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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. © 2012 International Society for Advancement of Cytometry
Adipose tissue is a rich source of multipotential stem cells, which are associated with small vessels and are easily recovered by mechanical and enzymatic digestion in a stromal-vascular fraction (SVF) (1, 2). Studies on whole adipose have revealed that the stem/progenitor components, organized around small vessels in an annular fashion, are dominated by a prevalent supra-adventitial layer of CD34+ cells of mesenchymal stromal cell (MSC)-like multipotentiality (1, 3, 4). These supra-adventitial adipose stromal cells (SA-ASC) surround arterioles and venules, which are colonized on their surfaces by CD146+ perivascular cells or pericytes (1, 5). A CD34+/CD31+ endothelial progenitor component is associated with the luminal layer, which in adipose small vessels is proliferative. As a heterogeneous mixture, the SVF is capable of differentiating in vitro along the mesodermal lineages (6–11) and forming vessel like tubules of CD31+/von Willebrand factor (vWF)+ cells in close association with α-smooth muscle actin (α-SMA) positive cells (11). In clinical application, the adipose stromal vascular fraction has been used to augment autologous lipoaspirate transfers for soft tissue reconstruction (12–16), under the premise that enrichment of stromal vascular cells will promote graft retention by promoting vascularization and adipogenesis (15, 17, 18). Since the plastic-adherent fraction of adipose stromal vascular cells, often referred to as adipose-derived stromal cells (ASC), resembles bone marrow derived culture-expanded MSC (7, 11, 19), the present study was designed to investigate the expression of MSC-associated markers on the discrete stem/progenitor subpopulations which comprise freshly isolated adipose stromal vascular cells.
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Adult stem/progenitor cells, especially those of mesodermal origin, have been the subject of intense work during the last decade, in part because of their proposed use for regenerative and anti-inflammatory therapy (23). In contrast to the hematopoietic system, where the differentiation hierarchy is well established, stromal stem cell biology remains unclear. MSC (24–26), the prototype for stroma-derived adult stem cells were identified and characterized first in bone marrow and then demonstrated in a variety of other organs (27). The similarities in phenotype, transcriptome, and potentiality between pericytes, which populate blood vessel adventitia in both fetal and adult human tissues, and mesenchymal stem cells have been noted (28, 29).
Adipose tissue is an attractive source of adult stem cells due to its abundance and surgical accessibility. Adherent fat progenitors were isolated in 1976 by two independent groups (30, 31). Cultured ASC were later shown to have the ability to differentiate along mesenchymal lineages (2) and express the MSC associated markers CD105 and CD44 (7). Culture expanded ASC, which like bone marrow derived MSC are expanded from plastic-adherent cells, share many properties with MSC. However, their characterization in situ in whole tissue resulted in the identification of several discrete populations organized around small vessels (1, 4). We were able to clarify the relationship between these populations by the combined use of immunohistostaining and multiparameter flow cytometry (1), demonstrating the histologic relationship between multipotent stem/progenitor populations (32), and inferring the existence of a highly proliferative transitional population between pericytes and SA-ASC (5). We proposed the term SA-ASC to replace pre-adipocyte or adipose stromal/stem cell, because the latter terms confounded several distinct populations (3, 33–36).
Recent studies have concluded, on the basis of retention of unique phenotype profiles in culture, that pericytes and SA-ASC are of distinct origin (37). In the present study, we used multidimensional flow cytometry to determine the relationship between adipose stromal populations and classically defined adult MSC. The salient findings were that the mesenchymal phenotype, as determined by the expression of CD73, CD105, and CD90, is expressed on a lower proportion of pericytes compared to SA-ASC which are essentially homogeneously positive with respect to mesenchymal markers. The existence of a rare highly proliferative population intermediate in phenotype between pericytes and SA-ASC, and also homogeneous for mesenchymal marker expression, suggests a transitional population between pericytes and SA-ASC. Interestingly, about half of the CD34+/CD31+ EPC detected in the adipose stromal vascular fraction expressed mesenchymal markers as well as CD146, consistent with a pericytic origin for these cells as well. All of the four populations had a detectable proportion of cells in cycle compared to passenger lymphocytes (Fig. 2, IV), indicating the dynamic nature of the stromal vascular fraction of adipose tissue. Since our data are phenotypic and cross-sectional, the developmental relationship between MSC, pericytes, EPCs, and adipose progenitor populations can only be inferred. Conclusive demonstration of the proposed progenitor/progeny relationships will require in vivo tracking studies.
Our working model for SVF differentiation, illustrated in Figure 3, is also supported by recent developmental studies. In the mouse, PPARγ+ fat progenitors develop during the first postnatal month from a pool of CD34+ precursors (38). These CD34+ cells proliferate and populate the adventitia of all adipose depot vasculature, where they display a pericytic phenotype (α-SMA, PDGFR-β, NG2), and lack expression of hematopoietic (CD45−) or endothelial (CD31−) markers. The coexpression of CD34 and pericyte markers is maintained in the adult mouse (39). This is reminiscent of the phenotype of perivascular progenitor populations in the human dermis (40), as well as the highly proliferative transitional subpopulation of human adult fat pericytes detected here (Fig. 2, IV) (1, 5, 20).
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 SA-ASC (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. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]
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Interestingly, while white adipocytes from the head are derived from the neural crest (41), most other fat depots seems to have a distinct, presumably mesodermal, origin (42). Similarly, during embryogenesis, primitive mesenchymogenic cells can arise from both the neural crest and the mesoderm (43, 44). Pericytes have been proposed to be the precursors of MSC in both fetal and adult tissues (29). According to in vitro hESC-based models, primitive MSC may express CD34 (45, 46), a phenotype shared in vivo by some rare adult bone marrow mesenchymal progenitors (47, 48) but also a larger subset of adipose pericytes (5). However, most classical definitions of adult human mesenchymal stem cells include the absence of CD34 expression (26, 49).
Recently, multipotent CD34+ mesodermal vasculogenic precursors have been identified using hESC/hiPSC-based methodologies (50, 51). These early CD34+ precursors can give rise simultaneously to mesenchymal/pericyte (CD146+/CD31−/CD45−) and endothelial (CD31+) lineages (52, 53), through the emergence of a common bipotent precursor, the “mesenchymoangioblast,” which exhibits a mesenchymal (CD105+/CD73+/CD90+) pericytic (CD140a+/CD146+) non-endothelial, non-hematopoietic (CD31−/CD43−/CD45−) phenotype. Notably, mesoangioblasts, a primitive CD34+ population of angiogenic and mesenchymal progenitors, emerge from the mouse dorsal aorta (54) during early embryogenesis and are believed to be the ancestors of postnatal multipotent pericytes. Thus, the progression documented in developmental biology is consistent with the concept that adult pericytes may give rise to both EPC and SA-ASC, possibly through a common CD146+/CD34+ intermediate (Fig. 3).