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Wnt pathway modulation generates blastomere-derived mouse embryonic stem cells with different pluripotency features

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Abstract

Purpose

This study aimed to determine the role of Wnt pathway in mouse embryonic stem cell (mESC) derivation from single blastomeres isolated from eight-cell embryos and in the pluripotency features of the mESC established.

Methods

Wnt activator CHIR99021, Wnt inhibitor IWR-1-endo, and MEK inhibitor PD0325901 were used alone or in combination during ESC derivation and maintenance from single blastomeres biopsied from eight-cell embryos. Alkaline phosphatase activity, FGF5 levels, expression of key pluripotency genes, and chimera formation were assessed to determine the pluripotency state of the mESC lines.

Results

Derivation efficiencies were highest when combining pairs of inhibitors (15–24.7%) than when using single inhibitors or none (1.4–10.1%). Full naïve pluripotency was only achieved in CHIR- and 2i-treated mESC lines, whereas IWR and PD treatments or the absence of treatment resulted in co-existence of naïve-like and primed-like pluripotency features. IWR + CHIR- and IWR + PD-treated mESC displayed features of primed pluripotency, but IWR + CHIR-treated lines were able to generate germline-competent chimeric mice, resembling the predicted properties of formative pluripotency.

Conclusion

Wnt and MAPK pathways have a key role in the successful derivation and pluripotency features of mESC from single precompaction blastomeres. Modulation of these pathways results in mESC lines with various degrees of naïve-like and primed-like pluripotency features.

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Data availability

Available upon request.

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Acknowledgments

We thank staff from Servei Estabulari from Universitat Autònoma de Barcelona for animal care, Jonatan Lucas for help with feeder cell culture and reagent preparation and Salvador Bartolomé for his assistance and advice in the design of the RT-qPCR experiments.

Code availability

Not applicable.

Funding

This work was supported by the Ministerio de Economia y Competitividad of Spain (AGL2014-52408-R) and the Generalitat de Catalunya (2014 SGR-524 and 2017 SGR-503). MVC and SAA were beneficiary of a PIF-UAB fellowship.

Author information

Author notes

  1. Marta Vila-Cejudo

    Present address: Tissue Engineering Unit, Centre for Genomic Regulation, Carrer del Dr. Aiguader 88, 08003, Barcelona, Spain

Authors and Affiliations

  1. Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain

    Marta Vila-Cejudo, Sandra Alonso-Alonso, Josep Santaló & Elena Ibáñez

  2. Department of Biochemistry and Molecular Biology, School of Veterinary Medicine and Center of Animal Biotechnology and Gene Therapy, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain

    Anna Pujol

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  1. Marta Vila-Cejudo
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Contributions

MVC: conceptualization, formal analysis, investigation, writing - original draft, visualization. SAA: formal analysis, investigation, visualization. AP: investigation. JS: conceptualization, writing – review & editing, funding acquisition. EI: conceptualization, writing – review & editing, funding acquisition.

Corresponding author

Correspondence to Elena Ibáñez.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval

Mouse care and procedures were conducted according to the protocols approved by the Ethics Committee on Animal and Human Research of the Universitat Autònoma de Barcelona and by the Departament d’Agricultura, Ramaderia, Pesca i Alimentació of the Generalitat de Catalunya (ref. 9995 and 9582).

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Vila-Cejudo, M., Alonso-Alonso, S., Pujol, A. et al. Wnt pathway modulation generates blastomere-derived mouse embryonic stem cells with different pluripotency features. J Assist Reprod Genet 37, 2967–2979 (2020). https://doi.org/10.1007/s10815-020-01964-7

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  • DOI: https://doi.org/10.1007/s10815-020-01964-7

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