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S. G. Lecaude, R. S. Zulliger, S. Wolf, V. Enzmann; In vitro Characterization of Retina-Committed Bone Marrow-Derived Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2637.
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The bone marrow contains hematopoietic stem cells but also other stem and progenitor cell types that are not committed to the hematopoietic lineage. The purpose of this study is to isolate and characterize bone marrow-derived progenitor cells and to determine their differentiation potential towards retinal pigment epithelial (RPE) cells.
Bone marrow was harvested from tibias and femurs of three weeks old GFP C57BL/6 mice. First, mature hematopoietic cells were removed using a cocktail of lineage antibodies coupled with paramagnetic beads (MACSTM) or by flow cytometry (FACS). Second, the cell populations of interest were separated by FACS. These cells were then grown in direct coculture with low passage murine RPE cells for 10 days. Expression profile of the progenitor cells in co-culture was assessed by immunocytochemistry and after additional FACS separation by real time qPCR.
Lineage depletion of the bone marrow performed by FACS and MACS yielded similar results with a lineage negative population representing 1.45±0.07% and 2.81±0.02% of the bone marrow population, respectively. Therefore the MACSTM system was adopted as the method of choice. After 10 days of co-culture the GFP+ progenitor cells adopted an RPE-like elongated morphology and immunocytochemistry showed expression of RPE markers such as RPE65 and MITF. The number of GFP+ cells harvested by FACS after co-culture ranged from 2.56±3.39% to 71.28±0.28% of the input cell number. The yield was depending on the progenitor cell type, its attachment properties to the culture dish and proliferation or lack thereof. RTqPCR analysis showed the expression of RPE markers (RPE65 and MITF) and neuronal markers (GFAP, TUBB3, MAP2).
The progenitor cells isolated from the bone marrow showed the expression of RPE markers and RPE-like morphology after co-culture with RPE cells. Further studies are pivotal to assess the functional properties of these progenitor cells pre-differentiated in vitro. These cells might be of interest for therapeutic use in degenerative pathologies such as AMD.
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