Journal: Signal transduction and targeted therapy

Article Title: Thiamine-modified metabolic reprogramming of human pluripotent stem cell-derived cardiomyocyte under space microgravity.

doi: 10.1038/s41392-024-01791-7

Figure Lengend Snippet: Fig. 1 Space microgravity leads to cardiomyocyte adaptative changes. a A schematic of all experimental processes, including somatic reprogramming, cardiomyocyte preparation, and cell treatment during the SZ-13 mission. b The representative pictures of culture flasks and apparatus for cell maintenance on the CSS. c The representative images of GCaMP-CMs under space microgravity (μg) and simulated 1 g (1g) conditions on the CSS. d The quantitative analyses on cell size of GCaMP-CMs in 1 g and μg groups. e The quantitative comparison on MFI (mean fluorescence intensity) for peak calcium density of GCaMP-CMs under 1 g and μg conditions. f The beating rate analyses of GCaMP-CMs under 1 g and μg conditions. g Representative calcium transients of GCaMP-CMs. The quantitative analyses of amplitude (h), time to peak (i), decay time (j) and tau (k) of GCaMP-CMs under 1 g and μg conditions. l Immunofluorescence staining of cytoskeleton proteins in hPSC-CMs under 1 g and μg conditions (α-Actinin, green; cTnT, red; Hoechst 33342, blue). m Representative polar histogram of myofibrillar alignment in hPSC-CMs under 1 g and μg conditions. The quantitative analyses on sarcomere length (n) and cTnT content (o) under 1 g and μg conditions. p The relative mRNA expression of cardiomyocyte-specific cytoskeleton genes (MYH6, MYH7, TNNT2, TNNI1, TNNI3) in hPSC-CMs under 1 g and μg conditions. q The protein expression of TNNI1 and TNNI3. r The relative mRNA expression of cardiomyocyte-specific calcium handling genes (RYR2, ATP2A2, SLC8A1, CACNA1C) in hPSC-CMs under 1 g and μg conditions. Data are presented as mean ± SEM; Student’s t test; *P < 0.05, **P < 0.01, ns not significant

Article Snippet: Immunofluorescence staining After returning to the earth, the fixed cardiomyocytes were adopted for immunofluorescence staining against α-Actinin (Abcam, USA) and cTnT (Proteintech, China) to illustrate the cytoskeleton morphology, and the nuclei were stained with Hoechst 33342 (Molecular Probes, USA).

Techniques: Comparison, Staining, Expressing

Journal: Signal transduction and targeted therapy

Article Title: Thiamine-modified metabolic reprogramming of human pluripotent stem cell-derived cardiomyocyte under space microgravity.

doi: 10.1038/s41392-024-01791-7

Figure Lengend Snippet: Fig. 3 Thiamine antagonist disturbs cardiomyocytes. a The beating rate analyses of GCaMP-CMs in the control group (CON) and thiamine antagonist group (amprolium, APL). b Representative calcium transients of GCaMP-CMs treated with CON and APL. c–e The quantitative analyses of amplitude (c), time to peak (d), decay time (e), and tau (f) of GCaMP-CMs treated with CON and APL. g Immunofluorescence staining of cytoskeleton proteins in GCaMP-CMs (α-Actinin, green; cTnT, red; Hoechst 33342, blue). h Representative polar histogram of the myofibrillar alignment in GCaMP-CMs. i, j The quantitative analyses on sarcomere length (i) and cTnT content (j) of GCaMP-CMs treated with CON and APL. k The thiamine content of cell culture supernatant of GCaMP-CMs. l The comparison of lactate content in the cell culture supernatant of GCaMP-CMs treated with CON and APL. m The quantitative analyses of ATP content in cell lysates of GCaMP-CMs treated with CON and APL. n Representative OCR traces of hiPSC-CMs treated with CON and APL were obtained using a Seahorse XFe24 Analyzer. Quantification of basal respiration (o), ATP production (p), and maximal respiration (q). r The α-ketoglutaric acid (AKG) content analysis under μg environment. Data are presented as mean ± SEM; Student’s t test or two-way repeated-measures ANOVA; *P < 0.05, **P < 0.01, ns not significant

Article Snippet: Immunofluorescence staining After returning to the earth, the fixed cardiomyocytes were adopted for immunofluorescence staining against α-Actinin (Abcam, USA) and cTnT (Proteintech, China) to illustrate the cytoskeleton morphology, and the nuclei were stained with Hoechst 33342 (Molecular Probes, USA).

Techniques: Control, Staining, Cell Culture, Comparison

Journal: Signal transduction and targeted therapy

Article Title: Thiamine-modified metabolic reprogramming of human pluripotent stem cell-derived cardiomyocyte under space microgravity.

doi: 10.1038/s41392-024-01791-7

Figure Lengend Snippet: Fig. 4 Thiamine supplementation relieves microgravity-induced cardiac adaption. a The thiamine content of cell culture supernatant of GCaMP-CMs under 1 g, simulated microgravity (μg) conditions. Partial microgravity-stimulated GCaMP-CMs were synchronously treated with thiamine (Thm+μg). b The beating rate analyses of GCaMP-CMs in 1 g, μg, and Thm+μg groups. c Representative calcium transients of GCaMP-CMs under 1 g, μg, and Thm+μg conditions. d–g The quantitative analyses of amplitude (d), time to peak (e), decay time (f), and tau (g) of GCaMP-CMs. h Immunofluorescence staining of cytoskeleton proteins in GCaMP-CMs under 1 g, μg, and Thm+μg conditions (α-Actinin, green; cTnT, red; Hoechst 33342, blue). i Representative polar histogram of myofibrillar alignment in GCaMP-CMs under 1 g, μg, and Thm+μg conditions. j, k The quantitative analyses on sarcomere length (j) and cTnT content (k) of GCaMP-CMs under 1 g, μg, and Thm+μg conditions. l The analyses of lactate content in cell supernatant of GCaMP-CMs under 1 g, μg, and Thm+μg conditions. m The quantitative analyses of ATP content in cell lysates of GCaMP-CMs. n Representative OCR traces of hiPSC-CMs in 1 g, μg, and Thm+μg groups were obtained using a Seahorse XFe24 Analyzer. Quantification of basal respiration (o), ATP production (p) and maximal respiration (q). Data are presented as mean ± SEM; one-way ANOVA or two-way repeated-measures ANOVA; * or #P < 0.05, **P < 0.01, ns not significant

Article Snippet: Immunofluorescence staining After returning to the earth, the fixed cardiomyocytes were adopted for immunofluorescence staining against α-Actinin (Abcam, USA) and cTnT (Proteintech, China) to illustrate the cytoskeleton morphology, and the nuclei were stained with Hoechst 33342 (Molecular Probes, USA).

Techniques: Cell Culture, Staining

Journal: Signal transduction and targeted therapy

Article Title: Thiamine-modified metabolic reprogramming of human pluripotent stem cell-derived cardiomyocyte under space microgravity.

doi: 10.1038/s41392-024-01791-7

Figure Lengend Snippet: Fig. 5 Thiamine treatment attenuates simulated microgravity-induced mouse cardiac dysfunction. a Representative M-mode echocardio- graphic images obtained from Sham, tail suspension (TS), and tail suspension + thiamine (TS+Thm) mice on day 28. b–f Assessment of echocardiographic parameters from the mice in Sham, TS, and TS+Thm groups. LVEF: ejection fraction, LVFS: fractional shortening, LV mass: left ventricle mass, LVPW.d: left ventricular posterior wall thickness at end-diastole, LVPW.s: Left ventricular posterior wall thickness at end systolic. g Immunofluorescence staining of cytoskeleton proteins (α-Actinin, green; cTnT, red; Hoechst 33342, blue). h The quantitative analyses on sarcomere length in three groups. i The quantitative analyses of SLC19A2 in heart tissue lysate. j, k The quantitative analyses of ATP production and lactic acid content in heart tissue lysate. l, m The cell size analysis by WGA staining in Sham, TS, and TS+Thm groups. Data are presented as mean ± SEM; one-way ANOVA; *P < 0.05, **P < 0.01, ns not significant

Article Snippet: Immunofluorescence staining After returning to the earth, the fixed cardiomyocytes were adopted for immunofluorescence staining against α-Actinin (Abcam, USA) and cTnT (Proteintech, China) to illustrate the cytoskeleton morphology, and the nuclei were stained with Hoechst 33342 (Molecular Probes, USA).

Techniques: Suspension, Staining

Journal: Stem cell research & therapy

Article Title: High glucose suppresses embryonic stem cell differentiation into cardiomyocytes : High glucose inhibits ES cell cardiogenesis.

doi: 10.1186/s13287-016-0446-5

Figure Lengend Snippet: Fig. 4 High glucose suppresses cardiomyocyte-specific marker expression. a mRNA levels of cardiomyocyte structure proteins, MEF2C, and TNNT2. b Representative images of TNNT2 immunofluorescent staining. c Quantitative fluorescent density data of TNNT2 immunofluorescent staining. d MEF2C and TNNT2 protein expression at differentiation day 9. Gene expression was assessed at EB formation day 5 and differentiation day 1, 3, 5, and 7. Experiments were repeated three times (n = 3). EB embryoid body derived from E14 or GR-E14 cells, HG high glucose (25 mM), LG low glucose (5 mM). Values were dedicated as mean ± SD. *Indicates significant difference compared with the other group

Article Snippet: Samples were blocked for 1 hour with 10% heat-inactivated donkey serum in PBS and incubated with the following antibodies: TNNT2 (1:200, Origene, Rockville, MD, USA), NKX2.5 (1:200, Sigma-Aldrich, St. Louis, MO, USA), and HCN1 (1:200, Sigma-Aldrich, St. Louis, MO, USA), overnight at 4 °C.

Techniques: Marker, Expressing, Staining, Gene Expression, Derivative Assay

Journal: Circulation Research

Article Title: Induction of Cardiomyocyte-Like Cells in Infarct Hearts by Gene Transfer of Gata4, Mef2c, and Tbx5

doi: 10.1161/circresaha.112.271148

Figure Lengend Snippet: Figure 5. Induction of cardiomyocyte-like cells by a polycistronic vector. A, Schematic representation of the 3F2A retrovirus containing GMT for polycistronic gene expression. B, Western blot analysis of 293 cells transfected with 3F2A for 2 days. C, Immunocytochemistry for Gata4 and vimentin in mouse fibroblasts transduced with 3F2A. D, Gata4 and Mef2c were expressed in the same cells after transduction of 3F2A. E, 3F2A retrovirus induced α-actinin expression and cross striations. The cell, indicated by arrows in the first and second panels, is enlarged in the third to fifth panels. White boxes are enlarged in the insets. F, Z-stack image of the GFP+/ α-actinin+ cell in E. G and H, Quantitative analyses of the α-actinin+ to GFP+ cell ratios (G) and the proportions of α-actinin+/GFP+ cells showing cross striations (H) (120 sections from 5 mice in control, 140 section from 5 mice in GMT, and 110 sections from 5 mice in 3F2A). I, The 3F2A-expressing cells also showed cTnT expression. White boxes in the first panels are enlarged in the second to fourth panels. J, Schematic representation to analyze mRNA expression in GFP- or 3F2A/GFP-infected cells. GFP+ cells were sorted by FACS after 1 week. K, 3F2A/GFP-transduced cells expressed significantly higher cardiac genes compared with controls determined by qPCR (n=3). Representative data are shown in each panel. All data are presented as mean±SEM. *P<0.05;**P<0.01 versus control. Scale bars, 50 μm.

Article Snippet: Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, blocked, and then incubated with primary antibodies against α-actinin, GFP, cTnT, vimentin, CD31, SMA (Sigma), collagen 1, Mef2c (Aviva Systems Biology), Gata4 (Santa Cruz Biotechnology), ANP (Chemicon), Nkx2.5 (Santa Cruz), or SM- MHC (Biomedical Technologies), with secondary antibodies conjugated to Alexa 488 or 546.

Techniques: Plasmid Preparation, Gene Expression, Western Blot, Transfection, Immunocytochemistry, Transduction, Expressing, Control, Infection

Journal: Cell stem cell

Article Title: Human Pluripotent Stem Cell-Derived Atrial and Ventricular Cardiomyocytes Develop from Distinct Mesoderm Populations.

doi: 10.1016/j.stem.2017.07.003

Figure Lengend Snippet: Figure 1. RA Signaling Promotes Atrial-like Cardiomyocyte Development (A) Schematic of the hPSC cardiomyocyte differentiation protocol indicating developmental stages and timing of RA addition. (B and C) qRT-PCR analysis of the expression levels of (B) a pan-cardiomyocyte gene and (C) ventricular-specific (MYL2), and atrial-specific (KCNJ3) genes in NKX2-5+SIRPa+CD90 cells isolated from day 20 EB populations induced with 10 ng/mL BMP4 and 6 ng/mL Activin A (10B/6A) and treated with RA at the indicated time points (n = 3) and in fetal tissue controls (n = 6) (t test, *p < 0.05 and **p < 0.01 versus DMSO control and ##p < 0.01 F-V versus F-A). (D) Heatmap comparing the gene expression profiles of NKX2-5+SIRPa+CD90 cells isolated from day 20 EBs (10B/6A induced) and treated with RA or DMSO (control) between days 3 and 5 (n = 5). Values represent log10 of expression levels relative to the housekeeping gene TBP. (E) Representative flow cytometric analyses of the proportion of NKX2-5+/CTNT+ and MLC2V+/CTNT+ cells in day 20 EB populations induced with 10B/6A and treated between days 3 and 5 with RA or DMSO (control).

Article Snippet: The following antibodies were used for staining: mouse anti-cardiac isoform of CTNT (ThermoFisher Scientific, 1:200), rabbit anti-human/rodent myosin light chain 2 (Abcam, 1:200), mouse anti-human COUPTF-II (R&D, 1:1000), or rabbit anti-human CTNT (Genway Biotech Inc., 1:1000).

Techniques: Quantitative RT-PCR, Expressing, Isolation, Control, Gene Expression

Journal: Cell stem cell

Article Title: Human Pluripotent Stem Cell-Derived Atrial and Ventricular Cardiomyocytes Develop from Distinct Mesoderm Populations.

doi: 10.1016/j.stem.2017.07.003

Figure Lengend Snippet: Figure 2. Induction of ALDH+ Cardiogenic Mesoderm (A) Representative flow cytometric analyses of ALDH activity in PDGFRa+ mesoderm in 10B/6A-induced EBs. ALDH inhibitor (DEAB)-treated cells were used as a control. (B and C) Representative flow cytometric analyses of day 4 ALDH activity and PDGFRa expression (left columns) and corresponding day 20 CTNT expression (right columns) following the manipulation (days 1–3) of (B) Activin A concentrations (0–10 ng/mL) in the presence of 10 ng/mL BMP4 or (C) BMP4 concentrations (1–10 ng/mL) in the presence of 2 ng/mL Activin A.

Article Snippet: The following antibodies were used for staining: mouse anti-cardiac isoform of CTNT (ThermoFisher Scientific, 1:200), rabbit anti-human/rodent myosin light chain 2 (Abcam, 1:200), mouse anti-human COUPTF-II (R&D, 1:1000), or rabbit anti-human CTNT (Genway Biotech Inc., 1:1000).

Techniques: Activity Assay, Control, Expressing

Journal: Cell stem cell

Article Title: Human Pluripotent Stem Cell-Derived Atrial and Ventricular Cardiomyocytes Develop from Distinct Mesoderm Populations.

doi: 10.1016/j.stem.2017.07.003

Figure Lengend Snippet: Figure 5. Optimization of CD235a+ Cardiogenic Mesoderm Induction (A and B) Representative flow cytometric analyses of day 4 ALDH activity and CD235a expression (left columns) and corresponding day 20 MLC2V and CTNT expression (right columns) following the manipulation (days 1–3) of (A) Activin A concentrations (2–20 ng/mL) in the presence of 10 ng/mL BMP4 or (B) BMP4 concentrations (3–20 ng/mL) in the presence of 12 ng/mL Activin A. (C) Representative flow cytometric plots showing the proportion of ALDH activity and CD235a expression in day 4 5B/12A- (blue) and 3B/2A-induced EBs (green). (D and E) Flow cytometric analyses of the proportion of (D) CTNT+ and (E) MLC2V+ cells in day 20 EB populations from 5B/12A- or 3B/2A-induced EBs treated with ROH, RA, or DMSO (control) for 48 hr (days 3–5) (t test, *p < 0.05 and **p < 0.01 versus DMSO control; n = 4).

Article Snippet: The following antibodies were used for staining: mouse anti-cardiac isoform of CTNT (ThermoFisher Scientific, 1:200), rabbit anti-human/rodent myosin light chain 2 (Abcam, 1:200), mouse anti-human COUPTF-II (R&D, 1:1000), or rabbit anti-human CTNT (Genway Biotech Inc., 1:1000).

Techniques: Activity Assay, Expressing, Control