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.

Hopes, Marina (2014) 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. Doctoral thesis, University of East Anglia.

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Abstract

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.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Mia Reeves
Date Deposited: 28 Jan 2016 09:27
Last Modified: 28 Jan 2016 09:27
URI: https://ueaeprints.uea.ac.uk/id/eprint/56815
DOI:

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