video-based motion edge detection and ca2+-recording system  (IonOptix)

Journal: Stem Cell Research & Therapy

Article Title: Exosomes secreted by endothelial cells derived from human induced pluripotent stem cells improve recovery from myocardial infarction in mice

doi: 10.1186/s13287-023-03462-w

Figure Lengend Snippet: In vitro cytoprotective effects of hiPSC-EC exosomes on hiPSC-CMs. A hiPSC-derived cardiomyocytes (hiPSC-CMs) were incubated for 24 h with PKH26-labeled hiPSC-EC exosomes (EC-Exo); then, the cardiomyocytes were fixed and immunofluorescently stained for α-actinin, and nuclei were counterstained with DAPI. Exosomes that had been taken up by the cardiomyocytes were identified using PKH26 fluorescence (bar = 100 μm). B–G hiPSC-CMs were cultured under normal or oxygen and glucose deprivation (OGD) conditions with PBS or hiPSC-EC exosome treatment for 48 h. B hiPSC-CMs were fixed, immunofluorescently stained for cardiac troponin I (cTnI) expression, and stained by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL); then, nuclei were counterstained with DAPI (bar = 100 μm). C Quantification for TUNEL + cardiomyocytes. D The activity of lactate dehydrogenase (LDH) released in the culture media was measured using a LDH release assay kit. E hiPSC-CM ATP content was analyzed using an ATP bioluminescence assay kit. F–G hiPSC-CMs were incubated with a Ca 2+ indicator (Fura-2 AM); then, Ca 2+ transients were recorded with continuous 0.5 Hz electric stimulation. F The representative traces of hiPSC-CMs are shown. G Quantification of Ca 2+ transient amplitudes. Quantitative data are presented as mean ± SEM. n = 4 independent experiments in C , D , and E and n = 5 independent experiments in G . Significance was evaluated via Student’s t -test in ( C and D ) and one-way ANOVA followed by Tukey’s post hoc test in ( E and G ). * p < 0.05 and ** p < 0.01

Article Snippet: Ca 2+ transients and cell shortening were detected simultaneously using a video-based motion edge detection and Ca 2+ -recording system (IonOptix) as previously described [ ].

Techniques: In Vitro, Derivative Assay, Incubation, Labeling, Staining, Fluorescence, Cell Culture, Expressing, TUNEL Assay, Activity Assay, Lactate Dehydrogenase Assay, ATP Bioluminescent Assay

Journal: Stem Cell Research & Therapy

Article Title: Exosomes secreted by endothelial cells derived from human induced pluripotent stem cells improve recovery from myocardial infarction in mice

doi: 10.1186/s13287-023-03462-w

Figure Lengend Snippet: Cell shortening and calcium transient assessments in isolated cardiomyocytes after MI. The rod-shaped cardiomyocytes were isolated from mouse left ventricles of the sham group, MI group, and MI + EC-Exo group on day 28 after MI or sham surgery. Cardiomyocytes were incubated with a Ca 2+ indicator (Fura-2 AM); then, cell shortening and Ca 2+ transients were recorded under continuous 0.5, 1, and 2 Hz electrical stimulation. A Representative traces of cell shortening. Quantification of B amplitude of cell shortening/cell resting length and C maximum upstroke velocity of cell shortening (+ dL/dt max )/cell resting length. D Representative traces of calcium transients. Quantification of E amplitude of Ca 2+ transient and F maximal ascending rate in cell contractile Ca 2+ transients (+ d[Ca 2+ ]/dt max )/resting calcium. Quantitative data are presented as mean ± SEM. A total of 40–44 cardiomyocytes from four different hearts were measured in each group. Significance was evaluated via one-way ANOVA followed by Tukey’s post hoc test in ( B , C , E , and F ). ** p < 0.01

Article Snippet: Ca 2+ transients and cell shortening were detected simultaneously using a video-based motion edge detection and Ca 2+ -recording system (IonOptix) as previously described [ ].

Techniques: Isolation, Incubation

Journal: Stem Cell Research & Therapy

Article Title: Exosomes secreted by endothelial cells derived from human induced pluripotent stem cells improve recovery from myocardial infarction in mice

doi: 10.1186/s13287-023-03462-w

Figure Lengend Snippet: Sarcoplasmic reticulum (SR) Ca 2+ content and Ca 2+ transport rate assessments in isolated cardiomyocytes after MI. Ca 2+ transients were evoked by 10 mM caffeine in ventricular myocytes from the sham group, MI group, and MI + EC-Exo group on day 28 after MI or sham surgery. A Representative traces of Ca 2+ transient during caffeine-induced contraction in 0 Na + /0 Ca 2+ Tyrode’s solution or normal Na + /Ca 2+ Tyrode’s solution (NT). B Quantification of the amplitude of caffeine-induced Ca 2+ transients in 0 Na + /0 Ca 2+ Tyrode’s solution. C Rate constant of SERCA-mediated Ca 2+ transport (the difference between the rate constant of caffeine-evoked Ca 2+ transients and electric stimulation-evoked Ca 2+ transients in NT solution). D Maximum upstroke velocity (V max ) of caffeine-induced Ca 2+ transient in 0 Na + /0 Ca 2+ Tyrode’s solution. E Rate constant of Na + /Ca 2+ exchanger-1 (NCX-1)-mediated Ca 2+ transport (the difference between delay rate constant of caffeine-evoked Ca 2+ transients in NT solution and in 0 Na + /0 Ca 2+ solution). F SERCA activity was accessed using a Ca 2+ -pump ATPase enzyme assay kit. Quantitative data are presented as mean ± SEM. A total of 40–44 cardiomyocytes from four different hearts were measured in each group. Significance was evaluated via one-way ANOVA followed by Tukey’s post hoc test in ( B – F ). ** p < 0.01

Article Snippet: Ca 2+ transients and cell shortening were detected simultaneously using a video-based motion edge detection and Ca 2+ -recording system (IonOptix) as previously described [ ].

Techniques: Isolation, Activity Assay, Enzymatic Assay

Journal: Stem Cell Research & Therapy

Article Title: Exosomes secreted by endothelial cells derived from human induced pluripotent stem cells improve recovery from myocardial infarction in mice

doi: 10.1186/s13287-023-03462-w

Figure Lengend Snippet: miR-100-5p loss abolishes, while miR-100-5p mimic reproduces, the part of protection of hiPSC-EC exosomes on cardiomyocytes against OGD injury. hiPSC-CMs were separately treated with PBS, mimic negative control (NC), miR-100-5p mimic, EC-Exo, EC-Exo NC , and EC-Exo anti−miR−100−5p under OGD conditions. A–B Protein samples were collected from the above-mentioned groups, and Western blot was performed to assess the protein expression levels of PP-1 β , p-Ser16-PLB, and PLB. A Representative immunoblots. Full-length blots are presented in Additional file : Fig. S10. B Quantitative analysis of protein expression levels. C SERCA activity was assessed using a Ca 2+ -pump ATPase enzyme assay kit. D hiPSC-CMs were incubated with a Ca 2+ indicator (Fura-2 AM) and stimulated at 0.5 Hz; then, Ca 2+ transients were recorded and quantified. E The activity of LDH released in the culture media was measured. F TUNEL + cardiomyocytes were assessed. Quantitative data are presented as mean ± SEM. n = 4 independent experiments in B and n = 5 independent experiments in C – F . Significance was evaluated via one-way ANOVA followed by Tukey’s post hoc test in ( B – F ). * p < 0.05 and ** p < 0.01

Article Snippet: Ca 2+ transients and cell shortening were detected simultaneously using a video-based motion edge detection and Ca 2+ -recording system (IonOptix) as previously described [ ].

Techniques: Negative Control, Western Blot, Expressing, Activity Assay, Enzymatic Assay, Incubation, TUNEL Assay