Differentiation Potential of Limbal Epithelium-derived and Fibroblast-derived iPSCs to Corneal Epithelial Cells

Abstract

Purpose: Induced pluripotent stem cells (iPSC) with unlimited proliferation capacity and autologous alternatives may offer new advances in cell therapy for limbal epithelial stem cell (LESC) deficiency. Our purpose was to directionally differentiate limbal epithelium-derived and fibroblast-derived iPSC into LESC using a combination of growth factors and Wnt modulators.

Methods: iPSCs were generated from fibroblast (iPSC-F) or primary LESC (iPSC-L) cultures using nonintegrating Yamanaka’s plasmids. iPSCs were kept on growth factor-reduced Matrigel in mTeSR1 medium. For differentiation, Accutase-treated iPSCs were seeded on a mixture of basement membrane (BM) proteins: fibronectin, type IV collagen, and laminin [FCL] in Epilife medium with defined growth supplements B-27 and N-2, human keratinocyte growth supplements, and an antibiotic-antimycotic mixture along with 10 µM ROCK inhibitor Y27632. iPSC-L and iPSC-F were treated with single or combined modulators. They were fixed in 4% p-formaldehyde on days 4, 8 and 15 after treatment onset, and indirect immunofluorescent staining was performed.<br /> <br />

Results: iPSCs were treated to differentiate into epidermal and eventually corneal epithelial lineage. Treatment with bone morphogenetic protein-4 (BMP-4) for two days gave rise to a relatively pure population of K18+ and PAX6+ cells by day 4. It was followed by increased expression of LESC markers, ΔNp63, and keratins 14, 15, and 17 at day 8 only in a subpopulation of treated cells. Addition of GSK-3β inhibitor CHIR99021 (Wnt activator) to BMP-4 promoted cell proliferation and survival. In contrast, TGF-β inhibition with SB-505124 at different time points caused cell loss; however, surviving cells were LESC marker-positive. A combination of EGF, KGF, BMP-4 and GSK-3β inhibitor was able to drive the expression of ΔNp63, keratin 14 and PAX6 in limbal iPSC-L markedly better than in fibroblast iPSC-F. This difference was augmented by the addition of limbal BM-expressed laminin-521 to the FCL substratum.

Conclusions: iPSC-L and iPSC-F were differentiated into corneal epithelial lineage with elevated expression of corneal epithelial markers by a combination of soluble factors without feeder cells. Limbal-derived iPSC showed a greater potential for differentiation into corneal epithelium both on FCL and FCL supplemented with a limbal BM component, laminin-521.