Supplementary MaterialsSupplementary Information 41467_2019_13868_MOESM1_ESM. are given as a Source Data file. Abstract The functions of the heart are achieved through coordination of different cardiac cell subtypes (e.g., ventricular, atrial, conduction-tissue cardiomyocytes). Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer unique opportunities for cardiac research. Traditional studies using these cells focused on single-cells and utilized mixed cell populations. Our goal was to develop clinically-relevant engineered heart tissues (EHTs) comprised of chamber-specific hPSC-CMs. Here we show that such EHTs can be generated by directing hPSCs to differentiate into ventricular or atrial cardiomyocytes, and then embedding these cardiomyocytes in a collagen-hydrogel to create chamber-specific, ring-shaped, EHTs. The chamber-specific EHTs display distinct atrial versus ventricular phenotypes as revealed by immunostaining, gene-expression, optical assessment of action-potentials and conduction velocity, pharmacology, and mechanical force measurements. We also establish an atrial EHT-based arrhythmia model and confirm its usefulness by applying relevant pharmacological interventions. Thus, our chamber-specific EHT models can be used for cardiac disease modeling, pathophysiological studies and drug testing. test is used for comparison. The HES3-NKX2C5egfp/w reporter hESC line was used to monitor cardiomyocyte differentiation. Flow-cytometry analysis for eGFP (identifying NKX2C5-expressing cells) and the cardiac-specific marker cardiac troponin T (cTnT) on d20 confirmed the efficiency of both ventricular and atrial differentiation protocols, resulting in 88??1% (test, Fig.?1e). The AP maximal upstroke velocity was also steeper in the ventricular cells (11.8??1.7?mV/ms, test. c, d Co-immunostaining of 30d atrial and ventricular EHTs for cardiac troponin I (cTnI) and either the ventricular-specific marker MLC2v (c) or the atrial marker sarcolipin (SLN) (d). Nuclei were stained with DAPI. Scale bars: 20?m. All eight additional immunostaining images were similar to the representative image shown. e Western blot densitometry of Cx40 and Cx43 protein expression in the atrial and ventricular EHTs (were all expressed in both the atrial and ventricular EHTs (Fig.?2b, top panel). The expression levels of and (responsible for the inward rectifier IK1 current), which can be used as surrogates for the degree of cardiomyocyte maturity, had been equivalent between your ventricular and atrial EHTs, suggesting a equivalent maturation level. However, expressions of and had been low in both chamber-specific EHTs in comparison with their amounts in charge adult individual heart-derived atrial and ventricular tissue (Fig.?2b, best -panel). We following compared the appearance degrees of genes, known through the books1,8,40C43 to become differentially portrayed either in atrial (Fig.?2b, middle sections) or ventricular cells (lower -panel). These research revealed significant differences in the expression degrees of such chamber-specific genes between your ventricular and atrial EHTs. Hence, the atrial-specific genes (encoding for the distance junction proteins connexin 40), (in charge of the appearance from the ultra-rapid potassium current (IKur) in atrial cells), (in charge of the appearance from the IKACh potassium current in atrial cells), (encoding for atrial natriuretic aspect), (encoding for the myosin regulatory light string 2, atrial isoform), and (encoding for the COUP transcription aspect 2 recognized to play a significant role in identifying atrial identification) had been all expressed considerably higher in the atrial EHTs in comparison using the ventricular EHTs. These genes had been also expressed considerably higher order Crizotinib in the control individual adult atrial tissues as compared using the control individual adult ventricular tissues. As opposed to the atrial-specific gene appearance, the appearance degrees of the mainly ventricular-specific markers (encoding for the myosin regulatory light string 2, ventricular isoform), (encoding for the beta-myosin large string), and (a cardiac-specific transcription aspect) had been considerably higher in the ventricular EHTs as compared with the atrial EHTs. This correlated with their different expression levels in the control adult human heart-derived atrial and ventricular samples (Fig.?2b, lower panel). The observed chamber-specific differences between the atrial and ventricular EHTs at mRNA levels were also noted at the protein levels. Thus, co-immunostaining studies targeting the general cardiac sarcomeric protein cTnI and the ventricular-specific marker MLC-2v revealed that the expression of the latter was significantly enriched in the order Crizotinib ventricular-EHTs as compared with atrial tissues (Fig.?2c). Morphometric analysis of the stained specimens, quantifying the relative Slc16a3 expression of MLC-2v (calculated as the percentage out the total EHT cellular volume that expresses cTnI that also expresses MLC-2v), revealed that it was significantly higher in the ventricular EHTs (88.8??2.7%, gene (encoding for Cx40) was significantly higher in the atrial EHTs (Fig.?2b, middle panel). In contrast, test, Fig.?3b). Open in a order Crizotinib separate windows Fig. 3 Electrophysiological characterization of the chamber-specific EHTs.a Confocal line-scan images showing examples of optical AP recordings of atrial and ventricular cells within the chamber-specific EHTs. b Comparison of mean APD30, APD50, and APD90 prices in the ventricular and atrial EHTs (check. c,.