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. 2007 Oct 31:5:55.
doi: 10.1186/1479-5876-5-55.

"In vitro" and multicolor phenotypic characterization of cell subpopulations identified in fresh human adipose tissue stromal vascular fraction and in the derived mesenchymal stem cells

Giuseppe Astori  1 , Francesca Vignati, Silvana Bardelli, Monica Tubio, Mauro Gola, Veronica Albertini, Franco Bambi, Giancarlo Scali, Damiano Castelli, Valeria Rasini, Gianni Soldati, Tiziano Moccetti
Affiliations

"In vitro" and multicolor phenotypic characterization of cell subpopulations identified in fresh human adipose tissue stromal vascular fraction and in the derived mesenchymal stem cells

Giuseppe Astori et al. J Transl Med. .

Abstract

Background: The stromal vascular fraction (SVF) is a heterogeneous cell population derived from the adipose tissue. There is still a lack of information concerning the characterization of the cell subpopulations constituting the SVF as well as its mesenchymal and haematopoietic potential. Furthermore there are great variations in its phenotypical characterization.

Methods: Composition of SVF was investigated by FACS analysis, cytological and "in vitro" assays. We studied CD34+ population by combining FACS with human CFC (colony-forming-cell haematopoietic assay). The endothelial fraction was investigated by quantifying the co-expression of specific markers (CD146, CD105, CD31 and UEA-1). Mesenchymal potential was assessed by CFU-F assay and cultured AT-MSC were characterized by a 5-color FACS analysis. The multipotent differentiation potential (osteogenic, adipogenic and chondrogenic) was investigated both at cellular and molecular level.

Results: We identified in the SVF two CD34+ populations with a marked difference in the intensity of antigen expression, the majority of the cells expressing CD34 at low intensity. Moreover, two CD146+ cell populations were clearly distinguishable in the SVF:a CD146 dim accounting for 9.9% of the total SVF cells and a CD146+ bright cell population accounting for about 39.3%. The frequency of CFC clones was comparable with the one reported for peripheral blood. Endothelial cells account for about 7.7% of the SVF cells. AT-MSC differenced in the osteogenic adipogenic and chondrogenic lineage.

Conclusion: The SVF is not a homogeneous cell population, and its final composition could be influenced both by the flow cytometric technique analysis and the SVF extraction steps. The CFU-F frequency in the SVF was 1/4880, a value about seven times greater than the data reported for bone marrow. The antigenic profile of AT-MSC was comparable with bone-marrow derived MSC. AT-MSC were able to differentiate along the osteogenic adipogenic and chondrogenic lineages. The data here reported, further contribute to the characterization of SVF, a tissue providing an alternative as a source of MSC for clinical applications.

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Figures

Figure 1
Figure 1
FACS analysis of haematopoietic and endothelial cell subsets in the SVF. (A) CD34+ cells were analyzed in co-expression with CD45 and CD133 cell markers on crude SVF cell population (n = 6) and (B) after CD34 cell selection (n = 3).
Figure 2
Figure 2
Endothelial cell markers expression on primary SVF cell population. The cells represented in the CD146 vs UEA-1 plot are CD45-.
Figure 3
Figure 3
expression of surface markers in AT-MSC cells derived from SVF cells expansion (n = 5).
Figure 4
Figure 4
osteogenic and adipogenic differentiation of adherent cells from SVF culture. A: osteogenic differentiation (Alizarin Red staining); B: control; C: adipogenic differentiation (Oil O Red staining); D: control (100×). E: gene expression profile of un-induced cells compared with cultured ASC following osteogenic and adipogenic induction. GAPDH: glyceraldehide phosphate dehydrogenase; OSC: osteocalcin; OSP: osteopontin; PPARγ: peroxisome proliferator-activated receptor γ; LPL: lipoprotein lipase; FABP4: fatty acid binding protein 4.
Figure 5
Figure 5
chondrogenic differentiation of cultured AT-MSC by micromass method. A: induced cells; B: control; Alcian Blue staining (20×).
Figure 6
Figure 6
SVF cells spotted on a cytospin slide. It is possible to observe the presence of immature cells (A) together with cells having a more differenciated morphology (B). May-Grunwald Giemsa staining (100×).
Figure 7
Figure 7
phase-contrast photomicrographs of haematopoietic clones in Methocult obtained from the SVF. A: large CFU-GM (50×); B: large BFU-E (50×).
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