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Tetsu Asami, Wendy Dailey, Mei Cheng, Sandra Galaforo, Michelle Dwyer, Kimberly A. Drenser; Hematopoietic Stem Cell Integration and Differentiation within the Murine Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3178.
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Several studies have shown that hematopoietic stem cells (HSC) have the ability to differentiate into non-hematopoietic cell types. We describe our experience with isolating a specific collection of HSCs called side population (SP) cells and exposing them to a factor known to activate the Wnt-signaling pathway, Norrin protein (NDP), to see if the activation of this pathway promotes integration and differentiation into other cell types.
All our experiments were conducted in compliance with the ARVO statement for the use of animals in ophthalmic and vision research. Flow cytometry was used to isolate the SP cells from the bone marrow of transgenic mice expressing enhanced green fluorescent protein (EGFP). Side population cells were added to the vitreous side of retinal explants of non-transgenic C57BL/6 mice pretreated with 5mU intravitreal injections of microplasmin. The explants were cultured for 3 weeks in B-27/N-2 serum free media with or without NDP for the first week. The explants were fixed at 1, 2 and 3 weeks and frozen sections were prepared and stained with various immunomarkers. We counted the number of SP cells in the ganglion cell layer, inner retinal layer and outer retinal layer of the explants and compared the 2 groups at different time points to assess stem cell survival. We counted the positive cells for various immunomarkers and compared the groups to each other.
The number of SP-derived cells in NDP treated retina was larger than that in non-NDP treated retina in all layers at 1, 2 and 3 weeks. It was also noted that many SP-derived cells had changed their morphology. In NDP treated retina, the number of positive cells for Nestin increased with longer culture duration and peaked at 3 weeks. In contrast, the number of Nestin positive SP cells in non-NDP treated retina peaked at 1 week. Immunostaining for activated microglia demonstrated microglia positive cells peaking at 2 weeks and 3 weeks in non-NDP treated and NDP treated retina, respectively.
The exposure of explants to NDP appears to help SP cells survive in retinal explants and appears to alter the differentiation pathways of stem cells.
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