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

• Published in: Biology open Vol. 7; no. 5; p. bio032102
• Main Authors: Wagoner, Michael D, Bohrer, Laura R, Aldrich, Benjamin T, Greiner, Mark A, Mullins, Robert F, Worthington, Kristan S, Tucker, Budd A, Wiley, Luke A
• Format: Journal Article
• Language: English
• Published: England The Company of Biologists Ltd 2018; The Company of Biologists
• Subjects: AP-2 protein; Aquaporin 1; c-Myc protein; Cell differentiation; Cell morphology; Cornea; Corneal endothelial cells; Cytology; Differentiation; Differentiation (biology); Endothelial cells; Endothelium; Fibroblasts; Induced pluripotent stem cells; KLF4 protein; Morphology; Myc protein; Na+/K+-exchanging ATPase; Nerve growth factor receptors; Neural crest; Neural crest cells; Oct-4 protein; Pathophysiology; Pax3 protein; Phenotypes; Platelet-derived growth factor; Platelet-derived growth factor BB; Pluripotency; Polymerase chain reaction; Proteins; Skin; Sox10 protein; Sox9 protein; Stem cells; Zonula occludens-1 protein; Corneal endothelial cells; Differentiation; Induced pluripotent stem cells; Neural crest cells
• ISSN: 2046-6390; 2046-6390
• DOI: 10.1242/bio.032102

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 , , and 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 , , , and 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 , , , and 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.