Protocol for modeling injury-induced regeneration in retinal organoids and isolating hNRSCs for subretinal transplantation in rd10 mice

Human retinal organoids (hROs) provide a platform for modeling injury and assessing regenerative capacity in vitro . Here, we present a protocol for stepwise generation of hROs from human embryonic stem cells (hESCs). We describe steps for microsurgical induction of neural retina (NR) injury, isolation of CPAMD8 + human neural retinal stem-like cells (hNRSCs) using flow cytometry, and their subretinal transplantation into rd10 mice. By isolating and subretinally injecting hESC-derived neural retinal stem-like cells, this protocol enables subsequent functional studies in retinal degeneration models. For complete details on the use and execution of this protocol, please refer to Liu et al. 1 • Step-by-step generation of retinal organoids with CMZ-like regions from hESCs • Dissection of neural retina and establishment of an injury-repair organoid model • Isolation of CPAMD8 + hNRSCs from injured hROs via flow cytometry • Subretinal transplantation of hNRSCs into the rd10 mouse retina Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Human retinal organoids (hROs) provide a platform for modeling injury and assessing regenerative capacity in vitro . Here, we present a protocol for stepwise generation of hROs from human embryonic stem cells (hESCs). We describe steps for microsurgical induction of neural retina (NR) injury, isolation of CPAMD8 + human neural retinal stem-like cells (hNRSCs) using flow cytometry, and their subretinal transplantation into rd10 mice. By isolating and subretinally injecting hESC-derived neural retinal stem-like cells, this protocol enables subsequent functional studies in retinal degeneration models.