Autocrine fibroblast growth factor 2 signaling is critical for self-renewal of human multipotent adipose-derived stem cells

Adipose tissue-derived stem cells offer tremendous potential for regenerative medicine. However, characterization of their self-renewal ability has not been performed yet, although it is a crucial feature for in vitro expansion of undifferentiated cells and in vivo maintenance of stem cell pools. We have undertaken the identification of molecular events that are involved in in vitro self-renewal of human multipotent adipose-derived stem (hMADS) cells from young donors, by assessing their proliferation rate, their ability to grow at the single-cell level (clonogenicity), and their differentiation potential. As hMADS cells are propagated in culture, cell morphology changes dramatically, concomitantly to a progressive decrease in proliferation, clonogenicity, and differentiation potential. This decrease is associated with a decrease in fibroblast growth factor 2 (FGF2) expression and can be circumvented by chronic treatment with exogenous FGF2. Moreover, analysis of FGF2 secretion revealed that it is exported to hMADS cell surface without being released into the culture medium, suggesting a strictly autocrine loop. Indeed, treatment of FGF2-expressing hMADS cells with PD173074, a specific FGF receptor inhibitor, decreases dramatically their clonogenicity and differentiation potential. Thus, hMADS cells express a functional autocrine FGF loop that allows maintenance of their self-renewal ability in vitro. Finally, inhibition of mitogen-activated protein kinase kinase 1 reduces the clonogenic potential of hMADS cells but does not affect their differentiation potential, indicating that the extracellular signal-related kinases 1/2 signaling pathway is partly involved in FGF2-mediated self-renewal. Together, our data clearly identify the key function of FGF2 in the maintenance of self-renewal of adipose tissue-derived stem cells.     

Nucleofection is a valuable transfection method for transient and stable transgene expression in adipose tissue-derived stem cells

Adipose tissue-derived stem cells are a powerful tool for in vitro study of adult stem cell biology. So far, they have not been extensively used for gain or loss of function studies since they are resistant to most common transfection methods. Herein, we tested several classic transfection methods on human multipotent adipose tissue-derived stem (hMADS) cells. Our results showed that lipofectants and calcium phosphate were poorly efficient for transgene delivery in hMADS cells. In contrast, nucleofection, an electroporation-based method that is assumed to target plasmid DNA directly to the cell nucleus, led to a significant transient transgene expression in hMADS cells (up to 76% enhanced green fluorescent protein [EGFP]-positive cells were detected). Furthermore, after selection of hMADS cells that were nucleofected with a selectable plasmid coding for EGFP, stable EGFP expressing clones could be propagated in culture and efficiently induced to differentiate into EGFP-positive adipocytes and osteoblasts. Finally, we verified that nucleofected hMADS cells could produce a functional, transgene-encoded, secreted protein. To this aim, hMADS cells were nucleofected with a plasmid coding for leukemia inhibitory factor (LIF). This protein was detected at high concentrations in supernatants from pCAG-LIF transfected hMADS cells. Moreover, supernatants were able to maintain mouse embryonic stem cells' undifferentiated phenotype, indicating that hMADS cells could secrete a functional LIF protein. Taken together, our data demonstrate that nucleofection allows both transient and stable gene expression in adipose tissue-derived stem cells, without impairing their differentiation potential.