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Shweta Singhal, Venkata Sudheer Babu Pakala, Milan N Mehta, Veluchamy A Barathi, Tien Yin Wong; Primary muller glial cultures of primate origin. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4208.
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© ARVO (1962-2015); The Authors (2016-present)
Age related retinal degeneration is a significant cause of irreversible blindness worldwide. An alternate approach to cell replacement therapy would be to enhance the ability of the retina to effect its own repair. Muller glia (MG) can mediate complete retinal regeneration after injury in zebrafish by transforming into stem cells. This regenerative capacity is attenuated in mammalian eyes where muller glia respond to injury with a gliotic response instead. Identifying key signals that activate/enhance the regenerative ability of MG could therefore be an elegant therapeutic strategy for irreversible retinal disease. However, differences between rodent and human eyes; and difficulty in sourcing/culturing human MG have hindered research in this area. The aim of this work was to investigate whether primate retinae can be used to establish reliable MG cultures, and ultimately help define critical regulators of the regenerative ability of higher mammalian MG.
Using previously defined protocols for MG isolation from rodent and human eyes, whole retinae of 6 globes from 4 different adult primates (sp. macaca fascicularis) were isolated after post mortem enucleation, digested using a trypsin based enzyme cocktail and cultured on fibronectin. Cell growth was monitored by phase contrast microscopy.
Retinal cell suspensions from all 6 globes demonstrated adhesion of scattered cells after one week in culture. These adherent cells expanded clonally to form colonies by 2 weeks and grew into a confluent monolayer by 6-8 weeks in culture. Once confluent the cells were amenable to passage and expansion in culture using trypsin digestion. They demonstrated a slow doubling time requiring up to 8-10 weeks to reach confluence per passage. We have been able to culture these cells for 9 passages thus far without loss of morphology or self-renewal capability. Their expression of muller glial markers (glutamine synthase, CRALBP), retinal markers (OTX2 and PKC) and stem cell marker LGR5 were characterised using immunocytochemistry and RT PCR.
Primary primate muller glial cultures can be established in vitro with a high success rate. While slow growing they are stable in culture and sufficiently expansible to enable molecular characterisation. To our knowledge this is the first report describing the behaviour of primate MG in culture, a potential resource to identify actionable targets for human retinal regenerative therapies.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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