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Ischemic heart disease (IHD) is a major factor influencing worldwide mortality rates. Furthermore, IHD has become a significant health problem among the Thai population. Stem cell therapy using mesenchymal stem cells (MSCs) is an alternative therapeutic method that has been applied to improve the quality of life of patients. Amniotic fluid (AF) contains a heterogeneous cell population, including MSCs, which are multipotent stem cells that have the capability to differentiate into mesenchymal lineages. The purpose of the present study was to evaluate the MSC characteristics of human (h)AF and determine its potency regarding cardiogenic differentiation. MSC characterization following flow cytometric analysis revealed that the cells expressed MSC markers, cluster of differentiation (CD)44, CD90, human leukocyte antigen-ABC and CD73. The results of the alamar blue assay demonstrated that cell proliferation rate continuously increased from the early cultivation phase up to 5-fold during days 1 to 5 of cell culturing. The highest rate of cell proliferation was observed on day 17 with a 30-fold increase compared with that on day 1. During the cardiogenic induction stage, morphological changes were observed between day 0 and day 21, and it was revealed that the hAF derived-MSCs in the cardiogenic-induced group exhibited myotube-like morphology after 7 days of cell culturing. Following cardiogenic induction, immunohistochemistry staining was performed on day 21, and reverse transcription-quantitative polymerase chain reaction on day 7 and 21. These steps were performed to detect the protein and gene expression levels of cardiac specific proteins (GATA4, cardiac troponin T, Nkx2.5 and Connexin43). The results of the present study indicated that hAF-MSCs possess the potential to differentiate into cardiomyocyte-like cells. Thus, it was concluded that hAF may be a suitable source of MSCs for stem cell therapy and tissue engineering.

Human amniotic fluid (hAF) mesenchymal stem cells (MSCs) are commonly cultured in medium containing FBS. However, there are concerns about using animal serum in therapeutic applications due to the potential for immunogenic reactions and the risk of transmission of pathogens. For safety reasons, human platelet lysate (hPL) has been suggested as a replacement for FBS because it appears to be a natural source of growth factors. In this present study, it was investigated whether FBS could be substituted with hPL in hAF-MSCs culture without affecting their properties. Pooled hPL was generated by the freeze-thaw method. The concentration of hPL was selected after evaluation by MTT assay. The hAF-MSCs were cultured in FBS- or hPL-supplemented conditions and shared a fibroblast-like morphology. Cell proliferation assays showed that the growth characteristic of hAF-MSCs cultured in 10% hPL-supplemented media was similar to those cultured in 10% FBS-supplemented media. The expression of MSC markers did not differ between the cells cultured in the different conditions. The endothelial differentiation potential was also investigated. Reverse transcription-quantitative (RT-q)PCR revealed that induced cells supplemented with hPL showed an increase level of endothelial specific gene expression compared to the FBS-supplemented cells. Immunofluorescence analysis showed specific protein localization in both induced cell groups. Additionally, induced cells supplemented with hPL had the potential to form networks on Matrigel. This present study indicated that hPL could be used to culture and enhance the endothelial differentiation potential of hAF-MSCs.