Modeling of Photoreceptor Donor-Host Interaction Following Transplantation Reveals a Role for Crx, Müller Glia, and Rho/ROCK Signaling in Neurite Outgrowth

Abstract

The goal of photoreceptor transplantation is to establish functional synaptic connectivity between donor cells and second-order neurons in the host retina. There is, however, limited evidence of donor-host photoreceptor connectivity post-transplant. In this report, we investigated the effect of the host retinal environment on donor photoreceptor neurite outgrowth in vivo and identified a neurite outgrowth-promoting effect of host Crx (−/−) retinas following transplantation of purified photoreceptors expressing green fluorescent protein (GFP). To investigate the noncell autonomous factors that influence donor cell neurite outgrowth in vitro, we established a donor-host coculture system using postnatal retinal aggregates. Retinal cell aggregation is sensitive to several factors, including plate coating substrate, cell density, and the presence of Müller glia. Donor photoreceptors exhibit motility in aggregate cultures and can engraft into established aggregate structures. The neurite outgrowth-promoting phenotype observed in Crx (−/−) recipients in vivo is recapitulated in donor-host aggregate cocultures, demonstrating the utility of this surrogate in vitro approach. The removal of Müller glia from host aggregates reduced donor cell neurite outgrowth, identifying a role for this cell type in donor-host signaling. Although disruption of chondroitin sulfate proteoglycans in aggregates had no effect on the neurite outgrowth of donor photoreceptors, disruption of Rho/ROCK signaling enhanced outgrowth. Collectively, these data show a novel role of Crx, Müller glia, and Rho/ROCK signaling in controlling neurite outgrowth and provide an accessible in vitro model that can be used to screen for factors that regulate donor-host connectivity. STEM CELLS 2019;37:529-541 SIGNIFICANCE STATEMENTPoor photoreceptor connectivity in transplants is an unresolved issue. Results of the study show that photoreceptor neurite extension is regulated by Rho/ROCK signaling and report the use of primary retinal cultures as an in vitro platform to investigate donor photoreceptor neurite outgrowth. The results could be used in future work to enhance the connectivity of transplanted photoreceptors.

Extensive incorporation, polarisation and improved maturation of transplanted human cones in a murine cone degeneration model

“…Whether the Müller glia directly enhance maturation of the transplant remains to be proven, however, it is well known that glia are important supporters of neuronal function. Müller glia are critical for photoreceptor neurite outgrowth in both 2D and 3D culture systems (34,35). Interestingly, Müller cells are also reported to play a role in organised outer segment assembly (36).…”

Photoreceptor nanotubes mediate the in vivo exchange of intracellular material

“…The formation of membrane protrusions, including neurites, is regulated by the coordinated activity of Rho GTPases, where in general, Rac1 and Cdc42 promote, and Rho inhibits (Faix & Rottner, 2006;Arkwright et al, 2010;Ridley, 2011;Chen et al, 2012) neurite outgrowth. We have shown previously that inhibiting Rho kinase, a downstream RhoA effector, increases neurite outgrowth in photoreceptor precursors (Tsai et al, 2019). Given the similarities between…”

Outer Retinal Cell Replacement: Putting the Pieces Together

“…Although there is evidence of some efficacy following hPSC-PRP delivery even in end-stage retinal degeneration—suggesting that host inner retinal circuitry remains viable for a time—the exact window of opportunity for effective cell replacement is currently unknown. 34 , 219 Treatments being studied seek to modulate a variety of naturally occurring processes that may act as barriers to donor PR integration in the degenerate outer retina, including glial scarring, 220 interneuron plasticity, 221 and neurite outgrowth, 222 which may in turn help create a more donor cell-receptive environment. Basic discovery research to better understand the molecular mechanisms involved in retinal circuit assembly, disassembly, and re-assembly will also be essential to address host-centered barriers to neuronal replacement.…”