Single blastomeres as a source of mouse embryonic stem cells: effect of genetic background, medium supplements, and signaling modulators on derivation efficiency

Marta Vila-Cejudo, Ot Massafret, Josep Santaló, Elena Ibáñez*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

1 Citation (Scopus)

Abstract

Purpose: 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. Methods: 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. Results: 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. Conclusions: 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.
Original languageEnglish
Pages (from-to)99-111
Number of pages13
JournalJournal of Assisted Reproduction and Genetics
Volume36
Issue number1
DOIs
Publication statusPublished - 15 Jan 2019

Keywords

  • Blastomere
  • ESC culture
  • ESC derivation
  • Mouse embryonic stem cells
  • Signal Transduction
  • Mice, Inbred C57BL
  • Male
  • Embryonic Development
  • Culture Media/pharmacology
  • Mouse Embryonic Stem Cells/cytology
  • Animals
  • Blastomeres/cytology
  • Embryo Culture Techniques/methods
  • Mice, Inbred CBA
  • Female
  • Cell Differentiation
  • Mice
  • Genetic Background

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