The combination of inhibitors of FGF/MEK/Erk and GSK3β signaling increases the number of OCT3/4- and NANOG-positive cells in the human inner cell mass, but does not improve stem cell derivation.
In embryonic stem cell culture, small molecules can be used to alter key signaling pathways to promote self-renewal and inhibit differentiation. In mice, small-molecule inhibition of both the FGF/MEK/Erk and the GSK3β pathways during preimplantation development suppresses hypoblast formation, and this results in more pluripotent cells of the inner cell mass (ICM). In this study, we evaluated the effects of different small-molecule inhibitors of the FGF/MEK/Erk and GSK3β pathway on embryo preimplantation development, early lineage segregation, and subsequent embryonic stem cell derivation in the humans. We did not observe any effect on blastocyst formation, but small-molecule inhibition did affect the number of OCT3/4- and NANOG-positive cells in the human ICM. We found that combined inhibition of the FGF/MEK/Erk and GSK3β pathways by PD0325901 and CHIR99021, respectively, resulted in ICMs containing significantly more OCT3/4-positive cells. Inhibition of FGF/MEK/Erk alone as well as in combination with inhibition of GSK3β significantly increased the number of NANOG-positive cells in blastocysts possessing good-quality ICMs. Secondly, we verified the influence of this increased pluripotency after 2i culture on the efficiency of stem cell derivation. Similar human embryonic stem cell (hESC) derivation rates were observed after 2i compared to control conditions, resulting in 2 control hESC lines and 1 hESC line from an embryo cultured in 2i conditions. In conclusion, we demonstrated that FGF/MEK/Erk and GSK3β signaling increases the number of OCT3/4- and NANOG-positive cells in the human ICM, but does not improve stem cell derivation.
Department for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, Ghent, Belgium. margot.vanderjeught@ugent.be
Margot
M
Van der Jeught
Thomas
T
O'Leary
Sabitri
S
Ghimire
Sylvie
S
Lierman
Galbha
G
Duggal
Karen
K
Versieren
Dieter
D
Deforce
Susana
S
Chuva de Sousa Lopes
Björn
B
Heindryckx
Petra
P
De Sutter
D001549:Benzamides / Q000494:pharmacology; D053624:Blastocyst Inner Cell Mass / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002452:Cell Count; D018929:Cell Culture Techniques; D019070:Cell Lineage; D004159:Diphenylamine / Q000031:analogs & derivatives / Q000494:pharmacology; D046149:Embryo Culture Techniques; D010064:Embryo Implantation / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D050993:GATA6 Transcription Factor / Q000235:genetics / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D020935:MAP Kinase Signaling System; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction; D013997:Time Factors
D001549:Benzamides; C473711:Chir 99021; D050993:GATA6 Transcription Factor; C494759:GATA6 protein, human; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D011725:Pyridines; D011743:Pyrimidines; C506614:mirdametinib; D004159:Diphenylamine; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases