共培养诱导人骨髓多能成体祖细胞分化为肝细胞样细胞

目的探索人骨髓来源多能成体祖细胞（ZHJ-MAPC）在与人肝细胞系L02在体外共培养条件下诱导分化为肝细胞的可行性。方法（1）间接共培养：将ZHJ-MAPC和人肝细胞系L02仅培养液相通行间接共培养。分别于培养第1、3、5、7天应用免疫细胞化学法鉴定ZHJ-MAPC的白蛋白、甲胎蛋白（AFP）、细胞角蛋白-18（CK-18）、细胞角蛋白-19（CK-19）等肝细胞特征性表型的表达情况。（2）直接共培养：将荧光染料羧基荧光素二醋酸盐琥珀酰亚胺酯（CFSE,绿色）标记的ZHJ-MAPC与L02细胞（均为1×10^4个／ml）按照各50％比例混合行直接共培养。5d后采用荧光免疫细胞化学SABC-Cy3法（红色）双标后在激光共聚焦显微镜下分别观察ZHJ-MAPC表达白蛋白、AFP、CK-18的情况。结果（1）间接共培养结果：AFP在ZHJ-MAPC间接共培养第1天即表现为强阳性,随后表达逐渐减弱;白蛋白在第5天达到高峰;CK-18在第5天开始出现阳性,第7天出现较强表达。CK-19在各时间点均为阴性。（2）直接共培养结果：共培养第5天在检测白蛋白和CK-18表达情况时呈黄色荧光的阳性细胞即向肝细胞分化的ZHJ-MAPC出现较多,而AFP阳性表达则较少。结论人骨髓来源的ZHJ-MAPC与人肝细胞系L02行间接或直接共培养均能够诱导其向成熟肝样细胞定向分化。

Multipotent adult progenitor cells from human bone marrow differentiate into hepatocyte-like cells induced by co-culture with human hepatocyte line

OBJECTIVE: To investigate the possibility of multipotent adult progenitor cells from human bone marrow ZHJ-MAPC to differentiate into hepatocytes by co-culture with human hepatocyte line L02 in vitro. METHODS: (1) Multipotent adult progenitor cells from human bone marrow, screened in Zhujiang hospital, named ZHJ-MAPC, were divided into 2 parts. Some ZHJ-MAPCs undergo co-culture with human hepatocytes of the line L-02 in the manner without cell to cell contact. On the days 1, 3, 5, and 7 immunocytochemistry was used to detect the expression of albumin (ALP), alpha-fetoprotein (AFP), cytokeratin (CK)-18, and CK-19, characteristic of hepatocyte. Some ZHJ-MAPC were labeled with Certified Functional Safety Expert (CFSE), a fluorescent reagent, and were mixed with L02 cells so as to undergo co-culture in the manner with cell to cell contact. Then the mixed cells were seeded on specialized dish for detection by Laser Scanning Confocal Microscope (LSCM). 5 days later, the cells were double stained with SABC-Cy3. The expression of ALP, AFP, and CK-18 in the ZHJ-MAPC were observed under LSCM. Separately cultured L02 hepatocytes served as positive control s and separately cultured ZHJ-MAPC served as negative controls. RESULTS: (1) Results of co-culture without cell to cell contact. On the first day, the ZHJ-MAPC expressed high level of AFP and then the AFP expression tapered daily. The expression of ALP was very weak on day 1 and then increased and peaked on day 5. The expression of CK-18 began to appear on day 5 and peaked on day 7. The expression of CK-19 was always negative. (2) Results of co-culture with cell to cell contact. On day 5 there were three colors of fluorescence under LSCM. Yellow cells represented the ZHJ-MAPC differentiating into hepatocytes; the green cells were undifferentiating ZHJ-MAPC; and the red cells were L02 hepatocytes. The result showed that ALP and CK-18 were expressed in many ZHJ-MAPC; AFP appeared only in a few cells. CONCLUSION: ZHJ-MAPC can be induced to differentiate into mature hepatocyte-like cells by co-culture, either with or without cell to cell contact.