模拟角膜缘干细胞微环境诱导人多潜能干细胞分化为角膜上皮细胞的研究

Potential of human induced pluripotent stem cells differentiating into corneal epithelial cells in simulated limbal stem cell microenvironment

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。

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.