A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells

A scalable, robust, and integrated differentiation platform for large-scale production of human pluripotent stem cell-cardiomyocyte (hPSC-CM) in a stirred suspension bioreactor as a single-unit operation was developed. This platform could become a valuable tool for mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications including drug discovery and toxicity assays.

Abstract

Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-β and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays.

Significance

Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease.

Introduction

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), can be considered an unlimited source for the production of cardiomyocytes (CMs) owing to their self-renewal and directed differentiation capabilities [1, 2]. Several approaches, including embryoid body formation [3], coculture [4], cytokine-directed differentiation [5], and protein transduction [6], have been introduced to generate hPSC-derived cardiomyocytes since the first report in 2001. Despite significant methodological advances, the hPSC-based production platforms still contain critical disadvantages, including high costs and low efficiency and reproducibility. This has limited the application of hPSC-derived cardiomyocytes for clinical and industrial applications such as drug discovery and toxicity testing [7].

Most of the promising applications of hPSCs-CMs, including those that are closer to commercialization such as high-throughput drug screening and toxicity testing, followed by clinical applications, necessitate the production of massive numbers of pure and functional CMs [8]. Therefore, developing robust and affordable technologies for large-scale expansion of hPSCs and their integrated differentiation into cardiomyocytes in scalable culture systems would largely facilitate their commercial applications.

To date, a number of different protocols and technologies have been introduced for the production of CMs from hPSCs with the aim of optimizing different aspects of their bioprocessing, including culture conditions that favor the production of mature and fully functional CMs, and robust, integrated, and scalable differentiation and purification methodologies [9, 10].

To develop fully defined culture conditions for generation of hPSC-CMs, the exploration of novel and efficient small molecules (SMs) for chemically induced cardiac differentiation has recently emerged as a viable alternative to recombinant cytokines and unknown factors in serum [11–13]. Manipulation of the signaling pathways required for normal heart development has guided the development of efficient hPSC-CM differentiation protocols [9, 14–16].

In contrast, the current experimental methods for the differentiation of hPSCs often rely on the production of heterogeneous cellular aggregates termed “embryoid bodies” (EBs) or two-dimensional (2D) small-scale static cultures [17–19]. These protocols are typically not scalable and/or result in the generation of EBs with a highly heterogeneous size and low CM yield. In order to establish more robust bioprocesses, different approaches, such as microwell-mediated control, microprinting technologies [20–23], and microcarrier cultures [20], have been applied to address these issues. However, these techniques have limited differentiation potential, scalability (microwell-mediated control, hanging drop, and microprinting technologies), universality, and/or reproducibility because of the differentiation protocol itself or the low throughput of the methods used such as forced aggregation techniques before transferring cells to dynamic culture conditions. In addition, most of the protocols depend on using expensive and complex media or reagents (mTeSR1; StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com; or StemPro-34; Thermo Fisher Scientific, Waltham, MA, http://www.thermofisher.com) or microcarriers for expansion of hPSCs and their directed differentiation to cardiomyocytes [24, 25].

Recently, efforts have been made to develop scalable culture systems for the large-scale production of hPSCs-CMs under three-dimensional (3D) culture conditions. Kempf et al. tried to generate hPSC-CMs in a 100-ml stirred suspension bioreactor with batch and perfused modes using mTeSR medium (StemCell Technologies) for the hPSC expansion phase and Roswell Park Memorial Institute (RPMI)/B27 medium (Thermo Fisher Scientific) for the differentiation phase. However, batch cultures failed to generate contracting EBs [24], and perfused cultures resulted in contracting EBs with a heterogeneous size (350–600 µm in diameter), which could limit their future application for clinical and industrial use.

In a previous study, we developed a robust and cost-effective culture system for mass production of size-controlled hPSC aggregate cultures in stirred suspension bioreactors. Our protocols have paved the way for mass production of these unique cells under xeno-free conditions with superior scalability (review available in [26]). Subsequently, we have shown that this platform can be easily integrated for large-scale generation of hepatocyte-like cells that improved hepatic failure in an animal model after transplantation [27, 28].

In the present report, we have used this platform for the development of an integrated, simplified process for large-scale production of highly homogenous hPSC-CM aggregates in a cost-effective single-unit operation with high efficacy, reproducibility, and universality. This scalable production system can be easily integrated with an efficient scalable purification system, including culture-based methods, such as those using lactate-enriched medium for selective purification of CMs, to generate a highly pure population of cardiac cells for biomedical applications [29]. The development of such integrated platforms can be considered an important step toward the commercialization of hPSCs-CM-based technologies for clinical, pharmaceutical, tissue engineering, and in vitro organ development applications.

Materials and Methods

Generation of Fibroblast Cultures

The Sydney Children’s Hospital Network Human Research Ethics Committee provided ethics approval (reference no. HREC/10/CHW/44). Suitable participants were selected from the Kids Heart Research DNA Bank, and the participants’ parents or guardians provided consent. Skin biopsies were performed by cardiothoracic surgeons and sampled from the upper arm of the participants. Fibroblasts were cultured from the skin biopsy specimens to establish cell lines for each participant.

Culture of hPSCs as Aggregates

hESC lines (RH5, RH6, R725.1, R661.5, and R662.2) [30, 31] and hiPSC lines (VC645-9, VC913-5, VC618-3, and VC646-1), the latter generated at the Victor Chang Cardiac Research Institute, Australia, were used in the present study. hPSCs were expanded using a previously described suspension culture method [27, 32]. In brief, to initiate suspension cultures, 2 × 10⁵ viable cells per milliliter were transferred to nonadhesive bacterial plates (60 mm; catalog no. 628102; Greiner Bio-One, Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) in 5 ml of hESC-conditioned medium containing 100 ng/ml basic fibroblast growth factor (bFGF; Royan Institute for Stem Cell Biology and Technology, Academic Center for Education Culture and Research, Tehran, Iran). The cells were incubated under standard conditions (37°C, 5% CO₂ and saturated humidity). The medium was renewed daily. The hESC medium included Dulbecco’s modified Eagle’s medium/F12 medium (catalog no. 21331-020; Gibco, Grand Island, NY, http://www.lifetechnologies.com) supplemented with 20% Knockout Serum Replacement (catalog no. 10828-028; Gibco), 2 mM l-glutamine (catalog no. 25030-024; Gibco), 0.1 mM β-mercaptoethanol (catalog no. M7522; Sigma-Aldrich), 1% nonessential amino acids (catalog no. 11140-035; Gibco), 1% penicillin and streptomycin (catalog no. 15070-063; Gibco), 1% insulin-transferrin-selenite (catalog no. 41400-045; Gibco).

Conditioned medium was prepared by overnight incubation of hESC medium (without bFGF) with confluent human foreskin fibroblasts in 75-cm² T flasks, previously inactivated by treatment with mitomycin C (catalog no. M0503; Sigma-Aldrich).

Optimizing Differentiation Process for Cardiac Differentiation in Static Suspension Culture

The hPSC aggregates cultured in static suspension mode (i.e., nonadhesive bacterial plates) were directly differentiated into CMs for optimization trials. In order to induce CM differentiation from hPSCs in suspension culture systems, 3-, 5-, and 7-day-old hPSC size-controlled spheroids (average size, 90 ± 30, 175 ± 25, and 250 ± 32 µm, respectively) were treated for 24 hours in differentiation medium (RPMI 1640; catalog no. 31870-022; Gibco) supplemented with 2% B27 without retinoic acid (catalog no. 12587-010; Gibco) or without insulin (catalog no. A18956-01; Gibco), 2 mM l-glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, 1% penicillin and streptomycin, and different concentrations (3, 6, 9, 12, and 15 µM) of the SM CHIR99021 (CHIR; catalog no. 041-0004; Stemgent, Cambridge, MA, http://www.stemgent.com) as glycogen synthase kinase inhibitor and canonical WNT/β-catenin pathway activator. The spheroids were washed with Dulbecco's phosphate-buffered saline (DPBS) and then maintained in fresh differentiation medium without SMs for 1 day. After 1 day, the medium was exchanged for new differentiation medium that contained 5 µM IWP2 (catalog no. 3533; Tocris Bioscience, Bristol, U.K., http://www.tocris.com) as a WNT antagonist, 5 µM SB431542 (catalog no. S4317; Sigma-Aldrich) as an inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase receptors, and 5 µM purmorphamine (Pur; catalog no. 04-0009; Stemgent) as a sonic hedgehog (SHH) agonist. Spheroids were cultured for 2 days in this medium. After washing the spheroids with DPBS, fresh differentiation medium without SMs was added and refreshed every 2–3 days until the end of the differentiation process at day 30.

Integrated Differentiation of hPSCs Toward CMs in a Stirred Suspension Bioreactor

In order to develop an integrated differentiation process to generate human CMs from hPSCs in a scalable manner, we expanded hPSCs as a single cell inoculated suspension culture in 125-ml spinner flasks (Cellspin; Integra Biosciences AG, Zizers, Switzerland, http://www.integra-biosciences.com) with a 100-ml working volume at a 40 rpm agitation rate, as previously described [27]. In brief, to initiate the dynamic cultures, 2 × 10⁵ cells per milliliter were transferred to 100 ml of hPSC medium, conditioned on human foreskin fibroblast, and containing freshly added 100 ng/ml bFGF [27] in the stirred bioreactor. Cell aggregates were treated with 10 µM ρ-activated kinase inhibitor (ROCKi; catalog nos. Y-27632 and Y0503; Sigma-Aldrich) 1 hour before enzymatic dissociation with Accumax cell aggregate dissociation medium (catalog no. AM105; Innovative Cell Technologies, Inc., San Diego, CA, http://www.accutase.com). The bioreactor was agitated at 35 rpm (increased to 40 rpm after 24 hours), and medium refreshing began after 48 hours of culture with the same medium, without ROCKi. The seeded spinner flasks were incubated under standard conditions. After passages in dynamic suspension culture, the cells were cryopreserved as previously described [28] or induced for differentiation.

To induce CM differentiation from hPSCs in dynamic suspension culture, the time points, differentiation media, and SMs were exactly similar to the static system, with the exception of the addition of 0.1% polyvinyl alcohol (PVA; catalog no. 363073; Sigma-Aldrich) for the first 48 hours and 10 µM ROCKi for the first 24 hours. In addition, aggregates were washed twice with 25 ml of DPBS after mesodermal and cardiac induction steps by stopping agitation for 5–10 minutes and removing the spent media containing SM cocktails. After washing the cardiac-induced aggregates, fresh differentiation medium (100 ml) was added to the culture vessel without SMs and totally refreshed every 2–3 days until the end of the differentiation process at day 30.

Exploring the Optimum hPSC Aggregate Size for Integrated Cardiac Differentiation

In order to achieve size-controlled hPSC aggregates and, subsequently, hPSC-CMs with high differentiation efficacy, the RH5 cell line was cultured in dynamic suspension culture for 3, 5, and 7 days under optimal hydrodynamic culture conditions. The diameters of size-controlled aggregates generated in each culture were quantified using a phase-contrast inverted microscope (model no. CKX41; Olympus, Center Valley, PA, http://www.olympusamerica.com) and Olysia Bioreport software (Soft Imaging System, Olympus). Next, hPSC aggregates of a defined size were transferred to differentiation media under static conditions to explore the optimum size of dynamic culture systems for differentiation.

Quantification of Beating Spheroids

We determined the cardiogenic differentiation efficiency using an inverted cell culture microscope (model no. CKX41; Olympus) to count the number of beating hESC and hiPSC spheroids throughout the experiment. The numbers of beating spheroids were normalized to the total numbers of spheroids at each time point. All quantification experiments and analyses were performed using at least three independent biological replicates.

RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction

We collected the hPSC spheroids at various time points in the differentiation process in both static and dynamic systems. Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). RNA quality and concentration were analyzed using a Biochrom WPA spectrophotometer (Biochrom, Holliston, MA, http://www.biochrom.com). Possible genomic DNA contamination was removed by DNase I (Invitrogen) treatment for 15 minutes at room temperature after which 2 µg of total RNA was used for reverse transcription with an oligo (dT)₂₀ primer and Super Script III First-Strand Synthesis System (Invitrogen), according to the manufacturer’s instructions. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed with the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) in triplicate for each sample and each gene. The PCR conditions included denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute for 35 cycles, with a 72°C extension for 7 minutes at the end. The expression of genes of interest was normalized according to GAPDH expression. The relative gene expression levels were quantified using the 2^(−ΔΔCt) method. The primer sequences are listed in supplemental online Table 1.

In order to analyze quantitative RT-PCR data, we used R statistical language (R Foundation for Statistical Computing, Vienna, Austria, http://www.r-project.org) [33]. Principal component analysis (PCA) was performed on the scaled data. For the time-course analysis, the genes were clustered according to the expression values in different samples using a K-means algorithm. Visualization of the data was performed using the R packages ggplot2 [34] and heatmap.

Flow Cytometry

RH5 hESC spheroids were collected at different time points after differentiation initiation in static and dynamic systems, washed twice with PBS, incubated with 0.05% trypsin-EDTA (catalog no. 25300-054; Gibco) at 37°C for 4–5 minutes and then pipetted 5–12 times. After neutralizing trypsin activity by the addition of medium, the cell suspension was passed through a 40-µm filter mesh (catalog no. 352340; BD Falcon, BD Biosciences, San Diego, CA, http://www.bdbiosciences.com) to remove clumps and undissociated spheroids. After trypsinization and achievement of single-cell suspensions, the cells were washed twice in ice-cold staining buffer (PBS supplemented with 1% heat-inactivated fetal bovine serum [FBS], 0.1% sodium azide, and 2 mM EDTA) and fixed in high-grade 4% paraformaldehyde (PFA) for 15 minutes at 4°C. The cells were washed again with staining buffer, permeabilized with 0.2% (vol/vol) Triton X-100 in PBS for 20 minutes, and blocked for 15 minutes at 4°C with a combination of 10% heat-inactivated goat serum in staining buffer. The cells were incubated overnight at 4°C (or 30 minutes at 37°C) with the suitable primary antibodies (1:100) or appropriate isotype matched controls, and then washed three times with staining buffer, after which secondary antibodies (1:500) were added to the cells. After 45 minutes of incubation at 4°C, the cells were washed three times with staining buffer and analyzed using a flow cytometer (FACSCalibur; BD Biosciences) and flowing software, version 2.5.1 (BD Biosciences). For each analysis, 0.5–1 × 10⁶ cells were used per sample. All experiments were replicated at least three times. The primary and secondary antibodies used for flow cytometry are listed in supplemental online Table 2.

Immunostaining and Imaging

hPSC differentiated beating spheroids were collected at different time points after differentiation initiation in static and dynamic systems. The collected spheroids were washed twice with PBS and dissociated into single cells with 0.05% trypsin-EDTA (catalog no. 25300-054; Gibco) at 37°C for 4–5 minutes. Individualized cells were cultured on gelatin-coated chamber slides (catalog no. 177437; Nunc; Thermo Scientific) in RPMI/B27 medium. After 5 days, the attached cells were washed once with PBS, fixed with 4% (wt/vol) PFA at room temperature for 15 minutes, washed once with washing buffer (PBS/0.1% Tween 20), permeabilized with 0.2% Triton X-100 in PBS for 15 minutes, and blocked with 5% (vol/vol) goat serum for 1 hour. Primary antibodies diluted in blocking buffer (1:100) were added to the cells, followed by an overnight incubation at 4°C. After incubation, the cells were washed three times with washing buffer, each for 5 minutes. Secondary antibodies diluted in blocking buffer (1:500) were added to cells, after which they were incubated for 1 hour at room temperature. The cells were subsequently washed three times with washing buffer, then covered with a Vectashield mounting medium that contained 4′6-diamidino-2-phenylindole (Vector Laboratories, Inc., Burlingame, CA, http://www.vectorlabs.com). Imaging was performed using an upright confocal microscope (Zeiss LSM700, Zeiss, Jena, Germany).

For immunohistochemistry analysis, the beating RH5 hESC spheroids were collected, washed with PBS, fixed with 4% (wt/vol) PFA at room temperature for 15 minutes, and prepared for paraffin-embedded tissue blocks. Paraffin-embedded spheroids were cut into 6-µm sections using a microtome (Microm HM325; Thermo Scientific) and kept at room temperature until use. For staining, we dewaxed and hydrated the spheroid section slides, followed by heat-mediated antigen retrieval using a Dako target retrieval solution (catalog no. S2367; Dako, Glostrup, Denmark, http://www.dako.com). Permeabilization, blocking steps, and incubation with primary and secondary antibodies were performed as described for individualized cultured cells. The primary and secondary antibodies used for cultured cells and staining the spheroids are listed in supplemental online Table 2.

Microelectrode Array Recording

We characterized the functional properties of hESC spheroid-derived CMs by performing an extracellular recording of field potentials (FPs) using a microelectrode array (MEA) data acquisition system (Multi Channel Systems, Reutlingen, Germany). The MEA plates contained a matrix of 60 titanium nitride electrodes (30 µm) with an interelectrode distance of 200 μm. MEA plates were sterilized and hydrophilized with FBS for 30 minutes, washed with sterile water and coated with 0.1% gelatin for 1 hour. For this analysis, areas of plated beating spheroids were mechanically dissected and plated on the middle of a sterilized MEA plate in medium that contained 20% FBS. On the day of the experiment, coated MEAs were connected to a head stage amplifier. Extracellular potentials were sampled at 50 KHz, and all recordings were performed at 37°C. Recordings were performed for 100 seconds at baseline and at 5 minutes after drug application. FP signals were analyzed for FP duration (defined as the interval between the minimum FP and maximum FP), interspike intervals, and beating frequency. Data were analyzed using AxoScope software (Molecular Devices, Sunnyvale, CA, http://www.moleculardevices.com).

Patch Clamp Electrophysiology

Action potentials (APs) were recorded from spontaneously beating hESC- or hiPSC-derived CMs using the current clamp in the whole cell patch clamp configuration. Beating spheroids were dissociated into single cells using 0.05% trypsin-EDTA (catalog no. 25300-054; Gibco) at 37°C for 4–5 minutes. Single beating cardiomyocytes were plated onto glass coverslips coated with ECM Gel (catalog no. E1270; Sigma-Aldrich) and maintained at 37°C. Before experimentation, the coverslips were transferred to a recording chamber mounted on the stage of an Olympus inverted microscope (catalog no. CKX41; Olympus). The extracellular bath solution within the chamber contained 150 mM NaCl, 5.4 mM KCl, 15 mM HEPES, 15 mM d-glucose, 1 mM MgCl₂, and 1.8 mM CaCl₂; adjusted to pH7.4 with NaOH. Recording pipettes were pulled from borosilicate glass capillaries using a P-97 horizontal puller (Sutter Instrument, Novato, CA, http://www.sutter.com) and had a tip resistance of 3–6 MΩ. The pipette solution contained 150 mM KCl, 10 mM NaCl, 2 mM CaCl₂, 5 mM EGTA, 10 mM HEPES, and 5 mM MgATP; adjusted to pH 7.2 with KOH. Data were acquired using a multiclamp 700B amplifier (Axon Instruments/Molecular Devices Corp., Union City, CA, http://www.moleculardevices.com), a Digidata 1440 analog-to-digital board and pClamp 10 software (Axon Instruments), at a sampling frequency of 10 kHz and low-pass filtered at 2 kHz. Data analysis was performed using Clampfit 10 (Axon Instruments) and Prism 6 (GraphPad Software, Inc., San Diego, CA, http://www.graphpad.com) software.

Statistical Analysis

All quantifications were performed using at least three independent replicates. Data are presented as mean ± SD, in which data with symmetrical (normal) or nonsymmetrical distributions were analyzed using one-way analysis of variance, followed by the post hoc least significant difference or Dunnett's significant difference test. Values of p < .05 were considered statistically significant.

Results

Modulation of WNT, Transforming Growth Factor-β, and SHH Signaling Pathways to Enhance Cardiac Differentiation

In order to develop an efficient protocol for large-scale generation of CMs from hPSCs, we attempted to manipulate the most important cardiogenic signaling pathways by using SMs in adherent culture. First, we optimized chemically induced CM differentiation methods using recently reported SM cocktails [14, 15]. After several optimization trials using multiple different combinations of SMs and treatment durations, we determined that the most effective protocol for CM differentiation was exposure of hESCs to 12 μM CHIR for 1 day, followed by a 1-day rest period in the same medium without SMs. Subsequently, IWP2, SB431542, and Pur (5 μM each) were used for an additional 2 days. The SMs inhibited WNT/β-catenin and transforming growth factor-β (TGF-β) and activated the SHH signaling pathways. This method led to the appearance of beating clusters in the adherent cultures at days 7–10 after the onset of differentiation (data not shown).

In order to transform the optimized adherent culture protocol to scalable suspension culture conditions, we induced 7-day spheroids of hPSCs that had been generated in low-attachment dishes under static suspension culture conditions with the optimized protocol (Fig. 1). For differentiation, the cells were treated the same as for the adherent culture protocol.

Figure 1.

Experimental design and optimization of chemically induced hPSC differentiation to cardiomyocytes (CMs) in a static suspension system. We used the human embryonic stem cell line RH5 in this step. (A): Five-day spheroids formed in suspension culture (spinner flasks) were transferred to low-attachment dishes that contained differentiation medium. For mesoderm induction, hPSC spheroids were treated for 1 day with 12 µM CHIR. Then, CHIR was removed, and the cells were cultured for 1 more day in differentiation media without small molecules. At the end of this stage, precardiac mesoderm had formed. To obtain cardiac progenitors, we treated the spheroids with IWP2, SB431542, and Pur (5 µM each) for 2 days, after which the media were renewed every 2–3 days until the end of the study (day 30). ∗, First beating at day 7 of differentiation. (B): The effect of spheroid size on CM differentiation. The first beating in the 5- and 7-day spheroids was observed at days 7 and 10. All the 5-day spheroids were beating by day 10. The percentage of beating spheroids increased slowly and had reached 100% by day 10 in the 7-day spheroids. (C): Evaluation of cardiac differentiation by counting the number of beating spheroids (%) and flow cytometry analysis of α-MHC-expressing cells (%) indicated that increasing the CHIR concentration to 12 μM resulted in increased CM differentiation. (D): Flow cytometry analysis of α-MHC-positive cells (%) and calculating the number of beating spheroids (%) showed that the combination of IWP2, SB431542, and Pur led to the most efficient CM differentiation in a static suspension system. All data are presented as mean ± SD (n = 3). Abbreviations: bFGF, basic fibroblast growth factor; CHIR, CHIR99021; DMEM, Dulbecco’s modified Eagle’s medium; hPSCs, human pluripotent stem cells; MHC, myosin heavy chain; Pur, purmorphamine; RPMI, Roswell Park Memorial Institute; SB, SB431542.

Daily analysis of differentiation in spheroids showed that the first beating appeared after 10 days of differentiation induction (Fig. 1B). To determine the optimal size of spheroids for CM differentiation, 3-, 5-, and 7-day hESC spheroids (average size, 90 ± 30, 175 ± 25, and 250 ± 32 µm, respectively) generated in dynamic suspension culture were used for differentiation induction in the static suspension system. In the case of the 3-day spheroids, cell viability was significantly decreased; the spheroids became disrupted and finally dispersed after CHIR treatment (data not shown). We excluded this group from further experiments. We observed that approximately 50% of the 5-day spheroids started beating only 7 days after differentiation induction; approximately 100% of spheroids formed beating structures after 10 days of differentiation induction and maintained spontaneous contractile activity for at least 30 days in culture (Fig. 1B). The average size of the 5-day spheroids was 175 ± 25 µm; therefore, this was chosen as the optimal size for the subsequent experiments in our study.

Optimization of Chemically Induced Cardiomyocyte Differentiation in Static Suspension System

Different concentrations of CHIR have been reported that favor mesendoderm induction. The 3D structure of spheroids might affect the diffusion rates of CHIR throughout the cell aggregates, which could, in turn, result in inefficient and heterogeneous differentiation. The sensitivity of cells in these compact structures might be completely different from that of cells in monolayer adherent culture. Therefore, to explore the optimal concentration of CHIR for induction of hPSC aggregates, we treated the 5-day aggregates with different concentrations of CHIR (3, 6, 9, 12, and 15 μM), and the spheroids were assessed after 10 days of differentiation.

Our data showed that by increasing the CHIR concentration, the percentage of beating spheroids also increased. Approximately 100% of the spheroids began beating at both 12 μM and 15 μM CHIR (Fig. 1C). Flow cytometry analysis showed that α−myosin heavy chain (αMHC) expression increased and reached a plateau (>90%) after treatment with 12 μM and 15 μM CHIR (Fig. 1C); this was also supported by the quantification of beating. These results suggested CHIR at 12 μM would be the optimal concentration for mesoderm induction. This concentration was used in the first step of our differentiation protocol in suspension cultures.

We sought to determine whether manipulating one or two signaling pathways was sufficient to induce efficient CM differentiation or whether the complete cocktail of three chemicals (IWP2, SB431542, and Pur) was necessary. We examined different combinations of these three chemicals and calculated the number of beating spheroids after 10 days of differentiation (Fig. 1D). The results indicated that treatment of cell aggregates with SB431542 or Pur resulted in the formation of only a few beating spheroids, and IWP2 treatment led to higher numbers of beating spheroids (up to 20%), confirming a role for WNT signaling in cardiogenesis in this static suspension system. The combination of SB431542 and Pur resulted in a lower percentage of beating spheroids compared with IWP2 and the other two chemical combinations. The combination of all three chemicals dramatically increased the number of beating spheroids to approximately 100% and was identified as the most effective combination at this step for CM differentiation in the suspension culture condition. Flow cytometry analysis of αMHC⁺ cardiac cells at day 10 after differentiation initiation supported the beating results and revealed that a large population of CMs (>90% positive for αMHC) formed when using all three chemicals (Fig. 1D). These results suggest that inhibition of WNT signaling is necessary at this step for CM differentiation in suspension cell aggregates. To achieve an efficient differentiation process, inhibition of TGF-β signaling and activation of SHH signaling was also required.

Gene Expression Pattern of Cardiogenic Genes in a Static Suspension System

We determined the expression profiles of cardiac lineage-specific genes throughout differentiation by performing quantitative RT-PCR on spheroids collected at different time points after differentiation initiation (days 0, 1, 2, 4, 6, 8, 10, 20, and 30). Rapid induction of T (Brachyury), a mesodermal marker, and Mesp1, a marker for the earliest steps of cardiovascular progenitor cell specification, was observed at the first 2 days of differentiation, confirming the role of WNT signaling in mesoderm lineage specification (supplemental online Fig. 1). Downregulation of these genes occurred simultaneously with significant upregulation of later cardiac progenitor transcription factors (HAND1, TBX5, ISL1, MEF2C, and NKX2-5) at day 4 of differentiation. The expression of GATA4 increased earlier than that of the other cardiogenic transcription factors and displayed a steady level of expression over time. The expression of cTNT, α-MHC, and β-MHC (structural protein-coding genes) were upregulated at day 6 of differentiation and maintained until the end of the experiment.

Flow cytometry analysis of differentiated cells at day 15 showed that most individualized cells (up to 90%) were cTNT⁺, and endothelial cells (von Willebrand factor-positive) and vascular smooth muscle cells (α−smooth muscle actin-positive) represented a small proportion of the differentiated cells (∼3% and ∼8%, respectively; supplemental online Fig. 2).

Taken together, these results show that CM differentiation in the static suspension system passed through the main steps of cardiogenesis seen in normal heart development, as well as in embryoid body- and monolayer-based differentiation methods.

Validation of Optimized Cardiomyocyte Differentiation for Different hPSC Lines in the Static Suspension Culture System

To test whether our developed protocol supported robust CM differentiation in different hPSC lines, we differentiated five hESC and four hiPSC lines in the static suspension system using an optimal induction procedure. Analysis of the percentage of beating spheroids showed that almost all lines started spontaneous beating at day 7 of differentiation, which increased rapidly to a plateau, with nearly 100% beating spheroids at days 10–15 (Fig. 2A). These data demonstrate the reproducibility and universality of our protocol for all tested hPSC lines. Flow cytometry analysis of dissociated beating spheroids demonstrated that approximately 90% of cells were cTNT⁺ in four evaluated cell lines at day 15 of differentiation (Fig. 2B). Subsequent experiments were performed with the RH5 hESC line.

Figure 2.

Reproducibility and cardiomyocyte (CM) differentiation pattern of human pluripotent stem cells in a static suspension system. (A): Five-day spheroids of different human ESC (hESC) and human iPSC (hiPSC) lines followed a similar differentiation pattern in which all cell lines showed a beating initiation point at day 7 and a plateau with nearly 100% beating spheroids at day 10. Error bars represent SD (n = 3). (B): Flow cytometry analysis of cTnT⁺ cells indicated that the efficiency of CM generation from hESCs (RH5) and hiPSCs (618-3, 913-5, and 616-1) had reached 90% by day 15 of differentiation. Error bars represent SD (n = 3). Abbreviations: D, day; ESC, embryonic stem cell; iPSC inducible pluripotent stem cell.

Integrated Generation of Human CMs in a Stirred Suspension Bioreactor

In order to develop an integrated platform for large-scale production of human CMs, we used our optimized static suspension differentiation strategy in a stirred bioreactor with a 100-ml working volume. Five-day hPSC spheroids that were 175 ± 25 µm in diameter were induced in a spinner flask by replacing hPSC expansion medium with the same volume of differentiation medium (100 ml) for differentiation induction (Fig. 3A). After several rounds of experiments, we found that the addition of 0.1% PVA and 10 µM ROCK inhibitor Y-27632 were essential for the first 2 days of differentiation culture to increase cell viability and spheroid integrity (data not shown). The efficiency of CM differentiation was determined by calculating the number of beating spheroids in the first 2 weeks of differentiation (Fig. 3B). Compared with the static differentiation system, the first spontaneous beating appeared at day 7. The percentage of beating spheroids increased progressively and reached a plateau with approximately 100% beating spheroids by day 10 (supplemental online Video 1). Immunostaining for cTNT, MLC2a, and NKX2-5 in sections of beating spheroids collected at day 10 showed cytoplasmic- and nuclear-localized immunolabeling for cTNT and MLC2a and NKX2-5, respectively, indicating the efficiency of CM differentiation (Fig. 3C). Furthermore, immunostaining for MLC2a and costaining for NKX2-5 and cTNT in cells from both hESC and hiPSC lines demonstrated the nuclear accumulation of NKX2-5 and defined the sarcomeric structures in differentiated CMs (Fig. 3D).

Figure 3.

Experimental design of chemically induced hESC differentiation to CMs in a dynamic system. (A): Five-day spheroids formed in suspension culture (spinner flasks) were transferred to a new spinner flask that contained differentiation medium (Fig. 1A). ROCK inhibitor was used for 1 day and PVA for the first 2 days. ∗, First beating at day 7 of differentiation. (B): Time course of development of spontaneously beating spheroids. The 5-day RH5 hESC spheroids began beating at 7 days after induction of differentiation. The maximum number of beating spheroids (>90%) was observed at day 10. Error bars represent SD (n = 3). (C): Immunostaining of hESC-derived beating spheroids sectioned at day 30 for CM-specific markers. (D): Dissociated beating spheroids were stained for CM-specific transcription factor (NKX2-5) and structural proteins (MLC2a and cTNT). Abbreviations: bFGF, basic fibroblast growth factor; CHIR, CHIR99021; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; hPSCs, human pluripotent stem cells; Pur, purmorphamine; PVA, polyvinyl alcohol; RPMI, Roswell Park Memorial Institute.

Cardiac-Specific Gene Expression Pattern and Cardiac Lineage Induction in a Dynamic Suspension System

We examined the pluripotency and cardiac-specific gene expression patterns and transcript enrichment in hPSCs during CM differentiation in a dynamic culture system by performing quantitative RT-PCR at eight time points (supplemental online Fig. 3). The results showed that the pluripotency genes OCT4 and NANOG were significantly downregulated and the mesodermal marker T was upregulated after differentiation induction. The highest level of MESP1 expression was observed at day 2. The expression of cardiac progenitor markers c-KIT, ISL1, and PDGFR-α increased during differentiation, with the highest level of expression at day 8. Late cardiac progenitor markers NKX2-5, TBX5, MEF2C, and GATA4 were upregulated exactly after differentiation induction, had peaked at days 6–8, and remained at a steady high level of expression until the end of the study (day 30). Cardiac structural and maturation markers such as cTNT, αMHC, and MLC2v began expression at day 6 and reached a plateau with sustained expression until the end of study at day 30.

We grouped the pluripotency and differentiation genes into three clusters according to their expression pattern (Fig. 4A). Cluster 1 included the pluripotency genes (OCT4 and NANOG) and mesodermal marker T, with all showing downregulation during CM differentiation. The genes in cluster 2 showed a later peak after differentiation induction, followed by downregulation over the differentiation process. Cluster 3 included genes that encode CM-specific transcription factors and structural proteins. These genes showed upregulation and sustained expression throughout CM differentiation. Replicated samples were clustered together in PCA that showed a clear roadmap of differentiation (Fig. 4B, left, arrow). This map started from day 0 (hPSCs) and terminated at day 30. Although the pluripotency genes NANOG and OCT4 were located in the same direction of the day 0 replicates, MLC2v was found at the end of the differentiation roadmap. These analyses demonstrated the quality of the obtained results and reproducibility of the experiment.

Figure 4.

Expression patterns of pluripotency and cardiac lineage-related markers in a dynamic suspension system. (A): Differentially expressed genes could be grouped into three clusters. Cluster 1 included pluripotency genes (OCT4 and NANOG) and a mesodermal marker (T) and showed a reduction in gene expression over the time course of cardiomyocyte (CM) differentiation. The genes collected in cluster 2 showed upregulation after differentiation induction followed by downregulation over the differentiation process. Cluster 3 included genes that encode CM-specific transcription factors and structural proteins. These genes showed sustained upregulation throughout CM differentiation. (B): Principal components analysis (PCA) of expression values in the dynamic suspension system. Left: Each point represents a replicate sample, and the colors show the days after differentiation. Right: The location of each gene in the same PCA plot. This analysis shows the expression of pluripotency and CM-related genes matched the days after differentiation induction. (C): Quantitative data on dissociated cells from RH5 human embryonic stem cell spheroid showing cardiac lineage markers at different time points during differentiation in the dynamic system. Error bars represent SD (n = 3). Abbreviation: MHC, myosin heavy chain.

To quantify the percentage of cardiac lineage cells, we performed flow cytometry analysis at different time points in which the cardiac lineage genes showed the highest level of expression (Fig. 4C). T⁺ mesodermal cells constituted a high fraction of spheroids cells (∼90%) at day 2 of differentiation, and this percentage decreased during differentiation. MEF2C⁺ cells represented ∼50% of total cells at day 6 and had increased to approximately 80% at day 8 of differentiation. A rapid increase in the percentage of αMHC⁺ was observed after day 6, which had increased further to 85% by day 10. Taken together, these flow cytometry analyses corroborated the gene expression results discussed and showed the stepwise induction of different cardiac lineage states in a dynamic differentiation system.

Electrophysiological Properties of Human CMs Differentiated in a Dynamic Suspension System

We investigated the electrophysiological properties of hPSC-derived CMs using MEA and patch clamp techniques. We sought to determine whether integrated differentiation in a dynamic suspension culture system will produce functional CMs. hPSC spheroid-derived CMs in a dynamic suspension system developed spontaneous electrical activity, indicated by the typical extracellular FPs formed at different areas of the beating spheroids. The presence of a sharp and a slow component of the FP was noted (Fig. 5A). The FPs could be divided into a rapid component, reflecting depolarization, a plateau phase, and a slow component, representing repolarization [35]. We observed the chronotropic response of the spheroid-derived CMs after administration of the β-agonist isoproterenol (Iso). Application of 100 nM Iso resulted in a typical and comparable increase in the FP frequency compared with the basal condition (Fig. 5B). Moreover, the FP duration was significantly shortened in the Iso-treated beating spheroids (525 ± 5 at baseline vs. 485 ± 6 at Iso treatment). Thus, the dynamic suspension culture system supported the development of spontaneous contractile activity and electrophysiological functionality.

Figure 5.

Extracellular field potentials recorded from beating spheroids using microelectrode array. (A): Microelectrode array recording showed typical electrical properties of human embryonic stem cell spheroid-derived cardiomyocytes (CMs). (B): Drug response of line RH5-derived CMs showed increased chronotropy when challenged with Iso, a β-adrenergic agonist. Abbreviations: FPD, field potential duration; Iso, isoproterenol.

We also studied action potentials in single beating cells using the whole cell mode of the patch clamp technique. A primary study of single beating cells at day 30 of differentiation showed nodal-like APs in most patched cells (supplemental online Fig. 4). This classification was based on the morphology of APs (supplemental online Fig. 4A), AP parameters (Vm <10 V/s and AP duration at 90% [APD₉₀] of ≤150) [36, 37] and mean beating rate >100 (supplemental online Fig. 4B, 4C). The application of quinidine at 100 µM resulted in deceleration (supplemental Fig. 4Db) of spontaneous APs followed by complete abolition of beating, demonstrating the contribution of I_Na (sodium current) in the depolarization phase of these APs. The cells showed partial recovery after washout (supplemental Fig. 4Dd). Quinidine also caused prolongation of the AP duration, showing the contribution of hERG channels in the repolarization phase of these cells. The effect of quinidine was reversible, although it had not fully reversed after 5 minutes, consistent with the slow dissociation kinetics for quinidine.

AP recording from single beating hPSC-derived CMs was also repeated at days 60 and 90 of differentiation. We observed APs of the three main cardiac cell types in cells differentiated from hPSCs in the dynamic suspension system (Fig. 6A–6C). Vm >10 (20.5 ± 1.9 V/s) and APD₉₀/APD₅₀ ≤1.6 (1.6 ± 0.09) was measured in morphologically ventricle-like APs, which further confirmed their subtype (Fig. 6A). Nodal-like APs showed a Vm <10 (8.7 ± 0.96 V/s) and APD₉₀/APD₅₀ >1.6 but <2 (1.8 ± 0.03; Fig. 6C). Atrial-like APs showed similar properties to ventricle-like APs but with shorter AP durations (Fig. 6B). Thus, integrated differentiation of hiPSCs in a dynamic suspension culture system supported the generation of different cardiac cell types.

Figure 6.

Action potentials (APs) recorded from single beating human pluripotent stem cell-derived cardiomyocytes (CMs). APs of single beating human embryonic stem cell-derived CMs: ventricle-like APs (A), atrial-like APs (B), and nodal-like APs (C). The classification of different cardiac cell types was based on the morphology of APs (left) and AP parameters. All AP recordings were performed at room temperature.

Discussion

Effective cardiac cell therapies and the development of pharmacology and toxicology screening platforms require large numbers of pure and functional cardiac cells that cannot be easily produced by most current protocols for the generation of CMs from hPSCs. For example, to achieve an effective cardiac cell therapy 1–2 × 10⁹ of hPSC-CMs are required for myocardial infarction (review provided in [38]). Therefore, developing flexible and robust protocols and platforms for cost-effective, reproducible, and large-scale generation of the desired hPSC-derivative cells is necessary to realize the great potential of hPSC-CMs for clinical and commercial applications.

To date, different CM differentiation protocols have been reported that manipulated the WNT, TGF-β, and SHH signaling pathways using different protein factors, matrix components, or SMs [14, 15]. However, most of the protocols resulted in large variations in CM differentiation efficacy among different cell types and lines, such as were experienced in our preliminary experiments. After numerous trials, we have developed a new procedure using CHIR (12 μM, WNT/β-catenin signaling activator) for 1 day to induce mesendodermal differentiation, followed by a 1-day rest period, and then 2 days of treatment with a SM cocktail composed of IWP1 (WNT/β-catenin inhibitor), SB431542 (TGF-β receptor inhibitor), and Pur (SHH agonist) to generate CMs. It is known that WNT signaling has a biphasic role in cardiogenesis [39]. The addition of the WNT protein or IWP2 can reduce endogenous WNT heterogeneity and provide a condition for stable expansion and efficient differentiation of WNT^high or WNT^low hESC populations, respectively [40]. However, our differentiation method has eliminated the variability in CM differentiation between hPSCs lines reported in other related studies. This universality of the production process will largely facilitate the future widespread application of the protocol and development of hPSC-CM-based therapeutic products.

More recently, another universal CM differentiation protocol for hiPSCs was developed using a chemically defined medium that consisted of three components—basal medium RPMI 1640, l-ascorbic acid 2-phosphate, and rice-derived recombinant human albumin. This protocol resulted in generation of CMs with high efficacy and productivity. However, the proposed culturing platform was based on a 2D adherent culture on peptide-modified surfaces, which are very expensive and offer poor scalability [9].

To overcome the complexity and limited scalability issues of the previously developed CM generation protocols, we have successfully developed a cost-effective and robust process that allows large-scale expansion of hPSCs as aggregates and their integrated differentiation for production of homogenous hPSC-CM aggregates. hPSCs were initially expanded in a stirred bioreactor as size-controlled homogenous aggregates. Next, the aggregates with different sizes were directly induced to differentiate into CMs by a simple stepwise protocol optimized for suspension culture under carrier-free conditions without the use of additional extracellular matrix or high-cost recombinant proteins or peptides. The time-course analysis of beating in variously sized spheroids (90 ± 30, 175 ± 25, and 250 ± 32 µm) showed that cardiac induction occurred in 5-day spheroids (175 ± 25 µm in diameter) at an earlier time and with greater efficiency than in 7-day spheroids (250 ± 32 µm); the 3-day spheroids disrupted after induction. These results support previous reports that recognized the size of hPSC aggregates or EBs as a key parameter influencing the induction efficiency of cardiac and other lineages [41, 42]. The low diffusion rate of growth factors, chemicals, and nutrients and of oxygen gradients inside the cell aggregates might underpin the delayed differentiation observed in the 7-day spheroids. However, treating cell aggregates with CHIR decreased hPSC aggregate integrity in dynamic culture conditions and increased cell loss after differentiation induction, explaining the disruption of 3-day aggregates after induction. Therefore, the generation of size-controlled aggregates in hPSC expansion cultures by controlling the hydrodynamic culture conditions and defining the optimum aggregate size for a specific differentiation protocol is crucial to achieving homogenous and efficient CM differentiation.

In optimized culture conditions, a stirred bioreactor containing hPSC aggregates (80–90 × 10⁶ of cells in a 100-ml working volume after 5 days of culture) directly differentiated into cardiomyocytes with optimized differentiation cocktails and strategy. With this approach, approximately 100% of the undifferentiated aggregates generated cardiospheres that showed spontaneous contractility and contained highly enriched CMs (up to 90% cTNT⁺ and MHC⁺ cells) with high functionality in vitro. This high purity and functionality could facilitate the development of an integrated, cost-effective purification system to generate the large-scale hPSC-CMs required for further applications.

Although our proposed strategy provides a robust, cost-effective, and universal platform for large-scale generation of hPSC-CMs, we believe the efficacy of the current proposed strategy could be improved further by optimization of the bioprocess parameters in fully controlled conditions. These parameters include oxygen tension, hydrodynamic culture conditions, feeding and media refreshment strategy, and the development of innovative SM delivery technologies to increase the differentiation efficacy and homogeneity and minimize hPSC cell loss after differentiation induction. More recently, it has been demonstrated that optimizing the bioprocess parameters, including oxygen tension (hypoxia), and bioreactor hydrodynamics can boost mouse iPSC differentiation toward CMs [43]. However, these finding should also be validated for differentiation of hPSC cell lines.

While our study was in preparation, Kempf et al. reported the development of a similar small molecule CM differentiation and culturing protocol applied to three hESC and hiPSC lines as aggregates in 100-ml stirred bioreactors [43]. Their study applied sequential CHIR99021 (7.5 µM compared with 12 µM in our study) and IWP2 (5 µM) treatments separated by 2 days (rather than the 1 day used in our study) and did not include the TGF-β signaling inhibitor SB431542 or SHH pathway inducer Pur. After scaling up the optimized differentiation protocol from 12-well plates to Erlenmeyer flasks and, finally, the stirred bioreactor, the hPSCs expanded in the batch stirred bioreactor culture as aggregates with a 283 ± 9.6-µm average diameter failed to produce hPSC-CMs in the differentiation phase. However, hPSC aggregates with a larger diameter (470–530 µm for different cell lines) that were produced in a continuous culture using a cyclic perfusion feeding strategy generated contracting hPSC-CMs after 6–10 days of differentiation induction. In the present study, we have demonstrated that functional and beating hPSC-CMs can be generated in simplified batch and single-unit operation from 5-day aggregates with a 175 ± 25-µm average diameter after 7 days of induction. This appears to be universally applicable, because it was reproducible for 9 hESC and hPSC cell lines. The simplified and batch operation mode of our protocol could offer an advantage over the strategy of continuous culture using perfusion feeding, which has a higher cost, greater complexity of process control in large-scale culture, and considerable cell loss during perfusion (∼25%). In addition, the hPSC-CMs produced in our culture system had a smaller average diameter (150–200 µm) compared with the hPSC-CMs generated from the perfusion feeding strategy (about 1 mm). The smaller and more homogenous cardiosphere sizes will facilitate their downstream processing (e.g., enzymatic dissociation and purification) and future use for cell therapy and drug discovery applications.

Regarding yield and universality, the continuous culture mode resulted in hPSC-CM yields that were similar to but lower than ours for the hESC line (67%–81% MHC⁺ cells in three trials after 10 days of induced differentiation), with yields apparently more variable with the hiPSC lines (range, 27%–83% MHC⁺ cells in three trials). Further work is required to determine the practical basis for these differences.

Conclusion

hPSCs likely have the potential to differentiate into all cell types of the body and constitute an extremely attractive tool for the generation of cells for cell therapy and other biomedical applications once the safety and scale-up issues have been overcome. An integrated, robust bioprocess for mass production of hPSC-CMs will pave the way for commercial and clinical applications.

To date, three reports [44–46] have been published, and 11 approved clinical trials involving hPSC-based therapies have been registered at the U.S. National Institutes of Health clinical trials website (http://www.clinicaltrials.gov), with 9 for ocular indications (8 from hESCs, 1 from hiPSCs [47]), 1 for diabetes, and 1 for severe heart failure. Although hPSC-CMs still seem to be some distance from clinical application, their large-scale production will also provide an opportunity for the development and refinement of screening platforms for drug toxicity and modeling of diseases, including congenital heart disease, long-QT syndromes, and Timothy syndrome (reviews provided in [48, 49]). The availability of massive numbers of human CMs will also provide broad scope for cardiac tissue engineering, in vitro organ development, molecular cardiovascular research, and the development of safer, more effective drugs for cardiovascular therapies using high-throughput technologies.

The protocol we have described allows the production of hPSC-CMs in a simplified and robust process from different hPSCs lines. It offers advantages over currently demonstrated suspension protocols, which are variously limited in scalability, complexity, affordability, efficacy, or CM functionality. Billions of hPSC-CMs can be produced using the proposed scale-out and scale-up strategies using hiPSCs or hESCs as the starting cells for production and purification (Fig. 7). Optimizing the key bioprocessing parameters is now an imperative that should be implemented.

Figure 7.

An integrated, robust bioprocessing platform for large-scale production of hPSC-CMs. Production of large-scale hPSC-CMs require hESCs (derived from human blastocysts) and hiPSCs (generated from patient fibroblasts) as starting material. Expansion of these cell lines as single-cell inoculated suspension cultures in stirred suspension bioreactors using scale-up and scale-out strategies resulted in a few billion to billions of undifferentiated hPSCs. The hPSCs could be easily differentiated in an integrated single-unit operation to hPSC-CMs during 10–15 days. Purification of hPSC-CMs could be further achieved by culturing the produced cells within lactate-enriched medium. The resultant cells offer tremendous advantages for developing different applications that require large numbers of cells such as high-throughput screening and drug discovery, in vitro organ development, and cardiac tissue engineering. Abbreviations: CMs, cardiomyocytes; hESC, human embryonic stem cell; hPSC, human pluripotent stem cell.

Author Contributions

H.F. and H.A.: experiment design, manuscript writing, cell culture, real-time polymerase chain reaction analysis, immunocytofluorescence, flow cytometry; S.A.: experiment design, manuscript writing; M.R.L.: human pluripotent stem cell suspension culture in bioreactor; S.K. and S.H.: electrophysiology; A.S.Z.: real-time polymerase chain reaction data analysis; A.B.: human induced pluripotent stem cell line generation, human induced pluripotent stem cell design; G.M.B.: clinical translation, provision of study material or patients, human ethics and sample procurement, fibroblast culture initiation; S.P. and M.P.: single cell electrophysiology experiment performance, manuscript writing; Y.O. and Y.M.: skin biopsy of subjects; J.V.: electrophysiology data analysis, manuscript writing; M.T.: manuscript writing; D.S.W.: clinical translation, provision of study material or patients, human ethics and sample procurement; R.P.H.: human induced pluripotent stem cell design, manuscript writing; N.A.: experiment design; H.B.: experiment design, manuscript writing.

Disclosure of Potential Conflicts of Interest

The authors indicated no potential conflicts of interest.