Biological Characterization And Pluripotent Identification Of Primordial Germ Cell And Amniotic Fluid Stem Cells

In this study,we successfully established the culture system of chicken embryos primordial germ cells in vitro,and identified PGCs from its biological characteristics.Next,we study chicken embryos meiosis and their potential to differentiate into sperm.besieds,the 8w sheep-derived amniotic fluid stem cells were isolated and their biological characteristics were identified,which provided a theoretical basis for cell transplantation therapy.Results:1.PGCs were isolated from chicken embryos that hatched for 5.5 days.The both side gonads were isolated from chicken embryos under the stereoscopic microscope.After digestion with 0.125% trypsin + 0.02% EDTA,the cells were cultured in H-DMEM medium with 10% fetal bovine serum,1 mM sodium pyruvate,1% GlutaMAX,5 ng/mL SCF,5 ng/mL LIF,10 ng/mL bFGF,10 ng/mL IGF and other factors to promote proliferation and inhibition differentiation of PGCs.After incubation for 24 h,the PGCs clones were observed under microscope,and the feeder layer were chicken embryonic gland stromal cells.After 48 h,the PGCs clones was significantly increased,which took on typical paving stone.The feeder layer used after passage was chicken embryo fibroblasts whcih were treated with mitomycin C.Both feeder layers were able to maintain the undifferentiated state of PGCs.In this study,PGCs could be cultured for 2 months in vitro and expression positive to AKP and PAS staining.Morphological observation showed that PGCs have a larger size,clear margin and strong in refraction;RT-PCR,immunocytochemistry and Flow cytometry analysis showed that PGCs express germ cells and embryonic stem cell specific markers.2.Chicken embryo PGCs can initiate meiosis under the action of Bone Morphogenetic Proteins,Activin A and Retinoic Acid,and promote the PGCs to differentiate into sperm under the action of Bovine Pituitary Extract,Follicle-stimulating hormone and testosterone.The results from the qPCR demonstrated that these hormones promote the differentiation of PGCs into sperm.3.Extracted 8 w embryonic amniotic fluid 10 mL in sterile condition,and centrifugal 15 min at a speed of 1500 RPM,eventually cultured in 12-well plates.The media was DMEM/F12 supplemented with 10 ng/mL b FGF.Due to amniotic fluid contained multi-type cells,selection the hole that cells took on long spindle-shaped to digestion and passage with 0.25% trypsin + 0.02% EDTA.After several passages,the purity of the cells was high,and identified the biological characteristics of AFSC.RT-PCR and immunocytochemistry showed that AFSCs express mesenchymal stem cell surface markers and embryonic stem cell specific markers.The growth curve showed a typical S type,indicating that AFSCs have a strong ability to proliferate.Karyotype analysis showed that the number of chromosomes in sheep AFSCs was 2n = 54,and more than 95% cells karyotype kept normal morphology and number.We induced the AFSCs into adipocyte?osteoblast and chondroblast.The results showed that AFSCs had the ability to differentiate into the three germ layers.