Differential phenotype and behavior in culture of CD34 positive cells from peripheral blood and adipose tissue

Abstract

The localization and quantification of endothelial progenitor cells (EPCs) are controversial. Circulating CD34 + cells in blood have been identified as EPCs and as biomarkers of cardiovascular disease. We discuss in this paper the current data describing differential phenotype and behavior in vitro of CD34 positive cells from the circulation and adipose tissue (AT). We also describe in brief our own findings from CD34 + cells isolated from leukopheresis cones derived from healthy platelet donors and from patients undergoing bariatric surgery. We conclude that CD34 + cells in blood and in AT are different in antigenic profile and behavior in culture. The findings described assert that CD34 + cells detected in blood previously identified as biomarkers of cardiovascular disease are predominantly HPCs rather than EPCs, and that true CD34 + EPCs can be readily identified and extracted from AT, supportive of the current evidence which suggests EPCs are resident in the tissue vasculature.

Keywords: Adipose tissue; Cell culture; Endothelial progenitor cells; Flow cytometry; Hematopoietic progenitor cells; Peripheral blood.

Figure 1

CD34+ cell isolation, culture and flow cytometry from human peripheral blood and adipose tissue. Top panels: Human omental adipose tissue was treated with collagenase solution and the liquid cell suspension phase (stromal vascular fraction/SVF) obtained. Buffy coat was obtained from leukopheresis cones. The cell suspensions were incubated with anti-CD34 monoclonal antibodies tagged with magnetic microbeads and the CD34 + fractions isolated by magnetic activated cell sorting. Left lower panel: CD34 + cells obtained from blood (leukopheresis cones) were analyzed by flow cytometry at baseline, and after 3 and 7 days in culture. Cells did not survive in endothelial growth medium and proliferated in hematopoietic medium only. Flow cytometry demonstrated persistent CD45 expression at isolation and at 3 and 7 days in culture, but negative for VEGFR2 expression throughout those time periods. At 7 days, CD34 expression is absent but CD45 expression persists, consistent with a more mature hematopoietic phenotype. A photomicrograph of these cells is shown (200x). Right lower panel: CD34 + cells obtained from adipose tissue were analyzed by flow cytometry at baseline, and after 7 days in culture. Cells proliferated in endothelial growth medium and when analyzed by flow cytometry were VEGFR2 positive but CD45 negative, similar to human vascular endothelial cells as shown. At 7 days in culture, CD34 + cells isolated from adipose tissue demonstrated persistent VEGFR2 expression and also expression of CD31, but no longer expressed CD34. They remained CD45 negative. A photomicrograph is shown of these AT derived cells in culture (200x magnification), demonstrating a cobblestone appearance of spindle shaped cells after 7 days. Confocal microscopy was also performed on these cells after 7 days in culture, demonstrating eNOS expression and confirming expression of VEGFR2 demonstrated by flow cytometry. Isotype control staining is in the panels immediately above and eNOS and VEGFR2 expression in the panels below.