Abstract
Purpose :
Cell replacement therapy is a promising approach for treating advanced optic neuropathies and restoring visual function. The present research aims to understand how pre-adhesion promotes stem cell-derived human ganglion cell (hRGC) survival and axon regeneration after transplants, and how hRGCs interact with host retinas.
Methods :
Day 40-60 retinal organoids generated from the Brn3b-tdTomato H9 cell line were used for transplant. Cells dissociated from retinal organoids were pre-adhered in culture for 7 days and intravitreally injected into mouse eyes or cultured in vitro in parallel for comparison. Two weeks after transplants, hRGCs were retrieved from host retinas by CD90 and human-specific CD47 immunopanning. Single-cell RNA sequencing (scRNA-seq) was performed on both the retrieved, transplanted hRGCs and the cultured cells. Retinas were collected at different time points for histology. All histological experiments were conducted at least three times independently. Data were analyzed by ANOVA and post hoc t-test with Tukey correction, with a P-value of <0.05 considered statistically significant.
Results :
After transplant in vivo, pre-adhesion of donor cells led to significantly more tdTomato-positive cell survival and more axon regeneration in the host retinas than those from organoids without pre-adhesion. ScRNA-seq of the donor cells before transplant demonstrated increased expression of axon extension-related genes and decreased expression of apoptotic genes in pre-adhered compared to floating organoids. In addition, pre-adhesion induced differentiation of a new cluster sharing similar gene profiles with mesenchymal cells. scRNA-seq showed improved RGC maturation and higher expression levels of axon cytoskeleton genes in both in vitro and in vivo cells than those before transplant. Furthermore, it revealed further up-regulation of axon guidance and pathfinding genes, including DCC, ROBO1/2, SLIT1/2, and UNC5C in the in vivo samples compared to the in vitro samples.
Conclusions :
Pre-adhesion of donor hRGCs is an effective way to promote cell survival and axon regeneration after transplant, and donor hRGCs react to the host environment by up-regulating receptors that facilitate axon pathfinding. These may enable improvements in axon guidance and circuit reconstruction, which may ultimately provide a strategy for therapeutics in glaucoma and other optic neuropathies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.