Abstract
Purpose :
Notch signaling pathway is a key determinant in the commitment of retinal ganglion cell (RGC) phenotypes from retinal progenitor cells. Based on the known role of the Notch signaling pathway in RGC development, numerous studies have used the gamma secretase inhibitor DAPT to inhibit Notch intracellular domain cleavage and enhance RGC differentiation from stem cells. Recently, the PEAK1 related, kinase-activating pseudokinase 1 (PRAG1) was identified as a critical mediator in the activation of the Notch transcriptional complex. Here, we evaluated an RGC differentiation approach from hiPSC-derived retinal organoids by comparing gamma secretase inhibition to inhibition of PRAG1 by a novel pharmaceutical compound.
Methods :
hiPSCs underwent retinal differentiation using an established retinal organoid strategy. To initiate RGC induction and maturation, Notch signaling was inhibited by blocking either the gamma-secretase complex or by inhibiting PRAG1. Retinal organoids were dissociated, and RGC-committed progenitors were isolated using a magnetic activated cell sorting approach. RT-qPCR and immunofluorescence staining were used to evaluate and identify the expression of RGC specific markers.
Results :
Use of either Notch inhibitors yielded RGC committed cells with significant upregulation of RGC-specific markers when compared to their parental iPSC line. However, RGCs derived via the inhibition of PRAG1 exhibited a significantly higher and earlier upregulation of RGC specifying markers when compared to RGCs derived from Notch inhibition through the gamma-secretase complex (p<0.05) and achieved neuronal morphologies indicative of mature phenotypes at an earlier stage of organoid lamination. Furthermore, Thy1 positive sorted cells expressed mature RGC markers such as Brn3, Atoh7 and neuronal markers such as beta-III tubulin and synaptophysin.
Conclusions :
An enhanced expression of phenotypic and morphological characteristics specific to RGCs was observed in the RGC-progenitors derived from the retinal organoids cultured with the PRAG1 inhibitor. We have developed a novel chemically mediated approach to enhance RGC differentiation from retinal organoids. The de novo generated RGCs could be employed as a model to analyze critical stages in RGC development, regenerative therapies, disease modeling or pharmacological screening.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.