Targeted Disruption of Outer Limiting Membrane Junctional Proteins (Crb1 and ZO-1) Increases Integration of Transplanted Photoreceptor Precursors into the Adult Wild-Type and Degenerating Retina

Abstract

Diseases culminating in photoreceptor loss are a major cause of untreatable blindness. Transplantation of rod photoreceptors is feasible, provided donor cells are at an appropriate stage of development when transplanted. Nevertheless, the proportion of cells that integrate into the recipient outer nuclear layer (ONL) is low. The outer limiting membrane (OLM), formed by adherens junctions between Müller glia and photoreceptors, may impede transplanted cells from migrating into the recipient ONL. Adaptor proteins such as Crumbs homologue 1 (Crb1) and zona occludins (ZO-1) are essential for localization of the OLM adherens junctions. We investigated whether targeted disruption of these proteins enhances donor cell integration. Transplantation of rod precursors in wild-type mice achieved 949 ± 141 integrated cells. By contrast, integration is significantly higher when rod precursors are transplanted into Crb1 rd8/rd8 mice, a model of retinitis pigmentosa and Lebers congenital amaurosis that lacks functional CRB1 protein and displays disruption of the OLM (7,819 ± 1,297; maximum 15,721 cells). We next used small interfering (si)RNA to transiently reduce the expression of ZO-1 and generate a reversible disruption of the OLM. ZO-1 knockdown resulted in similar, significantly improved, integration of transplanted cells in wild-type mice (7,037 ± 1,293; maximum 11,965 cells). Finally, as the OLM remains largely intact in many retinal disorders, we tested whether transient ZO-1 knockdown increased integration in a model of retinitis pigmentosa, the rho −/− mouse; donor cell integration was significantly increased from 313 ± 58 cells without treatment to 919 ± 198 cells after ZO-1 knockdown. This study shows that targeted disruption of OLM junctional proteins enhances integration in the wild-type and degenerating retina and may be a useful approach for developing photoreceptor transplantation strategies.

Defining the Integration Capacity of Embryonic Stem Cell-Derived Photoreceptor Precursors

“…7D) and around 60% of the subretinal cell masses contained GFP þ cells suggesting that these cells were viable. We examined the recipient retinas for the presence of increased gliosis by GFAP immunohistochemistry as we have previously shown that the induction of GFAP-labeled glial processes along the outer edge of the host retina can inhibit photoreceptor precursor cell integration into the ONL [15,25]. Although we found increased GFAP staining in Mül-ler glial processes spanning the retina, as expected following a subretinal transplantation, there was no indication of gliosis (Fig.…”

“…Adult mice were 6-15 weeks old and kept on a standard 12-hour lightdark cycle. Differentiated cells were dissociated and transplanted as previously described [1,[15][16][17], further details are provided in the Supporting Information Methods.…”

Migration, Integration and Maturation of Photoreceptor Precursors Following Transplantation in the Mouse Retina

“…Color images available online at www.liebertpub.com/scd other stages do not. Similarly, knowing the timing of maximal donor cell migration is of particular importance when designing strategies that transiently manipulate the recipient retina and/or donor cell behavior in order to promote donor cell migration and integration [31,32,41,42]. Here, we find that donor cell migration occurs within the first week posttransplantation, predominantly as single entities and involving a stereotyped sequence of morphologies, typical of locomoting cells elsewhere in the central nervous system (CNS).…”

“…4Cii, iii). Such events may arise as a result of small gaps or breaks in the recipient OLM since we have previously shown that donor cell integration is improved after transplantation into pharmacological or genetic models of OLM disruption, compared with wildtype [31,32]. Further inspection revealed that the apical attachments of cells in small columns are indeed frequently located at the same point (Fig.…”

Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures

“…A critical number of PRs need to be functionally reconnected into the retinal circuitry to restore vision. A number of elegant studies demonstrated the ability of some grafted PRs to integrate into the PR layer and synapse onto bipolar neurons [97][98][99]. However, only a limited number of PR progenitors, harvested at a narrow window of time (postnatal days 3-5 for mouse), showed successful integration [100].…”