The histone demethylase Jmjd3 sequentially associates with the transcription factors Tbx3 and Eomes to drive endoderm differentiation.

Stem cell differentiation depends on transcriptional activation driven by lineage-specific regulators as well as changes in chromatin organization. However, the coordination of these events is poorly understood. Here, we show that T-box proteins team up with chromatin modifying enzymes to drive the expression of the key lineage regulator, Eomes during endodermal differentiation of embryonic stem (ES) cells. The Eomes locus is maintained in a transcriptionally poised configuration in ES cells. During early differentiation steps, the ES cell factor Tbx3 associates with the histone demethylase Jmjd3 at the enhancer element of the Eomes locus to allow enhancer-promoter interactions. This spatial reorganization of the chromatin primes the cells to respond to Activin signalling, which promotes the binding of Jmjd3 and Eomes to its own bivalent promoter region to further stimulate Eomes expression in a positive feedback loop. In addition, Eomes activates a transcriptional network of core regulators of endodermal differentiation. Our results demonstrate that Jmjd3 sequentially associates with two T-box factors, Tbx3 and Eomes to drive stem cell differentiation towards the definitive endoderm lineage.

PTK7 marks the first human developmental EMT in vitro.

Epithelial to mesenchymal transitions (EMTs) are thought to be essential to generate diversity of tissues during early fetal development, but these events are essentially impossible to study at the molecular level in vivo in humans. The first EMT event that has been described morphologically in human development occurs just prior to generation of the primitive streak. Because human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are thought to most closely resemble cells found in epiblast-stage embryos prior to formation of the primitive streak, we sought to determine whether this first human EMT could be modeled in vitro with pluripotent stem cells. The data presented here suggest that generating embryoid bodies from hESCs or hiPSCs drives a procession of EMT events that can be observed within 24-48 hours after EB generation. These structures possess the typical hallmarks of developmental EMTs, and portions also display evidence of primitive streak and mesendoderm. We identify PTK7 as a novel marker of this EMT population, which can also be used to purify these cells for subsequent analyses and identification of novel markers of human development. Gene expression analysis indicated an upregulation of EMT markers and ECM proteins in the PTK7+ population. We also find that cells that undergo this developmental EMT retain developmental plasticity as sorting, dissociation and re-plating reestablishes an epithelial phenotype.

Defining the nature of human pluripotent stem cell progeny.

While it is clear that human pluripotent stem cells (hPSCs) can differentiate to generate a panoply of various cell types, it is unknown how closely in vitro development mirrors that which occurs in vivo. To determine whether human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) make equivalent progeny, and whether either makes cells that are analogous to tissue-derived cells, we performed comprehensive transcriptome profiling of purified PSC derivatives and their tissue-derived counterparts. Expression profiling demonstrated that hESCs and hiPSCs make nearly identical progeny for the neural, hepatic, and mesenchymal lineages, and an absence of re-expression from exogenous reprogramming factors in hiPSC progeny. However, when compared to a tissue-derived counterpart, the progeny of both hESCs and hiPSCs maintained expression of a subset of genes normally associated with early mammalian development, regardless of the type of cell generated. While pluripotent genes (OCT4, SOX2, REX1, and NANOG) appeared to be silenced immediately upon differentiation from hPSCs, genes normally unique to early embryos (LIN28A, LIN28B, DPPA4, and others) were not fully silenced in hPSC derivatives. These data and evidence from expression patterns in early human fetal tissue (3-16 weeks of development) suggest that the differentiated progeny of hPSCs are reflective of very early human development (< 6 weeks). These findings provide support for the idea that hPSCs can serve as useful in vitro models of early human development, but also raise important issues for disease modeling and the clinical application of hPSC derivatives.

The emergence of hematopoietic stem cells is initiated in the placental vasculature in the absence of circulation.

The mouse placenta was unveiled as an important reservoir for hematopoietic stem cells (HSCs), yet the origin of placental HSCs was unknown. By tracking developing HSCs by expression of Runx1-lacZ and CD41, we have found that HSCs emerge in large vessels in the placenta. Analysis of Ncx1(-/-) embryos, which lack a heartbeat, verified that HSC development is initiated in the placental vasculature independent of blood flow. However, fewer CD41+ hematopoietic cells were found in Ncx1(-/-) placentas than in controls, implying that some HSCs/progenitors colonize the placenta via circulation and/or HSC emergence is compromised without blood flow. Importantly, placentas from Ncx1(-/-) embryos possessed equal potential to generate myelo-erythroid and B and T lymphoid cells upon explant culture, verifying intact multilineage hematopoietic potential, characteristic of developing HSCs. These data suggest that, in addition to providing a niche for a large pool of HSCs prior to liver colonization, the placenta is a true site of HSC generation.