[关键词]
[摘要]
目的:探讨模拟角膜缘干细胞(LSCs)微环境诱导人多潜能干细胞(hiPSCs)分化为角膜上皮细胞的可行性。方法:体外建立hiPSCs细胞系,利用transwell体系将hiPSCs与角膜基质细胞共培养模拟角膜缘干细胞微环境,添加小分子骨形态发生蛋白4(BMP4)和特异性转化生长因子β抑制剂(SB431542),诱导hiPSCs向角膜上皮细胞分化。采用免疫荧光染色、流式细胞方法检测角膜上皮细胞特异标志物 CK3和CK12,角膜上皮细胞前体CK15,角膜缘干细胞标志物ABCG5的表达。结果:hiPSCs体外培养增殖活跃,免疫荧光染色显示干细胞特异性标志物OCT4、SOX2、TRA-1-60、NANOG呈阳性。采用transwell体系将hiPSCs与角膜基质细胞共培养,免疫荧光染色结果显示角膜缘干细胞标志物ABCG5及角膜上皮细胞前体标志物CK15阳性,角膜上皮细胞标志物CK3及CK12阴性; 在共培养的基础上添加小分子BMP4和SB431542,免疫荧光染色及流式细胞检测结果显示角膜上皮细胞特异性标志物CK3阳性表达,且随分化时间延长表达比例增高。结论:模拟角膜缘干细胞微环境同时添加小分子SB431542及BMP4,可成功诱导体外培养的hiPSCs向角膜上皮细胞分化。
[Key word]
[Abstract]
AIM: To investigate the potential of human induced pluripotent stem cells(hiPSCs)differentiating into corneal epithelial cells in the simulated limbal stem cells(LSCs)microenvironment.METHODS: The hiPSC cell lines were established in vitro, and hiPSCs were co-cultured with corneal stromal cells in transwell system, which simulated the LSC microenvironment. Bone morphogenetic protein 4(BMP4)and a specific transforming growth factor β inhibitor(SB431542)were added to improve the differentiation efficacy. The expression of corneal epithelial cell-specific markers CK3 and CK12, corneal epithelial cell precursor CK15, and the limbal stem cell markers ABCG5 were determined by immunofluorescence staining and flow cytometry.RESULTS: The hiPSCs were actively proliferated in vitro, and immunofluorescence staining showed positive stem cell-specific markers OCT4, SOX2, TRA-1-60 and NANOG. Furthermore, hiPSCs co-cultured with corneal stromal cells exhibited LSCs markers ABCG5 and corneal epithelial cell precursor markers CK15 were positive; however, corneal epithelial cell markers CK3 and CK12 were negative. With the addition of BMP4 and SB431542, hiPSCs showed positive expression of CK3, and the CK3 expression increased over the time.CONCLUSION: With the addition of SB431542 and BMP4, hiPSCs cultured in simulated LSCs microenvironment could differentiate into corneal epithelial cells.
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[基金项目]
深圳市科技创新委员会基础研究自由探索项目(No.JCYJ20180305124320418),深圳市科技创新委员会国际合作研究项目(No.GJHZ20200731095005016)