Aberrant gene expression yet undiminished retinal ganglion cell genesis in iPSC-derived models of optic nerve hypoplasia

ABSTRACT

Background

Optic nerve hypoplasia (ONH), the leading congenital cause of permanent blindness, is characterized by a retinal ganglion cell (RGC) deficit at birth. Multifactorial developmental events are hypothesized to underlie ONH and its frequently associated neurologic and endocrine abnormalities; however, environmental influences are unclear and genetic underpinnings are unexplored. This work investigates the genetic contribution to ONH RGC production and gene expression using patient induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs).

Materials and methods

iPSCs produced from ONH patients and controls were differentiated to ROs. RGC genesis was assessed using immunofluorescence and flow cytometry. Flow-sorted BRN3+ cells were collected for RNA extraction for RNA-Sequencing. Differential gene expression was assessed using DESeq2 and edgeR. PANTHER was employed to identify statistically over-represented ontologies among the differentially expressed genes (DEGs). DEGs of high interest to ONH were distinguished by assessing function, mutational constraint, and prior identification in ONH, autism and neurodevelopmental disorder (NDD) studies.

Results

RGC genesis and survival were similar in ONH and control ROs. Differential expression of 70 genes was identified in both DESeq2 and edgeR analyses, representing a ~ 4-fold higher percentage of DEGs than in randomized study participants. DEGs showed trends towards over-representation of validated NDD genes and ONH exome variant genes. Among the DEGs, RAPGEF4 and DMD had the greatest number of disease-relevant features.

Conclusions

ONH genetic background was not associated with impaired RGC genesis but was associated with DEGs exhibiting disease contribution potential. This constitutes some of the first evidence of a genetic contribution to ONH.

KEYWORDS:

• Optic nerve hypoplasia
• retinal ganglion cell
• human retinal organoids

Acknowledgments

We thank the brave and curious children and their families for generously donating the blood that launched this study. This work was assisted by the Stem Cell Analytics, Flow Cytometry, and SC2 Cores of the Saban Research Institute at Children’s Hospital Los Angeles. This study makes use of data generated by the DECIPHER community which includes Meena Balasubramanian of Sheffield Children’s NHS Foundation Trust, Diana Barelle of NIHR University of Southampton, Neeti Ghali of London Northwest University Healthcare NHS Trust, Sally Ann Lynch of Children’s Health Ireland at Crumlin, Sarah Smithson of University Hospitals Bristol NHS Foundation Trust, Rosalyn Jewell of Chapel Allerton Hospital, Hannah Titheradge of Birmingham Women’s and Children’s NHS Foundation Trust, Pradeep Vasudevan of University Hospitals of Leicester NHS Trust, and Ursula Moore of the Leeds Teaching Hospitals NHS Trust. Funding for the DECIPHER project was provided by Wellcome [grant number WT223718/Z/21/Z].

Authors’ contributions

JGA, DC and MB conceived of the study, designed experiments, and wrote the manuscript; JGA, HH, and NH produced ROs and conducted experiments; JGA analyzed and interpreted the data; CS recruited blood donors for iPSC generation and assembled clinical profile data. All authors read and approved the final manuscript.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Ethics approval and consent to participate

This study was approved by the Children’s Hospital Los Angeles’ Institutional Review Board (CCI-13-00369) and Stem Cell Research Oversight Committee (SCRO 13–0019). Informed consent was obtained from a parent or guardian of all subjects; assent was obtained from subjects aged >7 years if cognitively able. Permission to use information related to all Decipher entries in Table S1 has been granted.

Data availability statement

The RNA-Seq dataset generated during the current study is available via accession number GSE227975 at the GEO repository (https://www.ncbi.nlm.nih.gov/geo/).

Supplemenatry data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/13816810.2023.2253902.

Additional information

Funding

This work was supported with funding from the Neonatal Blindness Fund, the A.B. Reins Foundation, the Knights Templar Eye Foundation, an unrestricted grant to the USC Department of Ophthalmology from Research to Prevent Blindness, New York, NY, and NIH [grant R21EY025419] (MB and DC).