Device-free isolation of photoreceptor cells from patient iPSC-derived retinal organoids

ABSTRACT: Purpose: Autologous cell replacement shows great promise for the treatment of inherited retinal degeneration. While mature differentiation protocols exist to produce retinal organoids from patient-derived stem cells, not all cell types present in these organoids are desirable for transplant. To increase the potency of future cell therapies, methods for isolating photoreceptors from dissociated retinal organoids are needed. In this work, we show how partial dissociation can be used to exploit the spatial organization of retinal organoids to produce highly pure photoreceptor populations without the use of specialized sorting devices or reagents such as xenobiotic antibodies. Methods: Retinal organoids were generated as we have described previously. For flow cytometry experiments, organoids were dissociated using papain for 30 minutes or for 90 minutes followed by trituration. For scRNAseq experiments, liberated cells were collected from papain-dissociating organoids at 20-minute intervals. At the conclusion of the experiment, remaining tissue was fully dissociated. A control 60-minute full dissociation was performed in parallel. Photoreceptor purity was assessed via flow cytometry and scRNAseq. Results: Photoreceptors desired for transplant are typically found in the outer layers of retinal organoids, suggesting that partial dissociation could selectively release these cells. Flow cytometry results indicating an increase in purity of CD133+ cells from 21.1% to 91.7% when cells were partially dissociated as compared to full dissociation. A time dependent release of photoreceptors, with a maximum purity of 96% CRX+ cells at 40 minutes as compared to 66% pure for traditional dissociation. Conclusions: By timing the dissociation of retinal organoids, highly pure photoreceptor cell populations suitable for cell replacement therapies can be obtained under cGMP without the use of sorting reagents or equipment.

ORGANISM(S): Homo sapiens

PROVIDER: GSE266526 | GEO | 2024/09/02

REPOSITORIES: GEO