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Hui Lin, Lamei Deng, Ting Xie; A Small Population of Nestin Positive Cells from Adult Human Neural Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1123.
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© ARVO (1962-2015); The Authors (2016-present)
To establish human retinal cell lines with retinal stem cell potential fromprimary culture of neural retina.
A total of 28 donor healthy human eyes were obtained within 24h post mortem from the Tongren Eye Bank with full ethical approval, under the tenets of the Declaration of Helsinki. The neural retina was gently seperated for culture. Serum free medium with EGF and bFGF was used. When cells proliferated to form confluent monolayer colonies, different morphological colonies were digested and cultured respectively. Differentiation Media was maintained for 1 month for retinal neurons induction. Standard protocols were used for IF staning of the cultured cells at P2-P8 in serum-free medium and differentiation medium.
Single cell suspensions from adult neuroretina were plated on gelatin-coated plates. Only very few cells attached the plates within the first days, and even less went proliferation after 4-6 days. Several confluent colonies with different morphologies were formed after 2-3 weeks. For most colonies, cells could only proliferated limited days and arrest the growth at P1. For some other type of colonies, cells have short-term quick proliferation abilities and can be passaged for 2-3 times. For very few colonies (less than 0.5%) with the morphology (small size, obvious nuclei and short spindle shape), cells can proliferate and maintained for more than 8 passages. For this small polulation of cells, immunofluorescence staining showed positive staining of Nestin and A2B5 in cell surface and cytoplasma, and Sox2 and Islet1 in the nuclei. Pax6 staining was found both in the cytoplasma and nuclei in these cells. More interesting, the putative Müller glia marker, GS, which is expressed in the cytoplasma in Müller cells, was positive staining in the nuclei of these retinal cells. For the other colonies derived cells, few cells were positive staining with nestin and other stem cell marker. However, rare cells with differentiation retinal neuron markers were identified in these cells in differentiation media.
Although a small population of cells with retina stem cell markers expression can be isolated from adult human neural retina and expanded ex vivo, these cells are still hardly to be induced into retinal neurons.
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