Human embryonic stem cell-derived mesenchymal stem cells have potential of high differentiation into hepatocyte-like cells through increased level of hepatocyte growth factor

Background: As the aging society progresses, more patients are waiting for liver transplantation. However, the function of existing bio artificial liver is insufficient. Recently, research has been focused on stem cells to regenerate damaged liver. Most of all, the obstacle to the commercialization of widely used human adipose tissue-derived stem cells (hASCs) therapy is the limited supply of cells with consistent quality although most cell source of current stem cell therapy is hASCs. In this study, we attempt to differentiate human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) into hepatocyte-like cells and tested whether hESC-MSCs have a potential of liver regeneration as a source of cell therapy, considering that they have characteristics of high differentiation rates, unlimited proliferation possibility from a single colony, and homogenicity.Methods: hESC-MSCs and hASCs were cultured under condition of hepatogenic differentiation in vitro. And then various experiments including quantitative real-time PCR, immunoblotting, ELISA, immunofluorescence, Periodic acid-Schiff staining and flow cytometry analysis were performed to evaluate expression level of HGF and the extent of differentiation into hepatocyte-like cells.Results: We found that hepatocyte growth factor (HGF), important for maintaining hepatocyte activity, is highly expressed in hESC-MSCs. Besides, it was confirmed that the expression of ALB, TDO2, and CYP2E1, which are the hepatocyte-specific genes when hepatogenic differentiation was performed under conditions even without HGF in hESC-MSCs, was higher than that when hASCs was differentiated into hepatocytes. Likewise, we concluded that hESC-MSCs could be well-differentiated into hepatocyte-like cells compared to hASCs through staining data.Conclusions: We propose that hESC-MSCs have a promising potential as a commercial cell therapy source to treat liver failure in view of the limited available cell sources for stem cell therapy.

Human Hepatocytes and Differentiated Adult-Derived Human Liver Stem/Progenitor Cells Display In Vitro Immunosuppressive Properties Mediated, at Least in Part, through the Nonclassical HLA Class I Molecule HLA-G

One of the main challenges in liver cell therapy (LCT) is the induction of a tolerogenic microenvironment to promote graft acceptance in the recipient. Little is known about the immunomodulatory potential of the hepatic cells used in liver cell therapy. In this work, we wanted to evaluate the immunosuppressive properties of human hepatocytes and adult-derived human liver stem/progenitor cells (ADHLSCs), as well as the potential involvement of the immunomodulatory molecule HLA-G. We demonstrated that both cell types were capable of inhibiting the proliferative response of PBMCs to an allogenic stimulus and that the immune inhibitory potential of ADHLSCs, although lower than that of hepatocytes, increased after hepatogenic differentiation. We demonstrated that liver cells express HLA-G and that the immune inhibition pattern was clearly associated to its expression. Interestingly, HLA-G expression increased after the third step of differentiation, wherein oncostatin M (OSM) was added. A 48 hr treatment with OSM was sufficient to induce HLA-G expression in ADHLSCs and result in immune inhibition. Surprisingly, blocking HLA-G partially reversed the immune inhibition mediated by hepatocytes and differentiated ADHLSCs, but not that of undifferentiated ADHLSCs, suggesting that additional immune inhibitory mechanisms may be used by these cells. In conclusion, we demonstrated that both hepatocytes and ADHLSCs present immunomodulatory properties mediated, at least in part, through HLA-G, which can be upregulated following hepatogenic differentiation or liver cell pretreatment with OSM. These observations open up new perspectives for the induction of tolerance following LCT and for potential therapeutic applications of these liver cells.

Enhanced Hepatogenic Differentiation of Human Wharton’s Jelly–Derived Mesenchymal Stem Cells by Using Three-Step Protocol

Currently, human Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) are an attractive source of stem cells for cell-based therapy, owing to their ability to undergo self-renewal and differentiate into all mesodermal, some neuroectodermal, and endodermal progenies, including hepatocytes. Herein, this study aimed to investigate the effects of sodium butyrate (NaBu), an epigenetic regulator that directly inhibits histone deacetylase, on hepatic endodermal lineage differentiation of hWJ-MSCs. NaBu, at 1 mM, optimally promoted endodermal differentiation of hWJ-MSCs, along with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) supplementation (EGF + bFGF + 1 mM NaBu). CXCR4, HNF3β, SOX17 (endodermal), and GATA6 (mesendodermal) mRNAs were also up-regulated (p < 0.001). Immunocytochemistry and a Western blot analysis of SOX17 and HNF3β confirmed that the EGF + bFGF + 1 mM NaBu condition was appropriately pre-treated with hWJ-MSCs before hepatogenic differentiation. Furthermore, the hepatogenic medium + NaBu pre-treatment up-regulated hepatoblast (AFP and HNF3β) and hepatic (CK18 and ALB) markers, and increased the proportion of mature hepatocyte functions, including G6P, C/EBPα, and CYP2B6 mRNAs, glycogen storage and urea secretion. The hepatogenic medium + NaBu in the pre-treatment step can induce hWJ-MSC differentiation toward endodermal, hepatoblastic, and hepatic lineages. Therefore, the hepatogenic medium + NaBu pre-treatment for differentiating hWJ-MSCs could represent an alternative protocol for cell-based therapy and drug screening in clinical applications.