In vitro differentiation of W8B2+ human cardiac stem cells: gene expression of ionic channels and spontaneous calcium activity

Figures

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  Fig. 1. Effect of W8B2+ CSCs in vitro differentiation on cardiac markers. a Transcript coding for cardiac transcription factors analyzed by TLDA in differentiated vs undifferentiated W8B2+ CSCs. b Determination by immunostaining of protein expression profile of GATA4, MEF2C and NKX 2.5 (b1) and troponin T, a-actinin and b-MHC (b2) after in vitro differentiation of W8B2+ CSCs. Nuclei are represented in blue with TOPRO staining. Scale bar 100 μm. c Transcript coding for cardiac contractile structures analyzed by TLDA in differentiated vs undifferentiated W8B2+ CSCs. d Transcript coding for natriuretic peptides [atrial (ANP) and brain (BNP)] analyzed by TLDA in differentiated vs undifferentiated W8B2+ CSCs. The graphs illustrate the gene expression variations (expressed in log 2) of the differentiated cells versus the undifferentiated cells. Values between 1 and − 1 (horizontal red lines) indicate that gene expression does not vary (this corresponds to variations of + 2 times and − 2 times compared to undifferentiated cells). Each bar represents the mean value ± S.E.M. (standard error of the mean). (N = 4 patients)

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  Fig. 2. Evolution of spontaneous calcium activity parameters during W8B2+ CSC differentiation. a Types of spontaneous calcium activity recorded at the beginning of the differentiation process [day 2 after differentiation (1)] and at the end of the differentiation process [day 28 after differentiation (2)]. The graphs illustrate variations in [Ca2+]i represented by ΔF/F0 ratio, where F0 represents the basal fluorescence emitted by the GCaMP, and ΔF the difference between basal and actual fluorescence (F) at a given time point. b Bar graphs (1), (2), (3), (4) represent the evolution of the frequency, the peak amplitude, the duration and TTP (time to peak) of calcium oscillations, respectively, recorded during the 4 weeks of W8B2+ CSC differentiation. (N = 6 patients, n: represents the number of cells analyzed, AU: arbitrary unit, ns: not significant). *p < 0.05, **p < 0.01, ***p < 0.001 vs. week 1; one-way ANOVA followed by a Newman-Keuls test

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  Fig. 3. Effect of W8B2+ CSC in vitro differentiation on calcium channels, ion exchangers and contributors to calcium homeostasis. Transcript coding for players in the process of calcium homeostasis analyzed by TLDA in differentiated vs undifferentiated W8B2+ CSCs. Graphs illustrate gene expression variations (in log 2) of differentiated cells versus undifferentiated cells from N = 4 patients. Horizontal red lines correspond to 1 and − 1 values

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  Fig. 4. Role of L-type calcium channel and NCX exchanger activity in W8B2+ CSCs after 28 days of in vitro differentiation. a Effect of 5 µM nifedipine (L-type CaV inhibitor) and c 3 µM SEA-0400 (NCX exchanger inhibitor) on spontaneous calcium activity recorded in differentiated CSCs. Graphs illustrate variations in [Ca2+]i as represented by changes in the ΔF/F0 ratio. ΔF = F − F0 (F: fluorescence emitted by CaMP at a given time; F0: basal fluorescence emitted by GCaMP). Bar graphs (1), (2), (3) represent the evolution of the peak amplitude, the duration and frequency of calcium oscillations, respectively, recorded from differentiated W8B2+ CSCs after treatment with nifedipine (5 µM) (b) and SEA (3 µM) (d). (N = 3 patients, n: represents the number of events (oscillations) recorded, AU: arbitrary unit, ns: not significant). *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; t-test paired followed by a Wilcoxon test

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  Fig. 5. Role of SERCA and IP3 receptors on spontaneous calcium activity recorded in W8B2+ CSCs after 28 days of in vitro differentiation. a Effect of 5 µM xestospongin C (IP3 receptor inhibitor) and c 1 µM thapsigargin (SERCA inhibitor) on spontaneous calcium activity recorded in differentiated CSCs. Graphs illustrate variations in [Ca2+]i represented by changes in the ΔF/F0 ratio. ΔF = F − F0 (F: fluorescence emitted by GCaMP at a given time; F0: basal fluorescence emitted by GCaMP). Bar graphs (1), (2), (3) represent the evolution of the peak amplitude, the duration and the frequency of calcium oscillations, respectively, recorded from differentiated W8B2+ CSCs after treatment with xestospongin C (5 µM) (b) and thapsigargin (1 µM) (d). (N = 3 patients, n: represents the number of events (oscillations) recorded, AU: arbitrary unit, ns: not significant). *p < 0.05, **p < 0.01 vs. control; t-test paired followed by Wilcoxon test