fractionating exosomes  (Biofluids Inc)

Journal: Circulation research

Article Title: Exosomal microRNA-21-5p Mediates Mesenchymal Stem Cell Paracrine Effects on Human Cardiac Tissue Contractility

doi: 10.1161/CIRCRESAHA.118.312420

Figure Lengend Snippet: A, Heatmap and hierarchical clustering of expression of top 20 cardiac-related hMSC exosomal miRs compared with their expression in human foreskin fibroblasts (hFFs) and rat adult cardiac fibroblasts (rACFs). Expression levels represented as fold change (FC) relative to the average for hFF. Select miRs experimentally investigated are bolded and in blue. Grey boxes indicate data not available. B, PLSR score plot suggests cell type-dependent separation mainly across principal component (PC) 1 (x axis), not 2 (y axis). C, Correlation loading plot from PLSR suggests several PI3K/Akt-related (blue) and PI3K/Akt-unrelated (grey) miRs from (A) covary with human engineered cardiac tissue (hECT) developed force (DF), L-type calcium channel (LTCC) gene expression, and SERCA2a (sarcoendoplasmic reticulum calcium-ATPase) gene expression. D, Expression of miR-22-3p, miR-21-5p, and miR-181b-5p in hECTs 5 days after exosome-enriched fraction of the hMSC secretome (hMSC-exo) treatment relative to serum-free defined media (SFDM)-treated controls. *P<0.05, P values from unpaired t tests (n=4). E, Ingenuity Pathway Analysis (IPA, Qiagen) predictions suggest miR-21-5p positively regulates miR-181b-5p (top), but not vice versa (bottom). BAX indicates BCL2-associated × protein; and BCL2, B-cell lymphoma 2.

Article Snippet: 6 Baseline functional analysis was performed on culture day 5, followed by a single treatment with either (1) serum-free defined media (SFDM), (2) fresh hMSC conditioned SFDM media, (3) exosome-depleted hMSC conditioned SFDM media, (4) hMSC exosome-enriched SFDM (exosome-enriched fraction of the hMSC secretome [hMSC-exo]), (5) SFDM supplemented with 2 nmol/L of naked human miR mimic negative control (miR-scr-con; Thermo Fisher Scientific), (6) SFDM supplemented with 2 nmol/L of naked human miR-21-5p mimic (Thermo Fisher Scientific), (7) exosome-enriched fraction from naked human miR inhibitor negative control (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-scr-con), or (8) exosome-enriched fraction from miR-21-5p inhibitor (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-21-5p-KD).

Techniques: Expressing

Journal: Circulation research

Article Title: Exosomal microRNA-21-5p Mediates Mesenchymal Stem Cell Paracrine Effects on Human Cardiac Tissue Contractility

doi: 10.1161/CIRCRESAHA.118.312420

Figure Lengend Snippet: A, Expression of miR-22-3p, miR-21-5p, and miR-181b-5p in hECTs 5 days after miR-21-5p treatment relative to miR-scr-con. P values from unpaired t tests (n=3–5). B, Five days after miR-21-5p treatment, hECT developed force (DF) was significantly increased during 0.5 Hz pacing, whereas miR-scr-con negative controls had no significant effect. P values from repeated measures ANOVA followed by Bonferroni multiple comparisons test (n=5). Daily measurements of (C) DF and (D) beat rate in unpaced hECTs during spontaneous beating. P values from repeated measures ANOVA followed by Dunnett multiple comparisons test (n=4–5). In all panels, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

Article Snippet: 6 Baseline functional analysis was performed on culture day 5, followed by a single treatment with either (1) serum-free defined media (SFDM), (2) fresh hMSC conditioned SFDM media, (3) exosome-depleted hMSC conditioned SFDM media, (4) hMSC exosome-enriched SFDM (exosome-enriched fraction of the hMSC secretome [hMSC-exo]), (5) SFDM supplemented with 2 nmol/L of naked human miR mimic negative control (miR-scr-con; Thermo Fisher Scientific), (6) SFDM supplemented with 2 nmol/L of naked human miR-21-5p mimic (Thermo Fisher Scientific), (7) exosome-enriched fraction from naked human miR inhibitor negative control (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-scr-con), or (8) exosome-enriched fraction from miR-21-5p inhibitor (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-21-5p-KD).

Techniques: Expressing

Journal: Circulation research

Article Title: Exosomal microRNA-21-5p Mediates Mesenchymal Stem Cell Paracrine Effects on Human Cardiac Tissue Contractility

doi: 10.1161/CIRCRESAHA.118.312420

Figure Lengend Snippet: A, Expression of miR-22-3p, miR-21-5p, and miR-181b-5p in hECTs 5 days after exosome-enriched fraction of the hMSC secretome (hMSC-exo):miR-21-5p-KD treatment relative to hMSC-exo:miR-scr-con. P values from unpaired t tests (n=3). B, Five days after hMSC-exo:miR-scr-con treatment significantly increased hECT developed force (DF) during 0.5 Hz pacing in comparison to hMSC-exo:miR-21-5p-KD, which had no significant effect. P values from repeated measures ANOVA followed by Bonferroni multiple comparisons test (n=4). Daily measurements of (C) DF and (D) beat rate in unpaced hECTs during spontaneous beating. P values from repeated measures ANOVA followed by Dunnett multiple comparisons test (n=4). In all panels, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. # denotes statistical significance between groups at a given time point, #P<0.05, ##P<0.01.

Article Snippet: 6 Baseline functional analysis was performed on culture day 5, followed by a single treatment with either (1) serum-free defined media (SFDM), (2) fresh hMSC conditioned SFDM media, (3) exosome-depleted hMSC conditioned SFDM media, (4) hMSC exosome-enriched SFDM (exosome-enriched fraction of the hMSC secretome [hMSC-exo]), (5) SFDM supplemented with 2 nmol/L of naked human miR mimic negative control (miR-scr-con; Thermo Fisher Scientific), (6) SFDM supplemented with 2 nmol/L of naked human miR-21-5p mimic (Thermo Fisher Scientific), (7) exosome-enriched fraction from naked human miR inhibitor negative control (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-scr-con), or (8) exosome-enriched fraction from miR-21-5p inhibitor (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-21-5p-KD).

Techniques: Expressing

Journal: Circulation research

Article Title: Exosomal microRNA-21-5p Mediates Mesenchymal Stem Cell Paracrine Effects on Human Cardiac Tissue Contractility

doi: 10.1161/CIRCRESAHA.118.312420

Figure Lengend Snippet: hECTs treated with miR-scr-con vs miR-21-5p (A and B) and hMSC-exo:miR-scr-con vs hMSC-exo:miR-21-5p-KD (C and D) were snap-frozen for qRT-PCR on day 5 post-treatment, where expression of (A and C) cardiac-specific, calcium handling, and (B and D) apoptotic genes were studied. P values from 1-way ANOVA with post hoc Tukey test (n=3–5). E, Western blot of hPSC-CM monolayers treated for 48 h with miR-21-5p or miR-scr-con. In all panels, *P<0.05, **P<0.01, ***P<0.001. BAX indicates BCL2-associated × protein; BCL2, B-cell lymphoma 2; Casp, caspase; cTnT, cardiac troponin-T; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LTCC, L-type calcium channel; MHC, myosin heavy chain; SERCA2a, sarcoendoplasmic reticulum calcium-ATPase; and WCL, whole-cell lysate.

Article Snippet: 6 Baseline functional analysis was performed on culture day 5, followed by a single treatment with either (1) serum-free defined media (SFDM), (2) fresh hMSC conditioned SFDM media, (3) exosome-depleted hMSC conditioned SFDM media, (4) hMSC exosome-enriched SFDM (exosome-enriched fraction of the hMSC secretome [hMSC-exo]), (5) SFDM supplemented with 2 nmol/L of naked human miR mimic negative control (miR-scr-con; Thermo Fisher Scientific), (6) SFDM supplemented with 2 nmol/L of naked human miR-21-5p mimic (Thermo Fisher Scientific), (7) exosome-enriched fraction from naked human miR inhibitor negative control (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-scr-con), or (8) exosome-enriched fraction from miR-21-5p inhibitor (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-21-5p-KD).

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Circulation research

Article Title: Exosomal microRNA-21-5p Mediates Mesenchymal Stem Cell Paracrine Effects on Human Cardiac Tissue Contractility

doi: 10.1161/CIRCRESAHA.118.312420

Figure Lengend Snippet: A, Sample calcium transients from hPSC-CMs treated with miR-21-5 or miR-scr-con with or without LY294002 (LY), a PI3K inhibitor. B and C, Calcium transient amplitude of hPSC-CMs treated with miR-21-5p or miR-scr-con (B) without or (C) with LY. D and E, Calcium transient decay time constant (τCa) of hPSC-CMs treated with miR-21-5p or miR-scr-con (D) without or (E) with LY. For each panel, unpaired student t tests were performed between respective miR-scr-con and miR-21-5p experimental groups (n=5–6 per condition). *P<0.05, **P<0.01.

Article Snippet: 6 Baseline functional analysis was performed on culture day 5, followed by a single treatment with either (1) serum-free defined media (SFDM), (2) fresh hMSC conditioned SFDM media, (3) exosome-depleted hMSC conditioned SFDM media, (4) hMSC exosome-enriched SFDM (exosome-enriched fraction of the hMSC secretome [hMSC-exo]), (5) SFDM supplemented with 2 nmol/L of naked human miR mimic negative control (miR-scr-con; Thermo Fisher Scientific), (6) SFDM supplemented with 2 nmol/L of naked human miR-21-5p mimic (Thermo Fisher Scientific), (7) exosome-enriched fraction from naked human miR inhibitor negative control (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-scr-con), or (8) exosome-enriched fraction from miR-21-5p inhibitor (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-21-5p-KD).

Techniques:

Journal: Circulation research

Article Title: Exosomal microRNA-21-5p Mediates Mesenchymal Stem Cell Paracrine Effects on Human Cardiac Tissue Contractility

doi: 10.1161/CIRCRESAHA.118.312420

Figure Lengend Snippet: miR-21-5p effects on immature human cardiomyocyte L-type calcium channel (LTCC) and SERCA2a (sarcoendoplasmic reticulum calcium-ATPase) activity were experimentally calibrated. A, Scatter plots of the initial population (grey dots) filtered (white dots) to be within 1 SD (boxed region) of calcium transient decay time constant (τCa), and calcium transient amplitude ([Ca2+]i amplitude) metrics from Figure 5. B, Accepted sets of calibrated model parameters. C and D, Histograms illustrating distributions of the output simulation metrics—(C) [Ca2+]i amplitude and (D) τCa—resulting from the accepted population of calibrated models. E, Ten select accepted model parameters were subsequently input into ischemic adult human cardiomyocyte models to predict miR-21-5p effects on calcium transients (grey) in comparison to healthy (black line) and ischemic (dotted black line) adult human cardiomyocytes. ΔGLCa and ΔVmaxup denote fold changes in LTCC and SERCA2a maximal flux constants, respectively, because of miR-21-5p effects.

Article Snippet: 6 Baseline functional analysis was performed on culture day 5, followed by a single treatment with either (1) serum-free defined media (SFDM), (2) fresh hMSC conditioned SFDM media, (3) exosome-depleted hMSC conditioned SFDM media, (4) hMSC exosome-enriched SFDM (exosome-enriched fraction of the hMSC secretome [hMSC-exo]), (5) SFDM supplemented with 2 nmol/L of naked human miR mimic negative control (miR-scr-con; Thermo Fisher Scientific), (6) SFDM supplemented with 2 nmol/L of naked human miR-21-5p mimic (Thermo Fisher Scientific), (7) exosome-enriched fraction from naked human miR inhibitor negative control (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-scr-con), or (8) exosome-enriched fraction from miR-21-5p inhibitor (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-21-5p-KD).

Techniques: Activity Assay

Journal: Circulation research

Article Title: Exosomal microRNA-21-5p Mediates Mesenchymal Stem Cell Paracrine Effects on Human Cardiac Tissue Contractility

doi: 10.1161/CIRCRESAHA.118.312420

Figure Lengend Snippet: hMSCs (parent cells) release exosomes (exosome-enriched fraction of the hMSC secretome [hMSC-exo]) that are taken up by recipient human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). hMSC-exo delivery of microRNA-21-5p (miR-21-5p) increases hPSC-CM calcium handling gene expression, calcium handling, and thus contractility via the PI3K signaling cascade.

Article Snippet: 6 Baseline functional analysis was performed on culture day 5, followed by a single treatment with either (1) serum-free defined media (SFDM), (2) fresh hMSC conditioned SFDM media, (3) exosome-depleted hMSC conditioned SFDM media, (4) hMSC exosome-enriched SFDM (exosome-enriched fraction of the hMSC secretome [hMSC-exo]), (5) SFDM supplemented with 2 nmol/L of naked human miR mimic negative control (miR-scr-con; Thermo Fisher Scientific), (6) SFDM supplemented with 2 nmol/L of naked human miR-21-5p mimic (Thermo Fisher Scientific), (7) exosome-enriched fraction from naked human miR inhibitor negative control (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-scr-con), or (8) exosome-enriched fraction from miR-21-5p inhibitor (Thermo Fisher Scientific) treated hMSCs (hMSC-exo:miR-21-5p-KD).

Techniques: Derivative Assay, Expressing