Introduction. Mesenchymal stem cells (MSCs) are multipotent precursors able to differentiate into bone, cartilage and fat. In humans, the MCS were isolated from bone marrow, cord blood, peripheral blood, adipose tissue and the amniotic fluid (AF) in the second trimester of pregnancy (FA-MSC). The FA-MCS have immunosuppressive properties, extensive proliferative potential and self-renewal, and express the fetal stem transcription factor Oct4. The purpose of this work was to isolate and characterize cells isolated from human FA as alternative source of multipotent MSC for regenerative medicine and transplantation. Materials and Methods. Expansion. The FA cells (extracted from the first three milliliters of FA collected during diagnostic amniocentesis and can not be used for prenatal diagnosis), were cultured in serum free medium specific to the MSC (STEM CELL). Phenotypic analysis. On the FA-expanded MSC the presence of mesenchymal markers CD106, CD146 and CD105, and non-mesenchymal markers CD45 and CD34 have been evaluated by flow cytometry. The fetal origin of the FA-expanded MSC was verified by QF-PCR comparing FA-MSC DNA and maternal mononuclear DNA. Stemness analysis. The stemness, potential before and after expansion, was evaluated by expression analysis(RT-PCR) of Oct4 and Nanog gene. cDNA from adult peripheral blood and cDNA from chorionic villus at 10 weeks of gestation were used as control. The specific primers for the analysis of Oct4 and Nanog genes were designed in the laboratory on the basis of the gene maps (GENE ATLAS). Results and Conclusions. After 8 days of culture FA cells have produced CFU-F colonies. At 15 days of culture started the proliferation of fibroblasts, and at 28 days of culture the fibroblasts have reached 80% confluency. Phenotypic analysis. The flow cytometry assay allowed cultures immunophenotypic characterization. The flow cytometry analysis of the FA-MSC showed the presence of mesenchymal markers CD146, and CD105 and the absence of hematopoietic/endothelial markers CD34 and CD45. The QF-PCR confirmed the fetal origin of the expanded FA-MSC. Using medium serum-free adherence method, it has been developed a reproducible protocol for the isolation and expansion of AF-MSC. Phenotypic analysis results, confirmed the nature of the mesenchymal cells expanded AF. The presence of expression of Nanog (a homeobox playing a key role in embryonic development and in maintaining the pluripotency of stem cells), and Oct4 (a transcription factor involved in stem cells pluripotency and self-renewal maintenance) confirmed the stamness persistence of AF-MSCs after expansion. The expanded AF-MSCs could be used in different therapeutic fields: bone marrow transplantation in adults, transplant "in utero" and gene therapy for congenital blood diseases, treatment of chronic inflammatory bowel disease. The use of AF-MSCs also can open new perspectives for research in regenerative medicine because of their possible de-differentiation by "ex vivo" procedures. Disclosures No relevant conflicts of interest to declare.