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Li He, Run-Tao Yan, Shu-Zhen Wang; Generating photoreceptor-like cells by reprogramming cultured chick Müller glia with neurogenin. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):413.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptor regeneration bears important clinical implications and is a target of NEI Audacious Goal. Studies show that after retinal neurons are experimentally ablated Müller glia in higher vertebrates becomes activated and can produce a small number of neurons. Nonetheless, there lacks compelling evidence for Müller glia to generate photoreceptor cells. This study tests whether photoreceptor-like cells can be generated in chick Müller glia cell culture reprogrammed with neurogenin.
Müller glia cell culture was established with functionally mature retina isolated from day 19 chick embryos. During dissection, the peripheral region of retina was removed to minimize potential contamination by progenitor cells at the ciliary margin. Primary culture of Müller glia was passed once. At ~50% confluency, passage one culture was infected with RCAS retroviruses expressing neurongenin1 (ngn1), ngn3, or GFP as control. Immunocytochemistry and cell morphology were used to determine the presence of photoreceptor-like cells in the culture.
After two weeks in culture, a vast majority of the retinal neurons in the primary culture had died off and the culture became confluent with Müller glia cells. In passage one cultures, cells positive for visinin, a calcium-binding protein of photoreceptor cells, were abundantly detected in those infected with RCAS-ngn1 or RCAS-ngn3, whereas the control culture lacked such cells. Visinin+ cells exhibited morphologies typical of young photoreceptor cells - a compact cell body with a thin process on one side and a short, inner-segment-like compartment on the other. The cultures were also examined with RA4 antibody, which labels retinal ganglion cells and some unidentified progenitor/precursor cells. While RA4+ cells were present in all cultures, RA4+ cells with neuronal morphologies - displaying thin, long processes reminiscent of axons - were only present in cultures infected with RCAS-ngn1 or RCAS-ngn3, with those in the control culture overtly maintaining morphology of Müller glia (flat and lacking thin, long processes).
Our data show that photoreceptor-like cells could arise de novo in Müller glia cell cultures when reprogrammed with ngn1 or ngn3 and suggest a possibility of using ngn1 or ngn3 to guide reactive Müller glia towards the regeneration of photoreceptors and ganglion cells as well.
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