Investigation into the application of adult human Müller stem cells in retinal ganglion cell replacement therapy.
Doctoral thesis, UCL (University College London).
Müller stem cells were examined for expression of the retinal ganglion cell (RGC) developmental markers Atoh7, Brn3b, Isl1 and HuD. They expressed Atoh7 and Brn3b but very low levels of Isl1 and HuD, suggesting the existence of a population of RGC precursors among them. Expression of these markers varied with different extracellular matrices and growth factors used for cell culture. Progenicity of Müller stem cells was shown to be regulated by Notch activity and Notch inhibition with DAPT caused upregulation of RGC markers and acquisition of neural morphology. To identify the committed RGC precursor population and to follow its differentiation in culture, a Brn3b GFP transcriptional reporter was designed and transfected into Müller stem cells in vitro. Committed RGC precursors thus identified exit the cell cycle and upregulated expression of differentiated RGC markers as seen during development. Calcium imaging in response to neurotransmitters in vitro demonstrated the ability of the differentiated Müller cells to function as RGC. Functional activity of Müller stem cells in vivo was investigated by transplantation into animal models of retinal degeneration. Although undifferentiated Müller stem cells migrated and integrated into neonate retina, they failed to integrate in the degenerating RCS rat retina. The results showed that chondroitin sulphate proteoglycans (CSPGs) and microglia prevented migration of Müller stem cells in vivo and increased immunosuppression and treatment with chondroitinase ABC (a CSPG degrading enzyme) promoted migration of grafted cells. Using these conditions the behaviour of grafted Müller stem cells differentiated towards RGC fate was investigated in rat eyes depleted of RGC using NMDA and treated with TA to control microglial activation. These animals were examined for evidence of transplant cell migration and integration using histology, and for improvement in RGC function using ERGs. The results suggest that adult human Müller stem cells are similar to embryonic retinal progenitors in their intrinsic programming and can be directed using the same developmental cues to differentiate towards a RGC fate in vitro. They may therefore constitute a potential source of RGC for replacement therapy in glaucoma.
|Title:||Investigation into the application of adult human Müller stem cells in retinal ganglion cell replacement therapy|
|Additional information:||Authorisation for digitisation not received|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Ophthalmology|
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