TITLE

Isolation and enrichment of mouse female germ line stem cells

AUTHOR(S)
Khosravi-Farsani, S.; Fardin, A.; Habibi Roudkenar, M.; Sobhani, A.
PUB. DATE
June 2014
SOURCE
Iranian Journal of Reproductive Medicine;Jun2014 Supplement, Vol. 12, p30
SOURCE TYPE
Academic Journal
DOC. TYPE
Abstract
ABSTRACT
Introduction: The existence of female germ line stem cells (FGSCs) has been the subject of a wide range of recent studies. Successful isolation and culture of FGSCs could facilitate studies on regenerative medicine and infertility treatments in the near future. Our aim in the present study was evaluation of the most commonly used techniques in female germ line stem cell enrichment and establishing the best procedure. Materials and Methods: After digesting neonate ovary from C57B1/6 mice, we performed 2 different isolation experiments: Magnetic activating cell sorting (MACS) and preplating. MACS was done by two different antibodies against MVH and SSEA1 markers. Then to characterize colony forming cells by RT-PCR (for analysis of expression of Oct4, Nanog, C-kit, Fragilis, Mvh, Dazl, Scp3 andZp3), AP activity test and Immunocytochemistry, they were passaged and proliferated in vitro. Results: Data showed that colonies can be seen more frequently in preplating technique than that in MACs. Using the SSEA1 antibody with MACS, 1.98±0.49% (Mean±SD) positive cells were yield as compared to the total cells sorted. The colonies formed after preplating expressed pluripotency and germ stem cell markers (Oct4, Nanog, C-kit, Fragilis, Mvh and Dazl) but did not express Zp3 and Sep3 at the mRNA level. Immunocytochemistry in these colonies further confirmed the presence of OCT4 and MVH proteins and alkaline phosphatase activity measured by AP-kit showed positive reaction. Conclusion: We established a simple and efficient preplating technique that allows culture and enrichment of female germ line stem cells from neonatal mouse ovaries.
ACCESSION #
96841550

 

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