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

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.

Differentiation Protocol for 3D Retinal Organoids, Immunostaining and Signal Quantitation.

Structures resembling whole organs, called organoids, are generated using pluripotent stem cells and 3D culturing methods. This relies on the ability of cells to self-reorganize after dissociation. In combination with certain supplemented factors, differentiation can be directed toward the formation of several organ-like structures. Here, a protocol for the generation of retinal organoids containing all seven retinal cell types is described. This protocol does not depend on Matrigel, and by keeping the organoids single and independent at all times, fusion is prevented and monitoring of differentiation is improved. Comprehensive phenotypic characterization of the in vitro-generated retinal organoids is achieved by the protocol for immunostaining outlined here. By comparing different stages of retinal organoids, the decrease and increase of certain cell populations can be determined. In order to be able to detect even small differences, it is necessary to quantify the immunofluorescent signals, for which we have provided a detailed protocol describing signal quantitation using the image-processing program Fiji. © 2020 The Authors. Basic Protocol 1: Differentiation protocol for 3D retinal organoids Basic Protocol 2: Immunostaining protocol for cryosections of retinal organoids Support Protocol: Embedding and sectioning protocol for 3D retinal organoids Basic Protocol 3: Quantitation protocol using Fiji.

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

Retinoblastoma is a childhood tumor of the retina that is caused mostly by biallelic inactivation of the tumor suppressor gene RB1. To generate a research resource, we abrogated expression of RB1 in H9 hESCs by CRISPR/Cas9 induced deletion of the RB1 promoter, either on one or on both alleles. This enables studies on the role of RB1 loss during differentiation, for example in differentiation towards neural retina. The generation of three isogenic lines per deletion state enables validation of phenotypic results in independent clonal lines.