Abstract
Purpose :
To identify a time-dependent role of mTORC1 in retinal development using hiPSC-derived retinal organoids, especially retinal ganglion cell (RGC) differentiation, and retinal lamination process during early stages of retinal organoids (ROs).
Methods :
We utilized American Type Culture Collection (ATCC)-DYR0100 hiPSCs for retinal organoid differentiation. Rapamycin and MHY1485 were prepared at final concentrations of 100 nM and 10 μM, respectively. From 35 days of differentiation, organoids were transferred to the wells of a 96 well plate and treated with rapamycin and MHY1485 until 40 days of differentiation. The development of RGCs was assessed by the expression of various specific markers (HuC/D, AtoH7, Islet-1, and Brn3b) We examined the relative expression of total mTOR, Raptor, and Rictor, S6K1 to investigate the dynamics of mTOR activity in ROs. RGC markers HuC/D, AtoH7 and mTORC1 marker S2448, ps6 were detected in ROs by immunofluorescence staining.
Results :
mTORC1 activity in ROs was the highest at 40 days of differentiation. Hyperactivation of mTORC1 during this period using MHY1485 resulted in the significantly increased overall size of ROs compared to untreated controls and rapamycin-treated ROs, while showing markedly increased proliferative activity both in inner and outer layers of ROs. Also, MHY1485-treated ROs showed a significantly increased number of ectopic RGCs in outer layers, indicating disruption of retinal laminar structure, with robust expression of HuC/D in inner layers
Conclusions :
mTORC1 plays a critical role in the development of hiPSC-derived ROs, especially during the early stages of differentiation. Moreover, our results also outline the feasibility of using hiPSC-derived ROs as a tool for translational research on retinal development.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.