Gradual expansion of RPE-like cells in chick Müller glial culture

Abstract

Purpose : Müller glia is considered to have the potential to give rise to multipotent progenitor cells. Previously we detected cells that were darkly-pigmented and positive for RPE65 (RPE-like) arising from Müller glial cell cultures established with embryonic day (E) 13 and E14 chick retina. To shed light on this presumed “Müller-to-RPE” phenomenon, this study examined whether it could occur when more developed retina was used and whether and how RPE-like cells amplified in the culture.

Methods : Primary Müller glial cell cultures were established with central and central-peripheral regions of chick retina of E16, when cell proliferation has ceased, and E18, when the retina becomes functional. The neural retina was separated from attached RPE by treatment with a Ca²⁺-free and Mg²⁺-free solution. Dissociated retinal cells were cultured at low density with infrequent medium replacement.

Results : After 7 days in vitro (DIV), Müller glial cells started to constitute the majority of the cell population. From 0 DIV to 25 DIV, the culture lacked cells containing the dark pigment granules typically present in RPE cells. By 28 DIV, darkly-pigmented cells, albert low in number, were visible under microscope, but not yet visible to the naked eyes. More cells became darkly-pigmented as the culture aged. Many of these cells localized/clustered as colonies visible to the naked eyes. Sequential photographs of the same culture over time showed that individual colonies of darkly-pigmented cells increased in their sizes (enlargement), but their pattern of distribution within the culture and their shapes (marked by darkly-pigmented cells) remained more or less the same throughout the period.

Conclusions : The emergence of darkly-pigmented cells in E18 culture suggests a Müller-to-RPE transition occurring in culture derived from well-developed (functional) retina. The conservation of the shape of melanized colonies and their overall distribution pattern implies a presence of localized factor(s) promoting RPE-like cell “recruiting.” It remains to be studied whether the localized factor(s) is of “progenitor proliferation” or of molecular cues guiding existing, neighboring cells to become RPE-like.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.