Journal: World Journal of Stem Cells

Article Title: Wnt signaling pathway inhibitor promotes mesenchymal stem cells differentiation into cardiac progenitor cells in vitro and improves cardiomyopathy in vivo

doi: 10.4252/wjsc.v15.i8.821

Figure Lengend Snippet: Details of cardiac specific primers

Article Snippet: Untreated and IWP-4 treated MSCs were washed with PBS and fixed with 4% PFA, and then incubated in cardiac specific antibodies such as α-actinin (sc-17829, Santa Cruz Biotech, United States), Connexin-43 (C-43) (13-8300, Thermo scientific, United States), cardiac troponin I (cTnI) (Ab209809, Abcam, United Kingdom), Desmin (MAB3430 Chemicon International, United States), GATA-4 (sc-25310, Santa Cruz Biotech, United States), Nkx 2.5 (sc-376565, Santa Cruz Biotech, United States).

Techniques:

Journal: World Journal of Stem Cells

Article Title: Wnt signaling pathway inhibitor promotes mesenchymal stem cells differentiation into cardiac progenitor cells in vitro and improves cardiomyopathy in vivo

doi: 10.4252/wjsc.v15.i8.821

Figure Lengend Snippet: Cardiac markers gene expression analysis by quantitative real-time polymerase chain reaction. A: Seven days treatment of mesenchymal stem cells (MSCs) with inhibitor Wnt production-4 (IWP-4); B: Fourteen days treatment of MSCs with IWP-4 in comparison with untreated control showing significant increase in the expression of early cardiac markers, GATA-4, Nkx2.5, and late cardiac markers, MHC-β, MLC-2v , Mef-2D , cTnT , cTnC , cTnI , α-actinin , Ca-channel and Na-channel . Quantitative two fold (2 -ΔΔCT ) difference of mean is represented by ΔΔCt method. Statistical analysis was performed using an Independent sample t-test. Values are presented as mean ± SEM from three independent biological triplicates; level of significance is P < 0.05 ( a P < 0.05, b P < 0.01, and c P < 0.001). IWP-4: Inhibitor Wnt production-4; MSC: Mesenchymal stem cell; NS: No significant.

Article Snippet: Untreated and IWP-4 treated MSCs were washed with PBS and fixed with 4% PFA, and then incubated in cardiac specific antibodies such as α-actinin (sc-17829, Santa Cruz Biotech, United States), Connexin-43 (C-43) (13-8300, Thermo scientific, United States), cardiac troponin I (cTnI) (Ab209809, Abcam, United Kingdom), Desmin (MAB3430 Chemicon International, United States), GATA-4 (sc-25310, Santa Cruz Biotech, United States), Nkx 2.5 (sc-376565, Santa Cruz Biotech, United States).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Comparison, Control, Expressing

Journal: World Journal of Stem Cells

Article Title: Wnt signaling pathway inhibitor promotes mesenchymal stem cells differentiation into cardiac progenitor cells in vitro and improves cardiomyopathy in vivo

doi: 10.4252/wjsc.v15.i8.821

Figure Lengend Snippet: Analysis of fourteen days inhibitor Wnt production-4 treated mesenchymal stem cells. A: Morphology of untreated mesenchymal stem cells (MSCs), seven days inhibitor Wnt production-4 (IWP-4) treated MSCs and fourteen days IWP-4 treated MSCs; B: Immunocytochemical analysis of untreated MSCs, seven days and fourteen days IWP-4 treated MSCs showing positive expression of cardiac-specific proteins α-actinin , connexin-43 , cTnI , Desmin , GATA-4 , Nkx2.5 , secondary control, and DAPI control. Alexa fluor 488 secondary antibody was used for detection, then counterstained with DAPI to stain the nuclei; C: Quantification of fluorescence intensities in untreated MSCs, seven days, and fourteen days IWP-4 treated MSCs. For statistical analysis, One-way ANOVA was used followed by the Bonferroni post-hoc test. Data are presented as mean ± SEM with significance level P < 0.05 (where a P < 0.05, c P < 0.001). IWP-4: Inhibitor Wnt production-4; MSC: Mesenchymal stem cell; NS: No significant.

Article Snippet: Untreated and IWP-4 treated MSCs were washed with PBS and fixed with 4% PFA, and then incubated in cardiac specific antibodies such as α-actinin (sc-17829, Santa Cruz Biotech, United States), Connexin-43 (C-43) (13-8300, Thermo scientific, United States), cardiac troponin I (cTnI) (Ab209809, Abcam, United Kingdom), Desmin (MAB3430 Chemicon International, United States), GATA-4 (sc-25310, Santa Cruz Biotech, United States), Nkx 2.5 (sc-376565, Santa Cruz Biotech, United States).

Techniques: Expressing, Control, Staining, Fluorescence

Journal: World Journal of Stem Cells

Article Title: Wnt signaling pathway inhibitor promotes mesenchymal stem cells differentiation into cardiac progenitor cells in vitro and improves cardiomyopathy in vivo

doi: 10.4252/wjsc.v15.i8.821

Figure Lengend Snippet: Immunohistochemistry of heart tissue section. A: Immunohistochemical images of heart sections showing the transplanted untreated mesenchymal stem cells (MSCs) and fourteen days inhibitor Wnt production-4 (IWP-4) treated MSCs labeled with red fluorescent DiI dye. Cardiac specific proteins α-actinin, cTnI, and GATA-4 were immunostained for the expression of cardiac proteins. Alexa fluor 488 secondary antibody was used for detection; B: Quantification of the fluorescence intensities of the DiI labeled cells presented in bar graphs. As compared to normal MSCs, fluorescence intensity was significantly increased in case of alpha actinin, GATA-4, and cTnI in the fourteen days IWP-4 treated MSCs group in the infarcted myocardium. For statistical analysis, One-way ANOVA was used followed by the Bonferroni post-hoc test. Data are presented as mean ± SEM with significance level P < 0.05 where ( b P < 0.01, c P < 0.001). IWP-4: Inhibitor Wnt production-4; MSC: Mesenchymal stem cell; MI: Myocardial infarction.

Article Snippet: Untreated and IWP-4 treated MSCs were washed with PBS and fixed with 4% PFA, and then incubated in cardiac specific antibodies such as α-actinin (sc-17829, Santa Cruz Biotech, United States), Connexin-43 (C-43) (13-8300, Thermo scientific, United States), cardiac troponin I (cTnI) (Ab209809, Abcam, United Kingdom), Desmin (MAB3430 Chemicon International, United States), GATA-4 (sc-25310, Santa Cruz Biotech, United States), Nkx 2.5 (sc-376565, Santa Cruz Biotech, United States).

Techniques: Immunohistochemistry, Immunohistochemical staining, Labeling, Expressing, Fluorescence

Journal: Acta Cardiologica Sinica

Article Title: Analysis of Fatty Acid Metabolism in Fetal and Failing Hearts by Single-Cell RNA Sequencing Revealed SLC27A6 as a Critical Gene in Heart Maturation

doi: 10.6515/ACS.202307_39(4).20221219B

Figure Lengend Snippet: Altered cardiac gene expression and morphology of hiPSC-CMs with SLC27A6 deficiency. (A) mRNA levels of SLC27A6 during the cardiomyocyte (CM) differentiation process. (B) mRNA levels of SLC27A6 in NC-CMs and KD-CMs. (C) Representative western blots showing the SLC27A6 levels in NC-CMs and KD-CMs. Quantification was performed using ImageJ. (D) Line graphs depicting the ratios of MYH7/MYH6 and MYL2/MYL7, and the mRNA levels of TNNT2 and NKX2.5 in NC-CMs and KD-CMs. (E) Representative images of immunofluorescence staining for cTnT (green) in NC-CMs and KD-CMs. Nuclei were stained with DAPI (blue) (original magnification ×40; scale bar = 20 μm). (F) Quantitation of cardiomyocyte area, percentage of cells with disorganized sarcomere and multi-nuclear cells, respectively. Data are presented as mean ± SEM of at least three independent experiments, as analyzed using the two-tailed Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, relative to the control group. CM, cardiomyocyte; SEM, standard error of the mean; SLC27A6, solute carrier family 27 member 6.

Article Snippet: The cells were incubated overnight at 4 °C with primary anti-cardiac troponin T (cTnT) antibodies (Abcam).

Techniques: Gene Expression, Western Blot, Immunofluorescence, Staining, Quantitation Assay, Two Tailed Test, Control

Journal: Acta Cardiologica Sinica

Article Title: Analysis of Fatty Acid Metabolism in Fetal and Failing Hearts by Single-Cell RNA Sequencing Revealed SLC27A6 as a Critical Gene in Heart Maturation

doi: 10.6515/ACS.202307_39(4).20221219B

Figure Lengend Snippet: Expression pattern of key markers regulating in vitro cardiomyocyte directed differentiation. Line graphs depicting the mRNA levels of key markers at various stages of cardiomyocyte differentiation. Data are presented as the mean ± SEM of at least three independent experiments relative to the control group. NKX2.5, NK2 homeobox 5; SEM, standard error of the mean; TNNT2, cardiac troponin T (gene).

Article Snippet: The cells were incubated overnight at 4 °C with primary anti-cardiac troponin T (cTnT) antibodies (Abcam).

Techniques: Expressing, In Vitro, Control

Journal: Cell & Bioscience

Article Title: A neonatal rat model of pulmonary vein stenosis

doi: 10.1186/s13578-023-01058-8

Figure Lengend Snippet: Immunofluorescence of the upstream PV. A Representative images from the sham and PVS groups. White arrows indicate the smooth muscle layer; α-SMA ( red ), cTnT ( green ), DAPI ( blue ). a–h represent the α-SMA and c-TnT staining of the upstream PV of sham and PVS rats in the transverse direction. i–p represent the α-SMA and c-TnT staining of the upstream PV of sham and PVS rats in the longitudinal direction. B Quantification of α-SMA intensity; n = 30 slides from 6 samples. C Quantification of cTnT intensity; n = 30 slides from 6 rats. D Representative WGA and c-TnT staining of PVs and the local magnification. WGA (green); c-TnT (red); DAPI (blue). The white dashed rectangle shows the original region of the magnification. E Quantification of the cross-sectional area (CSA) of cardiomyocytes, n = 25 slides from five rats

Article Snippet: For immunofluorescence, after dewaxing, the slides were washed three times with phosphate-buffered saline, permeated with 0.5% Triton X-100 for 15 min, blocked with 10% donkey serum for 30 min, and stained with primary antibodies (anti-alpha smooth muscle actin (α-SMA) (ab7817, Abcam, Cambridge, UK) or anti-cardiac troponin T (cTnT) (ab459327, Abcam, Cambridge, UK)) overnight at 4 °C.

Techniques: Immunofluorescence, Staining

Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

Article Title: Human induced pluripotent stem cell-derived cardiac myocytes and sympathetic neurons in disease modelling

doi: 10.1098/rstb.2022.0173

Figure Lengend Snippet: Induction of cardiac myocytes and sympathetic neurons from human induced pluripotent stem cells. ( a,b ) Schematic summary of cardiac myocyte ( a ) and sympathetic neuron ( b ) induction protocols. ( c , d ) Flow cytometric results from day 20 cardiac myocytes ( c ) and day 50 sympathetic neurons ( d ). In ( c ) the right upper quadrant (Q2) shows positive population for ventricular myosin light chain 2 (MLC2v) and cardiac troponin (cTnT) in hiPSC-CM (from SFC854-03-02) of 80%. ( d ) Blue curve represents blank control and red curve represents hiPSC-SN (from UKKi007-A) stained with anti-tyrosine hydroxylase (anti-TH). Compared with negative control, 84.3% of hiPSC-SN expressed TH. ( e,f ) Structural characterization of day 20 cardiac myocytes ( e ) and day 42 sympathetic neurons ( f ) by immunofluorescence staining. The alignment of cTnT and α-actinin shows sarcomere structure within cardiac myocytes ( e ). Anti-TH-stained neuron cell body and axon; peripherin (PRPH) staining outlines the axon extension and formation of varicosities ( f ).

Article Snippet: Two to five days after seeding, cells were fixed with 4% formaldehyde, permeabilized and blocked in Tris-buffered saline (TBS) plus 0.5% Triton and 6% donkey serum. hiPSC-CM were then incubated in TBS with primary antibodies, anti-α-actinin (Sigma, A7811) and anti-cardiac troponin T (anti-cTnT, Abcam, ab45932), overnight at 4°C.

Techniques: Staining, Negative Control, Immunofluorescence

Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

Article Title: Human induced pluripotent stem cell-derived cardiac myocytes and sympathetic neurons in disease modelling

doi: 10.1098/rstb.2022.0173

Figure Lengend Snippet: Hypertrophic phenotype induced by angiotensin II (AngII) in hiPSC-CM (from one differentiation batch of hiPSC SFC854-03-02) versus Wistar neonatal cardiomyocytes. ( a ) The cartoon shows the induction of cardiac hypertrophy by exposing healthy hiPSC-CM to AngII for 48 h. Staining of α-actinin and cTnT shows disruption of cardiac structural protein by 1 µM AngII. Sarcomeres were fragmented and not organized into parallel myofilament bundles. ( b ) Treatment with AngII resulted in significant increase of cell size (left) and nuclear size (right). ( c ) Brain natriuretic peptide (BNP) mRNA expression significantly increased in 1 µM AngII-treated myocytes. Pretreatment of myocytes with AngII receptor blocker losartan antagonized BNP enhancement. Quantitative ELISA in cell culture supernatant revealed a tendency for increased proBNP secretion in hypertrophic groups. RT-qPCR repeated with iPS-OX1-19 hiPSC-CM confirmed significant increase of BNP and MYH7 (for myosin heavy chain 7), and an increased tendency for ANP and MYH6 . ( d , e ) Grouped data showing dynamic increases in cAMP in response to isoprenaline (Iso), IBMX and forskolin (FSK) in hiPSC-CM ( d ) and Wistar neonatal cardiomyocyte ( e ) models. One micromolar Ang II-treated group caused beta-adrenergic hyper-responsiveness and increased maximal cAMP level in both models.

Article Snippet: Two to five days after seeding, cells were fixed with 4% formaldehyde, permeabilized and blocked in Tris-buffered saline (TBS) plus 0.5% Triton and 6% donkey serum. hiPSC-CM were then incubated in TBS with primary antibodies, anti-α-actinin (Sigma, A7811) and anti-cardiac troponin T (anti-cTnT, Abcam, ab45932), overnight at 4°C.

Techniques: Staining, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR

Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

Article Title: Human induced pluripotent stem cell-derived cardiac myocytes and sympathetic neurons in disease modelling

doi: 10.1098/rstb.2022.0173

Figure Lengend Snippet: Functional phenotype of hiPSC-SN monoculture, hiPSC-CM monoculture and neurocardiac co-culture. ( a ) Cartoon showing cardiac myocytes and sympathetic neurons differentiated from a healthy hiPSC line and co-cultured together. ( b ) hiPSC-derived neurocardiac co-culture fixed and stained with cTnT (CM) and TH (SN) (left), cTnT (CM) and Syn (SN) (right). Overlay shows sympathetic neurons dispersed around cardiac myocyte clusters and extended axons towards myocytes, and dot-staining of anti-synapsin shows varicosities. ( c ) Representative normalized intracellular calcium ([Ca 2+ ] i ) trace of healthy hiPSC-SN monoculture (ReBl-PAT RYR2-WT ). [Ca 2+ ] i level increased significantly at the application of 100 or 200 nM angiotensin II (AngII) and 50 mM KCl. ( d ) Original Förster resonance energy transfer (FRET) traces showing dynamics of cytosolic cAMP changes in response to adenylyl cyclase activator forskolin (FSK, 0.1 and 0.3 µM) in healthy 20-day iPSC-CM. Saturation of the sensor was achieved by using FSK (10 µM) and IBMX (100 µM). ( e ) Comparison of myocyte cytosolic cAMP changes between iPSC-CM monoculture and co-culture with iPSC-SN. Baseline cAMP level showed no significant difference between monoculture and co-culture, while co-culture with SN activated myocytes' response to FSK with significantly higher increase of cytosolic cAMP. (( d , e ) from cell line iPS-OX1-19.)

Article Snippet: Two to five days after seeding, cells were fixed with 4% formaldehyde, permeabilized and blocked in Tris-buffered saline (TBS) plus 0.5% Triton and 6% donkey serum. hiPSC-CM were then incubated in TBS with primary antibodies, anti-α-actinin (Sigma, A7811) and anti-cardiac troponin T (anti-cTnT, Abcam, ab45932), overnight at 4°C.

Techniques: Functional Assay, Co-Culture Assay, Cell Culture, Derivative Assay, Staining, Förster Resonance Energy Transfer