Stem Cells Dev. 2015 Oct 1;24(19):2280-96.
Fibroblast-Negative CD34-Negative Cells from Human Adipose Tissue Contain Mesodermal Precursors for Endothelial and Mesenchymal Cells.
Authors: Amparo Navarro1, Severiano Marín2, Nicasia Riol3, Francisco Carbonell-Uberos3, María Dolores Miñana1.
1Regenerative Medicine Laboratory, Fundación Hospital General Universitario, Valencia, Spain.
2Department of Plastic and Reconstructive Surgery, Consorcio Hospital General Universitario, Valencia, Spain.
3Immunohematology Service, Centro de Transfusiones, Valencia, Spain.
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.
The stromal vascular fraction (SVF) of human adipose tissue is used to obtain mesenchymal cells which have shown to be useful for regenerative medicine or cell therapy. Nevertheless, the SVF is a very heterogeneous cell population containing more primitive cells than previously thought; and so, a population of pluripotent stem cells termed Multilineage Differentiating Stress-Enduring (Muse) cells with the ability to differentiate into cells of the three germ layers has been identified . We had previously identified a rare population of small non-adherent CD45–KDR+CD34+/- cells that displayed hemangioblastic properties in vitro . Moreover, we showed that the non-hematopoietic CD45- cells exhibited the ability to form hematopoietic colonies in vitro [2,3]. Therefore, we hypothesized that the SVF might contain endothelial precursors more primitive than the well described CD34+CD31+ endothelial progenitor cells.
To test this possibility, the SVF was first depleted CD45+ hematopoietic cells. Then, taking into consideration that in human bone marrow, fibroblast (FIB)-expressing cells give rise to fibroblastoid /stromal colonies, we separated the SVF CD45– cells into CD45–FIB– and CD45–FIB+ cell populations. We show that 70-80% of the SVF CD45– cells express the FIB antigen, and additionally that CD45–FIB+ cell population, unlike that of human bone marrow, expresses CD34 antigen; in fact, more than 90% of the CD34+ non-hematopoietic cells were contained in the CD45–FIB+ cell population. Next CD45–FIB– cells were separated according to CD34 expression into CD45–FIB–CD34+ and CD45–FIB–CD34– cells. The double-negative CD45– cells were seeded on uncoated plastic plates in endothelial growth medium (EGM), and after 7 days culture the non-adherent cells were re-plated in fibronectin/collagen coated plates in EGM (Figure 1). Cells cultured in these conditions gave rise to endothelial colonies; but additionally, adherent cells also generated stromal colonies.
Given the pluripotency marker expression of the CD45–FIB–CD34– cell population, these cells were plated onto Matrigel matrix-coated dishes in a defined stem cell expansion medium. Under these culture conditions, all the phenotypic heterogeneity present in freshly isolated SVF CD45– cells, including CD34+ and CD34+CD31+ cells, were generated.
These results indicate that CD45–FIB–CD34– cell population within the SVF is highly enriched in mesodermal precursors of mesenchymal and endothelial cells, and therefore can constitute a new potent cell source for tissue engineering and regenerative medicine.
Figure 1. Schematic illustration of cell isolation and cell culture procedures. The SVF of human adipose tissue was obtained by collagenase digestion. The SVF fraction depleted of erythrocytes was magnetically labeled with the following antibodies conjugated to microbeads: anti-CD45, anti-FIB and anti-CD34 microbeads to obtain CD45–FIB–CD34– cells. Sorted cells were plated in EGM in plastic dishes. After 7 days culture, the non-adherent cells were reseeded onto fibronectin/collagen coated dishes. Additionally, the CD45–FIB–CD34– cells were seeded onto Matrigel coated dishes in stem cell expansion medium.
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- Carbonell-Uberos F, Marin S and MD Miñana. (2013). Human adipose tissue contains erythroid progenitors expressing fetal haemoglobin. World J Stem Cells 5(4):205-216.