Amniotic fluid (AF) was described as a potential source of mesenchymal stem cells (MSCs) for biomedicine purposes. Therefore, evaluation of alternative cryoprotectants and freezing protocols capable to maintain the viability and stemness of these cells after cooling is still needed. AF stem cells (AFSCs) were tested for different freezing methods and cryoprotectants. Cell viability, gene expression, surface markers, and plasticity were evaluated after thawing. AFSCs expressed undifferentiated genes Oct4 and Nanog; presented typical markers (CD29, CD44, CD90, and CD105) and were able to differentiate into mesenchymal lineages. All tested cryoprotectants preserved the features of AFSCs however, variations in cell viability were observed. In this concern, dimethyl sulfoxide (Me2SO) showed the best results. The freezing protocols tested did not promote significant changes in the AFSCs viability. Time programmed and nonprogrammed freezing methods could be used for successful AFSCs cryopreservation for 6 months. Although tested cryoprotectants maintained undifferentiated gene expression, typical markers, and plasticity of AFSCs, only Me2SO and glycerol presented workable viability ratios.
Objectiveto examine growth, phenotypic characteristics, gene expression and differentiation of amniotic fluid stem cells (AFSC) using human serum in culture media.IntroductionAFSC are promising candidates for cell‐based therapies. They are obtained by prenatal screening, without raising the ethical concerns associated with human embryonic research. One major obstacle for their clinical use is the biosafety of fetal calf serum (FCS).MethodsAmniotic fluid cells obtained from 2nd trimester amniocentesis were grown in α‐MEM+human serum20%. Attached cells were cultured and counted under microscope, investigated about the expression of Oct‐4, SOX2 and Nanog, by RT‐PCR and about phenotype by FACS‐analysis. Induced osteogenic and chondrogenic differentiation were confirmed with Alizarine Red and H&E stains respectively.ResultsAFSC showed high proliferation ratios; expressed Oct‐4, SOX2 and Nanog; stained positive for CD29, CD44, CD90, CD105 and negative for CD14, CD31, CD34, CD45, CD106 and CD133; showed osteogenic and chondrogenic potential.ConclusionsGrowth of AFSC in a FCS‐free medium is feasible. Can be isolated easily, proliferate quickly, show a mesenchymal phenotype and have osteogenic and chondrogenic potential. Moreover, Oct‐4, SOX2 and Nanog, expressed by AFSC, are associated with the pluripotency and for the maintenance of stem cells.
Amniotic fluid (AF) present different cellular types in its composition and a specific group of these cells shows multipotent characteristics as high proliferation ratio and increased differentiation ability. The aim of our study is to better characterize these cells and evaluate their plasticity. AF cells were investigated regarding the expression of undifferentiation markers (Oct‐4, Sox‐2, Nanog) by RT‐PCR, surface protein identification by flow cytometry and differentiation assays into adipogenic, chondrogenic and osteogenic lineage, confirmed with Alizarin Red, H&E and Oil Red O stains respectively. AF cells showed high proliferation ratios and expressive doubling time. They also expressed pluripotent stem cell‐specific genes (Oct‐4, Sox‐2, Nanog) and were positive for mesenchymal membrane markers (CD29, CD44, CD133). They were also capable to differentiate into distinct lineages: adipocytes, osteocytes and chondrocytes. In conclusion, AF is a promising source of mesenchymal stem cells with high differentiation potential.Research governmental support: by CNPq.
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