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Jiagang Zhao, Christine Sutu, Joanne W Ho, Natalie A Afshari; Differentiation of Corneal Endothelial Cells from Human Pluripotent Stem Cells in Culture. Invest. Ophthalmol. Vis. Sci. 2016;57(12):881.
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© ARVO (1962-2015); The Authors (2016-present)
We seek to develop an alternative approach to corneal transplantation for treatment of diseases of endothelium. We aim to have unlimited corneal endothelia cells (CECs) that can be derived from human pluripotent stem cells (hPSCs) in a culture system.
The CEC induction is carried out under the defined small molecule-driven conditions and in a stepwise fashion. During the initial phase, the monolayer of hPSCs is massively converted into the eye field stem cells (hEFSCs). Subsequently, hEFSCs are further directed toward the fate of ocular neural crest cell. Finally, hEFSC-derived neural crest cells are induced and they formed a corneal endothelium-like sheet of cells.
Under the feeder-free and serum-free conditions, we were able to convert majority of hPSCs into neural crest cells via an eye field lineage. The eye field cell identity was confirmed by immunocytochemical labeling of key transcription factors. Over 80% of cells were positive for both PAX6 and LHX2, two critical fate restriction factors for eye field cells. This was further confirmed by QPCR on the elevated expression of a panel of early eye field transcription factors. The induction of neural crest cells was initiated by promoting Wnt signaling in hEFSCs culturing at low density. Within two weeks of induction, majority of cells expressed typical neural crest markers p75NTR and HNK-1. These cells also retained stemness as evidenced by the expression of NESTIN. hEFSC-derived neural crest cells can be propagated and cryopreserved. Finally, the CEC sheet formation was induced from the adherent neural crest cells in the presence of a ROCK inhibitor. The polygonal shaped CEC-like cells became visible after a week in culture. The expression of typical CEC markers such as ZO-1, N-cadherin and Na+/K+-ATPase were also detected.
Unlike previously reported induction methods, our work demonstrates a novel small molecule-based approach to derive human CECs from hPSCs under defined culture conditions. It mimics the lineage specification of CEC in vivo. It provides a robust and efficient experimental mean to generate ocular neural crest cells. By using the self-renewable neural crest cells as a source, unlimited CEC sheet can be generated in culture for drug discovery and therapeutic use.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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