Abstract
Purpose :
Knowledge of the mechanisms underlying photoreceptor loss in human CRB1-associated retinal disease (CD) is limited. Previous animal studies reported altered retinal cell proliferation and apoptosis in CD with potential involvement of the Hippo/Yap and NOTCH pathways. I hypothesize that CD leads to a prolonged proliferative state followed by increased cell death, leading to a lower photoreceptor population.
Methods :
Human retinal organoids (ROs) were generated from an induced pluripotent stem cell (iPSC) line derived from a patient with Leber congenital amaurosis homozygous CRB1 mutation (p.Q120X; p.Q120X), an iPSC line from a healthy donor, and a H9 embryonic stem cell line. Immunofluorescent staining for proliferating cells, cell type-specific markers, and CRB1-related proteins was performed on ROs every four weeks from weeks 8 to 20. The differences in distribution and number of retinal cells between diseased and healthy ROs were quantified from fluorescent images. RNA was extracted from whole ROs weekly between timepoints when healthy and diseased RO showed significant differences in retinal cell population. The mRNA levels of key proteins in the NOTCH (NOTCH1, NOTCH2, Hes1) and the Hippo/Yap (MST1, MST2, YAP) pathways were quantified using qRT-PCR.
Results :
Initial testing of H9 and healthy donor-derived ROs at week 8 displayed the expected order of differentiation with positive staining for retinal ganglion cells (Brn3a), retinal progenitor cells (PAX6, VSX2), and photoreceptor/ bipolar cell progenitors (Otx2, CRX), as well as negative staining for rod photoreceptors (NRL). As a baseline, healthy donor ROs showed mRNA expression of CRB1, PALS1, YAP, NOTCH2 and Hes1 at week 8. ROs were successfully grown from the CRB1-mutant cell line at week 3. Initial observations of mature CRB1-mutant ROs showed less rhodopsin staining, as well as less dense and shorter brush borders compared to H9 ROs at week 34, which implies alterations to the number and/or development of mature photoreceptors.
Conclusions :
Patient-specific retinal organoids derived from a CD patient showed differences in photoreceptor development compared to healthy ROs. Further study of protein and gene expression will help explore the effects of NOTCH1 and Hippo/Yap signaling on proliferation and cell death in CD. Understanding the disease mechanisms and development may facilitate the discovery of novel therapies and optimal treatment timings for CD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.