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Kaushali Thakore-Shah, Sophie Xiaohui Deng; Differentiation of Human Pluripotent Stem Cells towards Human Corneal Endothelial Cells via Neural Crest Lineage . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3464.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a defined protocol for efficient derivation of human corneal endothelial cells (HCECs) from human pluripotent stem cells (hPSC).
HPSCs were cultured on human recombinant Laminin-521 matrix in defined media. For differentiation of hPSCs, the cells were switched to a growth factor-free defined media conditioned on adult corneal stroma cells. Differentiation to neural crest cells (NCC) was induced by temporal modulation of BMP, TGF-beta, and WNT signaling. Differentiation status was assessed by staining for the neural crest markers p75NTR and HNK-1. NCC cells expressing p75NTR were harvested using FACS and cultured in growth factor-free defined media. Cells were induced to differentiate towards HCEC by addition of the epidermal and fibroblast growth factors (EGF and FGF), with or without concomitant activation of WNT signaling. Immunofluorescence analysis was used to test for expression of HCEC markers.<br />
By day 8 of NCC differentiation, cells exhibiting characteristic spindle shaped morphology of NCC were observed in culture. FACS analysis of day 11 cells revealed that majority of cells expressed the neural crest markers p75NTR and HNK-1. Five days after induction of p75NTR expressing NCCs, the cells exposed to WNT signaling activation were heterogeneous in morphology, with sporadic polygonal cells. In contrast, cultures not exposed to WNT activation had several compact cell clusters with polygonal HCEC-like morphology. Immunofluorescence analysis of the HCEC-like clusters revealed cell membrane staining of the HCEC markers ZO1 and N+K+-ATPase.
We have developed a robust protocol for efficient differentiation of hPSCs to HCECs using a serum-free defined system.
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