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 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.

FIG. 1.

(a) Immunofluorescent staining for OCT3/4 and GATA6 and a merged image with 4′,6-diamidino-2-phenylindole (DAPI) of day-6 blastocyst in the control (A), PD0325901 (B), and 2i (C) group. Note the difference in number of OCT3/4-positive inner cell mass (ICM) cells in the PD0325901 and 2i blastocysts compared to the control. Scale bar, 100 μm. (b) Immunofluorescent staining for NANOG and GATA6 and a merged image with DAPI of day-6 blastocyst in the control (D) and 2i (E) group. Note the difference in number of NANOG-positive ICM, cells in the 2i blastocyst compared to the control. Scale bar, 100 μm.

FIG. 2.

(a) Bright-field pictures of the early stages in human embryonic stem cell (hESC) derivation. (A) Control hESC line G11-1: day 6 postplating the blastocyst and day 2 postcutting the area of interest; (B) Control hESC line G11-2: day 2 and 4 postcutting the area of interest after plating the day-6 control blastocyst; (C) hESC line G11-3 (embryo culture in 2i): day 2 postcutting the area of interest after plating the day-6 control blastocyst and established hESC colony after 2nd cutting. Scale bar, 100 μm. (b) Immunofluorescent staining of all derived hESC lines for OCT3/4 and NANOG. (D) Control hESC line G11-1; (E) control hESC line G11-2; (F) hESC line G11-3 (embryo culture in 2i). Scale bar, 50 μm.

FIG. 3.

Example of G-banding, showing a normal karyogram in all lines.