Abstract
Purpose :
There is a gap in our knowledge about the growth and guidance of human retinal ganglion cells (hRGCs) axons. This information is essential for predicting the success of stem cell approach to glaucomatous degeneration. Here, we demonstrate that hRGCs recognize spatially distributed evolutionarily conserved chemotropic cues for intraretinal guidance and respond to them by improved regenerative potential.
Methods :
RGCs were directly differentiated from hESBrn3b-tdT cells (Sluch et al., 2017 SCTM 6:1972) by recapitulating developmental mechanism. hRGCs were co-cultured with embryonic day 16 (E16) rat central (chemo-attractive cues) or peripheral (chemo-repulsive cues) retinal cells to measure the response of axons to the differentially distributed guidance cues. Functional impact chemotropic cues on hRGCs was examined by electrophysiological. hRGCs were transplanted in neonatal rat retina to examine their ability to target their axons to the host optic disc. The impact chemotropic cues on axon regeneration was examined using our microfluidic model of regeneration.
Results :
The following observations were made: (1) hRGCs elaborated long neurites on E16 central retinal cells regulated by Netrin-DCC interactions, suggesting their ability to read the chemo-attractive cues. (2) hRGCs neurites were shorter on E16 peripheral retinal cells. The length of neurites were increased upon neutralization of the chemo-repulsive interactions, suggesting hRGCs ability to read the chem-repulsive cues. (3) These chemotropic cues differentially impacted the physiological maturity of hRGCs. (4) The transplanted hRGCs oriented their axons into the host optic disc, regulated by Netrin-DCC interaction. (4). The chemotropic cues regulated regeneration of hRGCs axons by recruiting the mTOR pathway.
Conclusions :
Human RGCs, directly differentiated from pluripotent cells by recapitulating developmental mechanism, have the whereabouts for recognizing spatially distributed evolutionarily conserved chemotropic cues for centripetal orientation toward the optic disc. In the process these molecules may influence the physiological status and regeneration potential of the ectopic hRGCs.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.