The epithelial‐mesenchymal transition (EMT) is involved in many different types of cellular behavior, including liver fibrosis. In this report, we studied a novel function of RAR‐related orphan receptor gamma (ROR‐γ) in hepatocyte EMT during liver fibrosis. To induce EMT in vitro, primary hepatocytes and FL83B cells were treated with TGF‐β1. Expression of ROR‐γ was analyzed by Western blot in the fibrotic mouse livers and human livers with cirrhosis. To verify the role of ROR‐γ in hepatocyte EMT, we silenced ROR‐γ in FL83B cells using a lentiviral short hairpin RNA (shRNA) vector. The therapeutic effect of ROR‐γ silencing was investigated in a mouse model of TAA‐induced fibrosis by hydrodynamic injection of plasmids. ROR‐γ expression was elevated in hepatocyte cells treated with TGF‐β1, and ROR‐γ protein levels were elevated in the fibrotic mouse livers and human livers with cirrhosis. Knockdown of ROR‐γ resulted in the attenuation of TGF‐β1‐induced EMT in hepatocytes. Strikingly, ROR‐γ bound to ROR‐specific DNA response elements (ROREs) in the promoter region of TGF‐β type I receptor (Tgfbr1) and Smad2, resulting in the downregulation of Tgfbr1 and Smad2 after silencing of ROR‐γ. Therapeutic delivery of shRNA against ROR‐γ attenuated hepatocyte EMT and ameliorated liver fibrosis in a mouse model of TAA‐induced liver fibrosis. Overall, our results suggest that ROR‐γ regulates TGF‐β‐induced EMT in hepatocytes during liver fibrosis. We suggest that ROR‐γ may become a potential therapeutic target in treating liver fibrosis. J. Cell. Biochem. 118: 2026–2036, 2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.

Mesenchymal stem cell (MSC) differentiation by growth factors may be improper due to possibility of clinical risk. We have previously developed a growth-factor-free coculture method and observed rat MSCs differentiated into hepatic progenitor cells. This study was aimed to validate hepatic differentiation potential in vivo. MSCs from bone marrow of green fluorescent protein-transgenic Sprague-Dawley rats were cocultured with hepatocytes from normal Sprague-Dawley rats, sharing growth-factor-free media. After 14 days, cells were implanted into the spleen of carbon tetrachloride (CCl 4 )-injured rats and kept for 4 weeks. Fibrosis remarkably decreased in CCl 4 / cocultured MSC at weeks 1, 3, and 4. Immunohistochemistry revealed that albumin, a-fetoprotein, and cytokeratin 19 (CK19) expression was high in CCl 4 /cocultured MSC only at week 1. Reverse transcription-polymerase chain reaction and Western blot revealed that CCl 4 /cocultured MSC had reduced a-fetoprotein expression at week 4, whereas CK18 and CK19 exhibited stronger expression. Albumin in CCl 4 /cocultured MSC increased at week 4 only in protein level. We assume that cocultured MSCs had stayed at hepatic progenitor stage until week 3, and differentiated into hepatocytes or bile-ductal epithelial cells afterward. Hepatic progenitor cells from MSC differentiation in the growth-factor-free coculture system may contribute to the therapeutic effect for liver disease in vivo.

The goal of this experiment was to generate insulin-producing human mesenchymal stem cells (hMSCs) as a therapeutic source for type I diabetes mellitus, which is caused by insulin deficiency due to the destruction of islet β cells. In various trials for the treatment of type I diabetes, cell-based therapy using adult stem cells is considered to be one of the most useful candidates for the treatment. In this experiment, a non-viral method called nucleofection was used to transfect hMSCs with pEGFP-C2 and furin-cleavable human preproinsulin gene (hPPI) to produce insulin-secreting cells as surrogate β cells. Transfection efficiency was determined using flow cytometry analysis. Expression and production of insulin were tested using RT-PCR and ELISA. The expression, production and maturation of insulin from the genetically engineered hMSCs showed an increase when compared with a non-transfected control group. Insulin expression from hMSCs using nucleofection in this study has shown the potential for type I diabetes therapy. For further study, an evaluation for in vivo experiments and clinical applications must be supplemented.

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Niche appears important for preventing the spontaneous differentiation or senescence that cells undergo during in vitro expansion. In the present study, it was revealed that human bone marrow-derived mesenchymal stem cells (hBM-MSCs) undergo senescence-related differentiation into the myocardial lineage in vitro without any induction treatment. This phenomenon occurred over the whole population of MCSs, much different from conventional differentiation with limited frequency of occurrence, and was accompanied by a change of morphology into large, flat cells with impeded proliferation, which are the representative indications of MSC senescence. By culturing MSCs under several culture conditions, it was determined that induction treatment with 5-azacytidine was not associated with the phenomenon, but the serum-starvation condition, under which proliferation is severely hampered, caused senescence progression and upregulation of cardiac markers. Nevertheless, MSCs gradually developed a myocardial phenotype under normal culture conditions over a prolonged culture period and heterogeneous populations were formed. In perspectives of clinical applications, this must be prevented for fair and consistent outcomes. Hence, the biomimetic 'niche' was constituted for hBM-MSCs by cultivating on a conventionally available extracellular matrix (ECM). Consequently, cells on ECM regained a spindle-shape morphology, increased in proliferation rate by two-fold and showed decreased expression of cardiac markers at both the mRNA and protein levels. In conclusion, the outcome indicates that progression of MSC senescence may occur via myocardial differentiation during in vitro polystyrene culture, and this can be overcome by employing appropriate ECM culture techniques.