Effect of Passage, Sorting, and Media on Differentiation Capacity and Marker Expression in Amniotic Fluid Stem Cells

Amniotic fluid-derived stem cells (AFSC) represent a promising source for tissue engineering applications; however, wide variation exists in the methods and medium used in AFSC culture. To address the maintenance of stem cell markers and differentiation capacity of AFSC, both c-kit sorted and unfractionated adherent AFSC from human patient samples were cultured in a variety of media and evaluated over six passages. Medium supplements included Chang medium, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and both bFGF and EGF. Protein and gene expression of pluripotency markers, including c-kit, SSEA4, Sox2, Oct4, and cMyc were measured in undifferentiated AFSC. Differentiation capacities of AFSC towards endothelial, osteogenic, and neurogenic lineages were analyzed at passages 5 and 6. Expression of c-kit was higher in c-kit sorted cells compared to unfractionated cells; however, SSEA4, Sox2, cMyc, Tra-1-60, and Tra-1-81 were higher in the unfractionated population. Pluripotency marker expression was significantly lower at passage 6 than earlier passages. Correspondingly, cells differentiated more efficiently at passage 5 than passage 6. Media had distinct effects on differentiation towards each lineage. These results indicate that AFSC should be utilized prior to passage 6 and that optimal isolation and culture conditions depend on final differentiation intent.