Feeder-free differentiation of cells exhibiting characteristics of corneal endothelium from human induced pluripotent stem cells

Abstract

The purpose of this study was to devise a strategy for the derivation of corneal endothelial cells (CEnCs) from adult fibroblast-derived induced pluripotent stem cells (iPSCs). IPSCs were generated from an adult human with normal ocular history via expression of <i>OCT4</i>, <i>SOX2</i>, <i>KLF4</i> and <i>c-MYC</i> Neural crest cells (NCCs) were differentiated from iPSCs via addition of CHIR99021 and SB4315542. NCCs were driven toward a CEnC fate via addition of B27, PDGF-BB and DKK-2 to CEnC media. Differentiation of NCCs and CEnCs was evaluated via rt-PCR, morphological and immunocytochemical analysis. At 17 days post-NCC induction, there were notable changes in cell morphology and upregulation of the neural crest lineage transcripts <i>PAX3</i>, <i>SOX9</i>, <i>TFAP2A</i>, <i>SOX10</i> and <i>p75NTR</i> and the proteins p75/NGFR and SOX10. Exposure of NCCs to B27, PDGF-BB and DKK-2 induced a shift in morphology from a spindle-shaped neural phenotype to a tightly-packed hexagonal appearance and increased expression of the transcripts <i>ATP1A1</i>, <i>COL8A1</i>, <i>COL8A2</i>, <i>AQP1</i> and <i>CDH2</i> and the proteins ZO-1, N-Cad, AQP-1 and Na⁺/K⁺ATPase. Replacement of NCC media with CEnC media on day 3, 5 or 8 reduced the differentiation time needed to yield CEnCs. IPSC-derived CEnCs could be used for evaluation of cornea endothelial disease pathophysiology and for testing of novel therapeutics.