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

为了开发肝再生治疗的新方法，我们尝试开发新的方法，通过该方法可以将人间充质干细胞（MSCs）分化为成熟的肝细胞。1.骨髓间充质干细胞的研究以UE 7 T-13细胞、逆转录病毒感染人乳头瘤病毒E7和人端粒酶逆转录酶（human telomerase reverse transcriptase，hTERT）的人骨髓间充质干细胞为实验材料，检测40种细胞因子、5-氮胞苷和IV型胶原包被对UE 7 T-13细胞肝分化的影响。因此，分化的最佳条件是IV型胶原包被/aFGF/bFGF/HGF。在此条件下，肝细胞特异性基因的表达最高。此外，该处理还极大地促进了糖原合成和尿素合成。在这种情况下，WISP 1和WISP 2被下调。利用UCBTERT-21细胞、逆转录病毒感染人脐带血来源的MSCs、8种细胞因子、5-氮杂胞苷、HDAC抑制剂对UCBTERT-21细胞肝分化的影响。结果表明，最佳分化条件为5-氮胞苷/HGF/aFGF/OSM。在此条件下，肝细胞特异性基因的表达最高。此外，该处理还显著促进了糖原和尿素的合成。在这种情况下，Wnt信号被下调。Fzd 8、Wnt受体、WISP 1的敲除也显著促进了MSCs的肝分化，这些结果为MSCs肝分化的研究提供了新的发现，并为体外扩增和促分化系统的开发奠定了基础。

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.