hASC and DFAT, multipotent stem cells for tissue engineering: comparison of their differentiation potential in vitro

Adipose tissue (AT) contains adipocytes, preadipocytes, fibroblasts, endothelial cells and mesenchymal stem cells (MSC) with a surface’s antigenic profile similar to MSC derived from bone marrow. DFAT (dedifferentiated fat cells), cells derived by dedifferentiation to mature adipocytes is another cell population with characteristics of stem cell isolated from adipose tissue. The aim of this research is to compare the characteristics of stemness, proliferation and differentiation of two cell populations obtained from the human subcutaneous AT to evaluate their potentiality in studies of tissue engineering and regenerative medicine. Stem cell population of vascular fraction of AT (hASC) and cells obtained from mature adipocytes cultured in a "ceiling" and dedifferentiated (DFAT) were analized for identification of surface markers. Cells were seeded in monolayer in presence of growth medium to evaluate the proliferative capacity. Cell populations were always cultured with osteogenic, adipogenic and condrogenic factors and the differentiation capacity was assessed by hystochemical techniques and molecular biology. HASC and DFAT were positive for MSC markers as CD13, CD73, CD90 and CD105 and negative for CD14, CD34 and CD45. Clonogenic test in methylcellulose exclude the acquisition of a pathological phenotype by DFAT for p10 next steps. The proliferative capacity expressed by doubling time obtained in vitro was similar in the populations. DFAT and hASC induced with specific factors into the culture medium had the ability to differentiate in three mesodermal lines. The DFAT were able to accumulate intracellular lipids after 7 days of adipogenic differentiation and gene expression of LPL and Adiponectin was much greater compared to that of hASC. The gene expression of ALP and Runx2 was greater at 14 Days in HASC respect to DFAT, where increases after 3 weeks, while at 21 days, the levels of protein expression are similar in both populations. Both cell populations were also able to differentiate in chondrocytes, showing a positive staining with Alcian blue and gene expression of Sox9 and Aggrecan. In conclusion, both stem cell populations derived from human adult AT have an high differentiation capacity and could be used for regenerative medicine. The results also suggests that DFAT are stem cells more committed to the adipose lineage and may be preferred as cell model in the study on the adipose tissue in vitro and in vivo.