Reduced oxygen concentration enhances conversion of embryonic stem cells to epiblast stem cells

Recently, an additional type of pluripotent stem cell-line derived from mouse embryos has been established and termed epiblast stem cell (EpiSC), and is expected to be an important tool for studying the mechanisms of maintenance of pluripotency since they depend on basic fibroblast growth factor-MAPK and Activin A-Smad2/3 signaling to maintain pluripotency, unlike mouse embryonic stem cells (ESCs). Further, because of the similarities between mouse EpiSCs and human ESCs, EpiSCs are expected to be effective experimental models for human stem cell therapy. Recently, study for conversion from ESC state to EpiSC state or reversion from EpiSC state to ESC state has attracted interest since these techniques may lead to increasing the potential of pluripotent stem cells and our knowledge about their developmental status. In the present study, we find that a low oxygen concentration in culture environment accelerated, improved, and stabilized the EpiSC state of the converted cells from the ESC state using Oct4ΔPE-GFP transgenic ESCs. Induced EpiSCs (iEpiSCs) in hypoxia possess closer gene expression patterns to native EpiSCs, and bisulfite sequences for the promoter regions of Stella and Oct4 genes have elucidated that the iEpiSC gain EpiSC-specific methylation patterns in hypoxia. Our data provide evidence that oxygen concentration is an important factor for establishment of the EpiSC-specific state.     

Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells

Human embryonic stem cells (hESCs) self-renew indefinitely while maintaining pluripotency. The molecular mechanism underlying hESCs self-renewal and pluripotency is poorly understood. To identify the signaling pathway molecules that maintain the proliferation of hESCs, we performed a microarray analysis comparing an aneuploid H1 hESC line (named H1T) versus euploid H1 hESC line because the H1T hESC line demonstrates a self-renewal advantage while maintaining pluripotency. We find differential gene expression for the Nodal/Activin, fibroblast growth factor (FGF), Wnt, and Hedgehog (Hh) signaling pathways in the H1T line, which implicates each of these molecules in maintaining the undifferentiated state, whereas the bone morphogenic protein (BMP) and Notch pathways could promote hESCs differentiation. Experimentally, we find that Activin A is necessary and sufficient for the maintenance of self-renewal and pluripotency of hESCs and supports long-term feeder and serum-free growth of hESCs. We show that Activin A induces the expression of Oct4, Nanog, Nodal, Wnt3, basic FGF, and FGF8 and suppresses the BMP signal. Our data indicates Activin A as a key regulator in maintenance of the stemness in hESCs. This finding will help elucidate the complex signaling network that maintains the hESC phenotype and function.     

Status of genomic imprinting in epigenetically distinct pluripotent stem cells

Mouse epiblast stem cells (EpiSCs) derived from postimplantation embryos are developmentally and functionally different from embryonic stem cells (ESCs) generated from blastocysts. EpiSCs require Activin A and FGF2 signaling for self-renewal, similar to human ESCs (hESCs), while mouse ESCs require LIF and BMP4. Unlike ESCs, EpiSCs have undergone X-inactivation, similar to the tendency of hESCs. The shared self-renewal and X-inactivation properties of EpiSCs and hESCs suggest that they have an epigenetic state distinct from ESCs. This hypothesis predicts that EpiSCs would have monoallelic expression of most imprinted genes, like that observed in hESCs. Here, we confirm this prediction. By contrast, we find that mouse induced pluripotent stem cells (iPSCs) tend to lose imprinting similar to mouse ESCs. These findings reveal that iPSCs have an epigenetic status associated with their pluripotent state rather than their developmental origin. Our results also reinforce the view that hESCs and EpiSCs are in vitro counterparts, sharing an epigenetic status distinct from ESCs and iPSCs.     

Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed

Human ESCs (hESCs) respond to signals that determine their pluripotency, proliferation, survival, and differentiation status. In this report, we demonstrate that phosphatidylinositol 3-kinase (PI3K) antagonizes the ability of hESCs to differentiate in response to transforming growth factor beta family members such as Activin A and Nodal. Inhibition of PI3K signaling efficiently promotes differentiation of hESCs into mesendoderm and then definitive endoderm (DE) by allowing them to be specified by Activin/Nodal signals present in hESC cultures. Under conditions where hESCs are grown in mouse embryo fibroblast-conditioned medium under feeder-free conditions, approximately 70%-80% are converted into DE following 5 days of treatment with inhibitors of the PI3K pathway, such as LY 294002 and AKT1-II. Microarray and quantitative polymerase chain reaction-based gene expression profiling demonstrates that definitive endoderm formation under these conditions closely parallels that following specification with elevated Activin A and low fetal calf serum (FCS)/knockout serum replacement (KSR). Reduced insulin/insulin-like growth factor (IGF) signaling was found to be critical for cell fate commitment into DE. Levels of insulin/IGF present in FCS/KSR, normally used to promote self-renewal of hESCs, antagonized differentiation. In summary, we show that generation of hESC-DE requires two conditions: signaling by Activin/Nodal family members and release from inhibitory signals generated by PI3K through insulin/IGF. These findings have important implications for our understanding of hESC self-renewal and early cell fate decisions.     

Synergistic effect of TGF‐β superfamily members on the induction of Foxp3+ Treg

TGF‐β plays an important role in the induction of Treg and maintenance of immunologic tolerance, but whether other members of TGF‐β superfamily act together or independently to achieve this effect is poorly understood. Although others have reported that the bone morphogenetic proteins (BMP) and TGF‐β have similar effects on the development of thymocytes and T cells, in this study, we report that members of the BMP family, BMP‐2 and ‐4, are unable to induce non‐regulatory T cells to become Foxp3⁺ Treg. Neutralization studies with Noggin have revealed that BMP‐2/4 and the BMP receptor signaling pathway is not required for TGF‐β to induce naïve CD4⁺CD25^? cells to express Foxp3; however, BMP‐2/4 and TGF‐β have a synergistic effect on the induction of Foxp3⁺ Treg. BMP‐2/4 affects non‐Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3⁺ Treg induced by TGF‐β. Data further advocate that TGF‐β is a key signaling factor for Foxp3⁺ Treg development. In addition, the synergistic effect of BMP‐2/4 and TGF‐β indicates that the simultaneous manipulation of TGF‐β and BMP signaling might have considerable effects in the clinical setting for the enhancement of Treg purity and yield.     

Activin and BMP4 Synergistically Promote Formation of Definitive Endoderm in Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) herald tremendous promise for the production of clinically useful cell types for the treatment of injury and disease. Numerous reports demonstrate their differentiation into definitive endoderm (DE) cells, the germ layer from which pancreatic β cells and hepatocytes arise, solely from exposure to a high dose of recombinant Activin/Nodal. We show that combining a second related ligand, BMP4, in combination with Activin A yields 15%-20% more DE as compared with Activin A alone. The addition of recombinant BMP4 accelerates the downregulation of pluripotency genes, particularly SOX2, and results in upregulation of endogenous BMP2 and BMP4, which in turn leads to elevated levels of phospho-SMAD1/5/8. Combined Activin A and BMP4 treatment also leads to an increase in the expression of DE genes CXCR4, SOX17, and FOXA2 when compared with Activin A addition alone. Comparative microarray studies between DE cells harvested on day 3 of differentiation further reveal a novel set of genes upregulated in response to initial BMP4 exposure. Several of these, including APLNR, LRIG3, MCC, LEPREL1, ROR2, and LZTS1, are expressed in the mouse primitive streak, the site of DE formation. Thus, this synergism between Activin A and BMP4 during the in vitro differentiation of hESC into DE suggests a complex interplay between BMP and Activin/Nodal signaling during the in vivo allocation and expansion of the endoderm lineage. STEM CELLS 2012; 30:631-642.     

BMP4/Smad Signaling Pathway Induces the Differentiation of Mouse Spermatogonial Stem Cells via Upregulation of Sohlh2

Spermatogonial stem cells (SSCs) capable of self‐renewal and differentiation are the foundation for spermatogenesis. Although several factors that govern these processes have been investigated, the underlying molecular mechanisms have not been fully elucidated. Here, we investigated the role of BMP4 in mouse SSC differentiation, and found that SSCs cultured in the presence of BMP4 underwent differentiation, characterized by downregulation of SSC self‐renewal markers, Plzf, and upregulation of SSC differentiation marker, c‐kit. Smad1/5/8 proteins were phosphorylated during BMP4‐induced differentiation. The effects of BMP4 on SSCs were blocked by BMP4 inhibitor (Dorsomorphin). The activation of BMP4/Smad signaling pathway in SSCs increased the expression of Sohlh2, which is involved in the early differentiation of spermatogonia. Knockdown sohlh2 expression by RNA interference abolished the effect of BMP4 on SSC differentiation and the upregulation of c‐kit expression. Overall, our results suggest that BMP4 plays an important role during the early differentiation of SSCs via upregulation of sohlh2. Anat Rec, 297:749–757, 2014. © 2014 Wiley Periodicals, Inc.     

Conditional expression of the dominant‐negative TGF‐β receptor type II elicits lingual epithelial hyperplasia in transgenic mice

Background: The transforming growth factor‐β (TGF‐β) signaling pathway is generally believed to be a potent inhibitor of proliferation. However, many epithelia lacking the essential Tgfbr2 gene still maintain normal tissue homeostasis. Here, transgenic mice expressing rtTA from the human keratin 14 (K14) promoter were used to generate an inducible dominant‐negative TGF‐β receptor type II (Tgfbr2) mutant model, which allowed us to distinguish between the primary and secondary effects of TGF‐β signaling disruption by Doxycycline treatment in K14+ epithelial stem cells. Results: We showed that in mice lacking TGF‐β signaling in K14+ cells, invasive carcinomas developed on the ventral surface of the tip of the tongue, while filiform papillae on the dorsal surface showed different pathological changes from the tip to the posterior of the tongue. In addition, acetylation levels of histone H4 and histone H3 rapidly increased, while pMAPK activity was enhanced and Jagged2 inactivated in lingual epithelia after disruption of TGF‐β signaling. Conclusions: Our results contribute to the understanding of TGF‐β signaling in regulating homeostasis and carcinogenesis in lingual epithelia. Developmental Dynamics 242:444–455, 2013. © 2013 Wiley Periodicals, Inc.