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Generation of mice derived from induced pluripotent stem cells

Academic Article
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Overview

related to degree

  • Nazor, Kristopher Lancaster, Ph.D. in Biology, Scripps Research 2008 - 2015
  • Hazen, Jennifer, Ph.D. in Neuroscience, Scripps Research 2006 - 2013

authors

  • Boland, M. J.
  • Hazen, Jennifer
  • Nazor, Kristopher Lancaster
  • Rodriguez, A. R.
  • Martin, G.
  • Kupriyanov, S.
  • Baldwin, Kristin

publication date

  • 2012

journal

  • Journal of Visualized Experiments : JoVE  Journal

abstract

  • The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution(2). Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types(2). This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications. The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)(3-5). Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage(6). Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line. Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC(1). These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs(3,4,7) and higher than that reported for most other iPSC lines(8-12). These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines(13-15). Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

subject areas

  • Animals
  • Cytological Techniques
  • Female
  • Mice
  • Pluripotent Stem Cells
  • Pregnancy
  • Stem Cell Research
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Research

keywords

  • Cellular Biology
  • Developmental Biology
  • Induced pluripotent stem cells
  • Issue 69
  • Medicine
  • Molecular Biology
  • Stem Cell Biology
  • developmental potential
  • iPSC
  • mouse
  • reprogramming
  • stem cells
  • tetraploid embryo complementation
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Identity

PubMed Central ID

  • PMC3565852

International Standard Serial Number (ISSN)

  • 1940-087X (Electronic) 1940-087X (Linking)

Digital Object Identifier (DOI)

  • 10.3791/4003

PubMed ID

  • 23222420
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Additional Document Info

start page

  • e4003

issue

  • 69

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