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Updated: Dec 24, 2025
Reconstruct Human Retinoblastoma In Vitro
Published on: October 11, 2022
Characterization of human-induced pluripotent stem cells carrying homozygous RB1 gene deletion
Xiaoyue Deng1, Toshiro Iwagawa1, Masaya Fukushima1
1Division of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
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.
Insights
Retinoblastoma, an infant cancer, arises from RB1 gene loss. Researchers found that deleting both RB1 alleles in human stem cells did not impact their maturation or proliferation.
Area of Science:
- Oncology
- Stem Cell Biology
- Genetics
Background:
- Retinoblastoma is an infant cancer linked to the loss of the RB1 tumor suppressor gene.
- The precise mechanism of RB1 loss in retinal cancer development remains unclear.
- Human-induced pluripotent stem cells (hiPSCs) offer a valuable model for studying retinoblastoma.
Purpose of the Study:
- To investigate the phenotypic consequences of homozygous RB1 gene deletion in human iPSCs.
- To determine if RB1 loss affects hiPSC maturation and proliferation, crucial for retinoblastoma research.
Main Methods:
- Utilized CRISPR/Cas9 gene editing to create hiPSCs with biallelic RB1 deletions.
- Analyzed RB1 transcript expression using RT-qPCR during retinal organoid differentiation.
- Assessed expression of pluripotency markers (SSEA4, OCT3/4, NANOG) and cell proliferation rates.
Main Results:
- RB1 transcripts were detected in immature hiPSCs and increased during retinal differentiation.
- Homozygous RB1 deletion did not alter the expression of key pluripotency markers.
- Cell proliferation rates remained unaffected in RB1-knockout hiPSCs compared to controls.
Conclusions:
- Homozygous deletion of the RB1 gene does not compromise the pluripotency, maturation, or proliferative capacity of human iPSCs.
- These findings support the use of RB1-deficient hiPSCs for mechanistic studies of retinoblastoma.
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