Derivation of LIF-Independent Embryonic Stem Cells Using Inducible OCT4 Expression

Methods Mol Biol. 2020:2117:229-234. doi: 10.1007/978-1-0716-0301-7_13.


Pluripotent mouse embryonic stem (ES) cells, which are derived from the inner cell mass (ICM) of preimplantation stage embryos, are capable of self-renewing indefinitely in the presence of the external signal leukemia inhibitory factor (LIF), activation of Wnt signaling through inhibition of GSK3, and inhibition of MAP kinase/ERK kinase signaling. The OCT4 transcription factor is expressed highly in pluripotent cells and is a central transcriptional regulator of the pluripotent state. Here, we describe a protocol to culture ES cells in LIF-independent and serum-free media using an inducible OCT4 (iOCT4) ES cell model system. This protocol is sufficient to sustain ES cell self-renewal in vitro in defined conditions in the absence of external signals. LIF-independent iOCT4 ES cells are fully capable of differentiating following deactivation of the inducible OCT4 transgene.

Keywords: Embryonic stem cells; LIF; OCT4; Pluripotent; Self-renewal.

MeSH terms

  • Animals
  • Cell Culture Techniques / methods*
  • Cell Differentiation
  • Cell Self Renewal
  • Cells, Cultured
  • Culture Media, Serum-Free / chemistry
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Glycogen Synthase Kinase 3 / metabolism
  • Leukemia Inhibitory Factor / metabolism*
  • Mice
  • Mouse Embryonic Stem Cells / cytology*
  • Mouse Embryonic Stem Cells / metabolism
  • Octamer Transcription Factor-3 / genetics
  • Octamer Transcription Factor-3 / metabolism*
  • Wnt Signaling Pathway


  • Culture Media, Serum-Free
  • Leukemia Inhibitory Factor
  • Octamer Transcription Factor-3
  • Pou5f1 protein, mouse
  • Glycogen Synthase Kinase 3