Jonathan Soucy, Volha Malechka, John Dayron Rivera, Petr Y Baranov | 2023
Abstract
Purpose : Cell transplantation has been proposed to replace retinal ganglion cells (RGCs) lost in glaucoma. Previously, we have shown that donor RGC survival and integration can be improved by altering the host microenvironment with neurotropic factors and chemokines. However, despite these improvements, most donor RGCs fail to integrate into the ganglion cell layer (GCL). Therefore, we hypothesized that some molecular barrier must be limiting donor RGC integration.
Methods : To identify this molecular barrier, we studied the donor RGC transcriptome following transplantation and found a 6-fold lower Down Syndrome Cell Adhesion Molecule (DSCAM) expression in integrated donor RGCs. DSCAM mediates neuronal self-avoidance to preserve mosaic spacing in the retina during development, which may prevent donor RGCs from migrating into the GCL. To inhibit DSCAM in donor RGCs, we established a Dox-inducible DSCAM shRNA human stem cell line (TetON-DSCAM-shRNA-hESC). RGCs were differentiated using 3D-retinal organoids, and doxycycline was used to induce DSCAM knockout (KO) ~24 hours before transplantation. For transplantation, 2x104 donor RGCs were injected subretinally, and SDF1 was injected intravitreally. Retinas were stained three days after transplantation and evaluated by tracking the position of donor RGCs in 3D reconstructions of retinal flat mounts (5-9 mice/group).
Results : DSCAM KO in donor RGCs resulted in the spontaneous migration of 92% of donor RGCs out of the subretinal space, compared to 52% without KO (Fig.). In agreement with our previous results, SDF1 treatment resulted in 69% of donor wild-type RGCs out of the subretinal space. Together, DSCAM KO and SDF1 treatment resulted in 86% of donor RGCs migrating out of the subretinal space, with 57% of donor RGCs migrating into the GCL compared to DSCAM KO alone with 37% of donor RGCs migrating into the GCL.
Conclusions : Our studies confirm that by controlling either the tissue microenvironment or intrinsic cell state, donor RGC integration can be improved. We validated our previous finding that SDF1 significantly improves the structural integration of donor RGCs, and showed for the first time that DSCAM is a major inhibitor of donor RGC migration within the retina. We have also demonstrated that this can be overridden with a temporary DSCAM KO in donor cells, enabling translatable therapeutic approaches to neuron replacement within the central nervous system.
Methods : To identify this molecular barrier, we studied the donor RGC transcriptome following transplantation and found a 6-fold lower Down Syndrome Cell Adhesion Molecule (DSCAM) expression in integrated donor RGCs. DSCAM mediates neuronal self-avoidance to preserve mosaic spacing in the retina during development, which may prevent donor RGCs from migrating into the GCL. To inhibit DSCAM in donor RGCs, we established a Dox-inducible DSCAM shRNA human stem cell line (TetON-DSCAM-shRNA-hESC). RGCs were differentiated using 3D-retinal organoids, and doxycycline was used to induce DSCAM knockout (KO) ~24 hours before transplantation. For transplantation, 2x104 donor RGCs were injected subretinally, and SDF1 was injected intravitreally. Retinas were stained three days after transplantation and evaluated by tracking the position of donor RGCs in 3D reconstructions of retinal flat mounts (5-9 mice/group).
Results : DSCAM KO in donor RGCs resulted in the spontaneous migration of 92% of donor RGCs out of the subretinal space, compared to 52% without KO (Fig.). In agreement with our previous results, SDF1 treatment resulted in 69% of donor wild-type RGCs out of the subretinal space. Together, DSCAM KO and SDF1 treatment resulted in 86% of donor RGCs migrating out of the subretinal space, with 57% of donor RGCs migrating into the GCL compared to DSCAM KO alone with 37% of donor RGCs migrating into the GCL.
Conclusions : Our studies confirm that by controlling either the tissue microenvironment or intrinsic cell state, donor RGC integration can be improved. We validated our previous finding that SDF1 significantly improves the structural integration of donor RGCs, and showed for the first time that DSCAM is a major inhibitor of donor RGC migration within the retina. We have also demonstrated that this can be overridden with a temporary DSCAM KO in donor cells, enabling translatable therapeutic approaches to neuron replacement within the central nervous system.