Abstract
Purpose :
Notable transplantation studies demonstrated intercellular transfer of cytoplasmic material including fluorescent labels between donor and host photoreceptors (PR). This phenomenon, material transfer (MT), confounds cell tracking analyses. We examined whether MT is PR-specific by conducting rigorous assessments for MT after retinal ganglion cell (RGC) transplantation.
Methods :
Organotypic retinal explants from B6 ACTb-EGFP mice were cocultured with tdTomato+ (tdT+) human embryonic stem cell derived RGCs (hRGCs) on the vitreous surface for 1 week. We assessed MT by inspecting for tdT labeling and human-specific lineage markers colocalized to host GFP+ retinal cells. We compared the incidence of MT in retinas with intact and enzymatically disrupted internal limiting membrane (ILM) using Pronase. We also assessed MT rates following in vivo intravitreal hRGC transplantation.
Results :
3D reconstructions of tiled confocal retinal flatmounts revealed tdT+GFP+ cells in the host inner nuclear layer (INL) with distinct Müller glial (MG) morphology. Immunostaining confirmed GFAP and tdT colocalization throughout the soma and radial processes in 85% of the atypical cells sampled (n=20). tdT+GFP+ cells were in contact with tdT+ hRGCs and retained a nuclear heterochromatin structure that was specific to mouse INL cells and distinct from hRGCs. We assessed recipient retinas for human-specific donor-derived nuclear antigen (HuNu) and mitochondrial antigen (HuMito), and found 92.7% of tdT+GFP+ MG were HuNu+ (n=68) and 72.7% were HuMito+ (n=11). Fluorescence activated cell sorting of tdT+GFP+ cells demonstrated greater expression of human-specific RGC transcripts, including BRN3B, RBPMS, and TUBB3, compared to tdT-GFP+ cells. We found 6.7% of BSS (n=15) and 35.5% of Pronase-treated (n=31) retinal explants exhibited tdT+GFP+ cells (p=0.037). Following in vivo transplantation, we observed tdT+GFP+ MG within the host INL exclusively in retinas with ILM disruption (0% in BSS-treated, n=32; 9.7% in Pronase-treated, n=31; p=0.071).
Conclusions :
We identified that hRGC xenografts into mouse retinas transferred donor-derived material, including cytoplasmic, nuclear, and organelle proteins and mRNA. The primary recipient cell type is MG, and MT is dependent on ILM disruption. We demonstrated that retinal MT is not unique to PRs and must be emphasized when interpreting hRGC transplantation results.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.