Investigation of stemness state in human amniocytes

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 stemness markers in 10 amniotic fluid cell lines. The genes investigated as stemness markers include OCT4, NANOG, SOX2, c-KIT (CD117), c-MYC, KLF4, FGF4 and THY1 (CD90).Materials and Methods: 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 37oC under 5% humidified CO2. 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 most remarkable results to emerge from the data is that all 10 amniotic fluid cell lines at 4th passage expressed at least one of the stemness markers. The OCT4 marker was positive in 6 samples and NANOG in 4 samples. All cell lines were negative for SOX2 as well as FGF4. Other interesting results have been revealed by c-KIT marker that was positive only in 2 lines.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.

کلیدواژه ها:

Amniotic fluid cell ، Amniocyte ، regenerative medicine ، Stemness marker