Background: Many studies have described amniotic fluid-derived stem cells (AFSCs) as a fascinating source of stem cells. Differentiation ability to all three embryonic germ layers, immunomodulatory properties, lacking tumorigenicity and less ethical and legal limitations rather than embryonic stem cells introduce an exciting choice for the future of regenerative medicine. Objective: The present paper aims to validate the expression of some stem cell markers in 14 amniotic fluid cell lines. The genes investigated as stemness markers include OCT4, NANOG, SOX2, c-KIT (CD117), c-MYC, KLF4 and THY1 (CD90). Material and Method: Amniotic fluid samples were donated by genetics laboratories of Yazd reproductive sciences institute. The amniocytes were cultured with a modified medium composed of 2:1 v/v DMEM:AmnioMAX II in which DMEM was supplemented by 4 mM L-Glu, 10 mM HEPES, 15% FBS, 1% PenStrp. Culture vessels were incubated at 37 C under 5% humidified CO 2. For RT-PCR, amniocytes were harvested by 0.25% Trypsin-EDTA and total RNA was isolated using the Qiagen RNeasy™ Mini Kit. cDNA synthesis was performed by means of the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) and finally, cDNA samples were amplified by human-specific primers. Results: The amniotic fluid cell lines isolated among nearly 50 individual cases were 14 in three distinct morphology, short spindle-fibroblastoid, long spindle-fibroblastoid and epitheloid cells. The most remarkable result to emerge from the RT-PCR data is that all isolates between passages 3 to 18 express at least one of the mentioned markers. Another interesting result was that the core regulatory circuit of pluripotency, i.e. SOX2, OCT4 and NANOG, were expressed at detectable levels in 7 isolates. Conclusion: The findings of this study indicate the idea that amniotic fluid cells have an intrinsic nature of heterogeneous phenotype and stemness potential that make them a valuable candidate to be used in the field of regenerative medicine.

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