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Fig. 4 | Stem Cell Research & Therapy

Fig. 4

From: Thyroid hormone enhances stem cell maintenance and promotes lineage-specific differentiation in human embryonic stem cells

Fig. 4

T3 promotes pluripotency under suboptimal conditions. A Impact of T3 on spontaneous differentiation. FGF2 and TGFβ were withdrawn from H1 cell culture at 30–40% confluence, and cells were allowed to spontaneously differentiate for 14 days. Medium was changed every two days, and pluripotency markers NANOG and POU5F1 were analyzed by RT-PCR on day 14. GAPDH was used as internal control, and the gene expression were normalized to the levels in E8 culture. Data are representative of three independent experiments. B Impact of T3 on pluripotency at low FGF2 concentrations. H1 cells were cultured in E8 medium with different concentrations of FGF2 with or without T3 for 3 passages before analysis by RT-PCR. Gene expression levels were normalized to GAPDH. Data are representative of two independent experiments. C Immunostaining of NANOG in H1 cells cultured for 3 passages in E8 medium with 1 ng/ml FGF2 in the presence or absence of T3. Scale bar, 200 μm. D Western blot analysis of ERK1/2 (Thr202/Tyr204) phosphorylation. H1 cells were cultured in E8 medium with or without T3 for 4 days with passaging on day 3 and then changed to E8 medium with or without FGF2 for another 24 h before collection for analysis. GAPDH was used as loading control. Data are representative of three independent experiments. E T3 promoted hESC growth in the absence of FGF2. H1 cells were cultured with or without FGF2 or T3 (500 nM) for 3 days. n = 6 biological repeats. *P < 0.05. F Impact of T3 on cell cycle profiles in the absence of FGF2. H1 cells were cultured with or without FGF2 or T3 (500 nM) for 3 days. Data are representative of three independent experiments. G, H T3 enhances hESC mitochondrial respiration in the medium without FGF2. H1 cells cultured with or without FGF2, the impact of T3 on oxygen consumption rate (OCR) measured by Mito Stress Test (G). Basal respiration, maximal respiration and ATP Synthase-Associated Respiration calculated from the assay results in panel G (H). n ≥ 3, *, P < 0.05

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