Recently, human mesenchymal stem cells (MSCs) are attracting attention as a useful source for regenerative therapy. Controlled production of cell therapy requires the establishment and management of an accurate isolation, characterization and monitoring for quality assurance of developing MSCs mediated. In this study, we were confirmed maintenance of potency of isolated and cultured human umbilical cord (hUC)-MSCs during ex vivo expansion or after cryopreservation. Expression of their cell specific marker was analyzed by flow cytometry and the differentiation potency was confirmed by guided differentiation of adipocyte, osteocyte, chondrocyte and hepatocyte after expanding over 15 doublings in vitro. Safe production of developing a cell therapy was proved by testing for microbial, mycoplasma, endotoxin, and adventitious agents. Also stability of cells in cultivation, preservation and/or differentiation was determined chromosomal assay. In developing using hUC-MSCs, cells showed an accurate isolation and stable expansion in ex vivo condition. The results of several management assay showed that the stem cell marker expression of CD31, CD34 and CD45 were under 10%, however CD90 was over 90% by FACS analysis. Any contamination and mutation in all tests weren't detected in specific points for safe or stable production of hMC-MSCs. Also the proliferation and differentiation potency maintains during in vitro culture and after cryopreservation of hUC-MSCs. These results could be used as standard methods of maintenance of hUC-MSCs for cell therapy products and clinical application.

Estrogens are ubiquitous signaling molecules that influence nearly every cell type, and exert profound effects on embryonic development, and differentiation. Wnt pathway, which recruits β-catenin into nuclei, and activates The Wnt-dependent transcription factors, also plays an important role in embryonic development and stem cell maintenance, and differentiation. Accumulating evidences indicate that potential convergence between these two pathways in carcinoma cells. However, physiological roles of estrogens in development and differentiation of human embryonic stem cells (hESCs) are relatively unknown. Here, we demonstrated that estrogenic compounds 17α-ethinylestradiol (EE2) and genistein (GEN) significantly increased β-catenin expression in undifferentiated hESCs cultured in feeder-free media. Interestingly, GEN treatement induced an increased trend of mesendodermal gene expressions, and significantly inhibited ectodermal gene expressions (Nestin and Pax6) in embrioid body (EB). Expectantly, GEN increased epithelial-mesenchymal transition (EMT) related gene expression (Snail2, and Twist), whereas decreased E-cadherin on day 6 of EB development. Taken together, these suggest that estrogens may in part the powerful effects on normal hESC differentiation. Mechanistic studies of estrogen signaling continue to suggest novel drug targets for stem cells and will also improve screening methods of developmental toxicity.

We previously reported that purified hepatocyte-like cells derived from human embryonic stem cell (hESC) promoted the liver tissue recovery not only by cell replacement, but also by delivering proteins (secretome) that enhance endogenous host liver regeneration. In this study, we investigated possible therapeutic effects of secretomes obtained from undifferentiated hESC and mesenchymal stem cell (hMSC), and explored the underlying mechanism in a mouse model of chronic liver injury. Mice pre-intoxicated with dimethylnitrosamine (DMN) were treated with single intraperitoneal injection of secretome or medium used to support the growth of hESCs or hMSCs. Both hESC- and MSC-secretomes induced robust host liver regeneration, as determined by biochemical and histological analyses. The expression of MMP2 was significantly increased in the liver that received hESC- or hMSC-secretome, compared to control groups. In contrast, expression of α-SMA, a hallmark of activated hepatic stellate cells, was profoundly decreased after administration of both secretomes. These results suggest that hESCs and MSCs may release soluble factors that support the host tissue regeneration of chronically injured liver.

The remarkable regenerative capacity of the adult liver provides a setting to test the functional consequences of grafting human cells generated from pluripotent stem cells. This presentation describes a procedure to differentiate hepatocytes from human embryonic stem and induced pluripotent stem cells using only defined factors. Two cell populations generated in vitro were grafted into the spleen of mice treated with the hepato-toxin carbon tetra-chloride. The population containing few hepatocytes generated few surviving cells that produced low levels of albumin and did not support regeneration of the host liver. The cells enriched in donor hepatocytes efficiently engrafted around the branches of the portal vein, expressed hepatic features for at least 5 weeks. These cells also contributed to the endogenous tissue regeneration and function of the host liver. These results show that the controlled differentiation of hepatocytes from human pluripotent cells provides new approaches to define the mechanisms of tissue regeneration and restore liver function.