Accelerated Limbal Epithelial Differentiation of Human Induced Pluripotent Stem Cells Using a Defined Keratinocyte Serum-Free Medium

Abstract

AbstractPurposeTreatment of bilateral limbal stem cell deficiency (LSCD) is challenging due to the limited autologous stem cell sources. This study aimed to differentiate human induced pluripotent stem cells (hiPSCs) into limbal epithelial stem cells (LESCs) using a defined keratinocyte serum-free medium (DKSFM).MethodsA fully characterized hiPSC line was committed to ectodermal differentiation using Essential 6 (E6) medium supplemented with 10 µM Y-27632 (Day 1), 10 µM SB-505124 plus 50 ng/ml bFGF (Day 2) and 25 ng/ml BMP-4 (Days 3 and 4). Differentiation was continued in DKSFM for an additional 21 days. Quantitative PCR (qPCR) and/or immunocytochemistry (ICC) for pluripotency, proliferation, LESC, and corneal epithelial markers were performed on samples collected at days 5, 10, 15, and 25 (D5 to D25) and compared with undifferentiated hiPSCs (UD).ResultsqPCR revealed a significant decrease in the expression ofOCT4andNANOGand a significant increase inABCG2andTP63following ectodermal induction (i.e., D5), compared with UD (P < 0.05). The expression levels ofKi67,ABCG2,TP63, andCK14were significantly higher at D10, compared with D5 and D25 (P < 0.05). The ratio of p63α-positive cells was 71% and 56% in D10 and D15 cells, respectively (P < 0.05).DiscussionOur method resulted in a limited but rapid differentiation of hiPSCs into LESC-like cells. The LESC-like cells appeared as early as 5 days following ectodermal induction and their population peaked after 10 days. Upon further optimization and validation, DKSFM can be used for rapid limbal epithelial differentiation of hiPSCs.