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Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange
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Disrupting Interactions Between β-Catenin and Activating TCFs Reconstitutes Ground State Pluripotency in Mouse

Abil Saj1, Sujash S Chatterjee2, Bowen Zhu1

  • 1Cancer Therapeutics and Stratified Oncology.

Stem Cells (Dayton, Ohio)
|June 4, 2017
PubMed

Insights

Replacing CHIR with iCRT3 in 2i-media preserves mouse embryonic stem cell (mESC) ground state pluripotency. This new method, iCRT3+PD, offers a stable alternative to 2i culture, reducing differentiation markers.

Area of Science:

  • Stem Cell Biology
  • Developmental Biology
  • Molecular Biology

Background:

  • The 2i medium (PD0325901 and CHIR99021) establishes naïve pluripotency in mouse embryonic stem cells (mESCs) by inhibiting MEK and GSK3 kinases.
  • While 2i promotes pluripotency, CHIR-induced β-catenin can activate TCFs, potentially leading to differentiation and compromising long-term culture stability.

Purpose of the Study:

  • To investigate if replacing CHIR with iCRT3, a β-catenin-TCF interaction inhibitor, can maintain ground state pluripotency in mESCs.
  • To assess the stability and efficacy of iCRT3+PD medium as an alternative to 2i for mESC culture.

Main Methods:

  • Mouse embryonic stem cells (mESCs) were cultured with iCRT3+PD medium, inhibiting MEK and β-catenin/TCF transcriptional activity.
  • Comparison of differentiation marker expression and pluripotency markers between iCRT3+PD and 2i cultures over multiple passages.
  • Assessment of mESC contribution to chimera generation and germline transmission.

Main Results:

  • iCRT3+PD co-inhibition significantly reduced differentiation marker expression compared to 2i medium over multiple passages.
  • mESCs cultured with iCRT3+PD demonstrated efficient contribution to chimera generation and germline transmission.
  • Inhibition of β-catenin's TCF-dependent transcriptional activity, not protein level, is key for retaining naïve pluripotency.

Conclusions:

  • Replacing CHIR with iCRT3 in combination with PD0325901 (iCRT3+PD) effectively maintains naïve ground state pluripotency in mESCs.
  • The iCRT3+PD medium serves as a viable and stable alternative to 2i for long-term mESC culture.
  • Targeting β-catenin's transcriptional activity via TCF interaction inhibition is crucial for stable naïve pluripotency.
Keywords:
2i-mediaMouse embryonic stem cellPluripotencyiCRT3β-catenin

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