School of Life Sciences, Beijing University of Chinese Medicine, Beijing, China.
J Cell Biochem. 2020 Jan;121(1):566-573. doi: 10.1002/jcb.29301. Epub 2019 Aug 12.
To investigate the possibility of inducing differentiation of human urine-derived stem cells (hUSCs) into hepatocyte-like cells by coculturing with human hepatocyte L02 cells in vitro.
HUSCs were isolated from fresh urine samples collected from healthy adult volunteers by centrifugation. Cells were observed under an inverted phase contrast microscope, and proliferative activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Stem cell surface markers were detected by flow cytometry. HUSCs were induced to differentiate into hepatocyte-like cells by coculturing with human hepatocyte L02 cells, which were confirmed by cellular morphology, messenger RNA expression of albumin (ALB), α-fetoprotein (AFP) and hepatocyte cytochrome P450 (CYP450) analyzed with quantitative reverse transcription polymerase chain reaction and the expression of glycogen detected by glycogen staining kits at 5, 10, and 15 days after coculturing.
HUSCs from urine were successfully isolated and cultured in vitro. At passages 3, the growth curve of hUSCs was S-shaped with good proliferation activity. Mesenchymal stem cell surface markers CD44 and CD90 were detected positive by flow cytometry. CD31 for endothelial cells and CD34 for hematopoietic stem cell markers were not detected. HUSCs gained the cellular morphology and function of hepatocyte cells including higher expression of several hepatocyte-specific genes such as ALB and some CYP450, lower expression of AFP and positive glycogen expression (P < .05) in coculturing with human hepatocyte L02 cells for 10-15d.
HUSCs can be induced to differentiate into hepatocyte-like cells by coculturing with human hepatocyte L02 cells for a certain number of days.
探讨体外共培养人尿液源干细胞（hUSCs）与人肝 L02 细胞诱导其向肝样细胞分化的可能性。
采用离心法从健康成人志愿者新鲜尿液中分离 hUSCs，倒置相差显微镜下观察细胞形态，噻唑蓝比色法检测细胞增殖活性，流式细胞术检测干细胞表面标志物。将 hUSCs 与 L02 细胞共培养诱导其向肝样细胞分化，于共培养后第 5、10、15 天，通过细胞形态、白蛋白（ALB）、甲胎蛋白（AFP）和细胞色素 P450（CYP450）信使 RNA 表达的定量逆转录聚合酶链反应分析，以及糖原染色试剂盒检测糖原表达情况，判断细胞向肝样细胞的分化情况。
成功分离并体外培养 hUSCs，第 3 代细胞呈 S 形生长曲线，增殖活性良好。流式细胞术检测 hUSCs 表达间充质干细胞表面标志物 CD44 和 CD90，不表达内皮细胞标志物 CD31 和造血干细胞标志物 CD34。hUSCs 与 L02 细胞共培养 10-15d 后获得了肝细胞的形态和功能，包括 ALB 等几种肝细胞特异性基因的高表达和 AFP 的低表达，以及阳性的糖原表达（P < .05）。
hUSCs 可通过与 L02 细胞共培养一定时间诱导分化为肝样细胞。
