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Journal of Assisted Reproduction and Genetics
Published in: Journal of Assisted Reproduction and Genetics 1/2019

01-01-2019 | Embryo Biology

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

Authors: Marta Vila-Cejudo, Ot Massafret, Josep Santaló, Elena Ibáñez

Published in: Journal of Assisted Reproduction and Genetics | Issue 1/2019

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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.
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Metadata
Title
Single blastomeres as a source of mouse embryonic stem cells: effect of genetic background, medium supplements, and signaling modulators on derivation efficiency
Authors
Marta Vila-Cejudo
Ot Massafret
Josep Santaló
Elena Ibáñez
Publication date
01-01-2019
Publisher
Springer US
Published in
Journal of Assisted Reproduction and Genetics / Issue 1/2019
Print ISSN: 1058-0468
Electronic ISSN: 1573-7330
DOI
https://doi.org/10.1007/s10815-018-1360-9

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