Fig 2: Differentiation of Capacitated Naive iPSCs(A) Flow cytometry analysis of SOX17 and CXCR4 expression after 3 days definitive endoderm induction of primed S6EOS and capacitated niPSC4 cells.(B) Immunostaining for FOXA2 and SOX17 after 3 days definitive endoderm induction of niPSC2.(C) qRT-PCR analysis of definitive endoderm markers after 3 days induction of niPSC2.(D) Immunostaining for SOX1 and PAX6 after 10 days neuroectoderm induction of niPSC2.(E) qRT-PCR analysis of neuroectoderm marker expression.(F) Immunostaining for TBX6 after 6 days of paraxial mesoderm induction of niPSC2.(G) qRT-PCR analysis of paraxial mesoderm markers.Scale bars, 100 µm. Error bars indicate SD of three technical replicates.See also Figure S4.

Fig 3: Cx43 protein expression and localization in iPSCs and cells of each germ lineage. (A) Representative immunofluorescent confocal micrographs, demonstrating Cx43 (green) localization in control iPSCs (SOX2, iPSCs: purple) and after directed differentiation toward the three germ lineages (Nestin, ectoderm; Brachyury, mesoderm; SOX17, endoderm: purple) and nuclei (Hoechst, nuclei: blue). Cx43 forms large puncta (white arrows) at the cell surface indicative of gap junction plaques. Scale bars = 50 µm. (B) Representative Western blots and (C) densitometric analysis of total Cx43 protein expression in PAX6-positive ectoderm, Brachyury-positive mesoderm, and SOX17-positive endoderm cells. * p < 0.05 compared to undifferentiated iPSCs. Data represent the standard error of the mean of 3–4 independent experiments.

Fig 4: MEG enhances definitive endoderm differentiation of hiPSCs and of variations of definitive endoderm induction conditions. (A) FACS analysis of SOX17 and FOXA2 in H9 hESCs cultured in mTeSR1 medium. Cells were stained with SOX17-APC and FOXA2-Alexa488 antibodies (SOX17-APC/FOXA2-Alexa488) or non-specific IgG isotype control antibodies conjugated with the same fluorophores (isotype control). Isotype IgG, when used as a gating control, gave rise to a high percentage of false-positive cells in the undifferentiated mTeSR1 culture (~50% APC+/Alexa488+ and ~98% Alexa488+ in the SOX17-APC/FOXA2-Alexa-488 stained sample). n > 10 independent experiments. (B) FACS analysis of SOX17 and FOXA2 in MMW2 hiPSCs cells induced to undergo definitive endoderm differentiation under AWS, MEG+AWS, and MEG->AWS conditions for 7 days. Cells were plated at 1.0 × 105 cells/cm2 for AWS and MEG+AWS conditions, and at 0.5 × 105 cells/cm2 for MEG->AWS condition. Cells cultured in mTeSR1 medium were used as a gating control. n = 2 independent experiments. (C) FACS analysis of SOX17 and FOXA2 in H9 hESCs induced to undergo definitive endoderm differentiation for 5 days in AW-B27, MEG+AW-B27, and MEG->AW-B27 conditions. B-27 supplement was used in these conditions instead of serum. Cells were plated at 1.0 × 105 cells/cm2. n > 3 independent experiments. (D) FACS analysis of SOX17 and FOXA2 in H9 hESCs induced to undergo definitive endoderm differentiation for 5 days in AW-ITS, MEG+AW-ITS, and MEG->AW-ITS conditions. ITS (insulin, transferrin, and selenium) supplement was used in these conditions instead of serum. Cells were plated at 2.0 × 105 cells/cm2. n = 2 independent experiments.

Fig 5: Cx43 knockout iPSCs successfully differentiate to cells of each germ lineage under directed differentiation conditions. Quantitative RT-PCR (qPCR) screen, representative Western blots, and densitometry of differentiated control and Cx43 knockout (GJA1-/-) iPSCs. (A) Ectoderm formation indicated by presence of NES and PAX6 transcripts and expression of PAX6 protein. (B) Mesoderm evaluated by identifying transcripts for T, MILX1, and NCAM1, and expression of Brachyury protein. (C) Endoderm formation confirmed by FOXA2 and SOX17 transcripts and expression of SOX17 protein. Data represent the standard error of the mean of 3–4 independent experiments. ns: not significant compared to control cells.