摘要
目的:探讨在体外诱导型多潜能干细胞(IPS)能否遵循正常肝脏发育途径定向分化为具备良好生理功能的肝细胞。方法:根据正常肝脏体内发育的规律,序贯应用肝脏发育所需的生长因子,设计特色培养基,诱导IPS细胞遵循正常发育途径定向高效分化为肝细胞:首先将IPS细胞定向分化为前层确定性内胚层细胞(ADE),再进一步分化为肝细胞。结果:在本研究中IPS细胞在体外可循正常发育通路定向诱导为肝细胞:先分化为前层内胚层细胞,再继续分化为肝细胞。分化的各时段实时定量RT-PCR检测显示:在mRNA表达的水平,IPS细胞经历了肝细胞在体内从胚胎干细胞的发育过程,内胚层细胞特异性RNA如CXCR4、Sox17和FOXA2在分化的早期明显升高,而早期肝细胞特异性RNA如HNF4和AFP在分化的中期开始升高,肝细胞成熟特异性RNA如白蛋白在分化的晚期才达高峰,同时IPS细胞的未分化标记RNA如OCT4在早期就明显下调。同mRNA表达一致,在分化的第20d,大部分的肝脏细胞表达甲胎蛋白和白蛋白(阳性率80%)。更为重要的是IPS来源肝细胞在体外具有典型成熟肝细胞的功能,能够摄取和释放靛青绿(ICG)并具有药物代谢酶细胞色素P450酶的活性。结论:IPS细胞在体外能够遵循正常肝脏发育通路,序贯表达肝脏发育各阶段的特色基因和蛋白,能够高效分化为有功能的肝细胞,本研究可能为临床终末期肝病进行细胞治疗提供肝细胞。
AIM：To directly differentiate the induced pluripotent stem cells （IPS cells） into hepatocytes in chemically-defined,monolayer conditions in vitro. METHODS：Based on the knowledge of mammalian hepatic development,we differentiated the IPS cells into hepatic lineage efficiently. The present study involves stepwise transition of IPS cells through a series of media designs to first specify cells towards anterior definitive endoderm,and secondly,to induce hepatic maturation. RESULTS：The IPS cells recapitulated the development of liver in vivo at RNA and protein level. Realtime PCR analysis was performed on cell preparations at various times during the culture process to determine the time course and degree to which the IPS cells differentiated toward a hepatocyte phenotype. Analysis included markers for endoderm-specific gene expression （Sox17; FoxA2,CXCR4）,hepatocytespecific gene expression （albumin and AFP）,and markers for undifferentiated IPS cells （OCT4 and Nanog）. The endodermspecific gene expression increased early while the expression of OCT4 and Nanog decreased and hepatocytespecific gene expression progressively increased over the course of the differentiation program. The resultant hepatocytelike cells（HLCs） showed phenotypic similarities with adult hepatocytes （expressing AFP and albumin） and additionally showed functionality expected to welldifferentiated hepatocytes such as indocyanine green（ICG） uptake and release. The P450 activity of cytochromsome was also positive. CONCLUSION：We successfully construct the IPS cells which efficiently differentiate into hepatocytes and recapitulate the hepatocytic development in vivo. The reproducible and efficient generation of HLCs from IPS cells represents a critical step towards the ultimate goal of producing functional HLCs for clinical use including cell transplantation or bioartificial liver support. Moreover,our system provide a valid way to study the liver development since it is monolayer,serumfree and mimic the in vivo liver
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2010年第5期947-951,共5页
Chinese Journal of Pathophysiology
基金
国家重点基础研究发展计划(973)项目基金资助(分课题)(No.2009CB522404)
国家自然科学基金联合重点资助项目(No.U0932006)
关键词
诱导型多潜能干细胞
肝细胞定向分化
Induced pluripotent stem cells
Hepatic efficient differentiation
