Promoted Expression of IGF-1, DNMT3a and OCT-4 in the Parthenogenetic Murine Blastocysts Developed in an Oil-Free Microtube Culture System may Support Stem Cell Generation

Abstract

Oil-free culture system was implemented to generate parthenogenetic embryonic stem cell (PESC) lines in mice. The oocytes at metaphase II stage were activated and the parthenogenetic embryos were cultured in two different culture systems: The oil-free microtube culture method (MTC), and the traditional micro-drop culture method (Drop). The gene expression of the blastocysts in each culture system was analyzed by real-time PCR, and the efficiency of stem cell generation of these two culture methods was compared. The blastocyst outgrowth rate was not significantly different between the MTC (75.0% and 42.9%) and the Drop culture group (65.4% and 48.0%) in both C57BL6 x DBA2 F1 hybrid (B6D2F1) and C57BL/6 inbred strains respectively, and after seeding on the feeder layers, outgrowing inner cell masses were found in both groups. However, three PESC lines (two from B6D2F1 and one from C57BL/6 strains) were only established from the MTC system after passed until ESC colonies were formed while no PESC line was obtained from the blastocysts cultured in Drop. Gene expression levels of IGF-1, DNMT3a and OCT-4 were also higher for MTC derived blastocysts. The PESCs in MTC maintained normal murine ESC morphology and were positive to pluripotent markers such as ALP, OCT-4 and NANOG. In conclusions, Our MTC system, involving oil-free microtube culture method, was effective for the generation of PESC lines in either F1-hybrid (B6D2F1) or C57BL/6 mice, and this may be due to the promoted expression of developmentally important genes, such as IGF-1, DNMT3a and OCT-4.