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. 2015 Sep 22;4(5):6.
doi: 10.1167/tvst.4.5.6. eCollection 2015 Sep.

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

Murilo Abud  1 , Petr Baranov  2 , Caroline Hicks  3 , Sara Patel  3 , Burke Lieppman  2 , Caio Regatieri  4 , John Sinden  3 , David Isaac  5 , Marcos Avila  5 , Michael Young  2
Affiliations

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

Murilo Abud et al. Transl Vis Sci Technol. .

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.

Keywords: cell therapy; photoreceptors; rapamycin; retina; retinal progenitor cells.

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Figures

Figure 1
Figure 1
Characteristics of pRPC at passage 6 in vitro. By immunocytochemistry (a) we observed positive nuclear staining for development transcription factors Sox2, Klf4, Pax6, Lhx2, cone-rod homeobox (Crx), rod-specific Neural retina-specific leucine zipper (Nrl), proliferation marker Ki-67, and intermediate filament Vimentin. Nuclei are counterstained with DAPI. Scale bar, 100 μm. RT-PCR analysis (b) confirmed the expression of these factors and absence of mature visual pigments (Rhodopsin, S-Opsin, M-Opsin), although Recoverin and rod outer membrane (ROM1) mRNA was expressed. We also have observed the expression GS.
Figure 2
Figure 2
Morphology of the retina at the transplantation site. Four weeks after transplantation, we did not observe any evidence of immune response or disruption of retinal laminar structure in either group. Here we show representative images from each animal in nonimmunosuppressed (a through d) and immunosuppressed (e through h) groups.
Figure 3
Figure 3
Survival and differentiation of pRPCs after subretinal transplantation to healthy recipients. Four weeks after surgery, we observed striking survival and integration of cells, with most of cells migrating into photoreceptor inner segment layer (IS; GFP, green). The bodies of several cells were found in the ONL (a, b, c) and inner nuclear layer (INL; b, c). We did not observe any signs of graft proliferation in any of the animals with only a single donor cell found to be Ki-67 positive (d, *), while host tissue showed some Ki-67 reactivity in INL (d, arrows). Most of the transplanted cells remained recoverin-positive (f), as can be seen by co-localization (yellow, arrows) of recoverin (red) and GFP (green). The proportion of cells obtaining rhodopsin expression (e) was significantly lower, with co-localization (yellow, arrows) observed in photoreceptor outer and inner segment layer. Nuclei are counterstained with Hoechst. Scale bar: 50 μm on all images.
Figure 4
Figure 4
The absence of immune response to transplant in nonimmunosuppressed (a) and immunosuppressed (b) animals 4 weeks after cell delivery. We have observed only single CD45 cells (arrow, b) in the inner retina with no difference between groups. Nuclei are counterstained with Hoechst. Scale bar: 50 μm.
Figure 5
Figure 5
Quantification of graft survival. Donor cell survival was assessed by counting GFP+ cells on five sections for each eye (mean ± SEM). Overall graft survival was graded on a scale from 0 (no cells) to 3. The t-test analysis shows no difference in graft survival in injected versus noninjected group.
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