Generation of cardiomyocytes from human-induced pluripotent stem cells resembling atrial cells with ability to respond to adrenoceptor agonists

Figures

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  Figure 1.. Gremlin2/RA-mediated differentiation of iPSCs into atrial cardiomyocytes (CMs). (a) Schematic of the atrial-specific differentiation protocols. Human iPSCs were dissociated into single CMs for the differentiation experiments. mTeSR1 is a defined tissue culture medium from Stemcell Technologies. Cardiac differentiation medium A (CDM A) including CHIR99021 and cardiac differentiation medium B (CDM B) were applied to both groups. From day 4, all CMs were maintained in CMM. In the Experimental group, Gremlin 2 (1 mg ml−1) was added at day 4, and of RA (1.0 mM) was added with Gremlin 2 still present at day 6, while the control group of cells was differentiated without the addition of Gremlin 2 and RA. (b) Functional analysis was performed after day 20 when both cell populations of hiPSC-derived CMs showed the expression of the CM-specific cytoskeleton markers α-actinin 2 (ACTN2) and TNNT2 (cardiac troponin C2), while the expression of the atrial CM-specific markers COUP-TFII and NPPA was restricted to the experimental group. (c) Immunofluorescence labelling of the cytoskeleton markers α-actinin 2 and F-actin for cells in the experimental group demonstrating an elongated morphology…

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  Figure 2.. Single-cell transcriptomic profile. (a) Plot showing the percentage of cardiomyocytes (CMs) expressing atrial-specific markers HEY1, MYL7, HOXA3 and SLN in the experimental and control groups. (b) Differential gene expression analysis showed many upregulated atrial-specific gene in experimental group with selected markers indicated. (c) Gene ontology (GO) plot showing the top 10 most enriched GO terms in the genes differentially expressed between the control and experimental group. Coloured by Bonferroni–Hochberg-adjusted p-value. Gene count along the x-axis marks the number of genes enriched within each GO term. (d) Heatmap plot showing the expression of cardiogenic and chamber-specific markers. Cells are coloured by relative expression. The annotation on the left indicates the specific fold change in expression from the control group to the experimental group. (e) (i) Heatmap plot showing the expression of genes coding for potassium handling proteins. Cells are coloured by relative expression. The annotation on the left indicates the specific fold change in expression from the control group to the experimental group. The associated currents for each channel subunit are…

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  Figure 3.. Electrophysiological characterization. (a) AP traces recorded from quiescent (i,ii) and spontaneous beating (iii,iv) cardiomyocytes (CMs) in the experimental group with an atrial-type morphology. (b) Mean values shown as bar graphs for (i) resting membrane potential (RP), (ii) APA and (iii) APD at 90% repolarization (APD90) and 50% repolarization (APD50), measured from seven cells prepared using the experimental protocol. (c) Records from cells prepared using the control protocol and having a ventricular phenotype showing (i) a representative AP trace, (ii) mean APD90 and APD50, (iii) APA and (iv) RP measured from five control hiPSC-derived CMs. Data are presented as mean ± s.e.m. (d) Currents induced by exposure to acetylcholine (ACh; 1 µM) under whole-cell voltage clamp conditions for voltage steps in the range −110 to +20 mV recorded from an example cell in the experimental group with an atrial morphology are shown as red traces and labelled as IKACh-AM. Corresponding traces from a control cell with a ventricular phenotype showing little or no current induced by ACh are shown in green and labelled as IKACh-VM. The I–V curve showing a plot of current density against membrane…

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  Figure 4.. Changes in Ca2+ transient (CaT) amplitude in response to α- and β-adrenoceptor agonists in experimental group iPSC-CM cultures treated with Gremlin 2/RA. (a) CaTs (measured using Fluo-4 as the Ca2+ probe) in one representative experiment before and after 10 min of exposure to the α-adrenoceptor agonist, PE (10 µM). In this and following panels, raw traces are shown in (i), averaged traces are shown in (ii), and graphs representing the mean increase in CaT amplitude are shown in (iii). Red traces in (i) and (ii) are in the presence of drugs while black traces are in their absence. A bar graph showing the mean increase in CaT in response to PE is shown in (iii). (b) CaTs in a representative experiment before and after 10 min of exposure to the β-adrenoceptor agonist isoproterenol (ISO; 100 nM). A bar graph showing the mean increase in CaT in response to isoproterenol is shown in (iii). (c) CaTs in response to PE (10 µM) in the presence of the α-adrenergic antagonist (1 µM prazosin). CaTs after 10 min exposure to PE were abolished by pretreatment with prazosin. Graph showing the mean changes in CaT amplitude in response to PE in the absence and presence of prazosin is shown in…