Chen Jie, Ou Qingjian, Wang Zhe, Liu Yifan, Hu Shuqin, Liu Yumeilan, Tian Haibin, Xu Jingying, Gao Furong, Lu Lixia, Jin Caixia, Xu Guo-Tong, Cui Hong-Ping
Department of Ophthalmology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China.
Department of Ophthalmology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, China.
Front Bioeng Biotechnol. 2021 Dec 15;9:788987. doi: 10.3389/fbioe.2021.788987. eCollection 2021.
Corneal endothelial cells (CECs) serve as a barrier and foothold for the corneal stroma to maintain the function and transparency of the cornea. Loss of CECs during aging or disease states leads to blindness, and cell replacement therapy using either donated or artificially differentiated CECs remains the only curative approach. Human induced pluripotent stem cells (hiPSCs) that were cultured in chemically defined medium were induced with dual-SMAD inhibition to differentiate into neural crest cells (NCCs). A small-molecule library was screened to differentiate the NCCs into corneal endothelial-like cells. The characteristics of these cells were identified with real-time PCR and immunofluorescence. Western blotting was applied to detect the signaling pathways and key factors regulated by the small molecules. We developed an effective protocol to differentiate hiPSCs into CECs with defined small molecules. The hiPSC-CECs were characterized by ZO-1, AQP1, Vimentin and Na/K-ATPase. Based on our small-molecule screen, we identified a small-molecule combination, A769662 and AT13148, that enabled the most efficient production of CECs. The combination of A769662 and AT13148 upregulated the PKA/AKT signaling pathway, FOXO1 and PITX2 to promote the conversion of NCCs to CECs. We established an efficient small molecule-based method to differentiate hiPSCs into corneal endothelial-like cells, which might facilitate drug discovery and the development of cell-based therapies for corneal diseases.
角膜内皮细胞（CECs）作为角膜基质的屏障和支撑，以维持角膜的功能和透明度。衰老或疾病状态下CECs的丧失会导致失明，而使用捐赠的或人工分化的CECs进行细胞替代疗法仍然是唯一的治愈方法。在化学成分明确的培养基中培养的人诱导多能干细胞（hiPSCs）通过双SMAD抑制诱导分化为神经嵴细胞（NCCs）。筛选小分子文库以将NCCs分化为角膜内皮样细胞。通过实时PCR和免疫荧光鉴定这些细胞的特征。应用蛋白质印迹法检测小分子调节的信号通路和关键因子。我们开发了一种有效的方案，用特定的小分子将hiPSCs分化为CECs。hiPSC-CECs通过紧密连接蛋白1（ZO-1）、水通道蛋白1（AQP1）、波形蛋白和钠钾ATP酶进行表征。基于我们的小分子筛选，我们鉴定出一种小分子组合，A769662和AT13148，其能够最有效地产生CECs。A769662和AT13148的组合上调蛋白激酶A（PKA）/蛋白激酶B（AKT）信号通路、叉头框蛋白O1（FOXO1）和垂体特异性转录因子2（PITX2），以促进NCCs向CECs的转化。我们建立了一种基于小分子的有效方法，将hiPSCs分化为角膜内皮样细胞，这可能有助于药物发现和角膜疾病细胞疗法的开发。
