Establishment of isolation procedure of human hepatic stem cell and tissue engineering technique to reconstitute hepatic tissue using the stem cell.

[目的]在本研究中，我们首先建立了从正常人肝脏中分离肝干细胞的方法。然后，我们利用秋田住友胶原胶株式会社开发的胶原凝胶，检测了肝干细胞在三维培养条件下是否有分化为肝细胞、胆管上皮细胞或胰腺细胞的潜能。最后，我们研究了肝干细胞体外移植重建的局部器官的临床应用。[成果](1)从健康大鼠肝脏的非实质部分建立了肝干细胞样细胞系，命名为肝干细胞样细胞。(2)肝干细胞样细胞体积小，直径20μm，核浆比高。这些细胞表达α-胎儿蛋白，但不表达白蛋白和细胞角蛋白19，表明该细胞具有不成熟的表型。(3)在胶原胶和肝星状细胞中培养HSL细胞，研究其分化潜能。因此，两种培养条件都诱导HSL细胞表达白蛋白，表明HSL细胞能够分化为成熟细胞。(4)最后，我们研究了如何处理HSL细胞以显示体内的肝功能。为此，我们将HSL细胞与胶原凝胶混合或包埋在胶原海绵中，移植到高蛋白血症大鼠体内。结果显示，移植胶原凝胶的HSL细胞组大鼠血清白蛋白升高67%，胶原海绵移植组大鼠血清白蛋白升高57%。这表明移植的HSL细胞已经分化为白蛋白产生细胞，并在体内具有肝细胞的功能。本研究为HSL细胞的临床应用提供了可能。

[Aim]ln this study, we first tried to establish the procedure for isolation ofhepatic stem cell from healthy human liver. Next, we examined whether the hepatic stem cell has potential to differentiate into hepatocyte, biliary epithelial cell, or pancreatic cell under tri-dimensional culture condition by using collagen gel developed by Akita Sumitomo Bakelite Co., Ltd. Finally, we aimed the clinical application of topo-organ reconstituted by the ex vivo transplantation of hepatic stem cell.[Achievements](1)We have established a liver epithelial cell line, designated hepatic stem like (HSL) cell, from nonparenchymal fraction of healthy rat liver.(2)The HSL cells are small with 20 μm in diameter and showed high nucleus/cytoplasm ratios. These cells expressed α-fetoprotein, but not albumin nor cytokeratin 19, indicating the cells have immature phenotype.(3)We cultured the HSL cells in collagen gels or with hepatic stellate cells to investigate their potential to differentiate. Consequently, both of culture conditions induced the HSL cells to express albumin, showing that the HSL cells are able to differentiate into mature cells.(4)Finally, we examined how we process the HSL cells to manifest the hepatic function in vivo. To this end, we transplanted the HSL cells which were mixed with collagen gel or embedded in collagen sponge into analbuminemic rats. As a result, analbuminemic rats that showed the increased levels of serum albumin were 67% in the rats transplanted the HSL cells with collagen gel and 57% in the rats with collagen sponge. This suggests that the transplanted HSL cells have differentiated into albumin producing cells and function as hepatocytes in vivo. The present study implies the possibility of clinical application of HSL cells.