Use this URL to cite or link to this record in EThOS:
Title: Human Organotypic Retinal Cultures as a model of human retinal ganglion cell degeneration in glaucoma : effects of epigenetic regulation and mesenchymal stem cell derived growth factors
Author: Hopes, Marina
ISNI:       0000 0004 5371 2765
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2014
Availability of Full Text:
Access from EThOS:
Access from Institution:
Purpose: Human Organotypic Retinal Cultures (HORCs) have been shown to be a useful experimental model to investigate retinal ganglion cell (RGC) fate in short term models of glaucomatous stress. The aim of the current work was to investigate the long-term fate of RGCs in HORCs and to develop culture conditions to promote the RGC survival. The potential neurotrophic effect of mesenchymal stem cell derived growth factors on the RGC survival was studied and the role of epigenetic regulation of retinal cell gene expression was examined. Methods: Quantitative real-time polymerase chain reaction (QRT-PCR) was used for assessment of retinal cell marker genes expression, immunohistochemistry and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) for quantitative assessment of apoptotic RGCs. LDH activity in culture media was used to assess total cell death in HORCs. Results: Serum-free (SF) DMEM/HamF12 with no added antibiotics was found to be the medium of choice for the human retina culture. A statistically significant loss of NeuN-labelled RGCs was documented after 2 weeks in culture using SF DMEM/Ham F12, whereas with Neurobasal medium, the loss was detected after week 1. The numbers of apoptotic RGCs were high under all culture conditions after 1 week. Vascular endothelial growth factor (VEGF) and platelet derived growth factors (PDGFs) conferred a protective effect on RGC survival in long-term HORCs, whereas leukaemia inhibitory factor (LIF) failed to exert this effect. The loss of RGC-derived gene markers expression was selectively altered by the histone deacetylase inhibitor (HDACI) trichostatin A (TSA). Conclusions: The timing for long-term HORCs use ex vivo is dependent on culture conditions. Long-term HORCs can be used for up to 2 weeks in order to prevent detectable RGC loss. Both VEGF and PDGF possess an ability to prolong RGC survival in long-term HORCs. HDAC inhibitor TSA selectively reverses the down-regulation of RGC-derived gene markers expression.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available