Generation of tetraploid complementation mice from embryonic stem cells cultured with chemical defined medium

Mouse embryonic stem cells(mESCs)derived from inner cell mass(ICM)of pre-implantation embryos,can maintain undifferentiated state when cultured in N2B27 medium supplemented with GSK3inhibitor CHIR99021 and MEK inhibitor PD0325901(‘‘2i’’)and leukemia inhibitor factor(LIF).Compare to conventional culture medium,all components of this medium are defined.With the N2B27 medium,‘‘2i’’and LIF,mESCs can contribute to the germline of the chimeric embryos,however,whether the‘‘all-ES cells’’mice can been generated by tetraploid complementation is unclear yet,while the tetraploid complementation serve as a golden standard to assess the pluripotency of ES cells.Here,our study showed that mESCs derived and cultured with the N2B27 complete medium could generate fertile mice by tetraploid complementation.In addition,the survival rate of tetraploid complementation mice produced by inbred mES cell lines is higher than the conventional culture condition,and increased the percentage of Oct4 positive cells contrast to conventional medium either.Therefore,the N2B27 medium supplemented with‘‘2i’’and LIF is an alternative choice forthe derivation and long-term culture of mouse embryonic stem cells.

Mouse embryonic stem cells （mESCs） derived from inner cell mass （ICM） of preimplantation embryos, can maintain undifferentiated state when cultured in N2B27 medium supplemented with GSK3 inhibitor CHIR99021 and MEK inhibitor PD0325901 （“2i”） and leukemia inhibitor factor （L1F）. Compare to conventional culture medium, all components of this medium are defined. With the N2B27 medium, “2i” and LIF, mESCs can contribute to the germline of the chimeric embryos, however, whether the “all-ES cells” mice can been generated by tetraploid complementation is unclear yet, while the tetraploid complementation serve as a golden standard to assess the pluripotency of ES cells. Here, our study showed that mESCs derived and cultured with the N2B27 complete medium could generate fertile mice by tetraploid complementation. In addition, the survival rate of tetraploid complementation mice produced by inbred mES cell lines is higher than the conventional culture condition, and increased the percentage of Oct4 positive cells contrast to conventional medium either. Therefore, the N2B27 medium supplemented with “2i” and LIF is an alternative choice for the derivation and longterm culture of mouse embryonic stem cells.