Single -cell transcriptomic profiling of stem cell derived retinal pigment epithelial cell monolayers after subretinal transplantation

Transplantation of stem cell derived retinal pigment epithelial (RPE) cells can potentially restore vision in patients with age-related macular degeneration (AMD). Several landmark Phase I/II clinical trials have demonstrated safety and tolerability of RPE transplants in AMD patients, albeit with limited efficacy. Currently, there is limited understanding of how the recipient retina regulates the survival, maturation and fate specification of transplanted RPE cells. To address this, we transplanted stem cell derived RPE into the subretinal space of immunocompetent rabbits for one month and conducted single-cell RNA sequencing analyses on the explanted RPE monolayers, compared to their age-matched in vitro counterparts. We observed an unequivocal retention of RPE identity, and a trajectory inferred survival of all in vitro RPE populations after transplantation. Furthermore, there was a unidirectional maturation trajectory towards the native adult human RPE state in all transplanted RPE cells, regardless of stem cell resource. Using adult human RPE cells as a reference, we reconstructed the gene regulatory networks in our transplanted stem cell derived RPE, and identified tripartite transcription factors (MAFF, JUND and FOS), known to modulate a broad array of RPE functions in vivo. This includes regulation of canonical RPE signature genes known to support host photoreceptors, and pro-survival genes required for adaptation to the host subretinal microenvironment. These findings shed insights into the transcriptional landscape of RPE cells after subretinal transplantation, with important implications for cell-based therapy for AMD. Two pluripotent stem cell sources, ESC and skin-derived iPSCs, were differentiated to RPE generated ESC-RPE and iPSC-RPE. They were further matured in vitro to day 60. Condition 1: At day 60, n=3 ESC-RPE and n=3 iPSC-RPE monolayers were were transplanted into the subretinal region of immunocompetent rabbits and follow-up for 30 days to day 90. Condition 2: At day 60, n=1 ESC-RPE and n=1 iPSC-RPE were further matured in vitro to day 90 and served as age-matched controls for transplanted RPE. Upon reaching day 90, both conditions were harvest and dissociated to single cells for single-cell RNA sequencing experiments.