Influences of embryo age and isolation method on amplification efficiency of mouse embryonic germ cells / 中国组织工程研究

ABSTRACT

AIM:Studies on totipotential stem cells using primordial germ cells (PGCs) have the same application prospect as embryonic stem cells, especially for the animals difficult to isolate embryonic stem cells from blastula. In vitro culture condition of embryo germ cells is the key to control this. This study designs a method to effectively amplify PGCs of mice in vitro and establishes an effective culture method of PGCs. METHODS: Experiments were carried out in the Department of Histology and Embryology of Chongqing Medical University and Key Laboratory of Biochemistry and Molecular Pharmacology from October 2006 to August 2007. ①Clean Kunming pregnant mice (embryos 0.5 dpc) were provided by Animal Experimental Center of Chongqing Medical University. Experimental procedures met the animal ethical standards. ②Allantois and surrounding tissues of 8.5 day post coitum (dpc) embryos, the hindhut and surrounding tissues of 9.5 dpc and 10.5 dpc embryos, the gonadal ridges and surrounding tissues of 11.5 dpc and 12.5 dpc embryos were collected and digested with 0.25%pancreatin-0.02%EDTA, then the cells were cultured in the plastic petridishes which are pretreated with 0.1% gelatine. The formation ratio of primary and the first passaged Embryonic germ (EG) clones were compared among those different time points. The SSEA-1 positive ratio of isolated cells was compared between the two days by immunohistochemical method. The assays of clone numbers counting and MTT were used to compare the different proliferation ability of PGCs from the 11.5 dpc and 12.5 dpc embryos. The effects of expanding PGCs from 11.5 dpc and 12.5 dpc embryos by those two ways above were compared. The EG clones were analyzed by the expression of alkaline phosphatase and the immunologic marker SSEA-1,the undifferentiated marker Nanog and the differentiation ability in vitro were also tested. RESULTS:①The formation ratio of primary and the first passaged EG clone from 11.5 and 12.5 dpc embryos was higher than that from 8.5 to 10.5 dpc embryos and efficiency of expansion was increased (P 0.05). On the third day, the number of the primary EG clones from 11.5 dpc embryos was higher than that from 12.5 dpc embryos (P 0.05), but the two groups had different characteristics. ④The EG clones have representative morphology with high level of alkaline phosphatase activity and SSEA-1, Nanog expression, which can differentiate into the cystic embryoid body. CONCLUSION: 11.5 dpc and 12.5 dpc mouse embryos, especially 11.5 dpc embryos are the optional time points to expand PGCs efficiently. Co-culturing with the tissue and isolating the gonadal ridges both of these two ways are practicable.