Abstract
Purpose :
No pre-clinical model exists to examine human-to-human donor-host cell interaction and engraftment in cell therapies for inherited retinal disease (IRD). We propose a novel chimeric retinal organoid (“chimeroid”) engraftment model comprised of human healthy stem cells and patient-derived, induced pluripotent stem cells (iPSCs). We hypothesize that addition of healthy progenitors in sufficient proportion will result in improved photoreceptor survival and rescue a normal phenotype in an organoid model of IRD.
Methods :
Control organoids developed from healthy human H9 embryonic stem cells and USH2A-or CRB1-patient-derived iPSCs (IRD-iPSCs) were cultured as positive and negative controls, respectively, to characterize pure healthy and diseased phenotypes at multiple stages of retinal organoid development. For chimeroid experiments, fluorescently-tagged H9 cells were co-cultured with IRD-iPSCs and mixed in various ratios of H9:IRD-iPSCs (1:1, 3:1, 1:3, 9:1, 1:9, 1:6, 6:1). The effect of mixing ratio on the rescue of diseased photoreceptors by healthy progenitors was assessed throughout organoid development.
Results :
H9 control organoids were grown to establish a healthy organoid phenotype against which chimeroids and control IRD-organoids were compared. H9 organoids displayed retinal lamination at Week 4 of development and maintained lamination at Week 6 and 8. Control CRB1- and USH2A-organoids were grown to establish pure diseased organoid phenotypes from each patient-derived line. Both lines of IRD-organoids were observed to have disorganized retinal lamination and smaller neural retina size at Week 4 compared to H9 controls. As well, both lines of IRD-organoids formed a lower yield of neural retinal vesicles at Week 4 compared to H9 controls. In co-cultures where neural induction stage of retinal organoid development has been reached (Week 3), H9 cells showed increased proliferation relative to IRD-iPSCs when in mixing ratios of 1:3 and higher (H9:IRD-iPSCs).
Conclusions :
Immunocytochemistry and fluorescent imaging will be used at early, mid and late timepoints to quantify and characterize changes in morphological and cellular organization of chimeroids compared to controls, as well as transcriptomic changes in diseased versus healthy cells. Together, these findings will provide new insight into non-cell autonomous mechanisms of cell rescue and donor-host retinal cell interactions.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.