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Title: Investigation into the application of adult human Müller stem cells in retinal ganglion cell replacement therapy
Author: Singhal, Shweta
ISNI:       0000 0004 2676 1182
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2007
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This work investigated the potential of adult human Muller stem cells to differentiate towards a retinal ganglion cell (RGC) fate in vitro and their ability to replace damaged RGC in vivo. Muller stem cells isolated from the adult human retina were examined for their expression of RGC developmental markers ATOH7, BRN3B, ISL1 and HUD, and extracellular matrix proteins and growth factors were used to modulate the expression of these RGC makers by these cells in vitro. The role of the Notch signalling pathway in Muller stem cell proliferation and differentiation was also examined and it was found that inhibition of Notch activity using gamma secretase inhibitor DAPT could further promote differentiation of these cells towards RGC fate in vitro. Cell preparations enriched for RGC developmental markers demonstrated neural morphology with extensive neurite formation. More significantly Muller stem cells differentiated into the RGC phenotype were able to demonstrate neuronal function as elicited by changes in their cytosolic calcium in response to neurotransmitter stimulation with nicotinic agonists. To further confirm the RGC phenotype, a BRN3B GFP transcriptional reporter was designed and transfected into the Muller stem cells to specifically identify the BRN3B expressing committed RGC precursors. Transduced cells expressing GFP were found to be post mitotic, expressed high levels of differentiated RGC markers and increased in numbers when cultured in the RGC differentiating conditions mentioned above. Transplantation of Muller stem cells in vivo into models of retinal degeneration revealed that microglia and chondroitin sulphate proteoglycans (CSPG) present a significant barrier to transplant cell migration. Use of enhanced immunosuppression and the CSPG degrading enzyme Chondroitinase ABC significantly improved transplant cell survival and migration of Muller stem cells in degenerating RCS rat retinae. These transplant conditions were used in eyes depleted of RGC by NMDA in the presence of Triamcinolone, to study the ability of differentiated Muller stem cells differentiated into RGC to replace these cells in vivo. The RGC differentiated Muller stem cells were able to migrate and integrate into the RGC depleted rat retina. More significantly they were able to partially restore lost retinal ganglion cell function as demonstrated by an improvement in the negative scotopic threshold response on the ERG. These results suggest that adult human Muller stem cells are similar to embryonic retinal progenitors in their intrinsic programming and can be directed using the same developmental cues to differentiate towards an RGC fate in vitro. Muller stem cells differentiated into RGC are also able to integrate and partially restore function in models of RGC depletion in vivo. They may therefore constitute a potential source of RGC for replacement therapy in glaucoma.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available