Retinoblastoma from human stem cell-derived retinal organoids

Jackie L Norrie,Michael A Dyer,Colleen Reilly,Jiakun Zhang,Matthew Wilson,Karen Lai,Sariah Allen,Lyra Griffiths,Xiang Chen,Xin Zhou,Elizabeth Stewart,Anjana Nityanandam,Hongjian Jin,Brent Orr,Dianna Johnson,Rachel Brennan,Gang Wu,Kyle Newman,Quynh Tran

doi:10.1038/s41467-021-24781-7

Abstract

Retinoblastoma is a childhood cancer of the developing retina that initiates with biallelic inactivation of the RB1 gene. Children with germline mutations in RB1 have a high likelihood of developing retinoblastoma and other malignancies later in life. Genetically engineered mouse models of retinoblastoma share some similarities with human retinoblastoma but there are differences in their cellular differentiation. To develop a laboratory model of human retinoblastoma formation, we make induced pluripotent stem cells (iPSCs) from 15 participants with germline RB1 mutations. Each of the stem cell lines is validated, characterized and then differentiated into retina using a 3-dimensional organoid culture system. After 45 days in culture, the retinal organoids are dissociated and injected into the vitreous of eyes of immunocompromised mice to support retinoblastoma tumor growth. Retinoblastomas formed from retinal organoids made from patient-derived iPSCs have molecular, cellular and genomic features indistinguishable from human retinoblastomas. This model of human cancer based on patient-derived iPSCs with germline cancer predisposing mutations provides valuable insights into the cellular origins of this debilitating childhood disease as well as the mechanism of tumorigenesis following RB1 gene inactivation.

Highlights

Retinoblastoma is a childhood cancer of the developing retina that initiates with biallelic inactivation of the RB1 gene
Offspring of the family cohorts presented with bilateral retinoblastoma (n = 4) and trilateral disease (n = 1) at diagnosis, and the parents had no evidence of tumors by ophthalmologic screening
Using the H9 ESC line and the induced pluripotent stem cells (iPSCs) lines produced from participants with germline RB1 alterations, we showed that molecular, cellular, functional, and anatomic features of retinal organoids produced by the Sasai method and the 3DRET method were indistinguishable in side-by-side comparisons retinal specification retinal differentiation

Summary

Introduction

Retinoblastoma is a childhood cancer of the developing retina that initiates with biallelic inactivation of the RB1 gene. Conditional inactivation of Rb1 and p107 or Rb1 and p130 can lead to retinoblastoma[10,11,12,13,14] in mice and more recently, a murine model of the MYCN amplified form of retinoblastoma was developed[15] While these genetically engineered mouse models (GEMMs) have provided important insights into retinoblastoma biology, there are some differences in the molecular and cellular features of retinoblastoma across species[11,14,16,17]. In a recent epigenetic study of the developing retina and retinoblastoma from mice and humans, the human tumor epigenome mapped to a later developmental stage than that of the mouse tumors[18] Those differences may be related to the initiation of tumorigenesis during retinal development, the cellular origin of the tumors, or both[19]. Parallel experiments were performed with targeted RB1 gene inactivation using CRISPR-

Methods

Results

Conclusion