Timed Notch Inhibition Drives Photoreceptor Fate Specification in Human Retinal Organoids

by Selleckchem | Jul 12 2023

Purpose: Transplanting photoreceptors from human pluripotent stem cell-derived retinal organoids have the potential to reverse vision loss in affected individuals. However, transplantable photoreceptors are only a subset of all cells in the organoids. Hence, the goal of our current study was to accelerate and synchronize photoreceptor differentiation in retinal organoids by inhibiting the Notch signaling pathway at different developmental time-points using a small molecule, PF-03084014 (PF).

Methods: Human induced pluripotent stem cell- and human embryonic stem cells-derived retinal organoids were treated with 10 µM PF for 3 days starting at day 45 (D45), D60, D90, and D120 of differentiation. Organoids were collected at post-treatment days 14, 28, and 42 and analyzed for progenitor and photoreceptor markers and Notch pathway inhibition by immunohistochemistry (IHC), quantitative PCR, and bulk RNA sequencing (n = 3-5 organoids from three independent experiments).

Results: Retinal organoids collected after treatment showed a decrease in progenitor markers (KI67, VSX2, PAX6, and LHX2) and an increase in differentiated pan-photoreceptor markers (OTX2, CRX, and RCVRN) at all organoid stages except D120. PF-treated organoids at D45 and D60 exhibited an increase in cone photoreceptor markers (RXRG and ARR3). PF treatment at D90 revealed an increase in cone and rod photoreceptors markers (ARR3, NRL, and NR2E3). Bulk RNA sequencing analysis mirrored the immunohistochemistry data and quantitative PCR confirmed Notch effector inhibition.

Conclusions: Timing the Notch pathway inhibition in human retinal organoids to align with progenitor competency stages can yield an enriched population of early cone or rod photoreceptors.

Comments:

In this study, the researchers aimed to enhance and synchronize the differentiation of photoreceptor cells in retinal organoids derived from human pluripotent stem cells. The ultimate goal was to develop a method for transplanting these photoreceptor cells to potentially reverse vision loss in individuals affected by retinal degenerative diseases.

To achieve this, the researchers utilized a small molecule called PF-03084014 (PF) to inhibit the Notch signaling pathway at various developmental time-points during the differentiation of retinal organoids. Human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs) were used as the starting cell sources for generating the retinal organoids.

The experimental design involved treating the retinal organoids with a concentration of 10 µM PF for a duration of 3 days, starting at different time-points during the differentiation process: day 45 (D45), D60, D90, and D120. After the treatment, the organoids were collected at post-treatment days 14, 28, and 42 for analysis.

The researchers employed multiple techniques to assess the effects of PF treatment on the organoids. Immunohistochemistry (IHC) was used to examine the expression of progenitor markers (such as KI67, VSX2, PAX6, and LHX2) and photoreceptor markers (including OTX2, CRX, and RCVRN). Quantitative PCR and bulk RNA sequencing were also utilized to investigate gene expression changes and confirm the inhibition of Notch pathway effectors.

The results of the study demonstrated that retinal organoids collected after PF treatment exhibited a decrease in progenitor markers and an increase in differentiated pan-photoreceptor markers, indicating enhanced photoreceptor differentiation. However, the effect was not observed in organoids treated at D120. Interestingly, organoids treated at D45 and D60 showed an increase in cone photoreceptor markers, while treatment at D90 resulted in an increase in both cone and rod photoreceptor markers.

The findings from the bulk RNA sequencing analysis aligned with the results obtained from IHC, providing further support for the effects of PF treatment on photoreceptor differentiation. Quantitative PCR data confirmed the inhibition of Notch pathway effectors, validating the mechanism of action of PF.

In conclusion, the study suggests that by inhibiting the Notch signaling pathway at specific developmental time-points, it is possible to accelerate and synchronize the differentiation of photoreceptor cells in human retinal organoids. This approach may lead to the generation of an enriched population of early cone or rod photoreceptors, which holds promise for potential transplantation therapies to treat vision loss in individuals with retinal degenerative diseases.