Study on hepatic differentiation of mesenchymal stem cells derived from human umbilical cord blood, and cell therapy for liver disease by use of mesenchymal stem cells.

在本次科研资助项目中，我们开展了“利用人脐带血间充质干细胞（CB-MSC）开发肝病细胞移植治疗模型”的研究项目，首先在原有的培养条件下，添加成纤维细胞生长因子-2（FGF-2），建立了稳定的CB-MSC细胞系，并成功进行了长期培养。我们的CB-MSC的细胞表面标志物被证实与以前的报道相似。接下来，我们尝试将CB-MSC细胞系分化为成熟的肝细胞。为了诱导成熟，从培养基中除去FGF-2，并向培养基中补充EHS凝胶。成熟步骤后，CB-MSC被诱导表达肝标志物，如谷氨酰胺合成酶和酪氨酸氨基转移酶（达特）。另外，我们构建了表达肝核因子-4（HNF-4）的逆转录病毒载体，并将其感染CB-MSC。表达HNF-4的CB-MSC也表达达特的转录本。最后，我们测试了CB-MSC细胞移植到慢性肝损伤小鼠中。我们采用免疫缺陷（SCID）小鼠慢性损伤的尿激酶型纤溶酶原激活剂转基因的白蛋白启动子/增强子（ALB-uPA/SCID小鼠）的控制下。这些小鼠是由经济振兴独特科技合作链接吉里项目提供的。移植的脐带血细胞在ALB-uPA/SCID小鼠的肝脏中的植入比先前报道的小鼠模型更有效。移植的CB细胞表现出类似于功能性肝细胞的表型，提示人脐血可为肝病患者提供可移植的肝细胞。该研究项目的成果将成为肝脏再生医学的基础，我们认为这项资助用于科学研究的目的已经实现。

In this grant in aid for scientific research, we carried out a research project on therapeutic-model development of cell transplantation for liver disease by use of mesenchymal stem cells derived from human umbilical cord blood (CB-MSC).At first, we established a stable cell line of CB-MSC under our original culture condition supplement with fibroblast growth factor-2 (FGF-2), and succeeded to culture these cells in long term. The cell surface markers of our CB-MSC were confirmed to be similar to those of previous reports.Next, we tried to differentiate CB-MSC cell line into mature hepatocytes. In order to induce maturation, FGF-2 was removed from media, and the medium was supplemented with EHS gel. After the maturation step, CB-MSC was induced to express hepatic markers, such as glutamine synthetase and tyrosine amino-transferase (TAT). Additionally, we constructed retroviral vector expressing hepatic nuclear factor-4 (HNF-4), then, CB-MSC was infected with the retrovirus. HNF-4 expressing CB-MSC also expressed the transcripts of TAT.Finally, we tested cell transplantation of CB-MSC into chronically liver-injured mice. We employed immunodeficient (SCID) mice damaged chronically by a urokinase-type plasminogen activator transgene under a control of albumin promotor/enhancer (ALB-uPA/SCID mice). These mice were kindly provided from the Yoshizato Project, Cooperative Link of Unique Science and Technology for Economy Revitalization. The transplanted cord blood cells engrafted in the liver of ALB-uPA/SCID mice more efficiently than previously reported mouse-models. The engrafted CB cells exhibited the similar phenotype of functional hepatocytes.Our results suggested that human cord blood can supply the transplantable hepatic cells for liver disease. The outcome of the research project will be a basis of liver regenerative medicine, and we think the purpose of this grant in aid for scientific research was accomplished.