Abstract
Purpose :
No pre-clinical model exists to investigate human-to-human donor-host cell interaction and engraftment in cell therapies for inherited retinal disease (IRD). We propose a chimeric retinal organoid (“chimeroid”) model, differentiated from the co-culture of healthy human stem cells and IRD patient-derived induced pluripotent stem cells (iPSCs). We hypothesize that chimeric association of diseased and healthy human photoreceptors within the chimeroid will promote improved diseased photoreceptor survival and rescue a normal outer retinal phenotype.
Methods :
Red fluorescent protein (RFP)-tagged H9 human embryonic stem cells were co-cultured with green fluorescent protein (GFP)-tagged patient-derived iPSCs (from patients with CRB1 or USH2A IRD mutations), in various H9-to-IRD cell mixing ratios (1:3,1:1,3:1). Throughout retinal organoid differentiation, immunohistochemistry was used to assess the rescue of diseased photoreceptors by healthy cells within the chimeroid outer retinal border.
Results :
H9- and disease-only organoids were first established to compare differences between pure healthy and pure disease phenotypes. At Wk24 of retinal organoid growth, CRB1-organoids had reduced photoreceptor outer segment (OS) formation compared to H9 organoids. In Wk34 USH2A-organoids, a significantly shorter OS length (49.35um±0.829) was observed compared to H9 organoids (60.93um±1.152; p<0.0001), with significantly lower cone Arrestin3 expression. For all chimeric experiments, cell mixing proportions (1:3,1:1,3:1) were maintained in early (Wk4) and mature (Wk24) H9:CRB1 and H9:USH2A chimeroids. By Wk24 in H9:CRB1 chimeroids, OS formation and expression of retinal cell-specific markers (Rhodopsin, Sox9) were increased in regions of the outer retinal border where H9-RFP photoreceptors were integrated.
Conclusions :
The co-culture of healthy and diseased cells promotes improved disease photoreceptor OS formation in mature human retinal chimeroids. As H9:USH2A and H9:CRB1 chimeroids reach late maturation (Wk34), fluorescence activated-cell sorting and qRT-PCR will be used to assess transcriptomic changes in diseased and healthy photoreceptors, elucidating mechanisms through which healthy cells rescue diseased cell OS formation. This will provide insight into human donor-host cell interactions during retinal cell engraftment to inform new IRD cures.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.