期刊
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
卷 57, 期 15, 页码 6878-6884出版社
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.16-20024
关键词
pluripotent stem cell; corneal endothelial cell; neural crest; eye field; small molecule
资金
- Research to Prevent Blindness
- National Institutes of Health (NIH
- Bethesda, MD, USA) [P30EY022589]
- [P30NS047101]
PURPOSE. We generate a renewable supply of corneal endothelial cells (CEC) from human pluripotent stem cells (PSCs) under defined culture conditions. METHODS. Corneal endothelial cell induction was driven by small molecules in a stepwise fashion of lineage specification. During the initial phase, PSC fate was restricted to the eye field-like state and became eye field stem cells (EFSCs). In the second phase, PSC-derived EFSCs were further directed toward either neural crest lineage or retinal lineage. The CECs were directly induced from ocular neural crest stem cells (NCSCs) by suppressing TGF-beta and ROCK signaling. RESULTS. Under chemically defined conditions, PSCs were massively converted into EFSCs and subsequently NCSCs. Eye field cell identity was characterized by the expression of key fate restriction factors for early eye field cells, such as PAX6, LHX2, and VSX2. The induction of ocular NCSCs was initiated by promoting WNT signaling in EFSCs. Within 2 weeks of induction, the majority of cells expressed the typical neural crest markers p75NTR and HNK1. Eye field stem cell-derived NCSCs can be propagated and cryopreserved. Subsequently, a CEC monolayer was induced from adherent NCSCs in the presence of small molecular inhibitors to suppress TGF-beta and ROCK signaling. The polygon-shaped CEC-like cells became visible after a week in culture. The NCSC-derived CECs expressed typical CEC markers, such as N-Cadherin and Na+/K+-ATPase. CONCLUSIONS. A novel small molecule-based approach was developed to derive human CECs from PSCs via ocular lineage specification. Moreover, EFSC-derived NCSCs could serve as an immediate source cell for rapid CEC induction in vitro.
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