Abstract
The in vivo progenitor of culture-expanded mesenchymal-like adipose-derived stem cells (ADSC) remains elusive, owing in part to the complex organization of stromal cells surrounding the small vessels, and the rapidity with which adipose stromal vascular cells adopt a mesenchymal phenotype in vitro. Immunohistostaining of intact adipose tissue was used to identify three markers (CD31, CD34, and CD146), which together unambiguously discriminate histologically distinct inner and outer rings of vessel-associated stromal cells, as well as capillary and small vessel endothelial cells. These markers were used in multiparameter flow cytometry in conjunction with stem/progenitor markers (CD90 and CD117) to further characterize stromal vascular fraction (SVF) subpopulations. Two mesenchymal and two endothelial populations were isolated by high speed flow cytometric sorting, expanded in short term culture, and tested for adipogenesis. The inner layer of stromal cells in contact with small vessel endothelium (pericytes) was CD146+/alpha-SMA+/CD90+/-/CD34-/CD31-; the outer adventitial stromal ring (designated supra adventitial-adipose stromal cells, SA-ASC) was CD146-/alpha-SMA-/CD90+/CD34+/CD31-. Capillary endothelial cells were CD31+/CD34+/CD90+ (endothelial progenitor), whereas small vessel endothelium was CD31+/CD34-/CD90- (endothelial mature). Flow cytometry confirmed these expression patterns and revealed a CD146+/CD90+/CD34+/CD31- pericyte subset that may be transitional between pericytes and SA-ASC. Pericytes had the most potent adipogenic potential, followed by the more numerous SA-ASC. Endothelial populations had significantly reduced adipogenic potential compared with unsorted expanded SVF cells. In adipose tissue, perivascular stromal cells are organized in two discrete layers, the innermost consisting of CD146+/CD34- pericytes, and the outermost of CD146-/CD34+ SA-ASC, both of which have adipogenic potential in culture. A CD146+/CD34+ subset detected by flow cytometry at low frequency suggests a population transitional between pericytes and SA-ASC.