Biallelic and monoallelic deletion of the RB1 promoter in six isogenic clonal H9 hESC lines

Döpper, Hannah; Horstmann, M.; Menges, Julia; Bozet, Morgane; Kanber, Deniz; Steenpaß, Laura

doi:10.1016/j.scr.2020.101779

Cited by 2 publications

(2 citation statements)

References 2 publications

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“…To relate the observed phenotype of RB1 ko to changes in gene expression, transcriptomes of organoids were generated. To validate and strengthen the obtained results, organoids heterozygous or homozygous for deletion of the entire exon 1 of RB1 were included in transcriptome analysis (Table S2) [30]. Analysis of such organoids showed absence of pRB and expression of retinal markers comparable to organoids with modifications in RB1 exon 3 (Figures S2 and S10-S12).…”

Section: Rna-seq Confirmed Aberrant Retinal Differentiation and Proli...mentioning

confidence: 87%

“…Cell splitting was performed with Gentle Cell Dissociation Reagent (STEM-CELL Technologies, Cologne, Germany) at a ratio of 1:8 every 4-5 days. Human ESCs were genetically modified by CRISPR/Cas9 as described [29,30]. Cell lines H9_RB1ex3_G12LS and H9_RB1ex3_C07, modified in RB1 exon 3, carry an identical deletion of seven basepairs (LRG_517t1:c.374_380del p.Glu125Valfs*9; GRCh38: Chr13:48342708-48342714) in heterozygous and homozygous states, respectively.…”

Section: Generation and Cultivation Of Hescsmentioning

confidence: 99%

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RB1-Negative Retinal Organoids Display Proliferation of Cone Photoreceptors and Loss of Retinal Differentiation

Kanber

Woestefeld

Döpper

et al. 2022

Cancers

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Retinoblastoma is a tumor of the eye in children under the age of five caused by biallelic inactivation of the RB1 tumor suppressor gene in maturing retinal cells. Cancer models are essential for understanding tumor development and in preclinical research. Because of the complex organization of the human retina, such models were challenging to develop for retinoblastoma. Here, we present an organoid model based on differentiation of human embryonic stem cells into neural retina after inactivation of RB1 by CRISPR/Cas9 mutagenesis. Wildtype and RB1 heterozygous mutant retinal organoids were indistinguishable with respect to morphology, temporal development of retinal cell types and global mRNA expression. However, loss of pRB resulted in spatially disorganized organoids and aberrant differentiation, indicated by disintegration of organoids beyond day 130 of differentiation and depletion of most retinal cell types. Only cone photoreceptors were abundant and continued to proliferate, supporting these as candidate cells-of-origin for retinoblastoma. Transcriptome analysis of RB1 knockout organoids and primary retinoblastoma revealed gain of a retinoblastoma expression signature in the organoids, characterized by upregulation of RBL1 (p107), MDM2, DEK, SYK and HELLS. In addition, genes related to immune response and extracellular matrix were specifically upregulated in RB1-negative organoids. In vitro retinal organoids therefore display some features associated with retinoblastoma and, so far, represent the only valid human cancer model for the development of this disease.

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Section: Rna-seq Confirmed Aberrant Retinal Differentiation and Proli...mentioning

confidence: 87%

Section: Generation and Cultivation Of Hescsmentioning

confidence: 99%

RB1-Negative Retinal Organoids Display Proliferation of Cone Photoreceptors and Loss of Retinal Differentiation

Kanber

Woestefeld

Döpper

et al. 2022

Cancers

Self Cite

View full text Add to dashboard Cite

show abstract

Comparative landscape of genetic dependencies in human and chimpanzee stem cells

She

Fair

Schaefer

et al. 2023

Cell

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Biallelic and monoallelic deletion of the RB1 promoter in six isogenic clonal H9 hESC lines

Cited by 2 publications

References 2 publications

RB1-Negative Retinal Organoids Display Proliferation of Cone Photoreceptors and Loss of Retinal Differentiation

RB1-Negative Retinal Organoids Display Proliferation of Cone Photoreceptors and Loss of Retinal Differentiation

Comparative landscape of genetic dependencies in human and chimpanzee stem cells

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