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Run-Tao Yan, Li He, Shu-Zhen Wang; RPE-like cells emerging from chick glial cell culture. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4570.
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© ARVO (1962-2015); The Authors (2016-present)
The demonstrations of Müller-originated retinal regeneration in fish and of glia-originated neurogenesis in adult mammalian brain have heightened the expectation for Müller glia being endogenous stem/progenitor cells for retinal regeneration in high vertebrates. This study tests whether the presumptive stemness/progenitorness of Müller glia would include the potential to develop towards RPE cells.
Primary Müller glial cell cultures were established using E13 and E14 chick retinas. These specific ages were used for two reasons: (i) the neural retina can still be readily separated from the RPE during dissection so as to avoid RPE carryover in harvested tissues; and (ii) retinogenesis has ceased in the central and central-peripheral regions so as to minimize the population of uncommitted cells. The peripheral region of retina was excluded to minimize potential contamination by progenitor cells. Trypsin/EDTA was used to dissociate cells of the harvested tissue, and the disassociated cells were cultured in 75-mL flasks. Frequency of medium replacements was reduced to promote neuronal cell death from malnutrition. Microscopy and immunocytochemistry were used to assess Müller-to-RPE transition.
After 1 week in culture, Müller glial cells started to constitute a significant portion of the cell population as they proliferated while neurons died off. This trend continued as culture time lengthened. At 2 weeks, the culture became confluent with glial cells, and the vast majority of the retinal neurons had died. At this point, sporadic, pigment granules-containing (RPE-like) cells were visible. As the culture aged, more cells exhibited pigment granules and the pigmentation enhanced. By 3 weeks, the culture contained many patches, large and small in size, of darkly-pigmented cells. By 4 weeks, some of the darkly-pigmented cells were cobblestone-like, similar to that of RPE cells. After 7 weeks in culture, pigmented cells appeared to cover the entire surface of the vessel, and some of the cells were immunopositive for RPE protein RPE65.
These observations indicated a Müller-to-RPE transition occurring in the culture and imply that those Müller glial cells retained certain stemness/progenitorness with default pathways that included RPE, similar to that of pluripotent stem cells, such as ESCs and iPSCs.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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