Journal: Europace

Article Title: Promoting differentiation of human-induced pluripotent stem cells into sinoatrial node-like cells through programmed regulation of AMPK signalling pathway

doi: 10.1093/europace/euaf288

Figure Lengend Snippet: The role of AMPK signalling pathway in NKX2.5 expression. ( A ) Pan-cardiomyocyte differentiation protocol. CHIR (Wnt agonist) and IWP2 (Wnt inhibitor) were used for directed differentiation of cardiac cells. ( B–D ) Expression trends of markers at various stages during cardiomyocyte differentiation including cardiac mesoderm marker MESP1, cardiomyocyte progenitor marker NKX2.5, and cardiomyocyte marker TNNT2. qRT–PCR analysis was conducted to observe the dynamic expression trends from Days 0 to 13 of induced differentiation. ( E–F ) KEGG analysis of signalling pathways enriched with NXK2.5 expression. ( G ) Different concentrations of the AMPK agonist AICAR (AA) were added on Day 4 of differentiation for 2 days. The expression level of NKX2.5 in each group was analysed by qRT–PCR on Day 6. ( H ) Different concentrations of AMPK inhibitor CC were added on Day 4 of differentiation for 2 days. The expression level of NKX2.5 in each group was analysed by qRT–PCR on Day 6. One-way ANOVA followed by Bonferroni post hoc test was performed, * P < 0.05, *** P < 0.001, **** P < 0.0001, and ns, not significant vs. control, N = 5. All PCR expression values were normalized to the housekeeping gene GAPDH. Data are presented as ‘mean ± SEM’.

Article Snippet: At Day 0, the medium was replaced with RPMI/B-27 containing 8 μM CHIR99021 (a GSK3 inhibitor) (S1263, Selleck, USA) without insulin and incubated for 48 h. Subsequently, the medium was refreshed with RPMI/B-27 containing 5 μM IWP2 (a Wnt inhibitor) (S7085, Selleck, USA) without insulin for 48 h. On Days 4–6, the medium was switched to RPMI/B-27 without insulin.

Techniques: Expressing, Marker, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: Differential Sarcomere and Electrophysiological Maturation of Human iPSC-Derived Cardiac Myocytes in Monolayer vs. Aggregation-Based Differentiation Protocols

doi: 10.3390/ijms18061173

Figure Lengend Snippet: High content cell imaging to analyze the cell size, nucleation and morphology using the cTnT and Dapi staining. ( A ) Design of the automated cellular imaging high content analysis; ( B ) Composite image of the detection of cTnT+ and Dapi+ cells. Detection setting for total, round and long cells ( C ) Cell surface comparison at day 27 ± 2. N > 1500 cells from at least three different differentiations with three different iPSC clones, **** p < 0.0001; ( D ) Distribution of cell surface area in generated cTNT+ cells from the 2D-IWR1 (green) and 2D-IWP2 (blue); ( E ) Frequency of number of nuclei in the total cells for each protocol ( F ) Distribution of round and long cells obtained with each protocol. ** p < 0.01.

Article Snippet: The cardiac progenitors were induced on day 3 with 2D differentiation medium supplemented with either a Wnt inhibitor IWR1 (Inhibitor of Wnt Response, 2.5 μM) or IWP2 (Inhibitor of Wnt Production, 5 μM) (Tocris, Bristol, USA) for 2 days.

Techniques: Imaging, Staining, High Content Screening, Comparison, Clone Assay, Generated

Journal: International Journal of Molecular Sciences

Article Title: Differential Sarcomere and Electrophysiological Maturation of Human iPSC-Derived Cardiac Myocytes in Monolayer vs. Aggregation-Based Differentiation Protocols

doi: 10.3390/ijms18061173

Figure Lengend Snippet: Quantification of sarcomere mRNA and proteins. ( A ) The mRNA expression of cTni and ssTni was quantified by quantitative RT PCR. ( n = 8 to 12 from four iPSC clones, p < 0.05 Kruskal-Wallis and Dunn’s Multiple Comparison Test, hVentriCM was excluded from statistical analysis); ( B ) Typical western blot of cTNi and ssTNi proteins; ( C ) Quantification of the ratio of cTNi/GAPDH on ssTNi/GAPDH proteins in hiPSC-CM generated with the 2D-IWR1 vs. 2D-IWP2 protocols. Quantifications from 4 differentiations with 4 different iPS clones, * p < 0.05, Mann–Whitney test.

Article Snippet: The cardiac progenitors were induced on day 3 with 2D differentiation medium supplemented with either a Wnt inhibitor IWR1 (Inhibitor of Wnt Response, 2.5 μM) or IWP2 (Inhibitor of Wnt Production, 5 μM) (Tocris, Bristol, USA) for 2 days.

Techniques: Expressing, Quantitative RT-PCR, Clone Assay, Comparison, Western Blot, Generated, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Differential Sarcomere and Electrophysiological Maturation of Human iPSC-Derived Cardiac Myocytes in Monolayer vs. Aggregation-Based Differentiation Protocols

doi: 10.3390/ijms18061173

Figure Lengend Snippet: Main electrophysiological parameters. (*) indicates significances levels of p < 0.05 and (**) p < 0.01 3D vs. 2D-IWR1. ( $ ) indicates significances levels of p < 0.05 and ( $$ ) p < 0.01 3D vs. 2D-IWP2 ( £ ) indicates significances levels of p < 0.05 and ( ££ ) p < 0.01 2D-IWR1 vs. 2D-IWP2.

Article Snippet: The cardiac progenitors were induced on day 3 with 2D differentiation medium supplemented with either a Wnt inhibitor IWR1 (Inhibitor of Wnt Response, 2.5 μM) or IWP2 (Inhibitor of Wnt Production, 5 μM) (Tocris, Bristol, USA) for 2 days.

Techniques: