Abstract
Purpose :
Glaucoma leads to the loss of retinal ganglion cells (RGCs). Cell transplantation has been proposed to restore RGCs; however, current attempts show limited donor RGC integration. We previously used stromal cell-derived factor-1 (SDF1) to direct mouse RGCs into the retina, but the migration mechanisms have not yet been studied.
Methods :
To improve clinical relevance, human RGCs were differentiated from Brn3b-tdTomato hESC in organoid cultures. RGCs were isolated by magnetic microbeads against CD90.2 at day 38-46 of differentiation. We studied RGC migration kinetics and modes in response to SDF1 gradient in vitro and in vivo using transwell setup and subretinal transplantation, respectively. For loss-of-function studies, we have applied: 1) small-molecule inhibitor of CXCR4 receptor on RGCs – AMD3100; 2) somal translocation inhibitor – CK666; 3) multipolar migration inhibitor – roscovitine.
Results :
Expression of CXCR4 on hESC-derived RGCs was confirmed by transcript- and proteomics, with ~83% of RGCs being CXCR4+. AMD3100, an SDF1 antagonist, treatment decreased the RGC recruitment in response to SDF1 from 42.2±10.5% to 12.7±1.8% (n=3; p<0.01) in vitro. Time-lapse studies in vitro show that inhibiting somal translocation decreases migration speed from 5.64±2.18 to 4.69±2.01µm/s (n=3, p<0.01), whereas inhibiting multipolar migration decreases speed to 3.20±1.52µm/s (n=3, p<0.01) in vitro – demonstrating hESC-RGCs migration via both modalities.
The short-term transplantation studies confirmed our previous observations that an SDF1 gradient significantly improves the migration and integration of human RGCs within three days after subretinal delivery. Blocking CXCR4 in donor RGCs also led to decrease in transplantation outcome: transplantation score (TS) of 3.3±0.3 (n=3, m=15) vs 1.3 (n=1, m=6).
Lastly, transplantation experiments with inhibitors suggest that multipolar migration is the major contributor to donor RGC migration in the retina, with donor RGCs migrating via multipolar migration having a TS of 3.2 (n=1, m=6) and RGCs migrating via somal translocation having a TS of 2.8 (n=1, m=5).
Conclusions :
Establishing an SDF1 gradient across the host retina enhances donor hESC-RGCs integration primarily through multipolar migration, and SDF1 acts through the CXCR4 receptor – suggesting the possibility that RGCs selected for these features before transplantation will improve donor cell integration.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.