The Effect of Transient Local Anti-inflammatory Treatment on the Survival of Pig Retinal Progenitor Cell Allotransplants

Abstract

Purpose: The development of photoreceptor replacement therapy for retinal degenerative disorders requires the identification of the optimal cell source and immunosuppressive regimen in a large animal model. Allotransplants are not acutely rejected in swine subretinal space, although it is not known if survival can be improved with immunosuppression. Here we investigated the survival and integration of expanded pig retinal progenitor cells (pRPCs) in normal recipients with and without transient anti-inflammatory suppression.Methods: pRPCs were derived from the neural retina of E60 GFP transgenic pigs, expanded for six passages, characterized, and transplanted into the subretinal space of 12 pigs. Six recipients received a single intravitreal injection of rapamycin and dexamethasone.Results: pRPCs expressed the photoreceptor development genes Sox2, Pax6, Lhx2, Crx, Nrl, and Recoverin in vitro. Transplanted cells were identified in 9 out of 12 recipients 4 weeks after the injection. pRPCs integrated primarily into the photoreceptor inner segment layer and outer nuclear layer with single cells present in the inner nuclear layer. Donor cells remained recoverin-positive and acquired rhodopsin. We did not observe any signs of graft proliferation. The immunosuppression did not affect the survival or distribution of grafts. No macrophage infiltration or loss of retinal structure was observed in either group.Conclusions: Local immunosuppression with rapamycin and dexamethasone does not improve the outcome of pRPC allotransplantation into the subretinal space.Translational Relevance: Survival and integration of pRPC together with the lack of graft proliferation suggests that allogeneic RPC transplantation without transient immunosuppression is a favorable approach for photoreceptor cell replacement.

Cellular regeneration strategies for macular degeneration: past, present and future

“…ReNeuron have developed an interesting technology, whereby foetal RPC can be expanded at least 40-fold in hypoxic conditions akin to that experienced in vivo, overcoming many of the supply problems in obtaining these cells from week 16 to week 18 human foetuses. Publications have suggested that in vitro expanded RPC have some ability to differentiate and express photoreceptor markers [ 97 – 99 ], but this is limited and differentiation after transplantation appears to be dependent on host conditions particularly maturity, with transplant into immature retinas being more successful than mature ones [ 100 − 102 ]. Furthermore, cell migration into the outer retina is variable and the predominant markers expressed by transplanted cells are glial [ 103 ].…”

In Situ Cross-linking Hydrogel as a Vehicle for Retinal Progenitor Cell Transplantation

“…For xenograft studies, green fluorescent protein-positive (GFPþ) pig RPCs (pRPCs) from fetal pigs, transfected with a retroviral vector containing the gfp gene following a bactin promoter, were thawed from cryovials and cultured as described previously 22 . GFPþ pRPCs were chosen for easier detection of transplanted cells via immunostaining for GFP.…”

“…Culture protocol was identical to hRPCs, with the same media on a fibrinogen-coated flask. Prior to transplantation, pRPCs were suspended in Hank's balanced salt solution (HBSS) with N-acetylcysteine (NAC) at previously published concentrations, which was shown to enhance transplant survival due to antioxidant effects 22 .…”

Restoring Vision by Regenerative Medicine Using Stem Cell: A Review

“…Especially, relocation of an Intermediary 4-week phase of RPE separation maximum reliably conserved vision matched to mature or newer RPE cells [88]. Afterward, relocation seems to be reliant on host situations mainly in adulthood, with relocating to young retinas existing more fruitful than matured ones [89]. It is likely, nevertheless, that distinction, immigration, and existence might be better by modifications in transfer and host atmosphere [90].…”