Comparative Transcriptome analysis of hESCs- and iPSCs-derived Corneal Endothelial Cells

Purpose: We previously reported the generation of corneal endothelial cells (CECs) using the human peripheral blood mononuclear cells (PBMC)-originated induced pluripotent stem cells (iPSCs). Herein, we extend our analysis through RNA-Seq based transcriptome profiling of H9 human embryonic stem cells (hESCs)- and iPSCs-derived CECs. Methods: The PBMC obtained from a healthy donor were subjected to generate iPSCs using Sendai-virus delivery system Cytotune 2.0 whereas the H9 hESCs were obtained commercially. Both hESCs and iPSCs were subjected to the 20-day procedure according to our previously published CECs generation method. The differentiated CECs were characterized by immunocytochemistry. Total RNA from hESCs- and iPSCs-derived CECs was used for the preparation of RNA-Seq libraries, which were sequenced on HiSeq 2500 system. The raw RNA-Seq reads were processed and analyzed using Lasergene Genomics Suite and the expression profiles were examined for differential expression using Spotfire DecisionSite with Functional Genomics. Results: The hESCs and iPSCs were differentiated into CECs through multipotent neural crest cells (NCCs) using a 20-day procedure. The differentiating CECs on day 20 (D20) exhibited a tightly packed hexagonal/polygonal shape, which expressed CECs-associated markers, zona occludens-1 and N-cadherin at the cell boundaries. A total of 180.18, and 179.01 million reads were obtained for hESCs- and iPSCs-derived CECs, respectively. Of these, >97% reads aligned to the human reference genome resulting in >250x sequence coverage for both hESCs- and iPSCs-derived CECs. Additional analysis identified expression (= 0.659 RPKM) of 13,561 and 13,551 genes in hESCs- and iPSCs-derived CECs, respectively, representing ~68% of the total human protein-coding transcriptome expressed in these CECs. Finally, a comparative analysis of both hESCs- and iPSCs-derived CECs transcriptomes identified >95% similarity at the gene level. Conclusion: These analyses reveal an overall similar transcriptional profile in both hESCs- and iPSCs-derived CECs. Overall design: The hESCs and iPSCs were differentiated into CECs through multipotent neural crest cells (NCCs) using a 20-day procedure. Subsequently, RNA-Seq based whole transcriptome profiling was performed for a comparitive analysis of hESCs and iPSCs-derived CECs.