Long-term cultivation of human amniotic fluid stem cells: The impact on proliferative capacity and differentiation potential

Human amniotic fluid mesenchymal stem cells (AF-MSCs) are a valuable, easily obtainable alternative source of SCs for regenerative medicine. Usually, amounts of cells required for the translational purposes are large thus the goal of this study was to assess the potency of AF-MSCs to proliferate and differentiate during long-term cultivation in vitro. AF-MSCs were isolated from amniotic fluid of healthy women in the second trimester of pregnancy and cultivated in vitro. AF-MSCs were cultivated up to 42 passages and they still maintained pluripotency genes, such as OCT4, SOX2, and NANOG, expression at a similar level as in the initial passages as determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Fluorescence-activated cell sorting analysis demonstrated that the cell surface markers CD34 (negative), CD44, and CD105 (positive) expression was also stable, only the expression of SCs marker CD90 decreased during the cultivation. The morphology of AF-MSCs changed over passage, acridine orange/ethidium bromide staining revealed that more cells entered into apoptosis and the first signs of aging were detected only at late passages (later than p33) using SA-beta-gal assay. Concomitantly, the differentiation potential towards cardiomyogenic lineage, induced with DNA methyltransferases inhibitors decitabine, zebularine, and RG108, was impaired when comparing AF-MSCs at p31/33 with p6. The expression of cardiomyocytes genes MYH6, TNNT2, DES together with ion channels genes of the heart (sodium, calcium, and potassium) decreased in p31/33 induced AF-MSCs. AF-MSCs have a great proliferative capacity and maintain most of the characteristics up to 33 passages; however, the cardiomyogenic differentiation capacity decreases to a certain extent during the long-term cultivation. These results provide useful insights for the potential use of AF-MSCs for biobanking and broad applications requiring high yield of cells or repeated infusions. Hence, it is vital to take into account the passage number of AF-MSCs, cultivated in culture, when utilizing them in vivo or in clinical experiments.