Fig 1.
Production of limbal-like cells (LiPSC) from human iPSC.
(A) Schematic figure displaying a multistep protocol of differentiating human iPSC to limbal-like cells. Time of treatment is illustrated by horizontal arrays. ROCK inhibitor (RI); TGF-beta inhibitor (SB431542). Morphology of the cells at each stage is illustrated at the top and compared to LEC (right). Scale bar, 50 μm. (B) Flow cytometry analysis of undifferentiated iPSC and LiPSC at P1 with anti-CD104 antibody. n = 3. **P <0.01 statistically significant by student's t-test.
Fig 2.
LiPSC express limbal-specific markers.
(A-B) Reverse transcriptase-quantitative PCR (RT-qPCR) on the expression of limbal-specific genes in LiPSC at P0 and P1 (A) and in LiPSC at P1 or HCE (B). The relative expression of each transcript was calculated as a fold-change relative to undifferentiated iPSC (A) or LEC (B). The figures A and B represent the mean of 4 independent biological samples, performed in triplicate. (C) Representative immunofluorescent staining of LiPSC at P2 for the expression of K14, Pax6, P63, E-Cadherin and K3 at 60% confluency or 100% confluency (bottom right). (D) P2 cells after thawing were immunostained with K14, E-cadherin and K3. Hoechst is shown in blue. Scale bar, 100 μm for all pictures.
Fig 3.
LiPSC at P1 are homogenous cell population.
Percentages of Pax6, K14 and E-cadherin positive cells in undifferentiated iPSC, LiPSC at P0 and P1 were determined by bar chart of flow cytometry analysis from 5 independent experiments, *P <0.001, **P <8E-7 statistically significant by student's t-test.
Fig 4.
(A). Cell numbering was used from 3 independent experiments to calculate the doubling time rate between LEC, LiPSC and HCE. (B,C). MMP-9 content was analyzed from conditioned medium of different cell types. ELISA (B) quantified its relative amount (ng/ml) and its activity was analyzed by zymography (C). HK: primary human keratinocytes; DF: primary human dermal fibroblasts.
Fig 5.
Cytotoxicity test for LiPSC as compared to LEC and HCE.
LEC, LiPSC (P0 to P2) and HCE were treated for 48H with SDS (A) or with benzalkonium (B) at different concentrations, as detailed in Methods section. Cell viability was calculated as compared to untreated cells in six replicate. *P <0.05, **P <0.01 statistically significant by student's t-test. n = 6.