Establishment And Characterization Of Cynomolgus Monkey Amniotic Fluid Stem Cell Line

Regenerative medicine,now the hot spot of clinical medicine research,is a medical discipline which focuses on repairing,replacing and regenerating human damaged tissues and organs.Amniotic fluid stem cells(AFSCs)represent an emerging source of stem cells with possible intermediate characteristics between embryonic stem cells and adult stem cells.They did not only possess the indefinite self-renewal potential and have the capability of differentiation to more types of specialized cells,but also avoid the ethical issues and do not form tumors in vivo.Beside,AFSCs have not been reported to have significant immunogenicity issue.All the advantages mentioned above make them become one of the promising stem cell sources in regenerative medicine.In this study,amniotic fluid cells were isolated from cynomolgus monkey amniotic fluid and cultured in vitro.After that,we optimized the culture condition of cynomolgus monkey amniotic fluid cells and identified the cells with RT-PCR and immunofluorescence.Furthermore,to detect the ability of differentiation,amniotic fluid cells were cultured in the certain induction condition.We analyzed and identified cynomolgus monkey amniotic fluid stem cells.1)The isolation and culture of cynomolgus monkey amniotic fluid stem cellAmniotic fluid was obtained from eight weeks cynomolgus monkey by amniocentesis,and centrifuged to collect AFSCs.Then we transferred the cells into culture dish added with prepared medium and got the fibroblast-like cells which were primarily determined as cynomolgus monkey amniotic fluid stem cells.We established 3 cynomolgus monkey amniotic fluid stem cells lines.In addition,we selected several basal culture medium and different concentration of FBS to optimize the culture condition for cmAFSCs.Cell growth dynamics was identified by MTT method and found that cmAFSCs accessed to exponential growth after 2 days,and became gently at day 8,which showed strong ability of proliferation.Moreover our cell lines maintained strong proliferation capacity after freezing and thawing.For the moment,we passaged the cmAFSCs to more than 20 generations.2)Identification of cynomolgus monkey amniotic fluid stem cellsAFSCs from 6th passages were analyzed by RT-PCR,and the results showed that mRNA expression of ES cell markers such as Oct-4 and NANOG,mesenchymal stem cell markers such as CD44 and CD90,and negative expression of hematopoietic stem cell marker CD34.Further we verify our results with immunofluorescence and confirmed the expression of stem cell markers Oct-4 and SSEA4.3)Differentiation potentiality of cynomolgus monkey amniotic fluid stem cellsTo assess differentiation ability of cmAFSCs,we induced cmAFSCs into osteogenic and adipogenic cell population by using particular inducing medium.We cultured in osteogenic induction medium for 3 weeks,calcium deposited on the surface of cells were positively stained by alizarin red.For dipogenic differentiation,endogenously lipid droplets in cytoplasmic appeared with inducing time were visualized by positive staining for oil red.RT-PCR for two kind of differentiated cell showed that both of them expressed specific marker genes.Above mention results showed the capacity of differentiate into particular type of cells.In conclusion,this study successfully established amniotic fluid stem cell line from cynomolgus monkey and laid the foundation for cell biology research and alternative treatment research.