The stromal-vascular fraction (SVF) of human adipose tissue contains, among other cell types, mesenchymal stem cells and precursors of adipocyte and endothelial cells. Here we show that, in addition, the nonhematopoietic fraction of the SVF has hematopoietic activity, since all types of hematopoietic colony-forming units (CFUs) developed when cultured in methylcellulose-based medium. This hematopoietic activity was restricted to the CD45 ؊ CD105 ؉ cell subset, well correlated with KDR ؉ cell content, and increased after culture with a combination of early-acting hematopoietic cytokines. Most of the CD45 ؊ KDR ؉ CD105 ؉ cells were nonadherent and did not express CD31, and this subset included both CD34؊ and CD34 ؉ cells. Moreover, these nonadherent cells migrated in response to KDR gradient, and when they were cultured in the presence of both hematopoietic and endothelial growth factors, a wave of CFUs was followed by a wave of mixed colonies comprising adherent elongated and nonadherent round hematopoietic cells. These mixed hematopoietic-endothelial (Hem-End) colonies were able to generate secondary Hem-End colonies and exhibited both hematopoietic and endothelial activity, as demonstrated by in vitro functional assays. These findings demonstrate for the first time the existence of primitive mesodermal progenitors within the SVF of human adipose tissue that exhibit in vitro hematopoietic and hemangioblastic activities, susceptible to being used in cell therapy and basic cell research.
IntroductionAdipose tissue has the unique property of expanding throughout adult life, and angiogenesis is required for its growth. However, endothelial progenitor cells contribute minimally to neovascularization. Because myeloid cells have proven to be angiogenic, and monocytes accumulate in expanding adipose tissue, they might contribute to vascularization.MethodsThe stromal vascular fraction (SVF) cells from human adipose tissue were magnetically separated according to CD45 or CD14 expression. Adipose-derived mesenchymal stromal cells (MSCs) were obtained from SVF CD45- cells. CD14+ monocytes were isolated from peripheral blood (PB) mononuclear cells and then cultured with SVF-derived MSCs. Freshly isolated or cultured cells were characterized with flow cytometry; the conditioned media were analyzed for the angiogenic growth factors, angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF) with Luminex Technology; their angiogenic capacity was determined in an in vivo gelatinous protein mixture (Matrigel) plug angiogenesis assay.ResultsCD45+ hematopoietic cells within the SVF contain CD14+ cells that co-express the CD34 progenitor marker and the endothelial cell antigens VEGF receptor 2 (VEGFR2/KDR), VEGFR1/Flt1, and Tie2. Co-culture experiments showed that SVF-derived MSCs promoted the acquisition of KDR and Tie-2 in PB monocytes. MSCs secreted significant amounts of Ang-2 and HGF, but minimal amounts of bFGF, G-CSF, or GM-CSF, whereas the opposite was observed for SVF CD14+ cells.Additionally, SVF CD14+ cells secreted significantly higher levels of VEGF and bFGF than did MSCs. Culture supernatants of PB monocytes cultured with MSCs contained significantly higher concentrations of VEGF, HGF, G-CSF, and GM-CSF than did the supernatants from cultures without MSCs. Quantitative analysis of angiogenesis at 14 days after implantation demonstrated that neovascularization of the implants containing SVF CD14+ cells or PB monocytes previously co-cultured with MSCs was 3.5 or 2 times higher than that observed in the implants with SVF-derived MSCs. Moreover, immunofluorescence of Matrigel sections revealed that SVF CD14+ cells differentiated into endothelial cells and contributed to vascular endothelium.ConclusionsThe results from this study suggest that adipose tissue-resident monocytes should contribute to tissue vascularization. Because SVF CD14+ cells were more efficient in inducing angiogenesis than SVF-derived MSCs, and differentiated into vascular endothelial cells, they may constitute a new cell source for cell-based therapeutic angiogenesis.
Group (GEM) guidelines for the management of patients with advanced melanoma.
There is considerable evidence that stem/progenitor cells reside in the vasculature during the prenatal and postnatal stages. The stromal vascular fraction (SVF) of human adipose tissue is markedly rich in blood vessels, and it is a source of mesenchymal/stromal cells (MSCs). Therefore, we hypothesized that, in addition to MSCs, the SVF may contain other mesodermal precursors. However, the SVF has a high content of CD34(+) cells with high proliferative capacity, which can prevent the growth of the most quiescent cells. By using an antifibroblast (FIB) antibody coupled to microbeads, we show that ∼ 90%-95% of the nonhematopoietic CD34(+) cells were retained in the CD45(-)FIB(+) fraction. Reverse transcription-polymerase chain reaction analysis revealed that the CD45(-)FIB(-)CD34(-) cell fraction expressed higher mRNA levels of KDR and GATA2 than its complementary CD45(-)FIB(-)CD34(+) cell fraction, which contained the SVF endothelial cells. Surprisingly, when CD45(-)FIB(-)CD34(-) cells were cultured in endothelial growth medium, they gave rise to endothelial colonies and mesenchymal colonies. Moreover, when CD45(-)FIB(-)CD34(-) cells were cultured in embryonic stem cell expansion medium, they gave rise to cells exhibiting the full range of phenotypes observed in the freshly isolated SVF, including CD34(+) and CD31(+) cells. Together, these results suggest that the CD45(-)FIB(-)CD34(-) cells within the SVF of human adipose tissue function as mesodermal precursors of mesenchymal and endothelial cells.
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