Human induced pluripotent stem cells (hiPSC)‐derived endothelial cells: new opportunity for corneal bioengineering

Abstract

AbstractPurpose hiPSCs can self‐renew indefinitely, while maintaining the capacity to differentiate into somatic cells. As such, they represent an essentially inexhaustible source of committed corneal endothelial cells (CECs) of potential use in cell‐based corneal therapies. We present a method of differentiation of hiPSCs into CE‐like cells.Methods hiPSCs reprogrammed from adipocytes by retrovirus (SBI) were expanded in mTSR1 medium. Differentiation was induced by sequential treatment mimicking the normal embryonic endothelial development, using co‐cultures during 3 weeks (not detailed because of ongoing patent application). Cells were sorted using magnetic beads with negative selection with anti‐K3/K12. Sorted cells were subsequently cultivated in maintenance medium for 3‐4 weeks. Differentiation was assessed by immunolabeling of ZO‐1, Glypican‐4, CD200R, Na/K/ATPase, CLCN3, MiTF, K3/K12. Stemcellness was assessed using anti‐OCT‐4.Results hiPSCs were successfully and stably differentiated into a monolayer of tightly packed cells expression typical ECs makers and neither keratocytes nor epithelial markers. We observed the consecutive formation of embryoïd bodies after 4 days (D), neural crest cells after 7D, mesenchymal cells after 10D and cells with CEC phenotype after 15D. They lost expression of OCT‐4.Conclusion hiPSCs are a promising source of CECs. In addition to the short‐circuit current measurement using a Ussing chamber, their ability to deswell corneas is being studied using our innovative bioreactor. Safe sources of cornea‐derived hiPSCs are also under study to facilitate clinical translation. Grants: ABM2013, IUF2012‐17, UJM2013