SDF1 directs donor retinal ganglion cell migration into the retina following allotransplantation in mice

Abstract

Purpose : Glaucoma and other optic neuropathies lead to permanent damage of the optic nerve and loss of retinal ganglion cells (RGCs). Cell transplantation has been proposed to restore the retinal neurocircuitry; however, current state-of-the-art studies show that most donor RGCs have limited capacity to integrate into the ganglion cell layer (GCL) following intravitreal delivery. Since a fully developed mammalian retina does not have the natural capacity to regenerate RGCs, we do not know the exact mechanisms and cues employed by stem cell-derived donor RGCs to migrate across the retina (XY plane) and into the GCL (Z-axis). Here, we investigate the migration of donor RGCs into and across the retina in the presence of a stromal cell-derived factor-1 (SDF1) gradient.

Methods : RGCs were differentiated from Thy1-GFP mouse iPSC (C57Bl/6 background) in 3D retinal organoid cultures. On day 21, RGCs were isolated from organoids by magnetic microbeads against CD90.2. The ability of stem cell-derived RGCs to migrate in vitro was tested using a microfluidic device. For transplantation RGCs were formulated at 5 x 10⁶ cells/mL with slow-release GDNF & BDNF particles. 2µL of Thy1-GFP+ RGC suspension was injected intravitreally (IVT), and 1µL 10ng/µL SDF1 was injected subretinally (SR) to establish a chemokine gradient across the retina to direct RGC migration. Two weeks after transplantation, whole mounted retinas were stained for GFP and RBPMS to assess donor RGC integration and distribution with respect to host RBPMS+ cells (GCL). RGC distribution across the retina was calculated as the percentage of 344 x 344 µm tiles within the whole mount that contained more than one integrated RGC.

Results : The expression of CXCR4, the receptor for SDF1, on donor cells was confirmed by qPCR and flow cytometry. Thy1-GFP+ donor RGCs integrate into GCL of host retinas with and without SR SDF1 and have the same morphology in both groups. The artificial SDF1 gradient increased the retinal coverage by donor cells from 54 ± 8% to 76 ± 4% (Figure 1), proposing a benefit of CXCR4-SDF1 interactions for enhanced donor RGC migration.

Conclusions : Establishing an SDF1 gradient across the host retina is an effective strategy to improve the distribution and structural integration of donor RGCs.

This is a 2021 ARVO Annual Meeting abstract.

 

Figure 1. SR injections of SDF1 improves donor RGC coverage within the host retina. Mean ± SD *p < 0.05