Mesenchymal markers on human adipose stem/progenitor cells

Abstract

The stromal-vascular fraction (SVF) of adipose tissue is a rich source of multipotent stem cells. We and others have described three major populations of stem/progenitor cells in this fraction, all closely associated with small blood vessels: endothelial progenitor cells (EPC, CD45-/CD31+/CD34+), pericytes (CD45-/CD31-/CD146+), and supra-adventitial adipose stromal cells (SA-ASC, CD45-/CD31-/CD146-/CD34+). EPC are luminal, pericytes are adventitial, and SA-ASC surround the vessel like a sheath. The multipotency of the pericytes and SA-ASC compartments is strikingly similar to that of CD45-/CD34-/CD73+/CD105+/CD90+ bone marrow-derived mesenchymal stem cells (BM-MSC). Here, we determine the extent to which this mesenchymal pattern is expressed on the three adipose stem/progenitor populations. Eight independent adipose tissue samples were analyzed in a single tube (CD105-FITC/CD73-PE/CD146-PETXR/CD14-PECY5/CD33-PECY5/CD235A-PECY5/CD31-PECY7/CD90-APC/CD34-A700/CD45-APCCY7/DAPI). Adipose EPC were highly proliferative with (14.3 ± 2.8)% (mean ± SEM) having >2N DNA. About half (53.1 ± 7.6)% coexpressed CD73 and CD105, and (71.9 ± 7.4)% expressed CD90. Pericytes were less proliferative [(8.2 ± 3.4)% >2N DNA)] with a smaller proportion [(29.6 ± 6.9)% CD73+/CD105+, (60.5 ± 10.2)% CD90+] expressing mesenchymal associated markers. However, the CD34+ subset of CD146+ pericytes were both highly proliferative [(15.1 ± 3.6)% with >2N DNA] and of uniform mesenchymal phenotype [(93.3 ± 3.7)% CD73+/CD105+, (97.8 ± 0.7)% CD90+], suggesting transit amplifying progenitor cells. SA-ASC were the least proliferative [(3.7 ± 0.8)%>2N DNA] but were also highly mesenchymal in phenotype [(94.4 ± 3.2)% CD73+/CD105+, (95.5 ± 1.2)% CD90+]. These data imply a progenitor/progeny relationship between pericytes and SA-ASC, the most mesenchymal of SVF cells. Despite phenotypic and functional similarities to BM-MSC, SA-ASC are distinguished by CD34 expression.

Figure 1

Expression of mesenchymal stromal cell associated markers CD90 and CD73 in an adipose tissue section. Panel A shows an adipose small vessel in cross-section. The lumen is marked by CD146+/CD90− endothelial cells, surrounded by CD146+/CD90+ pericytes. Panel B shows an adipose capillary in longitudinal section. The lumen is marked by Von Willebrand factor (vWF)+ cells; CD90+ cells are distinct and tightly associated with the capillary. Panels C and D show adipose vessels in cross section. vWF+ cells mark the lumen (panel C); two vessels are surrounded by CD73+/vWF− cells. A larger vessel (panel D) shows a discrete supra-adventitial layer of CD73+/CD146− cells.

Figure 2

Expression of mesenchymal stem cell markers on four stem/progenitor populations of the adipose stromal vascular fraction. I and II show the pregating cleanup strategy used to remove sources of artifact. I: Integral versus time of flight (DAPI, AA and forward light scatter, A) is used to identify singlet cells (A and AA), after which hypodiploid events were eliminated (B). Lymphocytes (C) and nonhematopoietic cells (D) are identified for further analysis. II: Autofluorescent cells (AB), identified by the logical gate P and Q and R, are removed from analysis. III: Four stem/progenitor populations are identified within clean non-hematopoietic cells. IV: The proportion of cycling cells (DNA >2N) is determined for lymphocytes and the four stem/progenitor populations: Endothelial progenitors (F), Pericytes (H), Transitional cells (J) and SA-ASC (I). IV: DNA profile and coexpression of mesenchymal associated markers CD90, CD73 and CD105 are shown for the four stem/progenitor populations. The lymphocyte DNA profile is shown for comparison (IV C). All fluorescence variables are scaled identically (4 decade), except DAPI vs DAPI TOF (2 log scale). A representative sample is shown; region statistics tabulate the mean and standard error of the mean for all eight samples analyzed in this series.

Figure 3

Schematic representation of the organization of adipose SVC and a working model of SVF differentiation. The upper figure is a schematic representation drawn from previously published work. Cells associated with the small vessels of adipose tissue are coded as follows: Endothelial cells (red), pericytes and smooth muscle cells (green), and supra-adventitial adipose stromal cells (yellow). The lower diagram is a working model based on the present results. For each cell type defined by phenotype, the bar graphs show the proportion of cells with a mesenchymal phenotype (CD73+/CD105+ as shown in Figure 2) and the proportion of proliferating cells.