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ABSTRACT: This year's International Society for Stem Cell Research (ISSCR) annual meeting was an opportunity for attendees to be inspired by early successes, to grapple with the challenges of applying emerging technologies, and to contemplate moving with increasing but cautious optimism toward the clinic.
Cell stem cell 10/2011; 9(4):311-5. · 23.56 Impact Factor
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ABSTRACT: 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 show here 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 endodermal and ectodermal stem cell compartments, which are critical for normal tissue regeneration and survival.
Cell stem cell 10/2011; 9(4):317-29. · 23.56 Impact Factor
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Nature 03/2011; 471(7339):453-5. · 36.28 Impact Factor
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ABSTRACT: While embryonic stem (ES) cells are well known to give rise to tissues comprising all three germ layers, only recently was
it shown that cells from the postnatal testis could produce embryonic-like stem cells in culture. The latter, arising in vitro from spermatogonia, can undertake most, if not all, the functions of ES cells. This chapter explores the potential predisposing
factors for postnatal germ cells to become pluripotent, including expression of pluripotency-associated genes and epigenetic
factors. The major published studies describing the production of ES-like cells from mice and human tissues are reviewed.
Finally, we assess the data demonstrating functionality of the differentiated derivatives of ES-like cells. The possible uses
of testis-derived stem cells for the study of pluripotency and for regenerative applications is also discussed in comparison
to other approaches using ES cells and induced pluripotent stem (ips) cells.
KeywordsSpermatogonia-Adult stem cells-Pluripotent stem cells-Testis-Cell transplantation
12/2010: pages 25-47;
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ABSTRACT: 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.
Cell 08/2010; 142(3):468-79. · 32.40 Impact Factor
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Jason M Butler,
Daniel J Nolan,
Eva L Vertes,
Barbara Varnum-Finney,
Hideki Kobayashi,
Andrea T Hooper, Marco Seandel,
Koji Shido,
Ian A White,
Mariko Kobayashi,
Larry Witte,
Chad May,
Carrie Shawber,
Yuki Kimura,
Jan Kitajewski,
Zev Rosenwaks,
Irwin D Bernstein,
Shahin Rafii
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ABSTRACT: 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 cocultures, 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. Interference 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.
Cell stem cell 03/2010; 6(3):251-64. · 23.56 Impact Factor
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Daylon James,
Hyung-song Nam, Marco Seandel,
Daniel Nolan,
Tyler Janovitz,
Mark Tomishima,
Lorenz Studer,
Gabsang Lee,
David Lyden,
Robert Benezra,
Nikica Zaninovic,
Zev Rosenwaks,
Sina Y Rabbany,
Shahin Rafii
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ABSTRACT: 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)beta at day 7 of differentiation increases hVPr-GFP(+) cells by tenfold. In phase 2, TGFbeta 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 TGFbeta 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 a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.
Nature Biotechnology 02/2010; 28(2):161-6. · 29.50 Impact Factor
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ABSTRACT: Efficient distribution of oxygen and nutrients are fundamental and universal requirements of tissue homeostasis. In adults,
angiogenesis from existing vessels occurs during routine tissue repair and is a prerequisite for the advance of tumor growth.
Indeed, proliferation of any solid tissue beyond microscopic scales, in vivo or in vitro, is inherently dependent on vascularization.
Endothelial ontogeny is hugely relevant to clinical science, as millions of patients each year succumb to vascular diseases
resulting in mortality and morbidity due to tissue ischemia. Medical interventions are only partially effective in revascularizing
ischemic tissues; moreover, recent clinical trials to induce revascularization using adult marrow-derived stem cells have
yielded only minor benefit. Human embryonic stem cells (ESCs) have the potential to provide an unlimited source of vascular
tissue for cell-based therapies, while also providing insight into the molecular events underlying human vascular development.
In order for human ESCs to fulfill this potential, however, it will first be necessary to define the developmental events
that influence endothelial differentiation and blood vessel formation. The molecular pathways involved in developmental vasculogenesis
in mouse have been well studied, yet many elements of this process are obscure, and the correlation of these studies to human
biology remains to be seen. Below, we review the current understanding of mammalian vascular development gained from studies
of mouse genetic models and ESCs, focusing on the cytokine-mediated signaling pathways that govern this process.
KeywordsEndothelial stem cells–Growth factor signaling–Angiogenesis–Vascular diseases–VEGF–TGF–BMP–Hedgehog–Notch–PDGF
12/2008: pages 319-328;
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Marco Seandel,
Jason M Butler,
Hideki Kobayashi,
Andrea T Hooper,
Ian A White,
Fan Zhang,
Eva L Vertes,
Mariko Kobayashi,
Yan Zhang,
Sergey V Shmelkov,
Neil R Hackett,
Sina Rabbany,
Julie L Boyer,
Shahin Rafii
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ABSTRACT: Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1(+) ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1(+) ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1(+) ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1(+) ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1(+) ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-kappaB pathways. Therefore, E4ORF1(+) ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis.
Proceedings of the National Academy of Sciences 12/2008; 105(49):19288-93. · 9.68 Impact Factor
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ABSTRACT: Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multipotent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. Disclosure of potential conflicts of interest is found at the end of this article.
Stem Cells 08/2008; 26(10):2516-22. · 7.78 Impact Factor
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ABSTRACT: 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.
Cancer cell 04/2008; 13(3):181-3. · 25.29 Impact Factor
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ABSTRACT: The undifferentiated spermatogonia of adult mouse testes are composed of both true stem cells and committed progenitors. It is unclear what normally prevents these adult germ cells from manifesting multipotency. The critical elements of the spermatogonial stem cell niche, while poorly understood, are thought to be composed of Sertoli cells with several other somatic cell types in close proximity. We recently discovered a novel orphan G-protein coupled receptor (GPR125) that is restricted to undifferentiated spermatogonia within the testis. GPR125 expression was maintained when the progenitor cells were extracted from the in vivo niche and propagated under growth conditions that recapitulate key elements of the niche. Such conditions preserved the ability of the cells to generate multipotent derivatives, known as multipotent adult spermatogonial derived progenitor cells (MASCs). Upon differentiation, the latter produced a variety tissues including functional endothelium, illustrating the potential applications of such cells. Thus, GPR125 represents a novel target for purifying adult stem and progenitors from tissues, with the goal of developing autologous multipotent cell lines.
Cell cycle (Georgetown, Tex.) 02/2008; 7(2):135-40. · 5.36 Impact Factor
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ABSTRACT: Among the molecular and cellular processes that orchestrate construction of new blood vessels, the distinct contribution of
circulating cells has recently become appreciated. The endothelial progenitor cell (EPC) was one of the first identified circulating
cell types found to directly contribute to neo-vessels walls. Subsequently, a complex network of signals between EPCs and
other circulating bone marrow-derived cell types has been described. Significant temporal and spatial heterogeneity in utilization
of circulating progenitor cells exists between tumor types, complicating analysis in both animal models and in patients. A
lack of standardized cell surface markers and techniques for quantitation of such rare cells has further complicated such
studies. Nonetheless, levels of EPCs and other bone marrow- derived progenitors may hold prognostic significance for cancer
patients or may be used in the future to guide therapy and EPCs may themselves represent a valid therapeutic target.
12/2007: pages 239-248;
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Marco Seandel,
Daylon James,
Sergey V Shmelkov,
Ilaria Falciatori,
Jiyeon Kim,
Sai Chavala,
Douglas S Scherr,
Fan Zhang,
Richard Torres,
Nicholas W Gale,
George D Yancopoulos,
Andrew Murphy,
David M Valenzuela,
Robin M Hobbs,
Pier Paolo Pandolfi,
Shahin Rafii
[show abstract]
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ABSTRACT: 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.
Nature 10/2007; 449(7160):346-50. · 36.28 Impact Factor
