Mu Ning, Wang Ming-yuan, Lin Yu-dong, Gao Yi
Department of General Surgery, 95th Clinical Department, PLA Fuzhou General Hospital, Fuzhou 351100, China.
Zhonghua Gan Zang Bing Za Zhi. 2007 Mar;15(3):174-8.
To investigate the possibility of marrow derived multipotent adult progenitor cells (MAPCs) differentiating into hepatocytes by co-culturing with human hepatocyte line L02, and to evaluate the potential use of MAPCs in tissue-engineering either experimentally or clinically.
(1) Co-culturing without cell-to-cell contact: MAPCs and L02 hepatocytes were spread on coverslips separately (both with a cell density of 1x10(5)/ml), and then they were put in a culture dish (10 cm). The expressions of Alb, AFP, CK18, and CK19 in MAPCs were detected by immunocytochemistry at different time points. A separate culture of L02 hepatocytes served as a positive control and a separate culture of MAPCs served as a negative control. (2) Co-culturing with cell-to-cell contact: MAPCs labeled with CFSE were mixed with L02 hepatocytes (both with a cell density of 1x10(4)/ml), and then the mixed cells were seeded on specific dishes for detection by laser scanning confocal microscope (LSCM). Five days later, the cells were double-stained with SABC-Cy3. The expressions of Alb, AFP, CK18 in MAPCs were observed under LSCM. Similarly, separately cultured L02 hepatocytes served as a positive control and separately cultured MAPCs served as a negative control.
(1) Results of co-culturing without cell-to-cell contact: On the first day, the MAPCs expressed a high level of AFP. Then AFP expression tapered daily and there was hardly any expression of AFP on day 7. The expression of Alb was very weak on day 1, but increased significantly by day 3, reached its peak on day 5, and still maintained a high level on day 7. The initial expression of CK18 appeared on day 5 and reached a higher level on day 7. The expression of CK19 was always negative. The positive control cells had a high expression of Alb and CK18, while there was a weak expression of AFP and a negative expression of CK19. The negative control cells had no expressions for the four markers. (2) Results of co-culturing with cell-to-cell contact: On day 5, there were three colors of fluorescence under LSCM: yellow cells were MAPCs differentiating into hepatocytes; green cells were undifferentiated MAPCs; red cells were L02 hepatocytes. The result showed that Alb and CK18 were expressed in many cells and AFP appeared in only a few cells.
Human MAPCs can be induced to differentiate into mature hepatocyte-like cells by co-culturing with L02 hepatocytes, either with or without cell-to-cell contact, but the former way may be more effective.
通过与人肝细胞系L02共培养，研究骨髓来源的多能成人祖细胞（MAPCs）分化为肝细胞的可能性，并从实验和临床角度评估MAPCs在组织工程中的潜在应用。
（1）非细胞接触共培养：将MAPCs和L02肝细胞分别接种于盖玻片上（细胞密度均为1×10⁵/ml），然后置于10 cm培养皿中。在不同时间点通过免疫细胞化学检测MAPCs中白蛋白（Alb）、甲胎蛋白（AFP）、细胞角蛋白18（CK18）和细胞角蛋白19（CK19）的表达。单独培养的L02肝细胞作为阳性对照，单独培养的MAPCs作为阴性对照。（2）细胞接触共培养：将用羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）标记的MAPCs与L02肝细胞混合（细胞密度均为1×10⁴/ml），然后将混合细胞接种于特定培养皿中，用激光扫描共聚焦显微镜（LSCM）检测。5天后，细胞用链霉亲和素-生物素复合物-异硫氰酸荧光素（SABC-Cy3）进行双重染色。在LSCM下观察MAPCs中Alb、AFP、CK18的表达。同样，单独培养的L02肝细胞作为阳性对照，单独培养的MAPCs作为阴性对照。
（1）非细胞接触共培养结果：第1天，MAPCs中AFP表达水平较高。随后AFP表达逐日下降，至第7天几乎无AFP表达。Alb在第1天表达较弱，但在第3天显著增加，第5天达到峰值，第7天仍维持较高水平。CK18在第5天开始表达，第7天达到较高水平。CK19的表达始终为阴性。阳性对照细胞Alb和CK18高表达，AFP弱表达，CK19阴性表达。阴性对照细胞这四种标志物均无表达。（2）细胞接触共培养结果：第5天，LSCM下可见三种荧光颜色：黄色细胞为分化为肝细胞的MAPCs；绿色细胞为未分化的MAPCs；红色细胞为L02肝细胞。结果显示，许多细胞表达Alb和CK18，只有少数细胞表达AFP。
人MAPCs与L02肝细胞共培养，无论有无细胞接触，均可诱导分化为成熟的肝细胞样细胞，但前一种方式可能更有效。
