ES Cell Culture Research Articles

Human ES Cell Culture Conditions Fail to Preserve the Mouse Epiblast State.

Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) are the pluripotent stem cells (PSCs), derived from the inner cell mass (ICM) of preimplantation embryos at embryonic day 3.5 (E3.5) and postimplantation embryos at E5.5-E7.5, respectively. Depending on their environment, PSCs can exist in the so-called naïve (ESCs) or primed (EpiSCs) states. Exposure to EpiSC or human ESC (hESC) culture condition can convert mESCs towards an EpiSC-like state. Here, we show that the undifferentiated epiblast state is however not stabilized in a sustained manner when exposing mESCs to hESC or EpiSC culture condition. Rather, prolonged exposure to EpiSC condition promotes a transition to a primitive streak- (PS-) like state via an unbiased epiblast-like intermediate. We show that the Brachyury-positive PS-like state is likely promoted by endogenous WNT signaling, highlighting a possible species difference between mouse epiblast-like stem cells and human Embryonic Stem Cells.

Abstract PHB01: The zebrafish as a mechanical filter: Using zebrafish xenografts to model Ewing sarcoma metastasis in vivo

Abstract Ewing Sarcoma (ES), a pediatric cancer driven by the oncogenic fusion protein EWS-FLI1, recurs or metastasizes in 1 in 3 patients with no clear correlation to accumulation of secondary mutations, raising the possibility that environmental cues may drive metastasis. Previous work in the field has observed distinct cellular morphologies of ES cells in cell culture as a function of EWS-FLI1 level and posited the idea that this may correlate with distinct functional cellular states. In this work, we use zebrafish xenografts to investigate the effect of variable microenvironments on cellular morphologies of ES cells. We use high resolution light-sheet microscopy to focus on specific recurring seeding sites within the 3-day old zebrafish larvae. We then implement a unique cell-shape classification method to distinguish discrete 3D cellular morphologies in these datasets. Using this workflow, we observe a variety of cellular morphologies within the fish, including elongated cellular protrusions, blebbing, and rounded cells. We demonstrate that ES cells take on distinct morphological states as a function of the region in which they reside. We find cell proliferation, but not cell death, vary greatly in the different regions, suggesting differences in the functional state of the ES cells. Interestingly, knockdown of EWS-FLI1 in xenografted cells leads to discrete switches between cellular morphologies, but only as a function of seeding site within the fish, suggesting a previously undescribed environmental cue that modulates cellular effects of EWS-FLI1. Overall, the xenograft model for ES in zebrafish embryos can be viewed as a “mechanical filter”, which provides unique, complex 3-dimensional environments to study cell behavior within an organism, while leveraging the advantages of the zebrafish system, namely higher throughput and high resolution in vivo imaging capabilities. This work establishes a pipeline for classification and distinction of morphological states within a living organism, and, more specifically, helps our understanding of cellular plasticity of ES cells in response to microenvironment. Citation Format: Dagan Segal, Bo-Jui Chang, Hanieh Mazloom-Farsibaf, Reto Fiolka, James Amatruda, Gaudenz Danuser. The zebrafish as a mechanical filter: Using zebrafish xenografts to model Ewing sarcoma metastasis in vivo [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PHB01.

Study of Differentiation Level by Three-dimensional Culture of Mouse ES Cells on Titanium Alloy Powder

Study of Differentiation Level by Three-dimensional Culture of Mouse ES Cells on Titanium Alloy Powder

Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells.

A Retinoic Acid-Hedgehog Cascade Coordinates Mesoderm-Inducing Signals and Endoderm Competence during Lung Specification

Organogenesis of the trachea and lungs requires a complex series of mesoderm-endoderm interactions mediated by WNT, BMP, retinoic acid (RA), and hedgehog (Hh), but how these pathways interact ina gene regulatory network is less clear. Using Xenopus embryology, mouse genetics, and human ES cell cultures, we identified a conserved signaling cascade that initiates respiratory lineage specification. We show that RA has multiple roles; first RA pre-patterns the lateral plate mesoderm and then itpromotes Hh ligand expression in the foregut endoderm. Hh subsequently signals back to the pre-patterned mesoderm to promote expression of the lung-inducing ligands Wnt2/2b and Bmp4. Finally, RA regulates the competence of the endoderm to activate the Nkx2-1+ respiratory program in response to these mesodermal WNT and BMP signals. These data provide insights into early lung development and a paradigm for how mesenchymal signals are coordinated with epithelial competence during organogenesis.

Phenotypic Screening of Drug Library in Actively Differentiating Mouse Embryonic Stem Cells

Phenotypic screening enables the discovery of new drug leads with novel targets. ES cells differentiate into different lineages by successively making use of different subsets of the genome’s possible macromolecular interactions. If a compound effectively targets just one of these interactions, it derails the developmental pathway to produce a phenotypical change. The OTRADI microsource spectrum library of 2000 approved drug components, natural products, and bioactive components was screened for compounds that can induce phenotypic changes in ES cell cultures at 10 µM after 3 days. Twenty-one compounds that induced specific morphologies also induced unique changes to an expression profile of a dozen markers of early embryonic development, indicating that each compound has derailed the molecular developmental process in a characteristic way. Phenotypic screens conducted with ES cultures differentiating along different lineages can be used to efficiently prescreen compounds able to regulate cell differentiation lineage.

Pluripotent and Metabolic Features of Two Types of Porcine iPSCs Derived from Defined Mouse and Human ES Cell Culture Conditions.

The domestic pig is an excellent animal model for stem cell research and clinical medicine. There is still no suitable culture condition to generate authentic porcine embryonic stem cells (pESCs) and high quality porcine induced pluripotent stem cells (piPSCs). In this study, we found that culture conditions affected pluripotent and metabolic features of piPSCs. Using defined human embryonic stem cell (hESC) and mouse ESC (mESC) culture conditions, we generated two types of piPSCs, one of which was morphologically similar to hESCs (here called hpiPSCs), the other resembled mESCs (here called mpiPSCs). Transcriptome analysis and signaling pathway inhibition results suggested that mpiPSCs shared more of mESC signaling pathways, such as the BMP pathway and JAK/STAT pathway and hpiPSCs shared more hESC signaling pathways, such as the FGF pathway. Importantly, the mpiPSCs performed embryonic chimera incorporation more efficiently than the hpiPSCs did. In addition, the mpiPSCs showed mitochondrial features of naive ESCs and lipid droplets accumulation. These evidences may facilitate understanding of the gene regulation network and metabolism in piPSCs and promote derivation of bona fide pESCs for translational medicine.

Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

Transcriptional program of Kpna2/Importin-α2 regulates cellular differentiation-coupled circadian clock development in mammalian cells.

The circadian clock in mammalian cells is cell-autonomously generated during the cellular differentiation process, but the underlying mechanisms are not understood. Here we show that perturbation of the transcriptional program by constitutive expression of transcription factor c-Myc and DNA methyltransferase 1 (Dnmt1) ablation disrupts the differentiation-coupled emergence of the clock from mouse ESCs. Using these model ESCs, 484 genes are identified by global gene expression analysis as factors correlated with differentiation-coupled circadian clock development. Among them, we find the misregulation of Kpna2 (Importin-α2) during the differentiation of the c-Myc-overexpressed and Dnmt1(-/-) ESCs, in which sustained cytoplasmic accumulation of PER proteins is observed. Moreover, constitutive expression of Kpna2 during the differentiation culture of ESCs significantly impairs clock development, and KPNA2 facilitates cytoplasmic localization of PER1/2. These results suggest that the programmed gene expression network regulates the differentiation-coupled circadian clock development in mammalian cells, at least in part via posttranscriptional regulation of clock proteins.

An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells.

ABSTRACTEmbryonic Stem cells derived from the epiblast tissue of the mammalian blastocyst retain the capability to differentiate into any adult cell type and are able to self-renew indefinitely under appropriate culture conditions. Despite the large amount of knowledge that we have accumulated to date about the regulation and control of self-renewal, efficient directed differentiation into specific tissues remains elusive. In this work, we have analysed in a systematic manner the interaction between the dynamics of loss of pluripotency and Activin/Nodal, BMP4 and Wnt signalling in fate assignment during the early stages of differentiation of mouse ES cells in culture. During the initial period of differentiation, cells exit from pluripotency and enter an Epi-like state. Following this transient stage, and under the influence of Activin/Nodal and BMP signalling, cells face a fate choice between differentiating into neuroectoderm and contributing to Primitive Streak fates. We find that Wnt signalling does not suppress neural development as previously thought and that it aids both fates in a context dependent manner. Our results suggest that as cells exit pluripotency they are endowed with a primary neuroectodermal fate and that the potency to become endomesodermal rises with time. We suggest that this situation translates into a “race for fates” in which the neuroectodermal fate has an advantage.

Developmental cardiotoxicity effects of four commonly used antiepileptic drugs in embryonic chick heart micromass culture and embryonic stem cell culture systems

Antiepileptic drugs (AEDs) are commonly used drugs in pregnant women with epilepsy. Prenatal exposure to AEDs increases the risk of major or minor congenital malformation during embryonic development. The precise mode of action and intracellular mechanisms of these AEDs during embryonic development remains unclear. To determine relative teratogenic risk of AEDs, four AED drugs including valproic acid (VPA), phenytoin (PHT), phenobarbital (PB), and trimethadione (TMD) were tested using two in vitro systems (the embryonic chick heart micromass (MM) culture and the in vitro differentiating mouse embryonic stem cells into cardiomyocytes culture systems). Cardiomyocyte cultures (the heart MM and ES cell-derived cardiomyocytes) were treated with or without different concentrations of PHT, PB, TMD (10–100μM), and VPA (100–2000μM). 5-Fluorouracil (5-FU) (1–10μM) and l-ascorbic acid (10–1000μM) were used as positive and negative controls. It was found that these four commonly used AEDs and 5-FU tested have the potential to inhibit embryonic heart cell differentiation (p<0.05) without inducing any cytotoxicity. VPA at higher concentrations (⩾800μM), and 5-FU at all doses proved to be cytotoxic in the differentiating ES cell culture system. The results demonstrated in this study suggest that the use of these four commonly prescribed AEDs during pregnancy may have an effect on embryonic heart development, and may be associated with congenital cardiovascular defects.

Zoledronic acid inhibits pulmonary metastasis dissemination in a preclinical model of Ewing’s sarcoma via inhibition of cell migration

BackgroundEwing’s sarcoma (ES) is the second most frequent primitive malignant bone tumor in adolescents with a very poor prognosis for high risk patients, mainly when lung metastases are detected (overall survival <15% at 5 years). Zoledronic acid (ZA) is a potent inhibitor of bone resorption which induces osteoclast apoptosis. Our previous studies showed a strong therapeutic potential of ZA as it inhibits ES cell growth in vitro and ES primary tumor growth in vivo in a mouse model developed in bone site. However, no data are available on lung metastasis. Therefore, the aim of this study was to determine the effect of ZA on ES cell invasion and metastatic properties.MethodsInvasion assays were performed in vitro in Boyden’s chambers covered with Matrigel. Matrix Metalloproteinase (MMP) activity was analyzed by zymography in ES cell culture supernatant. In vivo, a relevant model of spontaneous lung metastases which disseminate from primary ES tumor was induced by the orthotopic injection of 106 human ES cells in the tibia medullar cavity of nude mice. The effect of ZA (50 μg/kg, 3x/week) was studied over a 4-week period. Lung metastases were observed macroscopically at autopsy and analysed by histology.ResultsZA induced a strong inhibition of ES cell invasion, probably due to down regulation of MMP-2 and −9 activities as analyzed by zymography. In vivo, ZA inhibits the dissemination of spontaneous lung metastases from a primary ES tumor but had no effect on the growth of established lung metastases.ConclusionThese results suggest that ZA could be used early in the treatment of ES to inhibit bone tumor growth but also to prevent the early metastatic events to the lungs.

Telomere elongation in parthenogenetic stem cells

Telomere elongation in parthenogenetic stem cells

Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell–derived neocortex

Here, using further optimized 3D culture that allows highly selective induction and long-term growth of human ES cell (hESC)-derived cortical neuroepithelium, we demonstrate unique aspects of self-organization in human neocorticogenesis. Self-organized cortical tissue spontaneously forms a polarity along the dorsocaudal-ventrorostral axis and undergoes region-specific rolling morphogenesis that generates a semispherical structure. The neuroepithelium self-forms a multilayered structure including three neuronal zones (subplate, cortical plate, and Cajal-Retzius cell zones) and three progenitor zones (ventricular, subventricular, and intermediate zones) in the same apical-basal order as seen in the human fetal cortex in the early second trimester. In the cortical plate, late-born neurons tend to localize more basally to early-born neurons, consistent with the inside-out pattern seen in vivo. Furthermore, the outer subventricular zone contains basal progenitors that share characteristics with outer radial glia abundantly found in the human, but not mouse, fetal brain. Thus, human neocorticogenesis involves intrinsic programs that enable the emergence of complex neocortical features.

Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture

In the mammalian cortex, the dorsal telencephalon exhibits a characteristic stratified structure. We previously reported that three-dimensional (3D) culture of mouse ES cells (mESCs) can efficiently generate cortical neuroepithelium (NE) and layer-specific cortical neurons. However, the cortical NE generated in this mESC culture was structurally unstable and broke into small neural rosettes by culture day 7, suggesting that some factors for reinforcing the structural integrity were missing. Here we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. The addition of purified laminin and entactin (a laminin-associated protein), even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure even on culture days 12 and 15, and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence–like tissue formed in the region adjacent to the cortical NE. Thus, our results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D ESC culture, and supports the morphogenetic recapitulation of cortical development.

Feeder Independent Culture of Mouse Embryonic Stem Cells

Embryonic stem cell classically cultured on feeder layer with FBS contained ES medium. Feeder-free mouse ES cell culture systems are essential to avoid the possible contamination of nonES cells. First we determined the difference between ES cell and MEF by Oct4 population. We demonstrate to culture and to induce differentiation on feeder free condition using a commercially available mouse ES cell lines.

A Gene-Targeting Approach for the Biological Significance of AML1/Runx1 Arginine-Methylation

Abstract 1309Acute Myeloid Leukemia 1 (AML1; also called as Runx1: Runt-related transcription factor 1) belongs to the Class II group of leukemia-associated mutation-target genes, and it encodes the DNA-binding subunit of the hetero-dimeric transcription factor complex, Core-Binding Factor (CBF). CBF plays pivotal roles in initial hematopoietic development during embryogenesis and in cellular differentiation of thrombotic and lymphatic lineages throughout adult life. Recent researches revealed that cellular AML1 polypeptide is processed with post-translational modifications, including phosphorylation, acethylation, ubiquitination, and methylation. Biological significance of these modifications on the AML1's function as the hematopoietic regulator, however, largely remains to be elucidated. In this study, we focused on the arginine-methylation as an initial step towards the comprehensive understanding for the AML1-regulating mechanism through these modifications. Arginine residues just downstream to the Runt-domain, which is located at N-terminal region of the molecule and functions as the binding site to DNA and CBF beta: the hetero-dimerization partner, are recently reported to be methylated, resulting in the inhibition of the corepressor-binding thus enhancing its trans- activating activity. In order to elucidate biological significance of these methylations, we performed a series of genetic experiments: First, we generated the non-methylatable double arginine-to-lysine (RRKK) mutant of AML1 at these residues, which should keep AML1-corepressor-binding. When this mutant was subjected to the luciferase reporter-assay using a target-gene construct, it showed lower trans- activating activity in comparison to that for wild-type molecule, as expected. However, this loss-of-function mutation appeared to be dispensable at least for in vitro function for hematopoietic regulation in that this RRKK mutant did rescue hematopoietic differentiation of the AML1-deficient murine ES cells in culture when expressed from a knock-in allele as was the case for the wild-type cDNA of mouse AML1. To further evaluate the biological activity of this mutant in the context of an entire animal, we introduced this mutant cDNA into AML1/Runx1 locus of mouse ES cells by means of a targeted-insertion (knock-in) strategy. Germline mutant mouse lines were successfully established, following blastocyst-injection of these ES cell clones. Heterozygous mice were healthy and fertile, and genotyping for the live pups generated from heterozygotes-crossing revealed that this arginine-mutant allele segregated according to the Mendelian ratio. Homozygous AML1RRKK/RRKK mice were born alive and grew up adult, circumventing the mid-embryonic death due to hematopoietic block that was originally described for the AML1-deficient mice, thus the in vitro notion that these arginine-methylations were not essential for the early hematopoietic development described above was further underscored. There were no significant differences so far observed in peripheral blood cell counts among mice of the AML1RRKK/RRKK or AML1WT/WT genotypes, in comparison to their wild-type littermates. Preliminary studies revealed that AML1RRKK/RRKK mice showed imbalance of the peripheral T cell populations, implying that these methylations may have roles in these cellular lineages. We are currently focusing on further examination of these mutant mice, paying special attention to the cellular lineages where genetic manifestations were observed for AML1 haploinsufficient mice and/or conditional AML1-deficient mice. We hope that these efforts will unveil the biological significance of the AML1 methylation in hematopoietic regulation. Disclosures:No relevant conflicts of interest to declare.

Allogeneic Transplantation of Induced Pluripotent Stem Cells in a Porcine Model of Chronic Myocardial Ischemia Failed to Stimulate Myocyte Differentiation

Background: Recently, pig induced pluripotent stem cells (iPSCs) have been produced by by ectopic expression of four human transcriptional factors, OCT4, SOX2, KLF4, and cMYC in pig fetal fibroblasts through a lentiviral vector. The pig iPSCs showed pluripotency via in vitro differentiation assays and teratoma testing. However, these cells have failed to differentiate into beating myocytes in vitro. To determine whether in vivo conditions would drive iPSC differentiation into myocytes, we performed allogeneic transplantation of the pig iPSCs into areas of chronically ischemic myocardium. Methods: Pig iPSCs were expanded by standard ES cell culture method, and allogeneically transplanted into chronically ischemic myocardium of eight Yorkshire pigs by direct intramyocardial injection (four 10-cm culture dishes, total of 10-16 million cells, suspended in 2.5 ml of saline, with 25 injection sites). Cohorts of two animals were sacrificed at 2, 4, 8 wks and 3 months after injection to study the differentiation of the injected cells. Results: After transplantation, there were no signs of adverse side effects or graft versus host disease at any time point. Two weeks after injection, clusters of SSEA-4 positive cells were detected in the frozen sections of the injected area using immunofluorescent staining. Four to 8 weeks later, these clusters of cells started to proliferate, within the injected area, into spherical shaped small tumors surrounded by thin capsules. The cells inside these tumors showed homogeneous phenotype with no signs of differentiation into any lineage. Three months after the injection, no such small tumors were found; instead, similar SSEA-4 positive cell clusters were found in the injected area but in smaller numbers. By immunofluorescent staining, few smooth muscle actin or vWF positive cells were found inside the cell clusters, but no positive desmin cells were found, and by RT-PCR, the expression of VEGF, FGF, ANRT were significantly higher in the injected myocardium compared with none injected, suggesting that injected iPSCs were not differentiated into myocytes and might contribute to the formation of some new vessels. Conclusions: Allogeneic transplantation of iPSCs can be safely used to test the lineage differentiation of stem cells for the future iPSC-based therapy. Despite an ischemic environment, pig iPSCs continue to proliferate in vivo for three months after injection. However the proliferation ability of the iPSCs was limited within the immunoconpetent hosts. For future cell-based therapy, iPSCs with pro-myocyte differentiation will be needed to study their potential for myocardial regeneration.

Analysis of Molecular Expression Patterns of Bovine Embryonic Stem-Like Cells Derived by Conventional ES Cell Culture Condition.

Analysis of Molecular Expression Patterns of Bovine Embryonic Stem-Like Cells Derived by Conventional ES Cell Culture Condition.

Pou5f1/oct4 in pluripotency control: Insights from zebrafish

Gastrulation in vertebrates is a conserved process, which involves transition from cellular pluripotency to early precursors of ectoderm, mesoderm, and endoderm. Pluripotency control during this stage is far from being understood. Recent genetic and transcriptomic studies in zebrafish suggest that the core pluripotency transcription factors (TFs) Pou5f1 and TFs of the SoxB1 group are critically involved in large-scale temporal coordination of gene expression during gastrulation. A significant number of evolutionary conserved target genes of Pou5f1 in zebrafish are also involved in stem-cell circuit in mammalian ES cell cultures. Here, I will review the roles of Pou5f1 in development and discuss the evolutionary conservation of Pou5f1 functions and their relation to pluripotency control.