Gene expression of hematopoietic stimulating cytokines in human Adipose Derived Mesenchymal Stem Cells (hASC) sensitive and insensitive to TGF-beta

TANIA M. RODRÍGUEZ; ALEJANDRA CARREA; INES AVEDILLO DIEZ; ALEJANDRO SALDIAS; RAUL GALLO; MARCELO J. PERONE; RICARDO A. DEWEY

Ciudad Autonoma de Buenos Aires

Simposio; Simposio Internacional Sobre Investigacion en Celulas Madre; 2009

Ministerio de Ciencia, Tecnologia e Innovacion Productiva de Argentina

Human adipose stem cells (hASC) have arisen as a safe and abundant source of stem cells for regenerative medicine. Although they share many characteristics with bone marrow derived mesenchymal stem cells (hBM-MSC), they remain much less characterized.It is known that hBM-MSCs secrete hematopoietic stem and progenitor cells (HSPC) stimulating factors, but in hASCs these factors have not been characterized in detail yet. Transforming Growth Factor beta (TGF-b) modulates multiple biological functions including the expression of HSPC-stimulating cytokines by stromal cells. TGF-b1 maintains primitive hematopoietic stem/progenitor cells in a quiescent or slow cycling state, in part by down-regulating the expression of mitogenic cytokines. TGF-b1 responses are basically mediated by Type I (TBRI) and II (TBRII) surface receptors, which are expressed in most cell types. Here we show the biological effect of inhibition of the TGF-b cascade on the expression pattern of hematopoietic stimulating cytokines in hASC. To achieve this, we designed lentiviral vectors encoding two different TGF-b receptor II (TBRII) dominant negative mutants (DN): TBRIIA-DN and TBRIIB-DN; and with them, we transduced purified and characterized hASC from lipoaspirates. Activity of lentiviral encoded DN mutants was determined by MTT assay on the TGF-b sensitive Mv1-Lu mink cell line. Expression levels of Stem Cell Factor (SCF) (membrane bound and soluble), IL-6, Thrombopoietin (TPO) and Flt3-ligand (FL) were studied by semi-quantitative RT-PCR in TGF-b sensitive and insensitive cells. We found that hASC express either soluble or membrane bound SCF, but not TPO. Additionally, we observed that, TGF-b negatively modulates SCF and FL expression, and positively IL-6. We also detected that in genetically-modified hASC by lentiviral vectors expressing only the marker transgene GFP, the expression levels of all studied cytokines are significantly altered. A potential clinical application of hASC is to exploit their ability to secrete early acting cytokines (FL, SCF and TPO) to expand hemopoietic stem cells ex vivo. Our results suggest that to obtain full HSPC expansion ex vivo from hASC, TPO expression should be up-regulated and TGF-b action must be avoided.