Directed Differentiation of Human Pluripotent Stem Cells towards Corneal Endothelial-Like Cells under Defined Conditions

被引:15
|
作者
Gronroos, Pyry [1 ]
Ilmarinen, Tanja [1 ]
Skottman, Heli [1 ]
机构
[1] Tampere Univ, Fac Med & Hlth Technol, Arvo Ylpon Katu 34, Tampere 33520, Finland
基金
芬兰科学院;
关键词
corneal endothelial cells; human pluripotent stem cells; neural crest cells; differentiation; small molecule; retinoic acid;
D O I
10.3390/cells10020331
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
The most crucial function of corneal endothelial cells (CEnCs) is to maintain optical transparency by transporting excess fluid out of stroma. Unfortunately, CEnCs are not able to proliferate in vivo in the case of trauma or dystrophy. Visually impaired patients with corneal endothelial deficiencies that are waiting for transplantation due to massive global shortage of cadaveric corneal transplants are in a great need of help. In this study, our goal was to develop a defined, clinically applicable protocol for direct differentiation of CEnCs from human pluripotent stem cells (hPSCs). To produce feeder-free hPSC-CEnCs, we used small molecule induction with transforming growth factor (TGF) beta receptor inhibitor SB431542, GSK-3-specific inhibitor CHIR99021 and retinoic acid to guide differentiation through the neural crest and periocular mesenchyme (POM). Cells were characterized by the morphology and expression of human (h)CEnC markers with immunocytochemistry and RT-qPCR. After one week of induction, we observed the upregulation of POM markers paired-like homeodomain transcription factor 2 (PITX2) and Forkhead box C1 (FOXC1) and polygonal-shaped cells expressing CEnC-associated markers Zona Occludens-1 (ZO-1), sodium-potassium (Na+/K+)-ATPase, CD166, sodium bicarbonate cotransporter 1 (SLC4A4), aquaporin 1 (AQP1) and N-cadherin (NCAD). Furthermore, we showed that retinoic acid induced a dome formation in the cell culture, with a possible indication of fluid transport by the differentiated cells. Thus, we successfully generated CEnC-like cells from hPSCs with a defined, simple and fast differentiation method.
引用
收藏
页码:1 / 15
页数:15
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