Isolation of endothelial progenitor cells from human adipose tissue.

Background: Endothelial progenitor cells (EPCs) play an important role in angiogenic responses in multiple tissues and mediate a coordinate augmentation of the capillary network as adipose tissue (AT) expands in response to positive energy balance. However, the isolation and culture of EPCs from human AT has proven difficult so far. Here, we report the isolation and characterization of EPCs from human AT (AT-EPCs).

Methods: Omental and subcutaneous AT specimens (approximately 1-2 g) were obtained during abdominal surgery. Following AT digestion with collagenase, both the filtered (SVF-I) and unfiltered (SVF-II) stromal vascular fractions (SVF) of AT were used. Expression of endothelial markers, such as CD31 and VE-Cadherin, was analyzed by using flow cytometry. Both SVF-I and SVF-II fractions were used for magnetic-based enrichment of endothelial cells using anti-human CD31 beads. Immunofluorescence staining, immunoblotting, and quantitative real-time PCR were performed to analyze expression of endothelial markers. Functional assays, including matrigel-based capillary-like tube formation assay and acetylated LDL uptake assays, were also performed.

Results: CD31 and VE-Cadherin were more expressed in SVF-II than SVF-I. CD31+ cells from SVF-II exhibited an endothelial-like cobblestone morphology. The CD31+ fraction also expressed Von Willebrand Factor (vWF) and VE-Cadherin. High mRNA levels of E-selectin, e-NOS, VEGFR, and CD34 were found in CD31+ cells, and E-selectin and e-NOS proteins were readily detectable. In addition, CD31+ cells were able to form tubes and incorporate acetylated LDL in vitro.

Conclusions: Large amounts of AT-EPCs with distinct functional properties can be isolated from omental and subcutaneous adipose tissue.