Triple drug immunosuppression for xenotransplantation of human photoreceptor precursor cells in the canine retina.

Abstract

Purpose : To evaluate the effect of a systemic multidrug immunosuppressive protocol on the survival and integration of hESC-derived photoreceptor precursor cells (PRPCs) transplanted into the subretinal space (SRS) of normal and degenerated canine retinas.

Methods : Stage 2 retinal organoids (day 104-151 of differentiation) were generated from a CRX-tdTomato reporter line (WA09 CRX+/tdTomato, WiCell, Madison, WI) and dissociated to produce cell suspension containing fluorescent PRPCs for subretinal injection in 7 normal (12 eyes) and 3 mutant (rcd1/PDE6B) dogs (6 eyes) using a modified subretinal injector (RetinaJect, SurModics, CA). Seven dogs were placed under an immunosuppressive (IS) regimen (prednisolone, cyclosporine A, mycophenolate mofetil), 2 dogs were not medicated, and 1 dog had IS halted after 20 weeks. Survival of PRPCs was monitored by non-invasive multimodal imaging including color and fluorescence photography, cSLO (NIR and BAF modes), cross-sectional and en face OCT. Following termination, survival and integration of PRPCs and involvement of innate and adaptive cellular immune responses were assessed by IHC.

Results : In dogs under IS, loss of transplanted donor cells was seen within the first week post-injection (PI) but remaining cells survived up to 20 weeks PI. In normal retinas, most donor cells remained in the SRS, while in degenerated retinas clusters of cells were found to have migrated into the host’s ONL and inner retina. IHC analysis showed that most PRPCs expressed cone markers (hCA, M/L opsin) and some formed neurites and pedicle-like structures. In dogs that were not under IS, or that had IS halted, there was a rapid loss of transplanted cells. In these dogs, IHC revealed a mixed inflammatory infiltrate composed mostly of macrophages (CD18⁺) and resident microglia (Iba1⁺) and to a lesser extent of helper (CD4+) and cytotoxic (CD8+) T-cells as well as B-cells (CD20⁺).

Conclusions : Our triple drug IS regimen provided long-term (up to 5 months) survival of xenotransplanted hESC-derived PRPCs into the canine retina. Additionally, our results confirmed that degenerating retinas are more suitable for integration of donor PRPCs.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.