Journal: Frontiers in Endocrinology

Article Title: Culture in Glucose-Depleted Medium Supplemented with Fatty Acid and 3,3′,5-Triiodo- l -Thyronine Facilitates Purification and Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes

doi: 10.3389/fendo.2017.00253

Figure Lengend Snippet: High purity of cardiomyocytes can be obtained under glucose-depleted and fatty acid (with or without T 3 )-supplemented conditions. (A) The schematic for purification and maturation using metabolic selection. Glycogen synthase kinase 3 inhibitor CHIR99021 and Wnt pathway inhibitor IWP2 (gray boxes) were added to the differentiation medium on differentiation days 1 and 4, respectively. On differentiation day 10, cells were dissociated and plated on glass cover slips. After 3 days recovery, cells were cultured with metabolic selection medium (No-glucose DMEM medium supplemented with lactate, fatty acid, and fatty acid + T 3 ). On differentiation day 18, cells were collected for patch clamping experiments. On differentiation day 21, cells were collected for other experiments. (B) Representative fluorescence-activated cell sorting (FACS) analyses of cardiac Troponin T expression in the human pluripotent stem cell (hPSC)-derived cells on day 0 with routine culture medium, and on day 8 with metabolic selection medium. In the control group, the isotype control (mouse IgG) was used instead of the primary antibody. (C) Time courses of selection efficiency using the lactate-supplemented condition (red), the fatty acid-supplemented condition (green), and the fatty acid with T 3 -supplemented condition (purple); cells cultured with routine medium (blue) were set up as the control groups ( n = 3). The asterisk means each test group is significantly different from controls, P < 0.01.

Article Snippet: Briefly, hPSC were treated with small molecule CHIR99021 (Tocris, 4423, final concentration 10 μM) in the RPMI-BSA medium [RPMI 1640 Medium (HyClone, SH30027.01) supplemented with 213 μg/ml AA2P ( l -ascorbic acid 2-phosphate magnesium) (A8960, Sigma) and 0.1% bovine serum albumin (BSA) (A1470, Sigma)] for 24 h, then were incubated with RPMI-BSA medium for 48 h. On differentiation day 4, cells were treated with the small molecule IWP2 (Tocris, 3533, final concentration 5 μM) in RPMI-BSA medium.

Techniques: Purification, Selection, Cell Culture, Fluorescence, FACS, Expressing, Derivative Assay, Control

Journal: National Science Review

Article Title: Inhibition of Wnt activity improves peri-implantation development of somatic cell nuclear transfer embryos

doi: 10.1093/nsr/nwad173

Figure Lengend Snippet: Wnt inhibition greatly improves the development of SCNT embryos. (a) Comparison of Wnt-related genes in differential gene expression profiles of the ICM of IVF and SCNT blastocysts. The genes marked are Wnt antagonists or activators. (b) TCF/Lef: H2B-GFP IVF (left) and SCNT (right) embryos at late E4.5 stained for GFP, Oct4, Gata6 and DAPI. Scale bar, 20 μm. (c) Percentage of GFP-positive cells in the EPI of IVF and SCNT embryos. (d) Percentage of rosette-like SCNT embryos generated with no treatment, Wnt activator CHIR treatment and Wnt inhibitor IWP2 treatment. (e) Comparison of pluripotent marker gene expression among the IVF-, SCNT- and Wnti-treated SCNT embryos. (f) Absolute value comparison of the relative expression of markers in (d) to late-E4.5 IVF EPI between groups of IVF-, SCNT- and Wnti-SCNT embryos. P -values were determined using the Wilcoxon signed-rank test. (g) GSEA of specific genes expressed in naïve and primed ESCs between the late-E4.5 EPI of untreated and Wnti-SCNT embryos. NES, normalized enrichment score. (h) PCA comparison of gene expression profiles among the late-E4.5 EPI of IVF-, SCNT- and Wnti-SCNT embryos, naïve ESCs, primed ESCs and RSCs. Published transcriptome data of stem cell lines were obtained from GSE145727. (i) Hierarchical clustering analysis of the late-E4.5 EPI of IVF, untreated and Wnti-SCNT embryos, naïve ESCs, primed ESCs and RSCs. (j) PCA plot of E3.5 ICM, E5.5 EPI, E6.5 EPI, late-E4.5 EPI of IVF, SCNT and Wnti-SCNT embryos based on the H3K27me3 levels. (k) Heat map showing the H3K27me3 levels for the naïve and primed markers between late-E4.5 EPI of IVF, SCNT and Wnti-SCNT embryos. For (c) and (d), P -values were determined using the unpaired two-tailed t -test; error bars and means ± SD are shown for n ≥ 3 experiments.

Article Snippet: Small-molecule inhibitors IWP2 (Selleck, 686770-61-6), IWR1-endo (Selleck, 1127442-82-3) and Wnt activator CHIR99021 (Sigma, SML1046) were applied to inhibit or activate the canonical Wnt/β-catenin signaling pathway.

Techniques: Inhibition, Comparison, Gene Expression, Staining, Generated, Marker, Expressing, Two Tailed Test

Journal: Cell death discovery

Article Title: Transcriptome-based prediction of drugs, inhibiting cardiomyogenesis in human induced pluripotent stem cells.

doi: 10.1038/s41420-023-01616-6

Figure Lengend Snippet: Fig. 2 Transcriptome analysis of differentiated hiPSCs (IMR90) toward CMs. The hiPSCs were cultured as a monolayer on matrigel-coated plates for 2 days under pluripotent conditions and on day 0 exposed to GSK3 inhibitor, CHIR (10 µM) for 24 h. After 48 h exposed to Wnt inhibitor, IWP2 (5 µM). Spontaneously beating cardiac clusters were observed from day 9 onwards. Simultaneously, cells were exposed to test substances for a single exposure of 24 h (day1). The cells were harvested for gene array analysis on day1, day4 and day14 (Fig. 1A). Medium changes were done as indicated every alternate date. A Representative phase-contrast images of control and ISO treated hiPSC at day1-, 4 -and 14day. Scale bar, 100 µm. B PCA blot of 54,675 probe sets for three timepoints during the differentiation. C PCA blot of the 500 SPS with the highest variance across the mean of the condition‐wise samples. The respective day is indicated by the shape and the respective measured compound is indicated by the color of the dot, as labels are shown next to the plots. The distribution of the data points on the x-axis is given by the PC 1 and on the y-axis by PC2. The percentages in parentheses denote the proportion of explained variance for the respective PC.

Article Snippet: The medium was then changed to basal RPMI/B-27-ins medium and cells were kept for further 24 h. At day 2, RPMI/B-27-ins mediumwith small molecule WNT inhibitor IWP2 (Tocris, United Kingdom) 5 μM was added and cells were kept for 48 h (day 2 to day4).

Techniques: Cell Culture, Control