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Timothy Catchpole, Karl Csaky; An Improved Method to Study Circulating Vascular Progenitor Cells (VPC’s) in Patients with Neovascular Age-Related Macular Degeneration (nvAMD). Invest. Ophthalmol. Vis. Sci. 2013;54(15):316.
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Our previous studies showed that cultured late outgrowth endothelial cells (OEC’s), a type of VPC, are associated with nvAMD (Thill IOVS 2008) and demonstrate varying levels of expression of SM22, a marker for smooth muscle cells. The role of the differing types of OEC’s in nvAMD is unclear. As culturing of OEC cells is variable and time-consuming, we developed an alternative approach that analyzes variation in SM22 expression in circulating CD34 positive cells, another type of VPC.
Peripheral blood mononuclear cells (PBMC's) were isolated via the FICOLL Hypaque method from 50 ml of peripheral blood from subjects with nvAMD (n=8). The PBMC population was separated into CD34 positive and negative populations with magnetic bead sorting (Miltenyi Biotec). RNA was isolated from the sorted populations and quantitative SM22 gene expression was analyzed using Taqman qPCR.
An average of 1.73x106±7.5x105 PBMC's/ml of blood was obtained (range 8.4x105 - 3.36x106 cells/ml). The average amount of CD34 positive cells obtained via sorting was 1.5x105±6x104 cells, which provided 230±153 ng of RNA for gene expression analysis. The CD34 positive fractions had an average Relative Quantification (RQ) value of 5.86±7.28 (range 1 - 19.77) for SM22 expression when normalized to the subject demonstrating the lowest expression.
In these initial results we observed a large variance in the expression of SM22 in the isolated CD34 positive PBMC’s, similar to the earlier studies in cultured OEC’s. This suggests that this alternative approach is a viable way of studying gene expression in VPC’s. This technique will facilitate the study of the VPC population in nvAMD subjects, potentially leading to new insights into the disease.
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