ABSTRACT: OBJECTIVES:To characterize mesenchymal stem cell-like cells isolated from human amniotic fluid for a new source of therapeutic cells. MATERIALS:Fibroblastoid-type cells obtained from amniotic fluid at the time of birth. METHODS:The ability of ex vivo expansion was investigated until senescence, and stem cell-like characteristics were analyzed by examining differentiation potential, messenger RNA expression and immunophenotypes. RESULTS AND CONCLUSIONS:A morphologically homogenous population of fibroblastoid-type (HAFFTs) cells, similar to mesenchymal stem cells from bone marrow (BM-MSCs), was obtained at the third passage. The cells became senescent after 27 passages over a period of 8 months while undergoing 66 population doublings. Under appropriate culture conditions, by the 8th passage they differentiated into adipocytes, osteocytes, chondrocytes and neuronal cells, as revealed by oil red O, von Kossa, Alcian blue and anti-NeuN antibody staining, respectively. Immunophenotype analyses at the 17th passage demonstrated the presence of TRA-1-60; SSEA-3 and-4; collagen types I, II, III, IV and XII; fibronectin; alpha-SMA; vimentin; desmin; CK18; CD44; CD54; CD106; FSP; vWF; CD31; and HLA ABC. Reverse transcriptase-polymerase chain reaction analysis of the HAFFTs from passages 6-20 showed consistent expression of Rex-1, SCF, GATA-4, vimentin, CK18, FGF-5 and HLA ABC genes. Oct-4 gene expression was observed up to the 19th passage but not at the 20th passage. HAFFTs showed telomerase activity at the 5th passage with a decreased level by the 21st passage. Interestingly, BMP-4, AFP, nestin and HNF-4alpha genes showed differential gene expression during ex vivo expansion. Taken together, these observations suggest that HAFFTs are pluripotent stem cells that are less differentiated than BM-MSCs, and that their gene expression profiles vary with passage number during ex vivo expansion.
