Purchase this article with an account.
Kaushali Thakore-Shah, Sophie Xiaohui Deng; Effects of Extracellular Matrices in the Derivation of Human Corneal Endothelial-like Cells from Human Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5276.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To compare Matrigel and Laminin-521 as substrates for derivation of human corneal endothelial-like cells (HCEC) from human pluripotent stem cells (hPSC).
Differentiation of hPSCs to HCECs was carried out in two steps: 1) derivation of neural crest cells (NCC) from hPSC, and 2) derivation of HCEC from NCC . The derivation of NCC was performed using a previously published protocol, with the minor modification that hPSCs were cultured on either Matrigel or human recombinant Laminin-521 matrix. Efficiency of derivation was measured by flow cytometry using P75NTR, a marker of NCCs as well as proliferative corneal endothelial cells. P75NTR expressing NCCs as well as unsorted NCCs were then seeded onto Laminin-521 coated wells, and induced to differentiate towards HCEC by addition of growth factors. Expression of P75NTR and three additional HCEC markers was measured by qPCR.
NCCs derived on Matrigel (Mat-NCCs) had a 1.86 fold increase in the number of P75NTR expressing cells compared to NCCs derived on Laminin521 (Lam-NCCs). HCECs derived from Mat-NCCs and Lam-NCCs were tested for marker expression by qPCR. P75NTR expression was higher in HCECs derived from Lam-NCCs compared to HCECs derived from Mat-NCC. As expected, P75NTR expression was also higher in sorted compared to unsorted cells. The three additional corneal endothelial markers tested were expressed at higher levels in HCECs derived from Mat-NCCs compared to HCECs derived from Lam-NCCs. These three markers were also expressed at higher levels in unsorted cells compared to P75NTR sorted cells.
Differentiation of hPSCs to HCECs yields higher expression of HCEC markers when the first step of the differentiation protocol, namely NCC differentiation, is carried out on Matrigel.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
This PDF is available to Subscribers Only