Establishment of mouse embryonic stem cells from isolated blastomeres and whole embryos using three derivation methods

Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
Journal of Assisted Reproduction and Genetics (Impact Factor: 1.72). 12/2010; 27(12):671-82. DOI: 10.1007/s10815-010-9473-9
Source: PubMed


the aim of the present study is to compare three previously described mouse embryonic stem cell derivation methods to evaluate the influence of culture conditions, number of isolated blastomeres and embryonic stage in the derivation process.
three embryonic stem cell derivation methods: standard, pre-adhesion and defined culture medium method, were compared in the derivation from isolated blastomeres and whole embryos at 4- and 8-cell stages.
a total of 200 embryonic stem cell lines were obtained with an efficiency ranging from 1.9% to 72%.
using either isolated blastomeres or whole embryos, the highest rates of mouse embryonic stem cell establishment were achieved with the defined culture medium method and efficiencies increased as development progressed. Using isolated blastomeres, efficiencies increased in parallel to the proportion of the embryo volume used to start the derivation process.

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Available from: Elena Ibáñez, Jan 29, 2015
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    ABSTRACT: Efforts to efficiently derive embryonic stem cells (ESC) from isolated blastomeres have been done to minimize ethical concerns about human embryo destruction. Previous studies in our laboratory indicated a poor derivation efficiency of mouse ESC lines from isolated blastomeres at the 8-cell stage (1/8 blastomeres) due, in part, to a low division rate of the single blastomeres in comparison to their counterparts with a higher number of blastomeres (2/8, 3/8 and 4/8 blastomeres). Communication and adhesion between blastomeres from which the derivation process begins could be important aspects to efficiently derive ESC lines. In the present study, an approach consisting in the adhesion of a chimeric E-cadherin (E-cad-Fc) to the blastomere surface was devised to recreate the signaling produced by native E-cadherin between neighboring blastomeres inside the embryo. By this approach, the division rate of 1/8 blastomeres increased from 44.6% to 88.8% and a short exposure of 24 h to the E-cad-Fc produced an ESC derivation efficiency of 33.6%, significantly higher than the 2.2% obtained from the control group without E-cad-Fc. By contrast, a longer exposure to the same chimeric protein resulted in higher proportions of trophoblastic vesicles. Thus, we establish an important role of E-cadherin-mediated adherens junctions in promoting both the division of single 1/8 blastomeres and the efficiency of the ESC derivation process.
    No preview · Article · Dec 2010 · Stem cell reviews
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    ABSTRACT: The first event of differentiation in mammalian embryogenesis is the segregation of the inner cell mass and trophectoderm lineages in the blastocyst. Cellular and molecular events related to this process are still a controversial issue. During the years it was thought that first allocation of blastomeres before the blastocyst stage was done in the late eight-cell stage with the formation of inner and outer cells. Lately, many studies have pointed out that individual blastomeres at the four-cell stage differ in their developmental properties according to their position within the embryo. In this report, we wanted to elucidate whether these early decisions influence the production of mouse embryonic stem cell lines, so that a selective isolation of blastomeres at the four-cell stage to derive the lines could improve the efficiency of the derivation process. Results from blastomere tracking experiments support the idea of a different developmental potential of blastomeres within the four-cell stage embryo. However, we also show a high plasticity in the developmental pattern of blastomeres once isolated from the embryo, thus making all four-cell stage blastomeres equally competent to derive ESC lines.
    No preview · Article · Jul 2011 · Stem Cell Research
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