The canonical Wnt/β-catenin signaling has remarkably diverse roles in embryonic development, stem cell self-renewal and cancer progression. Here, we show that stabilized expression of β-catenin perturbed human embryonic stem (hES)-cell self-renewal, such that up to 80% of the hES cells developed into the primitive streak (PS)/mesoderm progenitors, reminiscent of early mammalian embryogenesis. The formation of the PS/mesoderm progenitors essentially depended on the cooperative action of β-catenin together with Activin/Nodal and BMP signaling pathways. Intriguingly, blockade of BMP signaling completely abolished mesoderm generation, and induced a cell fate change towards the anterior PS progenitors. The PI3-kinase/Akt, but not MAPK, signaling pathway had a crucial role in the anterior PS specification, at least in part, by enhancing β-catenin stability. In addition, Activin/Nodal and Wnt/β-catenin signaling synergistically induced the generation and specification of the anterior PS/endoderm. Taken together, our findings clearly demonstrate that the orchestrated balance of Activin/Nodal and BMP signaling defines the cell fate of the nascent PS induced by canonical Wnt/β-catenin signaling in hES cells.KEY WORDS: Primitive streak, Mesoderm, Endoderm, Stem cells, β-Catenin, Wnt Development 135, 2969Development 135, -2979Development 135, (2008 Lindsley et al., 2006), but precise roles of this signaling in human ES (hES) cells remains controversial (Dravid et al., 2005;Sato et al., 2004).We report here that the activation of canonical Wnt/β-catenin signaling in hES cells by conditional activation of stabilized β-catenin disrupted hES-cell self-renewal. Rather, the canonical Wnt/β-catenin and BMP signaling pathway in hES cells has significant roles in establishing the posterior PS/mesoderm progenitors, whereas attenuation of BMP signaling changes the cell fate to the anterior PS/endoderm progenitors. In addition, Activin and Wnt/β-catenin signaling pathways synergistically function in inducing undifferentiated hES cells to differentiate into the anterior PS/endoderm progenitors. This is the first in vitro model system that consistently recapitulates the human early embryogenesis and that enables us to analyze molecular events during the process of early embryogenesis from the epiblast to the PS formation, followed by lineage specification into the mesoderm and endoderm. More importantly, our findings will also be relevant to directed differentiation of specific tissue and cells from hES cells. MATERIALS AND METHODS Activation of β-catenin signaling in hES cellsThe ⌬Nβ-cateninER construct, in which the N-terminal 90 amino acids were deleted, was generated by in-frame insertion into the expression vector containing the hormone-binding domain of a mutant estrogen receptor (Littlewood et al., 1995;Sumi et al., 2007). Cell lines expressing ΔNβ-cateninER were obtained by transfection of hES cell lines KhES-1 and KhES-3 with ΔNβ-cateninER expression plasmids, followed by puromysin selection as described previously (Sumi ...

No abstract

ELABELA (ELA) is a peptide hormone required for heart development that signals via the Apelin Receptor (APLNR, APJ). ELA is also abundantly secreted by human embryonic stem cells (hESCs), which do not express APLNR. Here we show that ELA signals in a paracrine fashion in hESCs to maintain self-renewal. ELA inhibition by CRISPR/Cas9-mediated deletion, shRNA, or neutralizing antibodies causes reduced hESC growth, cell death, and loss of pluripotency. Global phosphoproteomic and transcriptomic analyses of ELA-pulsed hESCs show that it activates PI3K/AKT/mTORC1 signaling required for cell survival. ELA promotes hESC cell-cycle progression and protein translation and blocks stress-induced apoptosis. INSULIN and ELA have partially overlapping functions in hESC medium, but only ELA can potentiate the TGFβ pathway to prime hESCs toward the endoderm lineage. We propose that ELA, acting through an alternate cell-surface receptor, is an endogenous secreted growth factor in human embryos and hESCs that promotes growth and pluripotency.

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 b 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.

Due to an unfortunate miscommunication, the original version of this paper that was published online on September 17, 2015 contained misspellings in two of the authors' names. The corrected names, Iwona Szczerbinska and Yun-Shen Chan, now appear with this article online. We apologize for the confusion.

PRDM14 was identified by microarray analysis and was expressed in specifically undifferentiated human ES cells. PRDM14 protein is thought to regulate gene transcription in human ES cells, as it contains a PR domain, a subtype of the SET domain which catalyzes histone methylation. To analyze the function of PRDM14, we performed knock-down and forced expression of PRDM14 in human ES cells. Knock-down of PRDM14 by siRNA induced expression of early differentiation marker genes. Forced expression of PRDM14 suppressed expression of differentiation marker genes in the embryoid body. These results suggest that PRDM14 is involved in the maintenance of the self-renewal of human ES cells by suppression of gene expression.

SummaryInactivation of the Pancreatic and Duodenal Homeobox 1 (PDX1) gene causes pancreatic agenesis, which places PDX1 high atop the regulatory network controlling development of this indispensable organ. However, little is known about the identity of PDX1 transcriptional targets. We simulated pancreatic development by differentiating human embryonic stem cells (hESCs) into early pancreatic progenitors and subjected this cell population to PDX1 chromatin immunoprecipitation sequencing (ChIP-seq). We identified more than 350 genes bound by PDX1, whose expression was upregulated on day 17 of differentiation. This group included known PDX1 targets and many genes not previously linked to pancreatic development. ChIP-seq also revealed PDX1 occupancy at hepatic genes. We hypothesized that simultaneous PDX1-driven activation of pancreatic and repression of hepatic programs underlie early divergence between pancreas and liver. In HepG2 cells and differentiating hESCs, we found that PDX1 binds and suppresses expression of endogenous liver genes. These findings rebrand PDX1 as a context-dependent transcriptional repressor and activator within the same cell type.

Embryonic stem (ES) cells are self-renewing, pluripotent cell lines, characterized by their potential to differentiate into all cell types. The proto-oncogene product c-Myc has a crucial role in the self-renewal of mouse ES (mES) cells, but its role in human ES (hES) cells is unknown. To investigate c-Myc functions in hES cells, we expressed an inducible c-Myc fused to the hormone-binding domain of the estrogen receptor (c-MycER) protein that is activated by 4-hydroxy-tamoxifen. In contrast to its role in mES cells, activation of c-MycER in hES cells induced apoptosis and differentiation into extraembryonic endoderm and trophectoderm lineages concomitant with reduced expression of the pluripotent markers Oct4 and Nanog. Neither inhibition of caspase activity nor knockdown of p53 by RNA interference impaired the induction of differentiation markers induced by c-Myc activation. In addition, differentiation induced by c-Myc activation was associated with downregulation of a6 integrin expression, suggesting an important role for the integrin/extracellular matrix interaction in the regulation of ES cell behavior. None of these effects occurred with deletion of the c-Myc transactivation domain, indicating that c-Myc promotes both apoptosis and differentiation in a transcriptional activity-dependent manner. Together, our results provide new insights into the c-Myc functions regulating hES cell fate.