Wnt pathway modulation generates blastomere-derived mouse embryonic stem cells with different pluripotency features
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Wnt pathway modulation generates blastomere-derived mouse embryonic stem cells with different pluripotency features Marta Vila-Cejudo 1,2
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Sandra Alonso-Alonso 1
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Anna Pujol 3
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Josep Santaló 1
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Elena Ibáñez 1
Received: 1 June 2020 / Accepted: 4 October 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
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. Keywords Mouse embryonic stem cells . Single blastomeres . Wnt signaling pathway . MAPK signaling pathway . Pluripotency state
Introduction Mouse embryonic stem cells (mESC) can be derived and maintained in culture medium supplemented with fetal calf serum (FCS) [1, 2] or the more defined knockout serum replacement (KSR) [3], in the presence of leukemia inhibitory factor (LIF) [4, 5]. However, under these conditions, only
* Elena Ibáñez [email protected] 1
Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
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Present address: Tissue Engineering Unit, Centre for Genomic Regulation, Carrer del Dr. Aiguader 88, 08003 Barcelona, Spain
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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
embryos from permissive strains can successfully produce mESC lines. Moreover, while mESC lines cultured under these conditions are considered representatives of the naïve state of pluripotency, it is known that mESC grown in the presence of FCS + LIF form heterogeneous popula
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