TBX3 Promotes Human Embryonic Stem Cell Proliferation and Neuroepithelial Differentiation in a Differentiation Stage-dependent Manner

T-box 3 (Tbx3) is a member of the T-box family of genes. Mutations that result in the haploinsufficiency of TBX3 cause ulnar mammary syndrome in humans characterized by mammary gland hypoplasia as well as other congenital defects. In mice, homozygous mutations are embryonic lethal, suggesting that Tbx3 is essential for embryo development. Studies in mice have shown that Tbx3 is essential in the maintenance of mouse embryonic stem cell (ESC) self-renewal and in their differentiation into extraembryonic endoderm (ExEn). The role TBX3 plays in regulating human ESCs (hESCs) has not been explored. Since mouse and hESCs are known to represent distinct pluripotent states, it is important to address the role of TBX3 in hESC self-renewal and differentiation. Using overexpression and knockdown strategies, we found that TBX3 overexpression promotes hESC proliferation possibly by repressing the expression of both NFκBIB and p14(ARF) , known cell cycle regulators. During differentiation, TBX3 knockdown resulted in decreased neural rosette formation and in decreased expression of neuroepithelial and neuroectoderm markers (PAX6, LHX2, FOXG1, and RAX). Taken together, our data suggest a role for TBX3 in hESC proliferation and reveal an unrecognized novel role of TBX3 in promoting neuroepithelial differentiation. Our results suggest that TBX3 plays distinct roles in regulating self-renewal and differentiation in both hESCs and mouse ESCs. STEM Cells2012;30:2152-2163.     

Pedigreed primate embryonic stem cells express homogeneous familial gene profiles

Human embryonic stem cells (hESCs) hold great biomedical promise, but experiments comparing them produce heterogeneous results, raising concerns regarding their reliability and utility, although these variations may result from their disparate and anonymous origins. To determine whether primate ESCs have intrinsic biological limitations compared with mouse ESCs, we examined expression profiles and pluripotency of newly established nonhuman primate ESC (nhpESCs). Ten pedigreed nhpESC lines, seven full siblings (fraternal quadruplets and fraternal triplets), and nine half siblings were derived from 41 rhesus embryos; derivation success correlated with embryo quality. Each line has been growing continuously for approximately 1 year with stable diploid karyotype (except for one stable trisomy) and expresses in vitro pluripotency markers, and eight have already formed teratomas. Unlike the heterogeneous gene expression profiles found among hESCs, these nhpESCs display remarkably homogeneous profiles (>97%), with full-sibling lines nearly identical (>98.2%). Female nhpESCs express genes distinct from their brother lines; these sensitive analyses are enabled because of the very low background differences. Experimental comparisons among these primate ESCs may prove more reliable than currently available hESCs, since they are akin to inbred mouse strains in which genetic variables are also nearly eliminated. Finally, contrasting the biological similarities among these lines with the heterogeneous hESCs might suggest that additional, more uniform hESC lines are justified. Taken together, pedigreed primate ESCs display homogeneous and reliable expression profiles. These similarities to mouse ESCs suggest that heterogeneities found among hESCs likely result from their disparate origins rather than intrinsic biological limitations with primate embryonic stem cells.     

Phosphatase and tensin homolog regulates the pluripotent state and lineage fate choice in human embryonic stem cells

Understanding the intrinsic and extrinsic signals that regulate the molecular basis of the pluripotent state may improve our understanding of mammalian embryogenesis, different states of pluripotency, and our ability to tailor lineage differentiation. Although the role of the PI3K/Akt pathway in the self-renewal and maintenance of mESCs is well-established, the specific contribution of the pathway or of its negative regulator, PTEN, in the maintenance of the human pluripotent state is less understood. To explore the PI3K/AKT pathway in human embryonic stem cell (hESC) pluripotency and differentiation, we generated stable PTEN knockdown (KD) hESCs using short hairpin RNA. Similar to mESCs, we found that PTEN KD hESCs have increased self-renewal, cell survival, and proliferation over multiple passages compared to control cells. However, in contrast to mESCs, in vitro, PTEN KD hESCs differentiated inefficiently in directed differentiation assays, in part due to the continued maintenance of OCT4 and NANOG expression. In teratoma assays, PTEN KD hESCs generated tissues from the three germ layers, although with a bias toward neuroectoderm differentiation. These results demonstrate that PTEN is a key regulator of hESC growth and differentiation, and manipulation of this pathway may improve our ability to regulate and understand the pluripotent state.     

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 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.     

Engineered polymer-media interfaces for the long-term self-renewal of human embryonic stem cells

We have developed a synthetic polymer interface for the long-term self-renewal of human embryonic stem cells (hESCs) in defined media. We successfully cultured hESCs on hydrogel interfaces of aminopropylmethacrylamide (APMAAm) for over 20 passages in chemically-defined mTeSR?1 media and demonstrated pluripotency of multiple hESC lines with immunostaining and quantitative RT-PCR studies. Results for hESC proliferation and pluripotency markers were both qualitatively and quantitatively similar to cells cultured on Matrigel?-coated substrates. Mechanistically, it was resolved that bovine serum albumin (BSA) in the mTeSR?1 media was critical for cell adhesion on APMAAm hydrogel interfaces. This study uniquely identified a robust long-term culture surface for the self-renewal of hESCs without the use of biologic coatings (e.g., peptides, proteins, or Matrigel?) in completely chemically-defined media that employed practical culturing techniques amenable to clinical-scale cell expansion.     

Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response

MicroRNAs are important regulators in many cellular processes, including stem cell self-renewal. Recent studies demonstrated their function as pluripotency factors with the capacity for somatic cell reprogramming. However, their role in human embryonic stem (ES) cells (hESCs) remains poorly understood, partially due to the lack of genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified neural and mesenchymal derivatives. Using a combination of AGO cross-linking and microRNA perturbation experiments, together with computational prediction, we identified the targets of the miR-302/367 cluster, the most abundant microRNAs in hESCs. Functional studies identified novel roles of miR-302/367 in maintaining pluripotency and regulating hESC differentiation. We show that in addition to its role in TGF-β signaling, miR-302/367 promotes bone morphogenetic protein (BMP) signaling by targeting BMP inhibitors TOB2, DAZAP2, and SLAIN1. This study broadens our understanding of microRNA function in hESCs and is a valuable resource for future studies in this area.     

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.     

Human Embryonic Stem Cell-extracts Inhibit the Differentiation and Function of Monocyte-derived Dendritic Cells

Embryonic stem cells (ESC) possess inherent properties of immune privilege with the capacity to evade allogeneic immune responses. Moreover, ESCs have been shown to prevent immune activation in response to third party antigen presenting cells in vitro and have the capacity to promote allograft survival in vivo. However, clinical use of human ESCs to treat immunological disorders may risk teratoma or ectopic tissue formation. Here, we show that cellular extracts from both human and mouse ESCs retain the immune modulatory properties of intact cells. ESC-extracts that contained 12–24 μg of total protein effectively prevented T cell proliferation in allogeneic mixed lymphocyte reactions (MLR), whereas control fibroblast extracts did not affect proliferation. Cellular mechanisms underlying hESC extract-mediated immune modulation involve the maturation of monocyte derived dendritic cells (mDC). hESC extract-treated mDCs had reduced surface expression of co-stimulatory and maturation markers CD80, HLA-DR and CD83 and secreted lower levels of IL12p40. Accordingly, hESC extract-treated DCs were found to be poor stimulators of purified allogeneic T cells compared to those DCs treated with vehicle or fibroblast extracts. Our results demonstrate that ESC extracts retain the immune modulatory properties of ESCs and for the first time demonstrates that ESC derived factors can inhibit human mDC maturation and function.     

Use of xenofree matrices and molecularly-defined media to control human embryonic stem cell pluripotency: effect of low physiological TGF-beta concentrations

To monitor human embryonic stem cell (hESC) self-renewal without differentiation, we used quantitative RT-PCR to study a selection of hESC genes, including markers for self-renewal, commitment/differentiation, and members of the TGF-beta superfamily and DAN gene family. Indeed, low commitment/differentiation gene expression, together with a significant self-renewal gene expres sion, provides a better pluripotency index than self-renewal genes alone. We demonstrate that matrices derived from human mesenchymal stem cells (hMSCs) can advantageously replace murine embryonic fibroblasts (MEF) or hMSC feeders. Moreover, a xenofree molecularly-defined SBX medium, containing a synthetic lipid carrier instead of albumin, can replace SR medium. The number of selected differentiation genes expressed by hESCs in these culture conditions was significantly lower than those expressed on MEF feeders in SR medium. In SBX, the positive effect of a non-physiological concentration of activin A (10-30 ng/mL) to reduce differentiation during self-renewal could also be obtained by physiological concentrations of TGF-beta(100-300 pg/mL). In contrast, these TGF-beta concentrations added to activin favored differentiation as previously observed with TGF-beta concentrations of 1 ng/mL or more. Compared to SR-containing medium, SBX medium promoted down-regulation of CER1 and LEFTIES and up-regulation of GREM1. Thus these genes better control self-renewal and pluripotency and prevent differentiation. A strategy is proposed to analyze, in more physiological, xenofree, molecularly-defined media and matrices, the numerous genes with still unknown functions controlling hESCs or human-induced pluripotent stem cells (iPS).