Mesenchymal stem cells (MSCs), the archetypal multipotent progenitor cells derived in cultures of developed organs, are of unknown identity and native distribution. We have prospectively identified perivascular cells, principally pericytes, in multiple human organs including skeletal muscle, pancreas, adipose tissue, and placenta, on CD146, NG2, and PDGF-Rb expression and absence of hematopoietic, endothelial, and myogenic cell markers. Perivascular cells purified from skeletal muscle or nonmuscle tissues were myogenic in culture and in vivo. Irrespective of their tissue origin, long-term cultured perivascular cells retained myogenicity; exhibited at the clonal level osteogenic, chondrogenic, and adipogenic potentials; expressed MSC markers; and migrated in a culture model of chemotaxis. Expression of MSC markers was also detected at the surface of native, noncultured perivascular cells. Thus, blood vessel walls harbor a reserve of progenitor cells that may be integral to the origin of the elusive MSCs and other related adult stem cells.

The terms MSC and MSCs have become the preferred acronym to describe a cell and a cell population of multipotential stem/progenitor cells commonly referred to as mesenchymal stem cells, multipotential stromal cells, mesenchymal stromal cells, and mesenchymal progenitor cells. The MSCs can differentiate to important lineages under defined conditions in vitro and in limited situations after implantation in vivo. MSCs were isolated and described about 30 years ago and now there are over 55,000 publications on MSCs readily available. Here, we have focused on human MSCs whenever possible. The MSCs have broad anti-inflammatory and immune-modulatory properties. At present, these provide the greatest focus of human MSCs in clinical testing; however, the properties of cultured MSCs in vitro suggest they can have broader applications. The medical utility of MSCs continues to be investigated in over 950 clinical trials. There has been much progress in understanding MSCs over the years, and there is a strong foundation for future scientific research and clinical applications, but also some important questions remain to be answered. Developing further methods to understand and unlock MSC potential through intracellular and intercellular signaling, biomedical engineering, delivery methods and patient selection should all provide substantial advancements in the coming years and greater clinical opportunities. The expansive and growing field of MSC research is teaching us basic human cell biology as well as how to use this type of cell for cellular therapy in a variety of clinical settings, and while much promise is evident, careful new work is still needed.

We have identified a rare (0.05-0.1%) subset of human fetal bone marrow cells that contains muiltipotent hematopoietic precursors. The population of human precursor cells that express In bone marrow (BM), the main blood-forming organ in the developed mammal, cascades of stem-cell divisions give rise to most hematolymphoid cell populations (1-3). Of these, only totipotent hematopoietic stem cells (tHSCs) can reconstitute lethally irradiated animals by giving rise to all blood cells, including progeny HSCs.Isolation of candidate HSCs in the mouse required the development of assays for clonogenic precursors of the T [thyrnic colony-forming unit (CFU-T)], B, and myeloerythroid [splenic CFU (CFU-S)] lineages (4-6); such precursors lack detectable surface markers of the T, B, macrophage, granulocytic, and erythroid lineages [lineage-negative (Lin-)] (7) but express the Sca-1/Ly-6A antigen and low levels of the Thy-1 molecule (7-9). This Thy-11oLin-Sca-1+ population, representing -0.05% of mononucleated BM cells, is the only subset that initiates long-term BM stromal cultures; it is 1000-to 2000-fold enriched in the capacity to save lethally irradiated animals and reconstitute them long term with donorderived cells in all hematolymphoid lineages (7, 9). Independent attempts to isolate tHSCs have utilized other cellular properties (10-13) and demonstrated their activity in vitro or in vivo (14-16). In some studies, long-term reconstitution activity was separable from radioprotective and CFU-S activity (17). For example, the Thy-11OLin-Sca-l+Rh-123Io subset (comprising cells that take up little of the mitochondrial dye rhodamine 123) is enriched for self-renewal and longterm reconstitutive potential compared with its Rh-123 counterpart (18).Most attempts to identify human HSCs have utilized the high proliferative response of such cells in vitro in the presence ofhematopoietic cytokines and have revealed CD34 to be a potent cell surface marker of such progenitors (19,20); in mice, HSCs are not the only such cytokine-responsive cells (21). Several groups have developed human stromal cell-dependent long-term culture systems that have identified progenitors of the myeloerythroid type (22-24). The stromal culture system described here allows single human progenitor cells to differentiate into both the myeloerythroid and B-lymphoid lineages.Experimental in vivo hematopoietic assays are not practi- MATERIALS AND METHODSMonoclonal Antibodies. mAbs were purchased from Becton Dickinson (CD3, or from AMAC (CD35,. mAbs against HLA class I antigens were derived from hybridomas obtained from the American Type Culture Collection. mAbs to human CD34 (Tuk3) and to human Thy-1 (F15 421-5) were obtained from

Satellite cells are dormant progenitors located at the periphery of skeletal myofibers that can be triggered to proliferate for both self-renewal and differentiation into myogenic cells. In addition to anatomic location, satellite cells are typified by markers such as M-cadherin, Pax7, Myf5, and neural cell adhesion molecule-1. The Pax3 and Pax7 transcription factors play essential roles in the early specification, migration, and myogenic differentiation of satellite cells. In addition to muscle-committed satellite cells, multi-lineage stem cells encountered in embryonic, as well as adult, tissues exhibit myogenic potential in experimental conditions. These multi-lineage stem cells include side-population cells, muscle-derived stem cells (MDSCs), and mesoangioblasts. Although the ontogenic derivation, identity, and localization of these non-conventional myogenic cells remain elusive, recent results suggest their ultimate origin in blood vessel walls. Indeed, purified pericytes and endothelium-related cells demonstrate high myogenic potential in culture and in vivo. Allogeneic myoblasts transplanted into Duchenne muscular dystrophy (DMD) patients have been, in early trials, largely inefficient owing to immune rejection, rapid death, and limited intramuscular migration--all obstacles that are now being alleviated, at least in part, by more efficient immunosuppression and escalated cell doses. As an alternative to myoblast transplantation, stem cells such as mesoangioblasts and CD133+ progenitors administered through blood circulation have recently shown great potential to regenerate dystrophic muscle.

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Background The in vivo progenitor of culture-expanded mesenchymal-like adipose-derived stem cells (ADSC) remains elusive, owing in part to the complex organization of stromal cells surrounding the small vessels, and the rapidity with which adipose stromal vascular cells adopt a mesenchymal phenotype in vitro. Methods Immunohistostaining of intact adipose tissue was used to identify 3 markers (CD31, CD34, CD146) which together unambiguously discriminate histologically distinct inner and outer rings of vessel-associated stromal cells, as well as capillary and small vessel endothelial cells. These markers were used in multiparameter flow cytometry in conjunction with stem/progenitor markers (CD90, CD117) to further characterize stromal vascular fraction (SVF) subpopulations. Two mesenchymal and two endothelial populations were isolated by high speed flow cytometric sorting, expanded in short term culture and tested for adipogenesis. Results The inner layer of stromal cells in contact with small vessel endothelium (pericytes) was CD146+/α-SMA+/CD90±/CD34−/CD31−; the outer adventitial stromal ring (designated supra adventitial-adipose stromal cells, SA-ASC) was CD146−/α-SMA−/CD90+/CD34+/CD31−. Capillary endothelial cells were CD31+/CD34+/CD90+ (endothelial progenitor), while small vessel endothelium was CD31+/CD34−/CD90− (endothelial mature). Flow cytometry confirmed these expression patterns and revealed a CD146+/CD90+/CD34+/CD31− pericyte subset that may be transitional between pericytes and SA-ASC. Pericytes had the most potent adipogenic potential, followed by the more numerous SA-ASC. Endothelial populations had significantly reduced adipogenic potential compared to unsorted expanded SVF cells. Conclusions In adipose tissue perivascular stromal cells are organized in two discrete layers, the innermost consisting of CD146+/CD34− pericytes, and the outermost of CD146−/CD34+ SA-ASC, both of which have adipogenic potential in culture. A CD146+/CD34+ subset detected by flow cytometry at low frequency suggests a population transitional between pericytes and SA-ASC.

We elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermalhematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac-like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13 ؉ CD45 ؉ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/ fetal hemoglobin expression. A second wave of definitive-type erythroid burstforming units (BFU-e's), erythroid colonyforming units (CFU-e's), granulocytemacrophage colony-forming cells (GMCFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis. IntroductionClassic and contemporary anatomic studies of human embryos have revealed that human hematopoiesis begins in the second to third embryonic weeks with formation of mesoderm-derived blood islands in the extraembryonic mesoderm of the developing secondary yolk sac. 1,2 Blood islands develop foci of nucleated erythroblasts ("megaloblasts"), 3 intimately associated with and surrounded by endothelium. Yolk sac (primitive) blood cells consist of nucleated primitive erythrocytes expressing exclusively embryonic hemoglobins (eg, ⑀ 2 2 globin chains) and primitive macrophages that arise without detectable monocytic precursors. Following the onset of circulation at about 21 days of development, yolk sac cells are found in embryonic blood. The fetal liver subsequently replaces the yolk sac as the main hematopoietic organ 4,5 with appearance of definitive enucleate, macrocytic erythrocytes expressing fetal hemoglobins (eg, ␣ 2 ␥ 2 globin chains). Definitive blood cells and hematopoietic stem progenitor cells (HSPCs) can be detected in the fetal liver and embryo beginning at 5 to 6 weeks but have also been assayed as early as 4 to 5 weeks from human yolk sac, suggesting a gradual yolk sac/fetal liver HSPC transition. 1,6 The human adult long-term repopulating HSPC that ultimately seeds the fetal bone marrow and thymus is the legacy of fetal liver hematopoiesis.Both ...

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