| Summary: | 碩士 === 國立陽明大學 === 臨床醫學研究所 === 102 === During development, stem/progenitor cells could undergo self-renewal and be directed into specified tissues in response to various stimuli. Taking liver cells as an example, various protocols to differentiate propagated human stem cells, either from embryonic or adult origins, into functional hepatocytes have recently been proposed. Nevertheless, potential ethical dilemma by using human embryonic stem cells (hESCs) and unsatisfied differentiation efficiency limited their clinic uses. Numbers of investigations were reported suggesting that epigenetic modulators could be potentially important in promoting differentiation potential during hepatic differentiation although the detailed mechanisms remained poorly understood.
In order to determine the impact of epigenetic regulation for hepatic differentiation, two epigenetic modulators histone deacetylases inhibitor (HDACi) Trichostatin A (TSA) and DNA methyltransferases inhibitor (DNMTi) 5-azacytidine (5-AzaC) were applied, either alone or in combination, in human bone marrow derived mesenchymal stem cells (hBMSCs) during hepatic differentiation. Differential readouts including cell viability, dynamic expression of hepatic markers such as liver serum protein, live-enriched transcription factors (LETFs) and connexin junctional proteins as well as hepatic functional assays were performed to demonstrate bond-fide liver characteristics in hBMSCs derived hepatocytes. The results indicated that addition of TSA treatment exhibited greater cytotoxicity during differentiation compared with original protocol while combinational treatment of 5-azaC and TSA ameliorated cell mortality, partly by the improvement of apoptosis, and enhanced differentiation efficiency based on the detection of elevated expression of Albumin, Connexin protein Cx26/Cx32, Hepatocyte Nuclear Factor 4 alpha (HNF4a) and detoxification cytochrom p450 isoenzymes. In addition, various in vitro functional assays also showed that liver specific functions including glycogen storage, urea cycle activity and xenobiotics metabolism were evident in 5-azaC/TSA treated hBMSC derived hepatic cells. In order to extend potential clinical use of this model, further examination of the permissiveness for hepatitis C virus (HCV) was carried out. Although HCV entry proteins mRNAs were activated in hBMSC derived hepatic cells, no HCV core proteins was detected at 72 hours post-infection, possibly due to inefficient induction of HCV determinant microRNA-122 (miR-122). Taken together, we demonstrated that epigenetic modulators could facilitate stem cell mediated hepatic differentiation while underlying molecular machineries and the improvement of induction of miR-122 expression aiming to be sensitive to HCV infection in hBMSC derived hepatic cells remain to be determined.
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