Single blastomeres as a source of mouse embryonic stem cells: effect of genetic background, medium supplements, and signaling modulators on derivation efficiency
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To assess the role of the genetic background, the culture medium supplements, and the presence of modulators of signaling pathways on mouse embryonic stem cell derivation from single blastomeres from 8-cell embryos.
Mice from permissive and non-permissive genetic backgrounds, different culture media supplements, knockout serum replacement (KSR) and N2B27, and the presence or absence of 2i treatment were used to derive mouse embryonic stem cells (mESC) from single blastomeres isolated from 8-cell embryos and from control embryos at the blastocyst stage. After the sixth passage, the putative mESC were analyzed by immunofluorescence to assess their pluripotency and, after in vitro differentiation induction, their ability to differentiate into derivatives of the three primary germ layers. Selected mESC lines derived from single blastomeres in the most efficient culture conditions were further characterized to validate their stemness.
In control embryos, high mESC derivation efficiencies (70–96.9%) were obtained from permissive backgrounds or when embryos were cultured in medium complemented with 2i regardless of their genetic background. By contrast, only blastomeres isolated from embryos from permissive background cultured in KSR-containing medium complemented with 2i were moderately successful in the derivation of mESC lines (22.9–24.5%). Moreover, we report for the first time that B6CBAF2 embryos behave as permissive in terms of mESC derivation.
Single blastomeres have higher requirements than whole blastocysts for pluripotency maintenance and mESC derivation. The need for 2i suggests that modulation of signaling pathways to recreate a commitment towards inner cell mass could be essential to efficiently derive mESC from single blastomeres.
KeywordsMouse embryonic stem cells ESC derivation ESC culture Blastomere
We thank Jonatan Lucas for his technical assistance with feeder cell culture, the staff from Servei Estabulari from Universitat Autònoma de Barcelona for animal care, and Salvador Bartolomé for his assistance and advice in the design of the qPCR experiments.
This work has been supported by Ministerio de Economia y Competitividad (AGL2014-52408-R) and Generalitat de Catalunya (2014 SGR-524). MVC was beneficiary of a PIF-UAB fellowship and OM is beneficiary of a FI fellowship from the Generalitat de Catalunya.
Compliance with ethical standards
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. 8741).
Conflict of interest
The authors declare that they have no conflicts of interest.
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