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Efficient and user-friendly pluripotin-based derivation of mouse embryonic stem cells

Tim Pieters UGent, Lieven Haenebalcke UGent, Tino Hochepied UGent, Jinke D'Hont UGent, Jody Haigh UGent, Frans Van Roy UGent and Jolanda van Hengel UGent (2012) STEM CELL REVIEWS AND REPORTS. 8(3). p.768-778
abstract
Classic derivation of mouse embryonic stem (ES) cells from blastocysts is inefficient, strain-dependent, and requires expert skills. Over recent years, several major improvements have greatly increased the success rate for deriving mouse ES cell lines. The first improvement was the establishment of a user-friendly and reproducible medium-alternating protocol that allows isolation of ES cells from C57BL/6 transgenic mice with efficiencies of up to 75%. A recent report describes the use of this protocol in combination with leukemia inhibitory factor and pluripotin treatment, which made it possible to obtain ES cells from F1 strains with high efficiency. We report modifications of these protocols for user-friendly and reproducible derivation of mouse ES cells with efficiencies of up to 100%. Our protocol involves a long initial incubation of primary outgrowths from blastocysts with pluripotin, which results in the formation of large spherical outgrowths. These outgrowths are morphologically distinct from classical inner cell mass (ICM) outgrowths and can be easily picked and trypsinized. Pluripotin was omitted after the first trypsinization because we found that it blocks attachment of ES cells to the feeder layer and its removal facilitated formation of ES cell colonies. The newly established ES cells exhibited normal karyotypes and generated chimeras. In summary, our user-friendly modified protocol allows formation of large spherical ICM outgrowths in a robust and reliable manner. These outgrowths gave rise to ES cell lines with success rates of up to 100%.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
INHIBITION, SELF-RENEWAL, RAT BLASTOCYSTS, Pluripotin, Efficiency, GENERATION, Embryonic stem cell derivation, Inner cell mass, Blastocyst outgrowth
journal title
STEM CELL REVIEWS AND REPORTS
Stem Cell Rev. Rep.
volume
8
issue
3
pages
768 - 778
Web of Science type
Article
Web of Science id
000307333100013
JCR category
MEDICINE, RESEARCH & EXPERIMENTAL
JCR impact factor
4.523 (2012)
JCR rank
21/119 (2012)
JCR quartile
1 (2012)
ISSN
1550-8943
DOI
10.1007/s12015-011-9323-x
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
3009470
handle
http://hdl.handle.net/1854/LU-3009470
date created
2012-10-09 10:20:53
date last changed
2012-10-10 15:15:33
@article{3009470,
  abstract     = {Classic derivation of mouse embryonic stem (ES) cells from blastocysts is inefficient, strain-dependent, and requires expert skills. Over recent years, several major improvements have greatly increased the success rate for deriving mouse ES cell lines. The first improvement was the establishment of a user-friendly and reproducible medium-alternating protocol that allows isolation of ES cells from C57BL/6 transgenic mice with efficiencies of up to 75\%. A recent report describes the use of this protocol in combination with leukemia inhibitory factor and pluripotin treatment, which made it possible to obtain ES cells from F1 strains with high efficiency. We report modifications of these protocols for user-friendly and reproducible derivation of mouse ES cells with efficiencies of up to 100\%. Our protocol involves a long initial incubation of primary outgrowths from blastocysts with pluripotin, which results in the formation of large spherical outgrowths. These outgrowths are morphologically distinct from classical inner cell mass (ICM) outgrowths and can be easily picked and trypsinized. Pluripotin was omitted after the first trypsinization because we found that it blocks attachment of ES cells to the feeder layer and its removal facilitated formation of ES cell colonies. The newly established ES cells exhibited normal karyotypes and generated chimeras. In summary, our user-friendly modified protocol allows formation of large spherical ICM outgrowths in a robust and reliable manner. These outgrowths gave rise to ES cell lines with success rates of up to 100\%.},
  author       = {Pieters, Tim and Haenebalcke, Lieven and Hochepied, Tino and D'Hont, Jinke and Haigh, Jody and Van Roy, Frans and van Hengel, Jolanda},
  issn         = {1550-8943},
  journal      = {STEM CELL REVIEWS AND REPORTS},
  keyword      = {INHIBITION,SELF-RENEWAL,RAT BLASTOCYSTS,Pluripotin,Efficiency,GENERATION,Embryonic stem cell derivation,Inner cell mass,Blastocyst outgrowth},
  language     = {eng},
  number       = {3},
  pages        = {768--778},
  title        = {Efficient and user-friendly pluripotin-based derivation of mouse embryonic stem cells},
  url          = {http://dx.doi.org/10.1007/s12015-011-9323-x},
  volume       = {8},
  year         = {2012},
}

Chicago
Pieters, Tim, Lieven Haenebalcke, Tino Hochepied, Jinke D’Hont, Jody Haigh, Frans Van Roy, and Jolanda van Hengel. 2012. “Efficient and User-friendly Pluripotin-based Derivation of Mouse Embryonic Stem Cells.” Stem Cell Reviews and Reports 8 (3): 768–778.
APA
Pieters, T., Haenebalcke, L., Hochepied, T., D’Hont, J., Haigh, J., Van Roy, F., & van Hengel, J. (2012). Efficient and user-friendly pluripotin-based derivation of mouse embryonic stem cells. STEM CELL REVIEWS AND REPORTS, 8(3), 768–778.
Vancouver
1.
Pieters T, Haenebalcke L, Hochepied T, D’Hont J, Haigh J, Van Roy F, et al. Efficient and user-friendly pluripotin-based derivation of mouse embryonic stem cells. STEM CELL REVIEWS AND REPORTS. 2012;8(3):768–78.
MLA
Pieters, Tim, Lieven Haenebalcke, Tino Hochepied, et al. “Efficient and User-friendly Pluripotin-based Derivation of Mouse Embryonic Stem Cells.” STEM CELL REVIEWS AND REPORTS 8.3 (2012): 768–778. Print.