Abstract
Purpose :
Retinal ganglion cell (RGC) transplantation is an exciting solution to restore vision lost in glaucoma. Despite recent progress in improving the survival and integration of neurons following transplantation, a significant number of donor stem cell-derived RGCs remain structurally and functionally non-integrated.
Methods :
We have analyzed several available single-cell RNA sequencing datasets of grafted neurons (retinal ganglion cells, dopaminergic neurons, and photoreceptors) and non-neuronal donor cells (retinal pigment epithelium, hematopoietic cells) and identified DSCAM to be specific to the integration of RGCs. To study the effects of DSCAM block on RGC maturation and integration, we established an inducible DSCAM knockout stem cell line and co-delivered donor RGCs with an anti-DSCAM antibody. To study donor neuron integration, stem cell-derived RGCs were delivered subretinally or intravitreally in mice. For subretinal cell delivery, SDF1 was injected intravitreally to establish a chemokine gradient across the retina to further drive structural integration. Retinas were stained three days after transplantation and evaluated by identifying the position and maturation state of each donor RGC in 3D reconstructions of retinal flat mounts (5-9 mice/group).
Results :
Following transplantation, our transcriptome analysis showed that DSCAM expression was 6-fold lower in integrated donor RGCs. Silencing DSCAM and co-delivering SDF1 resulted in 56% of donor RGCs migrating from the subretinal space into the GCL, compared to 16% in the control (~40 donor RGCs per retina). Preliminary results also show more donor RGCs within the neural retina after blocking DSCAM following intravitreal delivery. Only those donor RGCs that integrated within the GCL expressed the mature RGC marker, RBPMS. Moreover, only RBPMS+ve donor RGCs were capable of spontaneous neural activity, indicated by an average peak burst firing rate of 3 Hz in vitro.
Conclusions :
Here, we confirmed that the integration of donor RGCs can be improved by controlling the microenvironment and cell state. We have demonstrated that blocking DSCAM can enhance donor RGC structural integration. This information will be invaluable for developing therapeutic approaches to replace neurons within the central nervous system.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.