Modeling human retinoblastoma using embryonic stem cell-derived retinal organoids.

Retinoblastoma (Rb) is the most prevalent intraocular malignancy in early childhood. Traditional models are unable to accurately recapitulate the origin and development of human Rb. Here, we present a protocol to establish a novel human Rb organoid (hRBO) model derived from genetically engineered human embryonic stem cells (hESCs). This hRBO model exhibits properties highly consistent with human primary Rb and can be used effectively for dissecting the origination and pathogenesis of Rb as well as for screening of potential therapies. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020).

Genotype-Phenotype Analysis and Mutation Spectrum in a Cohort of Chinese Patients With Congenital Nystagmus.

Purpose: Congenital nystagmus (CN) is a genetically and clinically heterogeneous ocular disorder that manifests as involuntary, periodic oscillations of the eyes. To date, only FRMD7 and GPR143 have been reported to be responsible for causing CN. Here, we aimed to identify the disease-causing mutations and describe the clinical features in the affected members in our study. Methods: All the subjects underwent a detailed ophthalmic examination. Direct sequencing of all coding exons and splice site regions in FRMD7 and GPR143 and a mutation assessment were performed in each patient. Results: We found 14 mutations in 14/37 (37.8%) probands, including nine mutations in the FRMD7 gene and five mutations in the GPR143 gene, seven of which are novel, including c.284G>A(R95K), c.964C>T(P322S), c.284+10T>G, c.901T>C (Y301H), and c.2014_2023delTCACCCATGG(S672Pfs*12) in FRMD7, and c.250+1G>C, and c.485G>A (W162*) in GPR143. The mutation detection rate was 87.5% (7/8) of familial vs. 24.1% (7/29) of sporadic cases. Ten mutations in 24 (41.7%) non-syndromic subjects and 4 mutations in 13(30.8%) syndromic subjects were detected. A total of 77.8% (7/9) of mutations in FRMD7 were concentrated within the FERM and FA domains, while all mutations in GPR143 were located in exons 1, 2, 4 and 6. We observed that visual acuity tended to be worse in the GPR143 group than in the FRMD7 group, and no obvious difference in other clinical manifestations was found through comparisons in different groups of patients. Conclusions: This study identified 14 mutations (seven novel and seven known) in eight familial and 29 sporadic patients with congenital nystagmus, expanding the mutational spectrum and validating FRMD7 and GPR143 as mutation hotspots. These findings also revealed a significant difference in the screening rate between different groups of participants, providing new insights for the strategy of genetic screening and early clinical diagnosis of CN.

Human embryonic stem cell-derived organoid retinoblastoma reveals a cancerous origin.

Retinoblastoma (Rb) is the most prevalent intraocular malignancy in children, with a worldwide survival rate <30%. We have developed a cancerous model of Rb in retinal organoids derived from genetically engineered human embryonic stem cells (hESCs) with a biallelic mutagenesis of the RB1 gene. These organoid Rbs exhibit properties highly consistent with Rb tumorigenesis, transcriptome, and genome-wide methylation. Single-cell sequencing analysis suggests that Rb originated from ARR3-positive maturing cone precursors during development, which was further validated by immunostaining. Notably, we found that the PI3K-Akt pathway was aberrantly deregulated and its activator spleen tyrosine kinase (SYK) was significantly up-regulated. In addition, SYK inhibitors led to remarkable cell apoptosis in cancerous organoids. In conclusion, we have established an organoid Rb model derived from genetically engineered hESCs in a dish that has enabled us to trace the cell of origin and to test novel candidate therapeutic agents for human Rb, shedding light on the development and therapeutics of other malignancies.

Towards stem cell-based neuronal regeneration for glaucoma.

Glaucoma is a neurodegenerative disease as a leading cause of global blindness. Retinal ganglion cell (RGC) apoptosis and optic nerve damage are the main pathological changes. Patients have elevated intraocular pressure and progressive visual field loss. Unfortunately, current treatments for glaucoma merely stay at delaying the disease progression. As a promising treatment, stem cell-based neuronal regeneration therapy holds potential for glaucoma, thereby great efforts have been paid on it. RGC regeneration and transplantation are key approaches for the future treatment of glaucoma. A line of studies have shown that a variety of cells can be used to regenerate RGCs, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and retinal progenitor cells (RPCs). In this review, we overview the current progress on the regeneration of pluripotent stem cell-derived RGCs and outlook the perspective and challenges in this field.