eagle medium (DMEM) after removal of leukemia inhibitory factor (LIF) for 3 days and then transferred in DMEM supplemented with retinoic acid or activin A. When the culture of embryoid bodies (EBs) derived from ES cells in DMEM added with activin A (2 × 10-9 M or 2 × 10-10 M) for 5 days was performed, endoderm marker genes, GATA4, Sox17 and Foxa2 were expressed in the EBs. However, at 6 days of culture, expression of Sox17 was not observed. When EBs were cultured with activin A (2 × 10-9 M) for 6 days, and followed by 6 days of culture with retinoic acid (10-6 M), expression of the pancreatic cell marker genes, GATA4 and Fxa2, were continued, and pancreatic islet genes, insulins 1 and 2, glucagons and somatostatin, w...

Understanding the first steps in embryonic stem cell exit from the pluripotent state

We are interested in understanding how a given cell type, in response to external cues from its environment, makes the decision to differentiate. In the case of mouse embryonic stem cells (mESCs), the key external factor that maintains their undifferentiated state is the cytokine leukemia inhibitory factor (LIF). LIF removal causes mESCs to exit their pluripotent state and differentiate into more restricted precursors. Although LIF is known to activate multiple different phosphorylation cascades, the mechanisms by which its removal leads to mESC differentiation are not well understood. In order to identify the molecular events that occur upon LIF removal, we developed a set of novel experimental approaches that allowed identification and quantification of global phosphorylation changes that occur when mESCs are deprived of LIF. These included growth of mESCs on permeable membranes and development of a robust and sensitive phospho-proteomics platform to quantify early signaling events. In addition to the well-characterized tyrosine 705 phosphorylation of STAT3, LIF removal results in the rapid phosphorylation of multiple other proteins known to regulate the mESC self-renewal on both tyrosine, serine, and threonine residues. We hypothesize that these unique posttranslational modifications help drive the exit of mESCs from the pluripotent state. Our data set the stage for future studies investigating the functional role of these phosphorylation events in mESCs. These studies were greatly facilitated by the National Blood Foundation, whose support in the crucial initiation phase of these studies was invaluable.

Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

Germ Cell Specification from Murine Embryonic Stem Cells: A Model for the Identification of Irreversible Meiotic Commitment.

Despite evidence indicating that differentiating mouse embryonic stem cells (mESC) can generate oocytes and follicle-like structures in vitro, fertilizable oocytes have not been produced. Unlike endogenous primordial germ cells (PGC), which have the innate ability to form oocytes that initiate and progress through meiosis, mESC-derived germ cells exhibit many meiotic abnormalities and fail to progress through prophase. As such, isolation of early stage (pre-meiotic) ESC-derived germ cells may prove useful for identifying key processes that lead to meiotic commitment and progression. Although the timing of meiotic entry in embryonic ovaries in vivo has been well characterized, the molecular events associated with irreversible meiotic commitment are not well defined. Induction of stimulated by retinoic acid gene 8 (Stra8) is associated with the early onset of meiotic commitment, and has been previously used to isolate male germ cells from mESC cultures. In the present study, we combined a dual reporter system with germline specification by mESC to identify two distinct populations of Stra8-positive germ cells that differ in their degree of commitment to meiosis. To track germ cells, we used a karyotypic XX mESC line derived in our lab from transgenic mice expressing green fluorescent protein (GFP) driven by a fragment of the mouse Oct4 promoter in which the epiblast-specific proximal enhancer has been inactivated (TgOG2 or ΔPE-Oct4-Gfp). When undifferentiated, TgOG2 mESC are GFP positive (GFP+). Following initiation of differentiation by removal of leukemia inhibitory factor (LIF) and mouse embryonic fibroblast (MEF) feeder cells, TgOG2 mESC were transiently transfected with a DsRed2 expression vector under control of the murine Stra8 promoter. In the continued absence of LIF and MEF, the level of GFP gradually decreased such that comparatively few GFP+ cells remained detectable after 5 days; however, DsRed2-expressing cells became identifiable by fluorescence microscopy 5 days after the induction of differentiation. Subsequent isolation by fluorescence-activated cell sorting (FACS) revealed two distinct DsRed2-expressing cell populations: one that was GFP+ and one that was GFP-negative (GFP-). The sorted populations were re-plated separately and while the DsRed+/GFP- population remained quiescent, DsRed+/GFP+ germ cells actively proliferated and maintained high levels of GFP expression. These findings indicate that mESC-derived Stra8+/Oct4- germ cells commit to meiosis and lose replicative capacity; however, there is a subset of Stra8- expressing germ cells that continue to express Oct4 and retain high proliferative potential. In light of recent evidence indicating that Stra8 binds to DNA and exhibits transcriptional activity, we hypothesize that irreversible meiotic entry is a transcriptionally defined event regulated by Stra8 in those germ cells that repress Oct4 expression. (Supported by Ruth L. Kirschstein National Research Service Award F32AG034809 to D.C.W, NIH MERIT Award R37-AG012279 to J.L.T, and Vincent Memorial Research Funds). (poster)

P56. Differential roles of Tcfs and Lef1 in the self-renewal or differentiation of mouse embryonic stem cells

Wnt/β-catenin signaling plays important roles in self-renewal and differentiation of embryonic stem (ES) cells. Tcfs and Lef1 are DNA-binding transcriptional factors for the canonical Wnt signaling pathway. In the absence of β-catenin, Tcf/Lef1 generally functions as transcriptional repressor with co-repressor proteins such as Groucho, CtBP, and HIC-5. But Tcf/Lef1 turns into transcriptional activator when they interact with β-catenin. Therefore, so far it was considered that the activity of Tcf/Lef1 is regulated by the absence or presence of β-catenin, however the intrinsic role of Tcf/Lef1 has not been examined. The purpose of this study was to determine if Tcf/Lef1 itself plays differential roles in the regulation of self-renewal and differentiation of mouse ES cells. Interestingly, the expression of TCFs and LEF1 was dynamically changed during various differentiation conditions, such as removal of LIF, EB formation and neuronal differentiation in N2B27 media, suggesting that the function of each Tcfs and Lef1 may be different in ES cells. Ectopic expression of Tcf1 or dominant negative form of Lef1 (dnLef1) sustains self-renewal of ES cells in the absence of leukemia inhibitory factor (LIF), while ectopic expression of Tcf3, Lef1 or dnTcf1 did not block differentiation. In addition, ectopic expression of Tcf1 or knockdown of Lef1 blocked differentiation into neural precursor and cardiomyocyte. Ectopic expression of either Lef1 or dnLef1 blocked neuronal differentiation, suggesting that transient induction of Lef1 seemed to be necessary for the initiation and progress of differentiation. ChIP analysis show that Tcf1 bound to Nanog promoter and ectopic expression of Tcf1 enhanced the transcription of Nanog. Overall, our data suggest that Tcf1 plays critical roles in the maintenance of stemness whereas Lef1is involved in the initiation of differentiation. (Supported by the National Research Foundation of Korea (2006-2004046) and Brain Korea 21 program.)

Heparan Sulfate Is Required for Embryonic Stem Cells to Exit from Self-renewal

Pluripotent embryonic stem cells (ESCs) must select between alternative fates of self-renewal and lineage commitment at each division during continuous proliferation. Heparan sulfate (HS) is a highly sulfated polysaccharide and is present abundantly on the ESC surface. In this study, we investigated the role of HS in ESC self-renewal by examining Ext1(-/-) ESCs that are deficient in HS. We found that Ext1(-/-) ESCs retained their self-renewal potential but failed to transit from self-renewal to differentiation upon removal of leukemia inhibitory factor. Furthermore, we found that the aberrant cell fate commitment is caused by defects in fibroblast growth factor signaling, which directly retained high expression of the pluripotency gene Nanog in Ext1(-/-) ESCs. Therefore, our studies identified and defined HS as a novel factor that controls ESC fate commitment and also delineates that HS facilitates fibroblast growth factor signaling, which, in turn, inhibits Nanog expression and commits ESCs to lineage differentiation.

272 LEUKEMIA INHIBITORY FACTOR SIGNALING PATHWAY IN PIG PARTHENOGENETIC PLURIPOTENT CELLS

Leukemia inhibitory factor (LIF), its receptor heterodimer (LRβ-gp130), and the related signal transducer and activator of transcription-3 (STAT3) constitute a system controlling self-renewal and pluripotency of embryonic stem cells (ESC) in the mouse, where LIF withdrawal or direct inhibition of STAT3 causes ESC differentiation. By contrast, several studies have demonstrated that LIF is not required to maintain human ESC pluripotency. Scattered information is available in other species, and data on the role of LIF in pig ESC are scanty. The aims of the present study were (a) to characterize the expression profile of gp130, LRβ, and STAT3 in pig parthenogenetic cell lines (ppC), previously derived in our laboratory and shown to be positive for the main pluripotency related markers; (b) to evaluate the role of LIF pathway in maintaining the pluripotency of these cells. To this purpose, ppC were cultured as previously described (Brevini et al. 2007 Theriogenology 68, 206–214) and screened by RT-PCR for the two LIF receptor subunits (LRβ and gp130) and STAT3. Pig granulosa cells were used as positive controls. To better investigate the possible role of LIF in maintenance of pluripotency in ppC, the formation of embryoid bodies (EB) was induced in the presence or in the absence of the cytokine. Undifferentiated cells were cultured in hanging drops either with or without LIF for 12 days. The EB formation and the expression of molecular markers specific for the three germ layers was evaluated at the end of the differentiation period. Molecular analysis allowed us to detect transcription of STAT3, whereas no signal for LRβ and gp130 was detected in ppC. These results seem to indicate that LIF does not play a role in the maintenance of pluripotency in the pig. However, after removal of LIF, ppC routinely formed EB that expressed molecular markers specific for the three germ layers. On the other hand, when LIF was added to the differentiation medium, pig cells were unable to form EB. They kept proliferating in an undifferentiated state, and no expression of molecular markers specific for the three germ layers was detected. Moreover, when re-plated on inactivated feeder-layers, they formed distinct colonies that maintained expression of pluripotency markers. Our results show that a role of LIF in pluripotency maintenance through a classical LRβ-gp130 and STAT3 activation pathway is unlikely. However, interaction with an alternative nonclassical activation signaling pathway cannot be ruled out. Indeed, the presence of the cytokine in the medium used for differentiation experiments actively inhibited EB formation, indicating a possible role in preventing differentiation in the porcine species. Further studies are needed to elucidate these aspects. Supported by: PRIN2005; PRIN2006; First 2006; First2007.

Animal Embryonic Stem (ES) Cells: Self-renewal, Pluripotency, Transgenesis and Nuclear Transfer

Mouse embryonic stem (ES) cells can be maintained indefinitely in the presence of leukemia inhibitory factor (LIF) and they express markers of self-renewal and pluripotency, which include the transcription factor Oct 4, STAT-3, stage-specific embryonic antigen (SSEA)-1, and alkaline phosphatase (AP). Upon removal of LIF, from the culture medium they cease to express markers such as Oct 4, rapidly losing the capacity for self-renewal and differentiating into a variety of cell types. Gene targeting is feasible in murine ES cells because these cells can be maintained in an undifferentiated state long enough to allow selection of properly targeted cell colonies with a high frequency of homologous recombination. Furthermore, blastocysts cloned from cultured murine ES cells develop to term at an efficiency (10-30%) that is three to ten times higher than blastocysts cloned from the nuclei of differentiated somatic cells. It seems likely that ES cells require less extensive reprogramming than do somatic cells, perhaps because in ES cells, many genes that are essential for early development are already active and thus do not require reactivation. Recently, we succeeded in isolating immortalized equine and bovine ES cells with a normal karyotype, that exhibit features similar to those of murine ES cells and express Oct 4, STAT-3, SSEA-1 and AP. We further confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells, as a source of nuclei for nuclear transfer (NT) and we generated cloned cattle with a higher frequency of pregnancies to term than has been achieved with differentiated somatic cells. Moreover, bovine ES cells that expressed enhanced green fluorescent protein (EGFP) were incorporated into both the inner cell mass (ICM) and the trophectdermal cells of developing blastocysts. These findings should facilitate targeted genetic manipulation of the genome and should allow production of cloned cattle in a single step after modification, as appropriate, of the genome.

Lymphoid Enhancer Factor 1-Mediated Wnt Signaling Promotes the Initiation of Trophoblast Lineage Differentiation in Mouse Embryonic Stem Cells

Embryonic stem (ES) cells can differentiate into all three embryonic germ layers but rarely into trophectoderm (TE) lineages that contribute to the placenta, although TE differentiation can be initiated by genetic manipulation of key genes involved in TE development. We demonstrate that Wnt signaling can initiate TE lineage differentiation by triggering an appropriate cue, caudal-related homeobox 2 (Cdx2). Overexpression and RNA interference knockdown studies indicate that Cdx2 induction in response to Wnt3a is mediated by lymphoid enhancer factor 1, whose expression is regulated by leukemia inhibitory factor (LIF) and bone morphogenetic protein. Removal of LIF, along with addition of Wnt3a, stimulated Cdx2 expression and induced formation of trophoblast stem (TS) cells. These TS cells were able to differentiate into cells with characteristics of spongiotrophoblast and trophoblast giant cells. This is, to our knowledge, the first evidence that TE lineage differentiation can be induced by Wnt signaling in mouse ES cells.