Summary The transcription factor Sox2 maintains the pluripotency of early embryonic cells and regulates the formation of several epithelia during fetal development. Whether Sox2 continues to play a role in adult tissues remains largely unknown. We here show that Sox2 marks adult cells in several epithelial tissues where its expression has not previously been characterized, including the stomach, cervix, anus, testes, lens and multiple glands. Genetic lineage tracing and transplantation experiments demonstrate that Sox2-expressing cells continuously give rise to mature cell types within these tissues, documenting their self-renewal and differentiation potentials. Consistent with these findings, ablation of Sox2+ cells in mice results in a disruption of epithelial tissue homeostasis and lethality. Developmental fate mapping reveals that Sox2+ adult stem cells originate from fetal Sox2+ tissue progenitors. Thus, our results identify Sox2 expression in numerous adult ectodermal and endodermal stem cell compartments, which are critical for normal tissue regeneration and survival.

Bone marrow endothelial cells (ECs) are essential for reconstitution of hematopoiesis, but their role in self-renewal of long term-hematopoietic stem cells (LT-HSCs) is unknown. We have developed angiogenic models to demonstrate that EC-derived angiocrine growth factors support in vitro self-renewal and in vivo repopulation of authentic LT-HSCs. In serum/cytokine-free co-cultures, ECs through direct cellular contact, stimulated incremental expansion of repopulating CD34−Flt3−cKit+Lineage−Sca1+ LT-HSCs, which retained their self-renewal ability, as determined by single cell and serial transplantation assays. Angiocrine expression of Notch-ligands by ECs promoted proliferation and prevented exhaustion of LT-HSCs derived from wild-type, but not Notch1/Notch2 deficient mice. In transgenic notch-reporter (TNR.Gfp) mice, regenerating TNR.Gfp+ LT-HSCs were detected in cellular contact with sinusoidal ECs and interfering with angiocrine, but not perfusion function, of SECs impaired repopulation of TNR.Gfp+ LT-HSCs. ECs establish an instructive vascular niche for clinical scale expansion of LT-HSCs and a cellular platform to identify stem cell-active trophogens.

Adult mammalian testis is a source of pluripotent stem cells. However, the lack of specific surface markers has hampered identification and tracking of the unrecognized subset of germ cells that gives rise to multipotent cells. Although embryonic-like cells can be derived from adult testis cultures after only several weeks in vitro, it is not known whether adult self-renewing spermatogonia in long-term culture can generate such stem cells as well. Here, we show that highly proliferative adult spermatogonial progenitor cells (SPCs) can be efficiently obtained by cultivation on mitotically inactivated testicular feeders containing CD34+ stromal cells. SPCs exhibit testicular repopulating activity in vivo and maintain the ability in long-term culture to give rise to multipotent adult spermatogonial-derived stem cells (MASCs). Furthermore, both SPCs and MASCs express GPR125, an orphan adhesion-type G-protein-coupled receptor. In knock-in mice bearing a GPR125-beta-galactosidase (LacZ) fusion protein under control of the native Gpr125 promoter (GPR125-LacZ), expression in the testis was detected exclusively in spermatogonia and not in differentiated germ cells. Primary GPR125-LacZ SPC lines retained GPR125 expression, underwent clonal expansion, maintained the phenotype of germline stem cells, and reconstituted spermatogenesis in busulphan-treated mice. Long-term cultures of GPR125+ SPCs (GSPCs) also converted into GPR125+ MASC colonies. GPR125+ MASCs generated derivatives of the three germ layers and contributed to chimaeric embryos, with concomitant downregulation of GPR125 during differentiation into GPR125- cells. MASCs also differentiated into contractile cardiac tissue in vitro and formed functional blood vessels in vivo. Molecular bookmarking by GPR125 in the adult mouse and, ultimately, in the human testis could enrich for a population of SPCs for derivation of GPR125+ MASCs, which may be employed for genetic manipulation, tissue regeneration and revascularization of ischaemic organs.

SUMMARY Hyperactivity of mTORC1, a key mediator of cell growth, leads to stem cell depletion although the underlying mechanisms are poorly defined. Using spermatogonial progenitor cells (SPCs) as a model system, we show that mTORC1 impairs stem cell maintenance by a negative feedback from mTORC1 to receptors required to transduce niche-derived signals. We find that SPCs lacking Plzf, a transcription factor essential for SPC maintenance, have enhanced mTORC1 activity. Aberrant mTORC1 activation in Plzf −/− SPCs inhibits their response to GDNF, a growth factor critical for SPC self-renewal, via negative feedback at the level of the GDNF receptor. Plzf opposes mTORC1 activity by inducing expression of the mTORC1 inhibitor Redd1. Thus, we identify the mTORC1-Plzf functional interaction as a critical rheostat for maintenance of the spermatogonial pool, and propose a model whereby negative feedback from mTORC1 to the GDNF receptor balances SPC growth with self-renewal.

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...

angiogenesis ͉ endothelium ͉ adenovirus ͉ tumor ͉ stem

The contribution of specific type I collagen remodeling in angiogenesis was studied in vivo using a quantitative chick embryo assay that measures new blood vessel growth into well-defined fibrillar collagen implants. In response to a combination of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), a strong angiogenic response was observed, coincident with invasion into the collagen implants of activated fibroblasts, monocytes, heterophils, and endothelial cells. The angiogenic effect was highly dependent on matrix metalloproteinase (MMP) activity, because new vessel growth was inhibited by both a synthetic MMP inhibitor, BB3103, and a natural MMP inhibitor, TIMP-1. Multiple MMPs were detected in the angiogenic tissue including MMP-2, MMP-13, MMP-16, and a recently cloned MMP-9-like gelatinase. Using this assay system, wild-type collagen was compared to a unique collagenase-resistant collagen (r/r), with regard to the ability of the respective collagen implants to support cell invasion and angiogenesis. It was found that collagenase-resistant collagen constitutes a defective substratum for angiogenesis. In implants made with r/r collagen there was a substantial reduction in the number of endothelial cells and newly formed vessels. The presence of the r/r collagen, however, did not reduce the entry into the implants of other cell types, that is, activated fibroblasts and leukocytes. These results indicate that fibrillar collagen cleavage at collagenase-specific sites is a ratelimiting event in growth factor-stimulated angiogenesis in vivo. IntroductionAngiogenesis is the process by which the preexisting vascular tree gives rise to new blood vessels. This process is tightly regulated during development and occurs only under highly specialized circumstances in the normal adult animal. Unregulated angiogenesis may be a pivotal element of disease etiology, such as during tumor growth and atherosclerosis. 1,2 All forms of angiogenesis, however, are thought to share certain basic features, including migration and mitogenesis of endothelial cells, lumen formation, connection of new vascular segments with the preexisting circulation, and extensive remodeling of the extracellular matrix by proteases. The detailed mechanisms by which proteolytic enzymes, such as the matrix metalloproteinases (MMPs), mediate some of these events in vivo remain unclear. 3,4 The MMPs constitute a large family of zinc-dependent endopeptidases that have been strongly implicated in both normal angiogenesis and tumor vascularization. 2,4 These enzymes are characteristically regulated at multiple levels, including gene expression, spatial localization, zymogen activation, and inhibition by the tissue inhibitors of metalloproteinases (TIMPs). 5 In vitro remodeling of extracellular matrices by cultured endothelial cells has consistently been shown to rely on MMPs. 4 However, it is unclear which aspects of cell culture models for angiogenesis may be directly extrapolated to the animal. In vivo studies also have foun...

Small numbers of proangiogenic bone marrow-derived cells (BMDCs) can play pivotal roles in tumor progression. In this issue of Cancer Cell, two papers, utilizing different tumor angiogenesis models, both find that activated MMP-9 delivered by BMDCs modulates neovessel remodeling, thereby promoting tumor growth. The changes in microvascular anatomy induced by MMP-9-expressing BMDCs are strikingly different between the preirradiated tumor vascular bed model employed by Ahn and Brown and the invasive glioblastoma model utilized by Du et al., likely mirroring the complexity of the real tumor microenvironment and the intricacy of roles of different BMDC populations in mediating tumor neoangiogenesis.