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.

Human pluripotent stem cell-derived retinal pigment epithelium (RPE) transplantation is currently under evaluation as treatment for macular degeneration. For therapeutic applications, cryostorage during cell production is typically needed with potential consequences to cell functionality. We have previously shown that the culture substrate affects human embryonic stem cell-derived RPE (hESC-RPE) properties in fresh cultures. Here, we aimed to further identify the role of RPE basement membrane proteins type IV collagen (Col-IV), laminin (LN), and nidogen-1 in the maturation and functionality of hESC-RPE after cryopreservation. In addition to cell attachment and morphology, transepithelial electrical resistance, expression of key RPE proteins, phagocytosis capacity and Ca2+ signalling were analysed. After cryostorage, attachment of hESC-RPE on culture surfaces coated with Col-IV alone was poor. Combining Col-IV and LN with or without nidogen-1 significantly improved cell attachment and barrier properties of the epithelium. Furthermore, functional homogeneity of the hESC-RPE monolayer was enhanced in the presence of nidogen-1. Our results suggest that the choice of coating proteins for the cell culture may have implications to the functional properties of these cells after cryostorage cell banking.