Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell-specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP + cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1 high VEGFR2 high VE-cadherin + ephrinB2 + . Using an Id1-YFP hESC reporter line, we showed that TGFβ inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides © 2010 Nature America, Inc. All rights reserved.Correspondence should be addressed to S.R. (srafii@med.cornell.edu).. 7 Present address: Weill Cornell Medical College, New York, New York, USA. 8 These authors contributed equally to this work. AUTHOR CONTRIBUTIONS D.J. designed and performed the experiments and wrote the manuscript. H.-s.N. and R.B. designed and created the Id1-YFP BAC transgenic vector. M.S. performed experiments and contributed to the manuscript. D.N. performed flow cytometric experiments. T.J. performed molecular cloning. M.T. and L.S. generated the Id1-YFP BAC transgenic hESC line. L.S. and G.L. generated the FD iPSC line. N.Z. and Z.R. generated the hESC lines WMC2, WMC8 and WMC9. D.L. and S.Y.R. designed experiments and performed data analysis. S.R. designed experiments and wrote the manuscript. Accession codes. GEO: GSE19735.Note: Supplementary information is available on the Nature Biotechnology website. COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests. Human embryonic stem cells (hESCs), which self-renew indefinitely 1 , offer a plentiful source of endothelial cells for therapeutic revascularization. However, few studies have identified specific developmental stimuli sufficient to support the specification and maintenance of large numbers of functional and vascular-committed endothelial cells from hESCs [2][3][4][5][6][7] . Although small numbers of hESC-derived endothelial cells have been generated in short-term cultures, these cells have not been subjected to sustained expansion, angiogenic profiling or interrogated as to the stability of vascular fate. As a result, molecular pathways that maintain vascular identity and long-term expansion of hESC-derived endothelial cells remain unknown. NIH Public AccessTo detect the emergence of endothelial cells from differentiating hESCs in real time, we generated a cell line for endothelial cell-specific lineage tracing. We cloned a 1.5-kilobase f...

We report that the erythropoietin receptor cytosolic juxtamembrane region is conformationally rigid and contains a hydrophobic motif, composed of residues L253, I257, and W258, that is crucial for Janus kinase 2 (JAK2) activation and receptor signaling. Alanine insertion mutagenesis shows that the orientation of this motif and not its distance from the membrane bilayer is critical. Intragenic complementation studies suggest that L253 is contained within an alpha helix functionally continuous to the transmembrane alpha helix. The alpha-helical orientation of L53 is required not for JAK2 activation but for activated JAK2 to induce phosphorylation of the erythropoietin receptor. This motif is highly conserved among cytokine receptors and couples ligand-induced conformational changes in the receptor to intracellular activation of JAK2.

Within high-grade gliomas, the precise identities and functional roles of stem-like cells remain unclear. In the normal neurogenic niche, ID (Inhibitor of DNA-binding) genes maintain self-renewal and multipotency of adult neural stem cells. Using PDGF- and KRAS-driven murine models of gliomagenesis, we show that high Id1 expression (Id1(high)) identifies tumor cells with high self-renewal capacity, while low Id1 expression (Id1(low)) identifies tumor cells with proliferative potential but limited self-renewal capacity. Surprisingly, Id1(low) cells generate tumors more rapidly and with higher penetrance than Id1(high) cells. Further, eliminating tumor cell self-renewal through deletion of Id1 has modest effects on animal survival, while knockdown of Olig2 within Id1(low) cells has a significant survival benefit, underscoring the importance of non-self-renewing lineages in disease progression.

SummaryLGR5 and BMI1 mark intestinal stem cells in crypt base columnar cells and +4 position cells, respectively, but characterization of functional markers in these cell populations is limited. ID1 maintains the stem cell potential of embryonic, neural, and long-term repopulating hematopoietic stem cells. Here, we show in both human and mouse intestine that ID1 is expressed in cycling columnar cells, +4 position cells, and transit-amplifying cells in the crypt. Lineage tracing revealed ID1+ cells to be self-renewing, multipotent stem/progenitor cells that are responsible for the long-term renewal of the intestinal epithelium. Single ID1+ cells can generate long-lived organoids resembling mature intestinal epithelium. Complete knockout of Id1 or selective deletion of Id1 in intestinal epithelium or in LGR5+ stem cells sensitizes mice to chemical-induced colon injury. These experiments identify ID1 as a marker for intestinal stem/progenitor cells and demonstrate a role for ID1 in maintaining the potential for repair in response to colonic injury.