Retinal Ganglion Cell (RGC) Precursors Derived From Adult Human Muller Stem Cells Exhibit Neural Function in vitro and Partially Restore RGC Function in vivo

Abstract

Purpose: : This study aimed to differentiate adult human Muller stem cells towards a retinal ganglion cell (RGC) phenotype in vitro using extracellular matrix proteins, growth factors and Notch inhibition (known to promote RGC differentiation during development). Differentiated cells were analysed for their neural phenotype and function in vitro and for their ability to restore RGC function in vivo.

Methods: : Muller stem cells were cultured on matrigel in the presence of FGF2 and the gamma secretase inhibitor DAPT (a Notch inhibitor). Cells were examined for expression of markers of RGC precursors and for changes in cytosolic calcium in response to glutamatergic, muscarinic and nicotinic stimulation using calcium imaging techniques. Enriched preparations of cells expressing RGC markers were transplanted intravitreally into 4 week old Lister Hooded rat eyes depleted of RGC by NMDA treatment. Migration and localisation of transplanted cells was examined immuno-histochemically at 3-4 weeks post-transplantation. Scotopic threshold response (STR) on the electroretinogram was used to assess RGC function in the transplanted animals, also at 3-4 weeks post-transplantation.

Results: : Muller stem cells cultured under the above conditions expressed the RGC markers BRN3B, ISL1 and HUD in vitro. They also acquired the ability to respond to nicotinic stimulation (characteristic of RGC) as seen by a rise in cytosolic calcium levels. Over 95% of the differentiated cells responded to nicotinic stimulation compared to less than 25% of the control cells. By contrast their ability to respond to glutamatergic stimulation (a Muller glial characteristic) was markedly decreased (from 70% in control cells to less than 5% in the differentiated cells). When transplanted in vivo the differentiated cells migrated into the RGC layer where they showed neural morphology and expressed ISL1 and HUD. More importantly, NMDA treated animals which received the differentiated Muller stem cell transplantation showed significant recovery of their negative STR amplitudes (p=0.0214, n=10) compared to NMDA treated animals which did not receive cell transplantation.

Conclusions: : These observations strongly suggest that Muller glial stem cells from the adult human retina have the ability to differentiate into functional RGC precursors with the potential to restore RGC function in vivo. These cells may constitute a valuable tool for the design of cell based therapies to treat RGC disease.

Keywords: Muller cells • ganglion cells • transplantation