Improvement of embryonic stem cell line derivation efficiency with novel medium, glucose concentration, and epigenetic modifications.
Although the first mouse embryonic stem (ES) cell lines were derived 2 decades ago, and standard protocols for ES cell derivation are widely used today, the technical difficulty of these protocols still pose a challenge for many investigators attempting to produce large numbers of ES cell lines, and are limited to only a few mouse strains. Recently, glucose concentration was shown to have a significant effect on the efficiency of ES cell derivation, but the mechanism(s) mediating this effect are still the subject of debate. In this report, we investigated the effect of glucose concentration on ES cell derivation efficiency from blastocysts in the context of a new medium, Minimum Essential Medium alpha (MEMalpha). Furthermore, we propose novel methods to improve mouse ES cell derivation efficiency using in vitro epigenetic modifications during early passages, combined with detection of Oct4-expressing cells. Based on the results reported here, modified MEMalpha containing high glucose improves the efficiency of ES cell derivation remarkably, compared with Knockout Dulbecco's-Modified Eagle Media (KDMEM). Epigenetic modifications are able to improve the efficiency even further.
Kim
Chul
C
Center for Regenerative Biology, University of Connecticut, Storrs, 06269-4243, USA.
Amano
Tomokazu
T
Park
Joonghoon
J
Carter
Mark G
MG
Tian
Xiuchun
X
Yang
Xiangzhong
X
D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000502:physiology; D018929:Cell Culture Techniques; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D044127:Epigenesis, Genetic; D015870:Gene Expression; D005947:Glucose / Q000737:chemistry / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000378:metabolism; D051379:Mice; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000378:metabolism
D003470:Culture Media; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D005947:Glucose