Optimized bioreactor differentiation protocol
We modified bioreactor and suspension cardiac differentiation protocols4,5 with goals of improving yield and reproducibility, increasing applicability across diverse hiPSC lines, reducing cost, and enabling cryopreservation and recovery of resulting hiPSC-CMs. Our optimized workflow (Fig. 1a and Supplementary Fig. 1a) built on previously described embryoid body suspension culture protocols4,5 by incorporating the following features: (1) use of quality-controlled master cell banks (MCBs) to ensure consistency of input hiPSCs; (2) use of a stirred bioreactor that continuously monitors and adjusts temperatures, O2, CO2, and pH; (3) use of small molecules rather than growth factors, which are more expensive and vulnerable to lot-to-lot variation, to guide differentiation; (4) optimization of the time point to initiate differentiation by Wnt activation; (5) optimization of the duration of Wnt activation and the timing of Wnt inhibition; and (6) incorporation of controlled freeze and thaw protocols.
a Schematic of the optimized bioreactor cardiac differentiation protocol. b, c Characteristics of successful bioreactor differentiations. Runs were categorized as failed when they yielded <90% TNNT2+ hiPSC-CMs. Runs were categorized as failed when they yielded <90% TNNT2+ hiPSC-CMs. Cultures with low frequency of SSEA4 by flow cytometry (b; n = 31 differentiations) or out of range mean EB diameter (c; n = 24 differentiations) had higher failure likelihood. d, e hiPSC-CM yield (d) and purity (e) at dd15 (bioreactor, n = 25 differentiations; monolayer, n = 8 differentiations). Percentage of cells positive for cardiomyocyte marker TNNT2 was measured by flow cytometry. f Spontaneous beating frequency of bCMs and mCMs at dd15 (n=number of differentiations; number of EBs or wells: bCM (n = 3; 71); mCM (n = 3; 46). g Timeline of bioreactor and monolayer cardiac differentiation monitored using TNNI1-GFP hiPSCs (bar: 200 µm). h–p. RT-qPCR analysis of marker gene expression during bioreactor and monolayer cardiac differentiation. Marker genes were: ACTN2, cardiomyocytes; VIM, non-cardiomyocytes; MYH7, MYL2 and MYL3, ventricular cardiomyocytes; MYL4 and MYL7, atrial cardiomyocytes; HCN4, developmentally repressed pacemaker channel. Expression is relative to dd5 bCM (h; n=number of differentiations: bCMs, n = 3; mCMs, n = 3) or dd15 bCM (i–p: bCMs, n = 3–4; mCMs, n = 3). Data are expressed as mean ± SEM. Points represent biological replicates for each independent differentiation, except for i‘-i“ in which points represent technical replicates from separate batches. d–f, h, i, j–p: two-tailed Welch’s unpaired t test. i‘-i“: one-way ANOVA with Tukey´s post-test. MCB, Master cell bank; hiPSC, human induced pluripotent stem cells; CM, cardiomyocyte; BF, bright field.
High quality input hiPSCs are critical for successful and consistent differentiation25. Towards that end, we implemented procedures to establish MCBs of quality-controlled hiPSCs (see Methods), including karyotyping (Supplementary Fig. 1b) and mycoplasma testing. To monitor undifferentiated status of hiPSCs input into the differentiation protocol, we measured pluripotency marker SSEA4 by FACS. High differentiation efficiencies (>90% expressing cardiomyocyte marker cardiac troponin T [TNNT2]) were correlated with high SSEA4 ( > 70%) values, and low SSEA4 ( < 70%) predetermined failed differentiation (<90% TNNT2+; Fig. 1b; Supplementary Fig. 1c).
In suspension culture, hiPSCs spontaneously aggregated to form embryoid bodies (EBs). We initiated mesoderm differentiation by addition of Wnt activator CHIR99021 (CHIR), as in monolayer differentiation protocols2. We defined the optimal time of CHIR addition based on EB diameter: EBs smaller than 100 μm fell apart upon CHIR incubation, and EBs bigger than 300 μm differentiated less efficiently (<90% TNNT2+; Fig. 1c; Supplementary Fig. S1c–e), likely due to inherent diffusion limits of larger EBs4,26. Therefore our protocol targets CHIR addition when EB diameter reaches 100 µm, which typically occurs at 24 hours.
We found that CHIR for 24 h followed by a gap of 24 h and then IWR-1 (IWR) for 48 h yielded optimal cardiac differentiation (Fig. 1a and Supplementary Fig. S1a). In 25 differentiations of 14 different hiPSC lines (6 lines from different donors and 8 gene-edited lines) treated with 7 μM CHIR and 5 μM IWR at these time points, we obtained on average ~1.21 million cells per mL (Fig. 1d) with >90% TNNT2+ cells (~2.4 hiPSC-CMs/input hiPSC; Fig. 1e). High percentages of TNNT2+ cells were further confirmed by analysis of cardiac markers TNNT2 and ACTN2 in cryosectioned bCMs at dd15 (Supplementary Fig. 1f; Supplementary Movie 1).
For functional comparison to bCMs, we used healthy control hiPSCs (WTC-Cas9; referred to as “control hiPSC-CMs”) and differentiated them in parallel in adherent monolayers using the same protocol, except for a 48 h incubation period with CHIR instead of 24 h (Supplementary Fig. 1a’). Incubation with CHIR for 24 h led to failed monolayer differentiation. We did not apply hiPSC-CM enrichment methods27 at the completion of either bCM or mCM differentiation protocols. Compared to bCMs, mCMs showed higher spontaneous beating frequency (Fig. 1f; Supplementary Movies 2 and 3), suggestive of lower maturity. Moreover, we obtained lower mCM yields (Fig. 1d) and higher intra- (Supplementary Movie 4) and inter-batch variability in cardiomyocyte purity (%TNNT2+ cells; Fig. 1e).
We first observed contraction in bCMs at differentiation day 5 (dd5; Supplementary Movie 5), versus dd7 in mCMs and previously reported suspension culture hiPSC-CM differentiations1,4,5,6 (Supplementary Table 1). To validate this observation, we differentiated hiPSCs in which GFP is fused to endogenous sarcomere protein TNNI128 in the bioreactor and visualized onset of GFP expression (Fig. 1g). We first observed GFP in bCMs on dd5, in contracting areas at the edges of EBs. mCMs first showed GFP expression at day 6 (Fig. 1g), but these GFP+ areas did not visibly contract until dd 7. By RT-qPCR, ACTN2 was expressed in dd5 bCMs, whereas its level in mCMs was far lower (Fig. 1h). Additionally, bCMs had less inter-batch variation in ACTN2 levels compared to mCMs at day 15 (Fig. 1i–i”). Mesenchymal marker vimentin (VIM) was lower in dd15 bCMs, although the difference did not reach significance due to variation in mCMs (Fig. 1j). Furthermore, we found significantly higher expression of ventricular markers MYH7, MYL2 and MYL3 (Fig. 1k–m) and a fraction of 83.4% bCMs stained for ventricular myosin light chain (MLC2v) using flow cytometry (Supplementary Fig. 1g). Atrial markers MYL4 and MYL7 were higher or unchanged, respectively, in bCMs compared to mCMs (Fig. 1n, o). Finally, HCN4, encoding a developmentally repressed pacemaker channel, was higher in some mCM batches with high interbatch variability (Fig. 1p). Finally, TNNT2 protein levels were significantly higher in dd15 bCMs compared to mCMs (Supplementary Fig. 1h).
The ability to cryopreserve and recover viable cells that retain functional properties is critical to incorporate large scale differentiation protocols into efficient workflows. We optimized freeze and thaw protocols by adjusting cell dissociation protocols, cryo-protectant media, freezing conditions, and thawing procedures, and used a dedicated computer-controlled cell freezer (Supplementary Table 5; see Methods). Our freeze/thaw protocol yielded ~94% viable cells (trypan blue negative) after cryo-recovery, with plating efficiencies of ~51% for bCMs and ~46% for mCMs (Supplementary Fig. 1i, j). Functional testing of cryo-recovered hiPSC-CMs is discussed in subsequent sections.
Together, we established an integrated bioreactor-based workflow that yields consistently high numbers of highly pure hiPSC-CMs and developed methods for efficient cryo-preservation and cryo-recovery.
Cell composition of bCMs and mCMs
To gain a better understanding of generated cell types and differences between bCMs and mCMs, we performed single cell RNA sequencing (scRNAseq) of freshly dissociated hiPSC-CMs at dd15. Using microdroplet technology, we captured single cell transcriptomes of bCMs and mCMs, each in biological duplicate. From a total of 5173 bCM and 2513 mCM high quality cell transcriptomes, unsupervised clustering on the most variable genes revealed 11 cell clusters and excellent agreement between biological duplicates (Fig. 2a and Supplementary Fig. 2a, b; Supplementary Data 1). Based on expression of canonical marker genes, the clusters were identified as cardiomyocytes (Clusters: 0, 1, 2, 4, 5, 8, 9), skeletal muscle cells (Cluster: 6), smooth muscle cells (Cluster: 76), non-cardiomyocytes (Cluster: 3), and endothelial cells (Cluster: 10). The cardiomyocyte fraction was markedly higher in bioreactor (88%) compared to monolayer (51%) differentiation (Fig. 2a, b and Supplementary Fig. 2b). We used canonical marker genes to classify the cardiomyocyte clusters. Clusters 0 and 1, highly enriched for ventricular marker genes MYH7, MYL2, and MYL3 were enriched in bioreactor (67% of cardiomyocytes) compared to monolayer differentiation (44% of cardiomyocytes; Fig. 2c; Supplementary Fig. 2b). Conversely, cluster 2, containing cardiomyocytes with high expression of atrial marker genes MYL4 and MYL7, were less frequent in bioreactor (8% of cardiomyocytes) compared to monolayer differentiation (36% of cardiomyocytes). Assignment of ventricular and atrial types was further supported by calculating chamber type scores that aggregated the expression of multiple marker genes (Fig. 2d). Comparison of the genes differentially expressed between bCM and mCM cells within the principal cardiomyocyte clusters (0, 1, 2; Fig. 2e) showed that genes more highly expressed in bCMs were strongly enriched for electron transport chain, mitochondrial respiratory chain complexes, and muscle contraction (Fig. 2f, left). In contrast, genes more highly expressed in mCMs were highly enriched for glycolysis, extracellular matrix organization, and heart development (Fig. 2f, right). We validated that bCMs expressed higher levels of mitochondrial metabolism genes HADHA and ACADVL than mCMs (Supplementary Fig. 2c, d), consistent with an upregulation of these genes in a prior report on suspension culture19. Upregulation of these genes was previously associated with a maturation protocol based on the induction of PPARdelta in hiPSC-CMs, which improved functional output29.
a scRNA-seq UMAP clustering of mCM and bCM cultures showing 11 clusters, marker genes, assigned cell types, and distribution in bCM and mCM cultures. b Stacked bar graph showing cellular composition of mCM (right) and bCM (left) cultures. Clusters are divided into cardiomyocytes (top) and non-cardiomyocytes (bottom). Color coding is the same as in a. c Violin-plots showing the relative expression of a subset of cardiac and non-cardiac marker genes (y-axis) across all clusters for bCMs (grey) and mCMs (red). d Composite ventricular and atrial cardiomyocyte (vCM, aCM) scores derived from multiple marker genes. Clusters 0 and 1 and greater vCM score, and cluster 2 had greater aCM score. e Differentially expressed genes (DEGs) between cardiomyocytes in bCM and mCM cultures. Significances were calculated using Wilcoxon rank-sum test (implemented by the Seurat findmarkers function), with p values adjusted using the Benjamini-Hochberg procedure (for DEGs, FDR < 0.05). f Top biological process gene ontology (GO) terms for DEGs more highly expressed in bCMs (left) or mCMs (right).
The non-cardiomyocyte fraction was dramatically lower in bioreactor compared to ML differentiation (12% vs. 49%; Fig. 2b, b’). An endothelial cell population marked by PECAM1, CDH5 and HLX was uniquely found in bioreactor differentiation (Fig. 2c; Supplementary Fig. 2b). mCMs were highly enriched for non-cardiomyocyte (non-CM) cluster 3 (bCMs: 3% vs mCMs: 20%), which mainly expressed fibroblast marker genes (COL3A1, COL1A1, FN1). (Fig. 2a, Supplementary Fig. 2b, Supplementary Data 1).
Taken together, scRNAseq analysis of cellular composition indicated that bioreactor differentiation yields a higher fraction of hiPSC-CMs, and these hiPSC-CMs have more mature gene expression profiles and greater ventricular identity, consistent with the RT-qPCR analysis (Fig. 1h–p). Additionally, all non-CM clusters showed robust marker expression in bCMs compared to mCMs, indicating higher cellular non-CM specification in bCMs (Supplementary Fig. 2b).
Functional characterization of bCMs in 2D assays
For morphological and functional analysis, cryopreserved control hiPSC-CMs were thawed and plated in 96-well plates pre-coated with diluted Geltrex (see Methods). After 7 days, unpatterned hiPSC-CMs were fixed and morphologically analyzed. Staining for ACTN2 showed that many bCMs had elongated morphology, reminiscent of the rod shape of mature, de facto human cardiomyocytes (Fig. 3a). Quantification of bCM circularity confirmed their consistent elongated morphology across multiple batches. In comparison, mCMs displayed greater circularity and higher inter-batch morphological variation (Fig. 3b; Supplementary Fig. 3a). bCM cell area was not significantly different than mCMs (bCMs: 1752 ± 112.3 μm2; mCMs: 1905 ± 246.6 μm; Supplementary Fig. 3b), and inter-batch variation in cell area was significantly less than mCMs (Fig. 3c). Measured cell areas were comparable to other hiPSC-CM control lines cultured for 718 and 30 days15,17. Mature, de facto human cardiomyocytes are 80% mononucleated30, and multinucleation tends to increase with cardiac disease10,17,31. Accordingly, both bCMs and mCMs were predominantly mononuclear. However, mCMs had elevated frequency of binucleated or multinucleated cardiac cells (Fig. 3d). Moreover, a greater fraction of mCM nuclei exhibited H2AFx immunoreactivity, a marker of DNA double strand breaks (Fig. 3e).
a–e Morphological characteristics of unpatterned hiPSC-CMs. Cryo-recovered bCMs and mCMs were cultured for 7 days on unpatterned Geltrex-coated dishes and then stained for sarcomere Z-line marker ACTN2. a Representative images illustrate elongated shape of bCMs compared to mCMs. Bar, 20 µm. b, c Circularity and cell area were quantified from 3 independent differentiation batches of bCMs and mCMs. Grey numbers indicate cells analyzed. Two-way ANOVA with Tukey’s post-test. d Nucleation of unpatterned bCMs and mCMs after 7 days in culture. Chi-squared p < 0.0001. e Unpatterned bCMs and mCMs stained for H2AFX, a marker of DNA damage response. Chi-squared p < 0.0001. f–k Cryo-recovered cells were plated on extracellular matrix rectangular islands. After 1, 3, and 7 days, samples were fixed and stained. f Representative images. Bar, 20 µm. g Quantification of single cell islands covered by bCMs or mCMs. Grey numbers indicate 10x fields analyzed. One-way ANOVA with Šidák´s post-test. h Sarcomere organization of micropatterned bCMs and mCMs measured using sarcomere packing density after 7 days on micropatterned substrates. Grey numbers indicate cells analyzed. Two-way ANOVA with Šidák´s post-test. i Nucleation of micropatterned bCMs and mCMs after 7 days in culture. Chi-squared p < 0.0001. j, k DNA damage response in micropatterned bCMs and mCMs. j Representative images. Bar, 20 µm. k Quantification of H2AFX staining in bCMs (n = 30) and mCMs (n = 41). Chi-squared p < 0.0001. l–n Ca2+ transients were recorded under 1 Hz electrical pacing 7 days after plating. l Average, normalized Ca2+ transients. m Maximum upstroke velocity. n Ca2+ transient amplitude. Kruskal–Wallis with Dunn’s multiple comparison test. o Average, normalized action potentials. p Maximum upstroke velocity. q action potential duration at 90% recovery (APD90). Kruskal–Wallis with Dunn’s multiple comparison test. r Mitochondrial stress test. Cells were cultured in 96 well dishes designed to measure oxygen consumption rate (OCR). Arrows indicate addition of oligomycin, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and antimycin/rotenone (AR). Sample sizes indicate number of differentiations/number of cells (b, c, g, h) or replicates (l–r). Data are expressed as mean ± SEM. Source data are provided as a Source Data file.
Plating hiPSC-CMs onto contact printed rectangular extracellular matrix (ECM) islands promotes their structural maturation, including alignment of sarcomeres perpendicular to the cell’s long axis32. We compared cryo-recovered bCMs to mCMs after seeding onto rectangular ECM islands with the 7:1 aspect ratio of mature adult human cardiomyocytes. Cells were fixed on day 1, 3 and 7 after plating and stained for cardiac marker ACTN2 (Fig. 3f and Supplementary Fig. 3c). Cell attachment and sarcomere alignment were quantified by unbiased computational image analysis33. bCMs better survived plating on the micropatterned substrates, as demonstrated by their markedly higher coverage at all timepoints compared to mCMs (Fig. 3g; Supplementary Fig. 3d). Sarcomere packing density and orientation order parameter, two different measures of sarcomere alignment33, were considerably higher in bCMs than in mCMs at all investigated timepoints (Fig. 3h and Supplementary Fig. 3e, f). Compared to unpatterned cells (Fig. 3d), a greater proportion of patterned bCMs and mCMs were bi- and multinucleated (Fig. 3i). Staining for DNA double strand break marker H2AFx indicated strikingly higher levels in patterned mCMs compared to patterned bCMs (Fig. 3j, k) or to unpatterned cells (Fig. 3e). Additionally, unbiased analysis of nuclear morphology34 identified a significantly higher fraction of nuclei with abnormal morphology in mCMs for all investigated timepoints (Supplementary Fig. 4a–g). These data indicate that micropatterned substrates increased morphological maturation of bCMs, in line with previous findings on fresh mCMs35,36. bCMs were more amendable to single cell micropatterning than mCMs, with greater survival and sarcomere assembly, and reduced manifestations of genotoxic stress.
Next, we analyzed the physiological properties of bCMs compared to mCMs. We recorded Ca2+ transients by loading hiPSC-CMs with the Ca2+ sensitive dye Fluo-4 and electrically pacing them for 10 seconds. mCMs failed to follow electrical pacing (1 Hz; n = 4 batches; Supplementary Fig. 5a) and were therefore excluded from the analysis. In contrast, bCMs were reliably captured by the same pacing protocol. Ca2+ transients showed high inter-batch reproducibility and consistent responses to beta-adrenergic stimulation by isoproterenol (Iso) (Supplementary Fig. 5b–f). Next, we recorded bCM action potentials (APs) using Fluovolt, a membrane voltage sensitive dye, and pacing at 1 Hz. bCMs displayed typical ventricular AP morphology (Supplementary Fig. 5g), corroborating our RT-qPCR (Fig. 1o, p) and scRNAseq findings (Fig. 2). As expected, adrenergic stimulation with Iso shortened action potential duration (Supplementary Fig. 5h).
Although mCMs failed to follow electrical pacing 4 days after cryo-recovery, we found that they did after 7 days and therefore repeated the Ca2+ transient and action potential measurements at this time point. Lactate treatment has been established as a method to enrich hiPSC-CMs and enhance their metabolic maturation37. Therefore we compared bCMs to mCMs without (-L) and with lactate treatment (+L), each with freezing and cryo-recovery (“cryo”) or without (“fresh”). mCMs included in these studies were already ~80% TNNT2+ prior to lactate treatment, which was slightly increased by lactate (Supplementary Fig. 6a). However, lactate did enhance mCM functional properties (see below). Immunostaining showed that bCMs formed a confluent monolayer of cardiomyocytes, whereas mCMs with or without lactate treatment formed cardiomyocyte patches surrounded by mesenchymal cells (Supplementary Fig. 6b).
We compared Ca2+ transients between fresh or cryo bCMs, mCMs-L, and mCMs+L (Fig. 3l–n and Supplementary Fig. 6c–g). Cryo bCMs had Ca2+ transients with the highest upstroke and recovery velocities, longest duration, and greatest amplitude. In comparison, cryo mCMs-L or mCMs+L had lower upstroke and recovery velocities, duration, and amplitude. Comparison of fresh to cryo showed different effects on bCMs compared to mCMs: Ca2+ transients in cryo bCMs had higher upstroke velocity, decay time, duration and amplitude than fresh bCMs, whereas Ca2+ transients in cryo mCMs+L had lower decay time, and duration than fresh mCMs+L. Cryo and fresh mCMs were comparable. The mCMs+L result was in keeping with a prior report of unchanged Ca2+ transient amplitude after cryo-recovery in some hiPSCs differentiated in monolayer conditions with lactate treatment, and contrasts with our finding that cryo-recovery increased bCM Ca2+ transient amplitude.
We similarly compared action potentials between these groups (Fig. 3o–q and Supplementary Fig. 6h–l). Cryo bCMs had higher action potential upstroke velocity and recovery velocity compared to cryo mCMs±L. Action potential upstroke velocity was also higher in cryo compared to fresh bCMs. Cryo and fresh mCMs were comparable. Unlike Ca2+ transient parameters, action potential parameters tended to be less affected by cryo-recovery across bCMs, mCMs+L, and mCMs-L.
To assess mitochondrial function, we measured cellular oxygen consumption rate (OCR) during the sequential addition of mitochondrial inhibitors (mitochondrial stress test). Cryo-recovered and freshly plated bCMs had higher basal respiratory OCR, maximal respiratory OCR, and ATP production than mCMs-L (Fig. 3r and Supplementary Fig. 6m–o). These observations are consistent with the higher level of mitochondrial metabolism genes HADHA and ACADVL in bCMs (Supplementary Fig. 2d, e). Lactate treatment significantly increased mCM mitochondrial function to a level comparable to bCMs (Fig. 3r and Supplementary Fig. 6m–o). These data indicate that bCMs intrinsically had metabolic maturation comparable to mCMs after lactate treatment.
Taken together, these functional data indicate that cryo bCMs have robust physiological cardiomyocyte properties.
Functional characterization of bCMs in EHTs
3D culture of hiPSC-CMs in fibrinogen gels subjected to anisotropic stress promotes cardiomyocyte maturation and sarcomere organization5,15. These engineered heart tissues (EHTs) also facilitate the measurement of cardiomyocyte force development and relaxation. We assembled EHTs using cryopreserved control bCMs (Supplementary Movie 6) and mCMs without lactate treatment (Supplementary Movie 7; Fig. 4a). From days 5 to 32 after EHT casting, we recorded EHTs during spontaneous beating. bCM and mCM EHTs had similar spontaneous beating frequencies (Fig. 4b). bCM EHTs generated greater force than mCM EHTs at all investigated timepoints (Fig. 4c) and considerably exceeded previously reported values using the same EHT constructs. In contrast, force generated by mCM EHTs was comparable to prior values5,15. Compared to mCM EHTs, bCM EHTs converged on similar contraction kinetics, as measured by the 50% contraction time (C50, Fig. 4d), and greater relaxation rates, as measured by the 50% and 90% relaxation time (R90, Fig. 4e and R50, Supplementary Fig. 7a).
a Representative images of EHTs after 29 days in culture (Scale bar, 1 mm). b–e Spontaneously beating EHTs assembled from cryo-recovered bCMs or mCMs were recorded in culture medium at 37 °C 5 to 32 days after EHT fabrication. b Spontaneous beating frequency. c EHT force. d Time to 50% contraction (C50). e Time to 90% relaxation (R90). Two-way ANOVA with Šidák´s post-test at each timepoint. f–h Analysis of EHTs in Tyrode solution without pacing (0 Hz) or with 1–3 Hz pacing. f EHT beat frequency in response to pacing. Only EHTs captured by pacing are shown. The percent of EHTs captured at each pacing rate is indicated. Two-way ANOVA with Šidák´s post-test. g Paced EHT force. h Paced EHT R90. i Histological characterization of bCM and mCM EHTs. After 34 days, EHT cryosections were stained for sarcomere Z-line protein ACTN2 and cardiac troponin T (TNNT2). Representative cryosections showed higher cellularity and greater sarcomere content and organization in bCM EHTs. j Sarcomere length. Quantification from 66 (bCM) or 59 (mCM) regions of interest in 4 (bCM) or 5 (mCM) EHTs from two independent differentiations. EHTs fixed at days 32, 34, and 39 were investigated and pooled for this analysis. Number of sarcomere intervals measured are indicated in the graph. Two-tailed Welch’s unpaired t-test. Data are expressed as mean ± SEM. Source data are provided as a Source Data file.
We recorded EHTs during optogenetic pacing at 1–3 Hz (Supplementary Movie 8). At any given pacing frequency, bCM EHTs were captured more frequently than mCM EHTs (Supplementary Fig. 7b). Under all pacing conditions, force was higher in bCMs EHTs compared to mCM EHTs (Supplementary Fig. 7c), and C50 did not significantly differ between these groups (Supplementary Fig. 7d). R90 was significantly lower in bCM EHTs at baseline and 2 Hz pacing; at 1 Hz and 3 Hz, bCM EHTs likewise had lower R90 values, although statistical significance was difficult to evaluate due to the low number of mCM EHTs captured at these rates (Supplementary Fig. 7e). An independent set of EHTs was similarly analyzed in Tyrode solution, with similar findings (Fig. 4f–h; Supplementary Fig. 7f). Under these conditions, both bCM and mCM EHTs generated greater force compared to culture medium, most likely due to higher calcium concentrations in Tyrode solution (bCMs: 0.374 mN vs 0.537 mN, mCMs: 0.104 mN vs 0.177 mN). Furthermore, in Tyrode solution a subset of bCM EHTs was successfully captured at 4 Hz pacing (Supplementary Fig. 7g–j; Supplementary Movie 9), notably faster than the previously reported maximal rates achieved for EHTs38,39,40 without physical conditioning41,42. Given greater force generation by bCMs in EHTs, we stained EHT cross-sections for cardiac sarcomere proteins ACTN2 and TNNT2. Consistent with the physiological data, we found greater sarcomere density in bCM compared to mCM EHTs (Fig. 4i). Furthermore, sarcomere length was higher in bCMs compared to mCMs EHTs (1.75 vs 1.64 µm; Fig. 4j), indicating greater maturity of bCMs sarcomeres.
Since lactate treatment enhanced physiological properties and metabolic maturation of mCMs, we also generated mCM+L EHTs and compared them to bCM and mCM-L EHTs. We made serial measurements at days 5, 20, 22, and 32. EHT spontaneous beating frequencies were higher in mCMs+L than mCMs-L or bCMs (Supplementary Fig. 8a). mCM+L and mCM-L EHTs generated comparable force, which was much lower than bCM EHTs after day 20 (Supplementary Fig. 8b). mCM+L EHTs showed shorter contraction time than bCM EHTs, likely reflecting markedly elevated bCM developed force, and comparable relaxation kinetics (Supplementary Fig. 8c, d).
Overall, greater function of bCMs EHTs was observed when compared to mCM EHTs with and without lactate treatment.
Suspension culture differentiation in spinner flasks
Compared to bioreactors, magnetically stirred suspension spinner flasks are more widely available and offer greater culture volume flexibility. However, spinner flasks are not capable of real time monitoring and adjustment of pH, oxygen, temperature, and CO2. We found that the suspension culture protocol performed well in spinner flasks with minor adaptations (Fig. 5a). hiPSCs subjected to our optimized workflow showed high SSEA4 values (>70%, Fig. 5b). In contrast to the bioreactor, optimal EB size ranged from 80–100 μm for CHIR addition (Fig. 5c). In six differentiations of two different hiPSC lines, differentiation with 7 μM CHIR and 5 μM IWR yielded on average 1.8 million cells/ml at dd15, although yield varied more for spinner flask-differentiated cardiomyocytes (sCMs) than bCMs (Fig. 5d). Cardiomyocyte purity of sCMs (94 ± 1.8% TNNT2+ cells; 3.58 hiPSC-CMs/input hiPSC; Fig. 5e) was comparable to bCMs. However, spontaneous beating frequency was higher for sCMs compared to bCMs (Fig. 5f).
a Schematic of the optimized protocol applied to magnetically stirred spinner flasks. Abbreviations as in Fig. 1. b, c hiPSC-CMs with low frequency of pluripotency marker SSEA4 by flow cytometry (b) or out of range mean EB diameter (c) had higher likelihood of failure, defined as <90% TNNT2+ cells (n = 8 differentiations). d, e hiPSC-CM yield (d) and purity (e) at bCM or sCM dd15 (bioreactor, n = 25 differentiations; sCMs, n = 6 differentiations). 2.5× and 3.8× scaling indicate 250 or 380 ml cultures, respectively. TNNT2+ cell percentage was measured by flow cytometry. f Spontaneous beating frequency of bCMs and sCMs at day 15 (n = number of differentiations; number of EBs: bCMs (n = 3/71); sCMs (n = 3/57). g–n RT-qPCR analysis of marker gene expression during bioreactor and spinner flask cardiac differentiation. Values are expressed as fold-change compared to bCMs day 5 or 15. n=number of differentiations: sCMs (n = 3). Points represent biological replicates for each independent differentiation, except for h‘ in which points represent technical replicates. o–q sCM Ca2+ transients properties under 1 Hz electrical pacing 7 days after cryo-recovery. o Average, normalized Ca2+ transients. p Maximum upstroke velocity. q Ca2+ transient amplitude. Kruskal–Wallis with Dunn’s multiple comparison test. n=number of differentiations/ number of wells for sCMs: cryo: n = 2/58; fresh: n = 3/40). r–t sCM action potential (AP) properties, optically recorded under 1 Hz electrical pacing 7 days after cryo-recovery. r Average, normalized APs. s Maximum upstroke velocity. t AP duration at 90% recovery (APD90). Kruskal–Wallis with Dunn’s multiple comparison test. u Representative image of an sCM EHT. Bar, 1 mm. Spontaneously beating EHTs were serially analyzed in culture medium. v Spontaneous beating frequency. w Force generated by EHTs. x Time for EHT 50% contraction (C50). y Time for EHT 90% relaxation (R90). Data are expressed as mean ± SEM. d–f two-tailed Welch’s unpaired t test. g–n one-way ANOVA with Dunnett’s post-test. bCM and mCM data from b–y were replotted from Figs. 1b–f, h, i, k–p, 3l–q and 4b–e to facilitate comparisons. Source data are provided as a Source Data file.
Taking advantage of the culture volume flexibility of spinner flasks, we explored the ability of the optimized suspension culture protocol to scale to larger volumes. Scaling from our standard culture volume of 100 ml by 2.5 (125 million hiPSC in 250 mL volume) or 3.8 fold (190 million hiPSC in 380 mL volume) resulted in greater than linear increases in yield: 250 ml yielded 600 million (2.4 million/ml; 2.9 sCMs/input hiPSCc) and 380 ml yielded 1320 million (3.4million/ml; 6.9 sCMs/input hiPSC) (Fig. 5d, e).
As with bioreactor differentiation, spontaneous beating was observed at dd5. Likewise, ACTN2 expression was expressed at comparable levels between sCMs and bCMs at dd5 (Fig. 5g). At dd15, sCMs expressed greater ACTN2 but with greater inter-batch variability (Fig. 5h–h’). Mesenchymal marker VIM was significantly lower in dd15 bCMs (Supplementary Fig. 9a). Ventricular markers MYH7 and MYL2 (Fig. 5i, j) were decreased in sCMs when compared to bCMs, although the MYH7 difference was not significant due to sCM variability. Atrial markers MYL4 and MYL7 were significantly lower or unchanged, respectively, in sCMs (Fig. 5l, m). The pacemaker gene HCN4 was not significantly different between bCMs and sCMs (Fig. 5n).
These data indicate that spinner flasks are an economical alternative to bioreactors and enable flexible culture volume scaling.
Comparison of sCMs and bCMs functional properties
For functional analysis of sCMs, we repeated Ca2+ transient and APD measurements with fresh and cryo-recovered control sCMs after 7 days of culture. Cryo-recovered sCMs were highly viable (~94.6%; Supplementary Fig. 9b) and formed confluent CM monolayers (Supplementary Fig. 9c), comparable to bCMs for both conditions (Supplementary Fig. 6b). In sCMs, Ca2+ transients and APDs had high upstroke velocities and Ca2+ transient amplitudes (Fig. 5o–q, S9d-h and Fig. 5r–t, Supplementary Fig. 9i–m) that were more similar to bCMs than mCMs. Consistent with the bCM results, Ca2+ transient amplitude was greater in cryo compared to fresh sCMs (Fig. 5q). However, no changes were found between cryo and fresh sCMs in upstroke velocities (Fig. 5p). Notably, we found higher reproducibility between cryo and fresh sCMs when compared to bCMs.
To compare mitochondrial function of sCMs to bCMs, we performed mitochondrial stress tests. These assays showed no significant differences between sCMs and bCMs (Supplementary Fig. 9n–q).
Next we compared the performance of EHTs assembled from cryo sCMs and bCMs (Fig. 5u). During continuous culture for 32 days, sCM EHTs generated less force than bCM EHTs (sCM 0.158 mN vs bCM 0.374 mN) but more than mCM EHTs (0.104 mN Supplementary Fig. 9r). EHT spontaneous frequency became lower for sCMs than bCMs at day 32. Times for 50% contraction (C50) or 90% relaxation (R90) were comparable (Fig. 5v–y).
These data show that sCMs share many of the functional characteristics of bCMs, although some parameters, particularly EHT force, were superior for bCMs.
Generation of bioreactor-derived cardiac organoids (bCOs)
By minor adjustment of our suspension culture differentiation protocol, we observed the emergence of larger spheroids that resembled previously published self-organized cardioids in size and morphology20,21. We named these spheroids “bioreactor-derived cardiac organoids” (bCOs; Supplementary Movie 10). Approximately 10% of all EBs formed at day 0–2 developed into spherical bCOs under these adjusted culture conditions (Supplementary Fig. 10a). The remaining EBs formed aspherical structures or aggregated during prolonged culture and were not analyzed further (Supplementary Fig. 10a). Assessment of spherical bCOs cultured for 15 days (Fig. 6a) revealed an average diameter of 1.8 ± 0.8 mm (Fig. 6b), spontaneous beating (16.3 ± 4.8 beats per minute; Fig. 6c), and 2-fold higher ACTN2 mRNA expression when compared to dd15 mCMs (Fig. 6d). bCOs formed wall-lined cavities divided by septae, reminiscent of cardiac chambers (Fig. 6e; Supplementary Fig. 10b Supplementary Movie 11). Most cells in bCO walls and septea expressed TNNT2 (Fig. 6f; Supplementary Fig. 10c). Closer analysis of the bCO walls showed sarcomere striation with anisotropic alignment (Supplementary Fig. 10d).
a Representative image of a bCO at dd15. b EB diameter of dd15 bCMs (40 EBs from three differentiations) and bCOs (31 bCOs from two differentiations). Two-tailed Welch’s unpaired t test. c Spontaneous beating frequency of dd15 bCOs (36 bCOs from three differentiations). d Expression of cardiac marker gene ACTN2 in dd15 mCMs, bCMs, and bCOs. ACTN2 transcript level was measured by RT-qPCR. One-way ANOVA with Sidak’s post-test. Points represent biological replicates for each independent differentiation. e Hematoxylin and eosin staining of a bCO section dd15. Boxed area is enlarged in e´. Scale bar in e and e´ = 200 µm. f bCO section stained with TNNT2 antibody, wheat germ agglutinin (WGA), and nuclei (Hoechst). Scale bar = 200 μm. g, h Cellular composition of dd15 bCOs determined by scRNAseq followed by UMAP clustering (g). Stacked bar graph (h) of the percentage of each cell state in bCOs. Cell states were grouped into cardiomyocytes (top) and non-CMs (bottom). Data are shown as mean ± SEM. Non-CMs, non-cardiomyocytes. Source data are provided as a Source Data file.
Morphological observation of bCOs indicated that most bCO spheroids initially remodel into biconcave discs (S10e,f) and then form “doughnut-shaped” bCOs by day 2 (S10g). This hole was then filled in, ultimately yielding a central chamber divided by a small number of septae that persisted throughout the culture duration of bCOs (Supplementary Fig. 10h), which could be extended to over 120 days.
We used scRNAseq to further interrogate the cellular composition and transcriptomic states of bCOs. 4032 high-quality single cell transcriptomes were integrated with bCM and mCM scRNAseq data. Similar to bCM cultures (Fig. 2b, c), 88% of cells were assigned to 6 cardiomyocyte clusters (0, 1, 2, 3, 6, 7), with the majority of cells in the ventricular cardiomyocyte clusters (0, 1), and 12% to non-CM clusters (4, 5, 8, 9, 10; Fig. 6g, h). Immunofluorescence staining of cryosectioned bCOs confirmed fibroblast (cluster 4: THY1+), endothelial (cluster 9: PECAM1+), and non-CM vimentin (VIM+) expressing cells (cluster 4, 5, 8, 9) at day 15 (Supplementary Fig. 10i). However, these non-CMs were very infrequent, in line with the scRNAseq results (Fig. 6h). Therefore, bCOs contain predominantly cardiomyocytes and a small fraction of non-CMs, comparable to previously published cardiac organoids generated in static culture without any further modification of basic cardiac differentiation media20,21,43. Taken together, bCOs represent the first self-organizing human heart organoid completely generated in suspension culture.
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- Source: https://www.nature.com/articles/s41467-024-50224-0