Background-Pericytes represent a unique subtype of microvessel-residing perivascular cells with diverse angiogenic functions and multilineage developmental features of mesenchymal stem cells. Although various protocols for derivation of endothelial and/or smooth muscle cells from human pluripotent stem cells (hPSC, either embryonic or induced) have been described, the emergence of pericytes in the course of hPSC maturation has not yet been elucidated. Methods and Results-We found that during hPSC development, spontaneously differentiating embryoid bodies give rise to CD105 ϩ CD90 ϩ CD73 ϩ CD31 Ϫ multipotent clonogenic mesodermal precursors, which can be isolated and efficiently expanded. Isolated and propagated cells expressed characteristic pericytic markers, including CD146, NG2, and platelet-derived growth factor receptor , but not the smooth muscle cell marker ␣-smooth muscle actin. Coimplantation of hPSC-derived endothelial cells with pericytes resulted in functional and rapid anastomosis to the murine vasculature. Administration of pericytes into immunodeficient mice with limb ischemia promoted significant vascular and muscle regeneration. At day 21 after transplantation, recruited hPSC pericytes were found incorporated into recovered muscle and vasculature. Conclusions-Derivation of vasculogenic and multipotent pericytes from hPSC can be used for the development of vasculogenic models using multiple vasculogenic cell types for basic research and drug screening and can contribute to angiogenic regenerative medicine. (Circulation. 2012;125:87-99.)Key Words: pluripotent stem cells Ⅲ ischemia Ⅲ mesenchymal stem cells Ⅲ pericytes Ⅲ vasculature I n adult tissues, the majority of blood vessels are composed of 3 layers including a luminal inner monolayer of endothelial cells (EC), tunica intimae, an intermediate muscular layer, tunica media, of smooth muscle cells (SMC) and an outer layer of fibroblast-like adventitial cells and connective tissue components, tunica adventitia. Microvessels, including capillaries, precapillary arterioles, postcapillary venues, and collecting venules are composed of internal endothelial layer surrounded by outer coverage of pericytes (also known as Rouget cells or mural cells). 1 Both perivascular SMC and pericytes have been shown to function as critical regulators of vascular development, stabilization, maturation, and remodeling mediated by transforming growth factor  (TGF-), platelet-derived growth factor B, or angiopoietin-1. 2,3 Although related in function and anatomic localization, pericytes can be distinguished from SMC on the basis of their characteristic morphology and specific cell marker expression: Whereas SMC form a separate layer of the tunica media in blood vessels, pericytes are physically embedded within the endothelial basement membrane to promote mutual communication with the underlying endothelium. 4 In addition, SMC and the majority of pericytes in multiple human and murine tissue types express ␣-smooth muscle actin (␣-SMA), which is involved in regulatio...
Induced pluripotent stem cells (iPSCs) represent an ideal cell source for future cell therapy and regenerative medicine. However, most iPSC lines described to date have been isolated from skin fibroblasts or other cell types that require harvesting by surgical intervention. Because it is desirable to avoid such intervention, an alternative cell source that can be readily and noninvasively isolated from patients and efficiently reprogrammed, is required. Here we describe a detailed and reproducible method to derive iPSCs from plucked human hair follicle keratinocytes (HFKTs). HFKTs were isolated from single plucked hair, then expanded and reprogrammed by a single polycistronic excisable lentiviral vector. The reprogrammed HFKTs were found to be very sensitive to human embryonic stem cell (hESC) growth conditions, generating a built-in selection with easily obtainable and very stable iPSCs. All emerging colonies were true iPSCs, with characteristics typical of human embryonic stem cells, differentiated into derivatives of all three germ layers in vitro and in vivo. Spontenaeouly differentiating functional cardiomyocytes (CMs) were successfully derived and characterized from these HFKT-iPSCs. The contracting CMs exhibited well-coordinated intracellular Ca²+ transients and contractions that were readily responsive to β-adrenergic stimulation with isoproterenol. The introduction of Cre-recombinase to HFKT-iPSC clones was able to successfully excise the integrated vector and generate transgene-free HFKT-iPSC clone that could be better differentiated into contracting CMs, thereby revealing the desired cells for modeling human diseases. Thus, HFKTs are easily obtainable, and highly reprogrammed human cell source for all iPSC applications.
Isolated microvessel-residing pericytes and pericytes from human pluripotent stem cells (hPSCs) exhibit mesenchymal stem cell-like characteristics and therapeutic properties. Despite growing interest in pericyte-based stem cell therapy, their immunogenicity and immunomodulatory effects on nonactivated T cells are still poorly defined, in particular those of vasculogenic hPSC pericytes. We found that tissue-embedded and unstimulated cultured hPSC-or tissue-derived pericytes constitutively expressed major histocompatibility complex (MHC) class I and the inhibitory programmed cell death-ligand 1/2 (PD-L1/2) molecules but not MHC class II or CD80/CD86 costimulatory molecules. Pretreatment with inflammatory mediators failed to induce an antigen-presenting cell-like phenotype in stimulated pericytes. CD146+ pericytes from hPSCs did not induce activation and proliferation of allogeneic resting T cells independent of interferon (IFN)-g prestimulation, similarly to pericytes from human brain or placenta. Instead, pericytes mediated a significant increase in the frequency of allogeneic CD25 2 T cells maintained a nonimmunogenic phenotype and mediated the development of functional regulatory T cells. Together, these findings reveal a novel feature of pericytemediated immunomodulation distinguished from immunosuppression, shared by native tissue pericytes and hPSC pericytes, and support the notion that pericytes can be applied for allogeneic cell therapy. STEM CELLS
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