Directed differentiation of periocular mesenchyme from human embryonic stem cells

Abstract Corneal tissue is the most transplanted of all body tissues. Currently, cadaveric donor tissues are used for transplantation. However, a global shortage of transplant grade material has prompted development of alternative, cell-based therapies for corneal diseases. Pluripotent stem cells are attractive sources of cells for regenerative medicine, because large numbers of therapeutically useful cells can be generated. However, a detailed understanding of how to differentiate clinically relevant cell types from stem cells is fundamentally required. Periocular mesenchyme (POM), a subtype of cranial neural crest, is vital for development of multiple cell types in the cornea, including clinically relevant cells such as corneal endothelium and stromal keratocytes. Herein, we describe protocols for differentiation of POM from pluripotent stem cells. Using defined media containing inhibitors of TGFβ and WNT signalling, we generated neural crest cells that express high levels of the POM transcription factors PITX2 and FOXC1. Furthermore, we identified cells resembling POM in the adult cornea, located in a niche between the trabecular meshwork and peripheral endothelium. The generation and expansion of POM is an important step in the generation of a number of cells types that could prove to be clinically useful for a number of diseases of the cornea.