Identification of stable housekeeping genes for induced pluripotent stem cells and -derived endothelial cells for drug testing

Abstract

There are no validated housekeeping genes in induced pluripotent stem cells (iPSC) and derived endothelial iPSC (iPSC-EC). This makes comparison of gene expression levels less reliable especially during drug treatments. Here, we utilized transcriptome sequencing data of iPSC and iPSC-EC with or without CRISPR-Cas9 induced translocation to identify a panel of 15 candidate housekeeping genes. For comparison, five commonly used housekeeping genes (B2M, GAPDH, GUSB, HMBS, and HPRT1) were included in the study. The panel of 20 candidate genes were investigated for their stability as reference genes. This panel was analyzed and ranked based on stability using five algorithms, delta Ct, bestkeeper, geNorm, Normfinder, and Reffinder. Based on the comprehensive ranking of Reffinder, the stability of the top, RPL36AL and TMBIM6, and bottom two genes, UBA1 and B2M, was further studied in iPSC-EC with or without genetic manipulation, and after treatment with telatinib. Using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), it was shown that gene expression of the top two housekeeping genes, RPL36AL and TMBIM6, remained stable during drug treatment. We identified a panel of housekeeping genes that could be utilized in various conditions using iPSC and iPSC derived endothelial cells as well as genetically modified iPSC for drug treatment.