Transplantation of cGMP compliant iPSC-derived retinal progenitor cells

Abstract

Purpose : Induced pluripotent stem cell (iPSC)-derived retinal progenitor cells are promising for restoring vision in patients with retinal degeneration. The purpose of this study was to evaluate local tolerability and cellular integration of human retinal cells generated under cGMP in a rat model of retinal degeneration following subretinal transplantation.

Methods : Patient-derived iPSCs were generated via Sendai viral (Cytotune 2) reprogramming of dermal fibroblasts. Retinal organoids were derived using a stepwise 3D differentiation protocol testing different cGMP compliant reagents and oxygen tension (5-20%) in a cGMP compliant Biospherix cell culture isolator. Organoids were characterized via light and confocal microscopy. At day 160, organoids were dissociated with papain and retinal progenitor cells were transplanted into immune suppressed Pde6b-/- rats (N=22). Immune response in rats and human donor cell survival, cellular identity, and synaptic integration were assessed at 3- and 30-days post-injection.

Results : IPSC reprogramming efficiency is highest under 5% oxygen tension, however, retinal organoid production was inefficient at this oxygen level. Increasing oxygen tension to 20% resulted in decreased pluripotency and increased ectoderm gene expression by day 7 of differentiation. By day 30, laminated retinal organoids were abundant in cultures differentiated under 20% oxygen tension, while they were smaller and less consistent at 5% oxygen tension. By 160 days of differentiation, retinal organoids generated at 20% oxygen tension under cGMP conditions contain abundant rod and cone photoreceptor precursor cells expressing markers such as NRL and cone arrestin, respectively. Human donor cells were identified in 100% of immunosuppressed rats. At both 3- and 30-days post-injection, cells expressing donor cell antigen (HNA) and photoreceptor cell markers (e.g., recoverin) were abundant at the injection site (Figure 1). Widespread neurite extension and expression of synaptic markers in conjunction with host bipolar cells was observed (Figure 1).

Conclusions : We have generated a cGMP compliant iPSC-derived retinal organoid differentiation protocol suitable for derivation of transplantable retinal progenitor cells.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Subretinal transplantation of clinical grade iPSC-derived retinal progenitor cells in Pde6b^-/- rats with advanced disease (i.e., no photoreceptor cells remaining at the time of transplantation).