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S. Hanekamp, A. Otani, M. Friedlander; Characterization of Hematopoietic Stem Cell (HSC) Subpopulations to Determine Mechanism of Targeting to Developing Retinal Vasculature . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1689.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify cell surface markers of bone marrow-derived hematopoietic stem cells (HSC) that functionally correlate with targeting of HSC subpopulations to developing vasculature in the mouse retina. Methods: Cell isolation. Bone marrow cells were extracted from adult BALB/cBYJ, ACTbEGFP, or C3H mice. Monocytes were purified by density gradient separation, labeled with various biotin-conjugated antibodies and separated into subpopulations using a magnetic cell-sorting device (MACS). Intravitreal Administration of Cells. An eyelid fissure was created with a fine blade to expose the P2 to P6 eyeball. Cells (~5 x 105 in 0.5µl) were then injected into the vitreous using a 33-gauge needle.Cell Characterization. Subpopulations of HSC were labeled with either PE- or FITC-conjugated antibodies to a variety of integrin subunits or endothelial cell markers as detailed below. Labeled cells were analyzed using Fluorescence Activated Cell Sorting (FACS). Sequential western blotting was used to identify heterodimer pairing. Results: Subpopulations of HSC differentially express a variety of cell surface markers that correlate with function. HSC subpopulations expressing the following markers were isolated by MACS: lin-; lin+; CD31-; CD31+; CD31, CD34 and Mac1-. These subpopulations were further analyzed by FACS for the presence of cell surface markers (Sca1, KDR, cKit, CD31, Tie-2) and various integrins (αv, α6, ß1, ß4, ß3 and ß5). These populations were also analyzed for differences in function. Each of the HSC subpopulations express different levels of individual integrins and cell surface markers. These differences correlate with observed differences in function (e.g., ability to incorporate into developing vasculature). Conclusions: Adult bone marrow contains cells have been shown to target and incorporate into developing mouse retinal vasculature. While the precise molecular basis of this targeting is not known, we demonstrate that subpopulations of HSC express cell surface markers that correlate with their ability to incorporate into developing vasulature. Such characterization of functionally important associations between cell surface markers and specific functions will assist in the identification of stem cells that may be useful in the treatment of degenerative and neovascular diseases of the eye.
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