Differences in the Activity of Endogenous Bone Morphogenetic Protein Signaling Impact on the Ability of Induced Pluripotent Stem Cells to Differentiate to Corneal Epithelial-Like Cells

Abstract

Cornea is a clear outermost layer of the eye which enables transmission of light onto the retina. The transparent corneal epithelium is regenerated by limbal stem cells (LSCs), whose loss/dysfunction results in LSCs deficiency (LSCD). Ex vivo expansion of autologous LSCs obtained from patient's healthy eye followed by transplantation onto the LSCs damaged/deficient eye, has provided a successful treatment for unilateral LSCD. However, this is not applicable to patient with total bilateral LSCD, where LSCs are lost/damaged from both eyes. We investigated the potential of human induced pluripotent stem cell (hiPSC) to differentiate into corneal epithelial-like cells as a source of autologous stem cell treatment for patients with total bilateral LSCD. Our study showed that combined addition of bone morphogenetic protein 4 (BMP4), all trans-retinoic acid and epidermal growth factor for the first 9 days of differentiation followed by cell-replating on collagen-IV-coated surfaces with a corneal-specific-epithelial cell media for an additional 11 days, resulted in step wise differentiation of human embryonic stem cells (hESC) to corneal epithelial progenitors and mature corneal epithelial-like cells. We observed differences in the ability of hiPSC lines to undergo differentiation to corneal epithelial-like cells which were dependent on the level of endogenous BMP signaling and could be restored via the activation of this signaling pathway by a specific transforming growth factor β inhibitor (SB431542). Together our data reveal a differential ability of hiPSC lines to generate corneal epithelial cells which is underlined by the activity of endogenous BMP signaling pathway. Stem Cells 2018;36:337-348.

Keywords: Bone morphogenetic protein 4; Corneal epithelial cells; Corneal epithelial progenitors; Epidermal growth factor; Human embryonic stem cell; Human induced pluripotent stem cell; Retinoic acid.

Figure 1

Schematic outline of the differentiation process. The differentiation process is divided into two stages, early (days 0–9) and advanced (days 10–20) (A). Endpoint analyses included quantitative real‐time polymerase chain reaction (qRT‐PCR), immunocytochemistry, and colony forming efficiency. List of differentiation induction media components for each of the eight groups (B). Downregulation of OCT4 from days 0 to 9 for all the groups in the three cell lines used (H9, SB‐Ad2, and SB‐Ad3) assessed by qRT‐PCR (C). Data are presented as mean ± SEM, n = 3. *, statistically different compared with day 0. ****, p < .0001. Abbreviations: BMP4, bone morphogenetic protein 4; CFE, colony forming efficiency; EGF, epidermal growth factor; ICC, immunocytochemistry; IWP‐2, Wnt antagonist II; qPCR, quantitative polymerase chain reaction.

Figure 2

Bone morphogenetic protein 4 (BMP4), retinoic acid, and epidermal growth factor improve early corneal/limbal differentiation process. Quantitative real‐time polymerase chain reaction analysis of BRACHYURY, RAX, BMP4, CK8, ECADHERIN, and ΔNp63 genes for groups G2–G8 compared with control group (G1) presented as z scores (A–F). z score was calculated using the following formula: z score = D/SEM where D is the difference between the two means and SEM is the standard error of mean (computed from the data). Dotted lines represent 90% confidence level. Abbreviation: BMP4, bone morphogenetic protein 4.

Figure 3

ΔNp63 protein expression and colony forming efficiency. Immunocytochemistry analyses for differentiated cells on day 9 for G1, G2, G3, and G5. Representative immunofluorescence images showing positive ΔNp63 and PAX6 nuclear staining in G3 of H9 cells (A) and percentage of ΔNp63 and PAX6 positive cells for H9, SB‐Ad2, and SB‐Ad3 (B). Colony forming efficiency assays for the three cell lines on day 9 for G1, G2, G3 G5, and human limbal epithelium (C). Scale bar = 50 µm. Data are presented as mean ± SEM, n = 3. *, statistically different compared with G1. *, p < .05; **, p < .01; ***, p < .001; ****, p < .0001. Abbreviations: CFE, colony forming efficiency; DAPI, 4′,6‐diamidino‐2‐phenylindole.

Figure 4

Cell morphology at advanced stage of differentiation and molecular characterization. Differentiated G1, G2, G3, and G5 of H9 cells morphological appearance on day 20 (A), and quantitative real‐time polymerase chain reaction analysis of corneal and limbal epithelial genes (B). Data are presented as mean ± SEM, n = 3. Scale bar = 50 µm. *, statistically different compared with G1. *, p < .05; **, p < .01.

Figure 5

ΔNp63 protein expression and colony forming efficiency at day 20 of differentiation. Percentage of ΔNp63 positive cells for H9, SB‐Ad2, and SB‐Ad3 cells on day 20 for G1, G2, G3, and G5 (A). Colony forming efficiency for the three cell lines on day 20 for G1, G2, G3 G5, and human limbal epithelium (B). Data are presented as mean ± SEM, n = 3. *, statistically different compared with G1 of the same cell line. *, p < .05; **, p < .01; ***, p < .001; ****, p < .0001. #, statistically different compared with the other group; ##, p < .01. Abbreviation: CFE, colony forming efficiency.

Figure 6

SB431542 exposure during early differentiation enhanced the hiPSC differentiation to corneal epithelial progenitors. Schematic outline for optimization experiment using SB431542 (A). Quantitative real‐time polymerase chain reaction analysis of the putative limbal stem cells (ΔNp63) gene for SB‐Ad3 cells from G5 subgroups on day 20 of optimization experiment following different durations of SB431542 exposure (B). Colony forming efficiency assays for the SB431542 exposed and unexposed G5 subgroups on day 20 (C). Data presented as mean ± SEM, n = 3. *, statistically different compared with untreated group. **, p < .01; ***, p < .001; ****, p < .0001. ns, no significant difference. Abbreviations: CFE, colony forming efficiency; ICC, immunocytochemistry; FBS, fetal bovine serum; qPCR, quantitative polymerase chain reaction.