Effect of different culture conditions on establishment of embryonic stem cells from BALB/cAJ and NZB/BINJ mice.
As the phenotype of a given single-gene mutation in mice is modulated by the genetic background of the inbred strain, embryonic stem (ES) cells derived from various inbred mouse strains are required to produce gene-targeted mice without the need for backcrossing and for detailed analysis of gene function in vivo. Here, we performed a comparative investigation of the effects of three culture conditions, LIF + KSR/ES medium described previously, High LIF + KSR/ES medium and iSTEM + LIF medium containing three inhibitors of glycogen synthase kinase 3, mitogen-activated protein kinase kinase, and fibroblast growth factor receptor signaling (3i), on the establishment of germline-competent ES cells derived from strains BALB/c and NZB mice. The results indicated that LIF + KSR/ES medium was permissive for the derivation of ES cells from NZB mice, which contribute to the somatic lineage in vivo, but not to the germline lineage. In contrast, ES cells that contribute to the makeup of chimeric mice were not propagated from blastocysts of BALB/c mice. Both germline and somatic competency were improved by increased LIF concentration in cultures of BALB/c ES cells, although we failed to establish germline-competent NZB ES cells using the same concentration of LIF. Unexpectedly, iSTEM + LIF medium containing 3i showed a negative effect on the derivation of NZB ES cells with normal chromosome numbers, but not on the maintenance of previously established ES cells. Our findings suggest that the stability of pluripotency in the inner cell mass isolated from blastocyst embryos may differ according to the genetic background of inbred mouse strains, and that although the concentration of LIF is a determinant for authentic pluripotency, including germline and somatic competency in BALB/c ES cells, additional factor(s) are required for commitment to germline lineage independent of somatic lineage in NZB ES cells.
Laboratory Animal Resource Center, University of Tsukuba , Tsukuba, Ibaraki, Japan.
Saori
S
Iijima
Yoko
Y
Tanimoto
Seiya
S
Mizuno
Yoko
Y
Daitoku
Satoshi
S
Kunita
Fumihiro
F
Sugiyama
Ken-ichi
K
Yagami
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D002678:Chimera; D018920:Coculture Techniques; D003470:Culture Media / Q000737:chemistry*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D005260:Female; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008814:Mice, Inbred NZB
D003470:Culture Media; D053605:Leukemia Inhibitory Factor