Characterization of human‐induced pluripotent stem cells carrying homozygous RB1 gene deletion. (28th April 2020)

• Record Type: Journal Article
• Authors: Deng, Xiaoyue; Iwagawa, Toshiro; Fukushima, Masaya; Watanabe, Sumiko
• Is Part Of: Genes to cells. Volume 25:Number 7(2020)
• Page Start: 510
• Page End: 517
• Publication Date: 2020-04-28
• Subjects: genome editing -- hiPSC -- organoid -- proliferation -- RB1 -- retina -- retinoblastoma; Cytogenetics -- Periodicals; Cells -- Mechanical properties -- Periodicals; Molecular genetics -- Periodicals; Genes -- Periodicals; Molecular biology -- Periodicals; Cytology -- Periodicals; Biomechanics -- Periodicals; 571.6
• DOI: 10.1111/gtc.12771
• Languages: English
• ISSNs: 1356-9597

Abstract

Abstract: Retinoblastoma is an infant cancer that results from loss of RB1 expression in both alleles. The RB1 gene was the first reported cancer suppressor gene; however, the mechanism by which RB1 loss causes cancer in the retina has not yet been clarified. Human‐induced pluripotent stem cells (iPSCs) provide an ideal tool for mechanistic research regarding retinoblastoma. However, because RB1 is a tumor suppressor, loss of both alleles of RB1 in human iPS cells may affect the phenotype of the cells. To examine this possibility, we established human iPSCs with deletions in both alleles of RB1 by CRISPR/Cas9 technique to characterize the associated phenotype. We first examined the expression of RB1 transcripts by RT‐qPCR, and RB1 transcripts were expressed in immature hiPSCs and then the expression levels of RB1 transcripts consistently increased during retinal organoid differentiation in human iPSCs. Expression levels of immature markers including SSEA4, OCT3/4 and NANOG were indistinguishable between control iPSCs and RB1 knockout iPSCs. Proliferative activity was also unaffected by homozygous RB1 deletion. Taken together, we showed that homozygous deletion of RB1 did not affect the maturation and proliferation statuses of human iPSCs. Abstract : RB1 expression increased when human iPSC differentiate into retinal lineage. Deletion of RB1 in human iPSC did not affect their proliferation. RB1 ablation did not affect immature marker expression.