Exploring the survival of transplanted hiPSC-derived photoreceptors in the 13-lined ground squirrel

Abstract

Purpose : To assess the survival of transplanted human induced pluripotent stem cell (hiPSC)-derived photoreceptors in the cone dominant 13-lined ground squirrel (13-LGS; Ictidomys tridecemlineatus).

Methods : Cone-rich 3D-retinal organoids were differentiated from hiPSC with GFP knocked in at the AAVS1 safe harbor locus. 13-LGS retinas were degenerated via experimental retinal detachment (RD, n=18) or by intravitreal chemical injection of either adenosine triphosphate (ATP, n=16) or tunicamycin (Tm, n=6). 13-LGS were immunosuppressed using cyclosporine A for 14 days. Organoids were dissociated with papain and cell viability was assessed by flow cytometry. Cells were transplanted into the subretinal space of all eyes (0.7-1.4x10⁶ cells/eye) at least two weeks post-damage. To track survival of transplanted cells, eyes were imaged in vivo immediately post-transplantation and at 1 week, and 1-,2-,3-, and 4-months later, using SLO, OCT, and AOSLO. AOSLO images were acquired at 3-mo to capture high resolution images of the cell patches. After the 4-mo imaging time point, retinas were analyzed by immunohistochemistry (IHC) with marker antibodies to GFP (reporter), Otx2 (photoreceptor), and HuNu and LMNB2 (human-specific).

Results : Cell viability using flow cytometry was 61.2%-93.5%. Locations of transplanted cells were observed with OCT, and initial transplantation was deemed successful when cells were deposited and retained subretinally. 15 of 40 transplants failed for technical reasons: reflux of cells into the vitreous space (ATP, RD; n=3); degeneration of GFP signal over time (ATP, RD; n=7); and hemorrhage that obstructed imaging (Tm; n=5). GFP signal was detected with SLO at all time points post-transplantation in 25 of 40 eyes (ATP, n=11; Tm, n=1; RD, n=13), demonstrating survival of transplanted cells. AOSLO showed clumps of transplanted cells settled above the surviving cones of the host retina. IHC confirmed the expression of GFP, Otx2, HuNu, and LMNB2 in the transplanted cells. Integration of cells in RD retinas was restricted to the outer retina/subretinal space, whereas chemically degenerated retinas exhibited additional signal integration into the inner retina.

Conclusions : hiPSC-derived photoreceptors survived in the outer retina of 13-LGS eyes for up to 4-mo post-transplantation. Further studies are needed to examine the functional properties of these cells.

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