Kazemnejad S, Allameh A, Seoleimani M, Gharehbaghian A, Mohammadi Y, Amirizadeh N, Esmaeili S
Department of Clinical Biochemistry, Faculty of Medical Science, Tarbiat Modares University, Tehran.
Int J Artif Organs. 2008 Jun;31(6):500-7. doi: 10.1177/039139880803100605.
To supporting growth and functional differentiation of adult stem cells into hepatocytes in a well-controlled manner, we performed differentiation of human bone marrow mesenchymal stem cells (hBMSCs) to hepatocytes-like cells on a constructed 3-dimensional (3D) nanofibrous biocompatible scaffold.
After characterization of the hBMSCs isolated from human bone marrow, the performance of the cells seeded and their proliferation on the scaffold was evaluated by scanning electron microscopy (SEM) and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Different approaches such as immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), and biochemical assays were used to estimate the ability of hBMSC-derived cells to express hepatocyte-specific markers.
Scanning electron micrographs and MTT analysis revealed the cells were able to expand and remained biologically and metabolically active for 21 days. Immunocytochemical analysis of albumin and alfa-fetoprotein showing the accumulation of these markers in differentiated cells was confirmed by RT-PCR. Additional markers such as cytochrome P450 3A4, cytokeratin-18, and cytokeratin-19 detected by RT-PCR showed progressive expression during 3 weeks of differentiation on 3D scaffold. The hepatocyte-like cells displayed several characteristics of metabolic functions as judged by production of albumin, urea, transferrin, serum glutamic pyruvic transaminase (SGPT), and serum oxaloacetate aminotransferase (SGOT). Levels of above-mentioned markers, except SGOT in differentiated cells on scaffold, were found to be significantly greater than in the 2D culture system (p<0.05).
Overall data suggest that the engineered nanofibrous scaffold is a conductive matrix for functional hBMSC-derived hepatocyte-like cells and is promising for maintenance of hepatocytes suitable for implantation.
为了以可控方式支持成体干细胞向肝细胞生长和功能分化，我们在构建的三维（3D）纳米纤维生物相容性支架上，将人骨髓间充质干细胞（hBMSCs）诱导分化为类肝细胞。
对从人骨髓中分离出的hBMSCs进行鉴定后，通过扫描电子显微镜（SEM）和3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法评估接种在支架上的细胞性能及其增殖情况。采用免疫细胞化学、逆转录聚合酶链反应（RT-PCR）和生化分析等不同方法，评估hBMSC来源细胞表达肝细胞特异性标志物的能力。
扫描电子显微镜图像和MTT分析显示，细胞能够扩增并在21天内保持生物学和代谢活性。通过RT-PCR证实了免疫细胞化学分析中白蛋白和甲胎蛋白在分化细胞中的积累。RT-PCR检测到的其他标志物，如细胞色素P450 3A4、细胞角蛋白-18和细胞角蛋白-19，在3D支架上分化的3周内呈逐渐表达。通过白蛋白、尿素、转铁蛋白、血清谷丙转氨酶（SGPT）和血清谷草转氨酶（SGOT）的产生判断，类肝细胞表现出多种代谢功能特征。发现除支架上分化细胞中的SGOT外，上述标志物的水平均显著高于二维培养系统（p<0.05）。
总体数据表明，工程化纳米纤维支架是功能性hBMSC来源类肝细胞的导电基质，有望维持适合植入的肝细胞。
