Concurrent generation of functional smooth muscle and endothelial cells via a vascular progenitor.

Smooth muscle cells (SMCs) and endothelial cells (ECs) are typically derived separately, with low efficiencies, from human pluripotent stem cells (hPSCs). The concurrent generation of these cell types might lead to potential applications in regenerative medicine to model, elucidate, and eventually treat vascular diseases. Here we report a robust two-step protocol that can be used to simultaneously generate large numbers of functional SMCs and ECs from a common proliferative vascular progenitor population via a two-dimensional culture system. We show here that coculturing hPSCs with OP9 cells in media supplemented with vascular endothelial growth factor, basic fibroblast growth factor, and bone morphogenetic protein 4 yields a higher percentage of CD31(+)CD34(+) cells on day 8 of differentiation. Upon exposure to endothelial differentiation media and SM differentiation media, these vascular progenitors were able to differentiate and mature into functional endothelial cells and smooth muscle cells, respectively. Furthermore, we were able to expand the intermediate population more than a billion fold to generate sufficient numbers of ECs and SMCs in parallel for potential therapeutic transplantations.

Viral-mediated coexpression of Pdx1 and p48 regulates exocrine pancreatic differentiation in mouse ES cells.

Embryonic stem cells (ES) can spontaneously activate a pancreatic differentiation program in vitro, although with low efficiency. The aim was to improve such process by using viral mediated gene transduction. In this study, we have examined the suitability of using viral vectors to express key transcriptional factors involved in pancreatic development. ES cell lines that constitutively express Pdx1, a homeodomain protein involved in both exocrine and endocrine pancreatic development and differentiation, were established using a lentiviral vector. These cells were additionally infected with an adenovirus expressing p48, a bHLH factor that is also crucial for pancreatic development and acinar differentiation. Quantitative RT-PCR analysis demonstrated an increase in the expression of exocrine genes, including those coding for both digestive enzymes and transcription factors. Immunocytochemical staining also revealed an increase in the number of amylase-expressing cell clusters. However, other important genes involved in acinar cell maturation (i.e., Mist1) were not modulated under these conditions, suggesting that the cells display features of immature exocrine cells or because of an uncoupled gene expression of the exocrine differentiation program. Importantly, this effect was selective for the acinar lineage as the expression of a large set of endocrine markers remained unchanged. Therefore, combined expression of key genes involved in pancreatic development may be a promising approach to generate mature pancreatic exocrine cells.

Human bone marrow mesenchymal stem cells can express insulin and key transcription factors of the endocrine pancreas developmental pathway upon genetic and/or microenvironmental manipulation in vitro.

Multipotential stem cells can be selected from the bone marrow by plastic adhesion, expanded, and cultured. They are able to differentiate not only into multiple cell types, including cartilage, bone, adipose and fibrous tissues, and myelosupportive stroma, but also into mesodermal (endothelium), neuroectodermal, or endodermal (hepatocytes) lineages. Our goal was to characterize the multipotential capacities of human mesenchymal stem cells (hMSCs) and to evaluate their ability to differentiate into insulin-secreting cells in vitro. hMSCs were obtained from healthy donors, selected by plastic adhesion, and phenotyped by fluorescence-activated cell sorter and reverse transcription-polymerase chain reaction analysis before and after infection with adenoviruses coding for mouse IPF1, HLXB9, and FOXA2 transcription factors involved early in the endocrine developmental pathway. We found that native hMSCs have a pluripotent phenotype (OCT4 expression and high telomere length) and constitutively express NKX6-1 at a low level but lack all other transcription factors implicated in beta-cell differentiation. In all hMSCs, we detected mRNA of cytokeratin 18 and 19, epithelial markers present in pancreatic ductal cells, whereas proconvertase 1/3 mRNA expression was detected only in some hMSCs. Ectopic expression of IPF1, HLXB9, and FOXA2 with or without islet coculture or islet-conditioned medium results in insulin gene expression. In conclusion, our results demonstrated that in vitro human bone marrow stem cells are able to differentiate into insulin-expressing cells by a mechanism involving several transcription factors of the beta-cell developmental pathway when cultured in an appropriate microenvironment.