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

Abstract

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 GSK3b 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 GSK3b 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 smallmolecule 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 GSK3b 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 GSK3b 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 GSK3b signaling increases the number of OCT3/4-and NANOG-positive cells in the human ICM, but does not improve stem cell derivation.

Inhibition of Transforming Growth Factor β Signaling Promotes Epiblast Formation in Mouse Embryos

“…Even under seemingly identical conditions, we still observed hypoblast cells in embryos although their number was significantly reduced compared with controls. Our results are in line with what was reported in human embryos cultured in 2i [9]. Therefore, we propose that additional mechanisms governing hypoblast formation events do exist besides FGF signaling, which need to be further investigated.…”

“…The increased number of epiblast cells and decreased number of hypoblast cells in embryos cultured in the presence of an FGF inhibitor is neither the result of selective proliferation of epiblast lineage nor the outcome of apoptosis of the hypoblast lineage but is due to selective lineage choice in the presence of these inhibitors [5]. Similarly, it was shown that 2i supplementation significantly increased the number of epiblast cells in human embryos [9]. In contrast to mice, FGF signaling appeared not to be involved in human hypoblast formation [9,10].…”

“…Similarly, it was shown that 2i supplementation significantly increased the number of epiblast cells in human embryos [9]. In contrast to mice, FGF signaling appeared not to be involved in human hypoblast formation [9,10]. Another study showed that the epiblast cell number can be increased by the addition of insulin in the culture medium [11].…”

Mouse and human blastocyst-derived stem cells: vive les differences

“…When human pre-implantation embryos were grown in the presence of ERK inhibitors to the blastocyst stage, there was no apparent effect on the formation of the PE (Kuijk et al, 2012;Roode et al, 2012;Van der Jeught et al, 2013), as would have been predicted from the mouse, suggesting that the formation of EPI versus PE is not under the tight control of FGF levels in the human. In addition, levels of FGF receptors are low in the human TE, which does not proliferate in response to FGF (Kunath et al, 2014).…”

Influence of Activin A Supplementation During Human Embryonic Stem Cell Derivation on Germ Cell Differentiation Potential

“…M4287; Sigma). For standard hESC lines UGent11-5, UGent11-6, and UGent11-7, culturing from blastocyst to hESC stage was carried out in a low-oxygen environment at 37°C, consisting of 6% CO2, and 5% O2, using standard hESC medium [26,33]. Within approximately 7 days, hESCs outgrowths were cut mechanically, transferred to freshly inactivated MEFs, and continually mechanically passaged until several colonies appeared.…”