stemdiff ventricular cardiomyocyte differentiation kit  (STEMCELL Technologies Inc)

Journal: Advanced Science

Article Title: Targeting miRNA‐1a and miRNA‐15b: A Novel Combinatorial Strategy to Drive Adult Cardiac Regeneration

doi: 10.1002/advs.202414455

Figure Lengend Snippet: Screening of co‐miRNAi that can promote cardiomyocyte proliferation in the P1 and P7 rat cardiomyocytes. A) Schematic diagram of the research. A set of 17 LNAs have been screened and then all combinations of the top hits will be tested in the neonatal P1 and P7 rat cardiomyocytes. The effects of the best combination will then be validated in vitro and in vivo. B,C) Effect of the removal of individual LNA from the pool of 17 LNAs in the neonatal P1 rat cardiomyocytes. Neonatal P1 rat cardiomyocytes were treated with different combinations of LNAs and stained for EdU (green) and cardiomyocyte‐specific α‐actinin (red). (B) Heat map shows the fold change of EdU + cells in α‐actinin + cardiomyocytes. Data are normalized to All (17 LNAs). The red and green colors indicate high and low values, respectively. n = 3. (C) Representative images of EdU and α‐actinin in cardiomyocytes. Scale bar is 50 µM. D,E) Neonatal P1 rat cardiomyocytes were treated with different combinations of LNAs for 2 days, and then cardiomyocytes were stained for Aurora B (green), cardiomyocyte‐specific α‐actinin (red), and DAPI (blue). (D) Heat map shows the quantification of the percentage of Aurora B + cells in telophase in α‐actinin + cardiomyocytes. The red and green colors indicate high and low values, respectively. (E) Representative images of Aurora B, α‐actinin, and DAPI in cardiomyocytes. Means of n = 3 biological replicates per group. Scale bar is 100 µm. F,G) P7 rat cardiomyocytes were treated with different combinations of LNAs for 48 h and stained for Aurora B (green) and α‐actinin (red), and DAPI (blue). (F) Heat map shows the quantification of rat α‐actinin + cardiomyocytes that were Aurora B + in telophase as a percentage of total α‐actinin + cells. Data are normalized to LNA‐NC. The red and green colors indicate high and low expression values, respectively. n = 3. G) Representative images of Aurora B, α‐actinin, and DAPI.

Article Snippet: The human iPSC‐derived cardiomyocytes (hiPSC‐CMs) were reprogrammed using the STEMdiff Cardiomyocyte Differentiation Kit (STEMCELL Technologies) according to the manufacture's protocol.

Techniques: In Vitro, In Vivo, Staining, Expressing

Journal: Advanced Science

Article Title: Targeting miRNA‐1a and miRNA‐15b: A Novel Combinatorial Strategy to Drive Adult Cardiac Regeneration

doi: 10.1002/advs.202414455

Figure Lengend Snippet: The effects of combinatorial miRNA‐1a/15b interference on proliferation in cardiac tissue and in non‐cardiac tissues in vivo. A) Representative immunofluorescence images of Ki67 on heart sections of adult hearts injected with LNA‐1a/15b or control 28‐days post MI injury. Ki67 labels proliferating cells (green); cardiomyocyte‐specific α‐actinin (red) and DAPI (Blue). B) Representative immunofluorescence images of Aurora B on heart sections of adult hearts injected with LNA‐1a/15b or control 28‐days post MI injury. Aurora B labels proliferating cells (green); cardiomyocyte‐specific α‐actinin (red) and DAPI (Blue). C) Quantification of percentages of Ki67 + cardiomyocytes. Data are expressed as means ± SEM; N = 3 mice per Control group, n = 5 mice per MI group; * P < 0.05 versus Control LNA‐NC, % P < 0.05 versus MI LNA‐NC. Two‐tailed unpaired t‐test. Scale bar is 100 µm. D) Quantification of percentages of Aurora B + cardiomyocytes. Data are expressed as means ± SEM; N = 3 mice per Control group, N = 5 mice per MI group; % P < 0.05 versus MI LNA‐NC. Two‐tailed unpaired t‐test. Scale bar is 100 µm. E) Representative images of Ki67 (green), Phalloidin (red), and DAPI (blue) in liver. Scale bar is 100 µm. F) Representative images of Ki67 (green), Phalloidin (red), and DAPI (blue) in SKM. Scale bar is 100 µm. G) Quantification of percentages of Ki67 + liver cells. Data are expressed as means ± SEM; n = 3 mice per group. Two‐tailed unpaired t‐test. H) Quantification of percentages of Ki67 + SKM cells. Data are expressed as means ± SEM; n = 3 mice per group. Two‐tailed unpaired t‐test. I) Representative images of Ki67 (green), Phalloidin (red), and DAPI (blue) in kidney. Scale bar is 100 µm. J) Representative images of Ki67 (green), Phalloidin (red), and DAPI (blue) in WAT. Scale bar is 100 µm. K) Quantification of percentages of Ki67 + kidney cells. Data are expressed as means ± SEM; n = 3 mice per group. Two‐tailed unpaired t‐test. L) Quantification of percentages of Ki67 + WAT cells. Data are expressed as means ± SEM; n = 3 mice per group. Two‐tailed unpaired t‐test. SKM, Skeletal Muscle; WAT, White Adipose Tissue. M) ASAT and N) ALAT levels in plasma of C57BL/6J mice injected with wither LNA‐1a/15b or Control LNA‐NC 28‐days post MI injury. Data are expressed as means ± SEM; n = 5‐6 mice per group. MI, Myocardial Infarction; ASAT, Aspartate aminotransferase; ALAT, Alanine aminotransferase.

Article Snippet: The human iPSC‐derived cardiomyocytes (hiPSC‐CMs) were reprogrammed using the STEMdiff Cardiomyocyte Differentiation Kit (STEMCELL Technologies) according to the manufacture's protocol.

Techniques: In Vivo, Immunofluorescence, Injection, Control, Two Tailed Test, Clinical Proteomics

Journal: Advanced Science

Article Title: Targeting miRNA‐1a and miRNA‐15b: A Novel Combinatorial Strategy to Drive Adult Cardiac Regeneration

doi: 10.1002/advs.202414455

Figure Lengend Snippet: The effects of combinatorial miRNA‐1a/15b interference on cardiomyocyte proliferation and cardiac function in human cardiac tissue mimetics. A) Representative immunofluorescence images of EdU incorporation on sections of 40‐day‐old human cardiac mimetics treated with LNA‐1a/15b or LNA‐NC in both normoxia and hypoxia. EdU labels proliferating cells (Magenta); Wheat‐Germ‐Agglutinin (WGA) marks cell surfaces (green); cardiomyocyte‐specific α‐actinin (red) and DAPI (Blue). B) Quantification of percentages of EdU + cardiomyocytes. Data are expressed as means ± SEM; n = 3 per group; ** P < 0.01 versus Normoxia LNA‐NC, % P < 0.05 versus Hypoxia LNA‐NC. Two‐tailed unpaired t‐test. Scale bar is 100 µm. C) Representative immunofluorescence images of pHH3 on sections of 40‐day‐old human cardiac mimetics treated with LNA‐1a/15b or LNA‐NC in both normoxia and hypoxia. pHH3 labels proliferating cells (Magenta); cardiomyocyte‐specific α‐actinin (red) and DAPI (Blue). Scale bar is 100 µm. D) Quantification of percentages of pHH3 + cardiomyocytes. Data are expressed as means ± SEM; n = 3 per group; ** P < 0.01 versus Normoxia LNA‐NC, % P < 0.05 versus Hypoxia LNA‐NC. Two‐tailed unpaired t‐test. E) Representative traces of contractile human cardiac mimetics. Human cardiac mimetics were treated with LNA‐1a/15b or LNA‐NC for 3 days in both normoxia and hypoxia, and then the contractility assays were performed by determining the amplitude peak of contracting human cardiac mimetics. F) Quantification of the amplitude peak of contracting human cardiac mimetics. Data are expressed as means ± SEM; 4 – 5 organoids per group; * P < 0.05 versus Normoxia LNA‐NC, % P < 0.05 versus Hypoxia LNA‐NC. Two‐tailed unpaired t‐test.

Article Snippet: The human iPSC‐derived cardiomyocytes (hiPSC‐CMs) were reprogrammed using the STEMdiff Cardiomyocyte Differentiation Kit (STEMCELL Technologies) according to the manufacture's protocol.

Techniques: Immunofluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting miRNA‐1a and miRNA‐15b: A Novel Combinatorial Strategy to Drive Adult Cardiac Regeneration

doi: 10.1002/advs.202414455

Figure Lengend Snippet: Changes in gene expression and metabolites with inhibition of miRNA‐1a and miRNA‐15b. A) The heatmap shows the variant genes. X‐axis: samples of LNA‐NC, LNA‐1a, LNA‐15b, LNA‐1a/15b; The red and blue colors indicate upregulation and downregulation, respectively. Means of n = 3 biological replicates per group. B) Gene Ontology (GO) biological process terms enriched in LNA‐1a/15b‐regulated genes. C) Top 7 GO biological process terms related to the heart development enriched in LNA‐1a, LNA‐15b, and LNA‐1a/15b. Red, genes regulated specifically by miRNA‐1a inhibition; Blue, genes regulated specifically by miRNA‐15b inhibition; grey, genes regulated in‐common by both miRNA‐1a and miRNA‐15b depletion; yellow, genes regulated unique to combinatorial inhibition of miRNA‐1a and miRNA‐15b. D) Pathway analysis bubble plot in KEGG shows the enriched metabolic pathways in P1 rat cardiomyocytes treated with LNA‐1a/15b under hypoxia. The horizontal axis is the pathway impact, which represents the importance of differential metabolites in metabolic pathways. The vertical axis represents the results of pathway enrichment analysis. The dot size represents the centrality of the metabolites involved in the corresponding metabolic pathway. The color of the dot represents the impact factor; large sizes and dark colors represent the central metabolic pathway enrichment and pathway impact values, respectively. E) Heat map of relative metabolite abundance in Neonatal P1 rat cardiomyocytes treated with either control LNA‐NC or LNA‐1a/15b, and then incubated in the hypoxia and hypoxia for 2 days. Depicted are metabolites with log2(fold change) > 0.5 compared to Normoxia LNA‐NC treatment and adjusted p value < 0.01 in at least one treatment group compared to the corresponding control LNA‐NC. n = 4 biological replicates per group.

Article Snippet: The human iPSC‐derived cardiomyocytes (hiPSC‐CMs) were reprogrammed using the STEMdiff Cardiomyocyte Differentiation Kit (STEMCELL Technologies) according to the manufacture's protocol.

Techniques: Gene Expression, Inhibition, Variant Assay, Control, Incubation