Efficient derivation of pluripotent stem cells from siRNA-mediated Cdx2-deficient mouse embryos.
In the early mammalian embryo, lineage separation of and subsequent crosstalk between the trophectoderm (TE) and inner cell mass (ICM) are required to support further development. Previous studies have shown that the homeobox transcription factor Cdx2 is required for TE differentiation and that lack of Cdx2 expression causes death of embryos at the peri-implantation stage. In this study, we effectively eliminated Cdx2 transcripts by microinjection of siRNA into embryos and evaluated the effect on efficiency of deriving embryonic stem cells (ESCs). By this approach, we successfully created nonviable embryos similar to reported knockout embryos. Accordingly, the efficiency of ESC derivation dropped from 19.1% in control blastocysts to 2% in Cdx2-deficient blastocysts, indicating loss of pluripotency in the ICM. Strikingly, when 8-cell stage embryos were cultured under ESC culture conditions before lineage separation, fully functional pluripotent stem cell lines were obtained, with efficiency even greater than that for control embryos. These results demonstrate that Cdx2 plays an essential role within the microenvironment created by the TE to support ICM pluripotency but that the ESC culture system, with mouse embryonic fibroblasts, could rescue the pluripotent cell population for efficient ESC derivation.
Wu
Guangming
G
Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany.
Gentile
Luca
L
Do
Jeong Tae
JT
Cantz
Tobias
T
Sutter
Julien
J
Psathaki
Katherina
K
Araúzo-Bravo
Marcos J
MJ
Ortmeier
Claudia
C
Schöler
Hans R
HR
D000818:Animals; D053624:Blastocyst Inner Cell Mass / Q000166:cytology* / Q000378:metabolism; D000071616:CDX2 Transcription Factor; D002478:Cells, Cultured; D004475:Ectoderm / Q000166:cytology* / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000637:transplantation; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D008845:Microinjections; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000637:transplantation; D011247:Pregnancy; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013724:Teratoma / Q000473:pathology; D014157:Transcription Factors / Q000172:deficiency* / Q000235:genetics; D014158:Transcription, Genetic
D000071616:CDX2 Transcription Factor; C487514:Cdx2 protein, mouse; D018398:Homeodomain Proteins; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins