Development of expanding and differentiation-promoting system ex vivo for liver regeneration

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For the purpose of development of liver regeneration therapy, we attempted to develop the novel methods, by which human mesenchymal stem cells (MSCs) can be differentiated to mature hepatocytes. We used bone marrow-derived MSCs and cord blood-derived MSCs.1.Study by using bone marrow-derived MSCsUsing UE7T-13 cells, human bone marrow-derived MSCs which were retrovirally infected with human papilloma virus E7 and human telomerase reverse transcriptase (hTERT), 40 conditions of several cytokines, 5-azacytidine and type IV collagen coating were examined on hepatic differentiation of UE7T-13 cells. As a result, the optical condition for differentiation is type IV collagen-coating/aFGF/bFGF/HGF. By this condition, expression of hepatocyte-specific genes was highest. In addition, glycogen synthesis and urea synthesis was also greatly promoted by this treatment. Under this circumstance, WISP1 and WISP2 were down-regulated. Knockdown of WISP1 and WISP2 by siRNA also greatly enhanced hepatic differentiation of of MSCs.2.Study by using cord blood-derived MSCsUsing UCBTERT-21 cells, human cord blood-derived MSCs which were retrovirally infected with human telomerase reverse transcriptase (hTERT), 8 conditions of several cytokines, 5-azacytidine, HDAC inhibitor were examined on hepatic differentiation of UCBTERT-21 cells. As a result, the optical condition for differentiation is 5-azacytidine/HGF/aFGF/OSM. By this condition, expression of hepatocyte-specific genes was highest. In addition, glycogen syntheisis and urea syntheisis was also greatly promoted by this treatment. Under this circumstance, Wnt signal was down-regulated. Knockdown of fzd8, the receptor of Wnt, WISP1 also greatly enhanced hepatic differentiation of of MSCs.These results indicated new findings of hepatic differentiation of MSCs ; and contribute to the development of expanding and differentiation-promoting system ex vivo for liver regeneration.