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Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair

Originally publishedhttps://doi.org/10.1161/CIRCULATIONAHA.116.024145Circulation. 2017;135:1832–1847

Abstract

Background:

Advancing structural and functional maturation of stem cell–derived cardiomyocytes remains a key challenge for applications in disease modeling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions.

Methods:

We systematically investigated cell composition, matrix, and media conditions to generate EHM from embryonic and induced pluripotent stem cell–derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological, functional, and transcriptome analyses to benchmark maturation of EHM.

Results:

EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical β1- and β2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition, we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair.

Conclusions:

We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell–derived cardiomyocytes under defined, serum-free conditions.

Introduction

Editorial, see p 1848

The availability of human embryonic stem cells (ESCs)1 and human-induced pluripotent stem cells (iPSCs)2 and the scalability of their directed differentiation into bona fide cardiomyocytes, as well,37 have facilitated the rapid evolution of myocardial tissue engineering. Early tissue-engineering studies in chick embryo and rodent models have established electromechanical stimulation as an important engineering paradigm,810 which has now been translated to human models.1116 The accumulating evidence for advanced maturation in 3-dimensional versus monolayer cultures provides a solid rationale for applications in phenotypic screens11 and heart repair.17,18 As the use of myocardial tissue engineering increases in academia and industry, it is essential to establish conditions readily adaptable to current good manufacturing practice. To achieve this goal, it is imperative to define the essential elements required for the structural and functional maturation of tissue-engineered myocardium under defined, serum-free conditions. Last, robust and reproducible utility in ESC- and iPSC-based models is of pivotal importance.

In this study, we report a systematic approach for the design of engineered human myocardium (EHM) with structural and functional properties observed in the postnatal heart. Unbiased transcriptome profiling provided evidence for advanced maturation in EHM in comparison with parallel monolayer cultures. To demonstrate the applicability of EHM for the modeling of “human heart failure in the dish,” we introduce a catecholamine overstimulation protocol with outcomes similar to what is typically observed in clinical heart failure. Last, we provide proof-of-concept for the scalability and in vivo applicability of defined EHM as an important step toward clinical translation of tissue-engineered heart repair.

Methods

Human Pluripotent Stem Cell Lines

We utilized: H9.219; HES3 (Embryonic Stem Cell International) including the transgenic derivative HES3-ENVY20; HES2 (Embryonic Stem Cell International) including the transgenic derivative HES2-RFP21; H71 (WiCell); hiPS-G1 (generated in-house using Sendai Virus reprogramming, Cytotune Kit, Thermo Fisher); hiPS-BJ (Dr Toshiyuki Araki, New York), approved according to the German Stem Cell Act by the Robert-Koch-Institute to W.-H.Z.: permit #12; reference number: 1710-79-1-4-16.

Cardiomyocyte Differentiation and Purification

Differentiated embryoid bodies (H9.2, HES3, HES3-ENVY, HES2, hiPS-BJ) were shipped to Göttingen at room temperature and arrived within 72 to 96 hours. Cardiomyocytes from H7 (L. A. Couture, City of Hope) were shipped at –80°C. Frozen human cardiomyocytes were stored at –152°C. Most experiments were performed with HES2-RFP and hiPS-G1 lines differentiated in monolayers according to Hudson et al22 with modifications. In brief, pluripotent stem cells (PSCs) were plated at 5×104 to 1×105 cells/cm2 on 1:30 Matrigel in phosphate-buffered saline (PBS)–coated plates and cultured in Knockout DMEM, 20% Knock-out Serum Replacement, 2 mmol/L glutamine, 1% nonessential amino acids, 100 U/mL penicillin, and 100 µg/mL streptomycin (all Life Technologies) mixed 1:1 with irradiated human foreskin fibroblast (HFF)–conditioned medium with 10 ng/mL fibroblast growth factor-2 (FGF2) or TeSR-E8 (STEMCELL Technologies). After 1 day the cells were rinsed with Roswell Park Memorial Institute (RPMI) medium and then treated with RPMI, 2% B27, 200 µmol/L l-ascorbic acid-2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich), 9 ng/mL Activin A (R&D Systems), 5 ng/mL BMP4 (R&D Systems), 1 µmol/L CHIR99021 (Stemgent), and 5 ng/mL FGF-2 (Miltenyi Biotec) for 3 days. Following another wash with RPMI medium, cells were cultured from day 4 to 13 with 5 µmol/L IWP4 (Stemgent) followed by RPMI, 2% B27, 200 µmol/L l-ascorbic acid-2-phosphate sesquimagnesium salt hydrate. Where indicated, cardiomyocytes were metabolically purified by glucose deprivation23 from day 13 to 17 in RPMI without glucose and glutamine (Biological Industries), 2.2 mmol/L sodium lactate (Sigma-Aldrich), 100 µmol/L β-mercaptoethanol (Sigma-Aldrich), 100 U/mL penicillin, and 100 µg/mL streptomycin. Please refer to online-only Data Supplement Table I for an overview of the different cardiac differentiation protocols3,17,19,22,24 used in this study.

EHM Generation

An overview of the protocols to generate human EHM is displayed in Table. Details can be found in the online-only Data Supplement Material.

Table. Overview of EHM Protocols

ComponentStarting ProtocolMatrix ProtocolSerum-Free Protocol
EHM reconstitution mixture
 Collagen rat (research grade), mg /EHM0.4
 Collagen bovine (medical grade), mg /EHM0.40.4
 Matrigel, %, v/v10
 Base mediumDMEMDMEMRPMI
 Horse serum, %10
 Chick embryo extract, %2
 Fetal bovine serum, %20
 B27 (without insulin), %4
EHM culture medium
 Base mediumIscoveIscoveIscove*
 Fetal bovine serum, %2020
 B27 (without insulin), %4
 IGF-1, ng/mL100
 FGF-2, ng/mL10
 VEGF165, ng/mL5
 TGF-β1, ng/mL5
 Nonessential amino acids, %1%1%1%
 Glutamine, mmol/L222
 Penicillin, U/mL100100100
 Streptomycin, µg/mL100100100
 β-Mercaptoethanol, µmol/L100100

DMEM indicates Dulbecco modified Eagle medium; EHM, engineered human myocardium; FGF-2, fibroblast growth factor-2; IGF-1, insulin-like growth factor 1; RPMI, Roswell Park Memorial Institute medium; TGF-β1, transforming growth factor-β1; and VEGF165, vascular endothelial growth factor 165.

*Alternatively other basal medium with ≥1.2 mmol/L calcium.

Analyses of Contractile Function

Contraction experiments were performed under isometric conditions in organ baths at 37°C in gassed (5% CO2/95% O2) Tyrode solution (containing: 120 NaCl, 1 MgCl2, 0.2 CaCl2, 5.4 KCl, 22.6 NaHCO3, 4.2 NaH2PO4, 5.6 glucose, and 0.56 ascorbate; all in mmol/L). Spontaneous beating frequency was determined at 2 mmol/L calcium after 10 minutes of equilibration of EHMs. EHMs were electrically stimulated at 1.5 to 2 Hz with 5 ms square pulses of 200 mA. EHMs were mechanically stretched at intervals of 125 μm until the maximum systolic force amplitude (force of contraction [FOC]) was observed according to the Frank-Starling law. Responses to increasing extracellular calcium (0.2–4 mmol/L), increasing stimulation frequencies (1, 2, 3 Hz), and adrenergic stimulation with isoprenaline (1 μmol/L) followed by functional antagonism by the muscarinergic agonist carbachol (10 µmol/L) at ≈EC50 calcium of individual EHMs were investigated. Where indicated, an isoprenaline concentration response curve was performed in the presence or absence of specific β1-adrenoceptor antagonist CGP-20712A (300 nmol/L, Sigma-Aldrich) or specific β2-adrenoceptor antagonist ICI-118551 (50 nmol/L, Sigma-Aldrich). Postrest potentiation was assessed after 2 minutes of stimulation at 1.5 to 2 Hz and pauses of 10 s. The last stimulated beat amplitude was compared with the first stimulated beat amplitude after the pause. Only EHMs without spontaneous contractions during the stimulation pause were included in the analysis.

EHM Heart Failure Model

l-Norepinephrine hydrochloride (NE) and endothelin-1 were prepared in distilled water containing 200 µmol/L l-ascorbic acid-2-phosphate sesquimagnesium salt hydrate (all from Sigma-Aldrich). EHMs were treated with indicated concentrations for 7 days. N-Terminal pro B-type natriuretic peptide was measured by using the Elecsys kit (Roche Diagnostics).

EHM Dissociation

To isolate single cells, EHMs were incubated in collagenase 1 solution (2 mg/mL in calcium-containing PBS in the presence of 20% fetal bovine serum) at 37°C for 60 to 90 minutes. EHM were washed with PBS (without calcium) and further incubated in Accutase (Millipore), 0.0125% Trypsin (Life Technologies), 20 µg/mL DNase (Calbiochem) for 30 minutes at room temperature. Cells were then mechanically separated and transferred into PBS with 5% fetal bovine serum for live cell flow cytometry. To preserve rod-shaped morphology of EHM-derived cardiomyocytes, 30 mmol/L 2,3-butanedione monoxime was added to the collagenase solution, and the final cell suspension was quickly transferred to 4% formaldehyde (Histofix, Roth). EHM-derived cells were spread out on glass slides (Superfrost plus, Menzel-Gläser) in distilled water and air dried.

Human Samples

Human fetal heart tissue (3 biopsies from a single donation) was obtained after elective abortion material (vacuum aspiration) without medical indication following informed consent. The collection of fetal material was approved by the Ethical Committee of the Leiden University Medical Center (MEC-P08.087). Human heart samples were collected from the left ventricles of nonfailing donor hearts (n=4 donor hearts) not suitable for transplantation as approved by the Ethical Committee of the University Medical Center Göttingen (31/9/00). Gingiva samples were obtained from otherwise healthy donors during elective periodontal surgical treatment as approved by the Ethical Committee of the University Medical Center Göttingen (16/6/09). Cardiac fibroblasts were purchased from Lonza. The study was conducted in accordance with the Declaration of Helsinki by the World Medical Association.

RNA Sequencing

RNA was prepared using Trizol (Life Technologies) following the manufacturer’s instruction. RNA integrity was assessed with the Agilent Bioanalyzer 2100. Total RNA was subjected to library preparation (TruSeq Stranded Total RNA Sample Prep Kit from Illumina) and RNA-sequencing on an Illumina HighSeq-2000 platform (SR 50 bp; >25 Mio reads/sample). Sequence images were transformed with the Illumina software BaseCaller to bcl files, which were demultiplexed to fastq files with CASAVA (v1.8.2). Fastq files were mapped to GRCh38/hg38 using STAR 2.4 or TopHat225 and reads per kilobase of transcript per million (RPKM) were calculated based on the Ensembl transcript length as extracted by biomaRt (v2.24). We only considered protein_coding transcripts for further analysis. Gene ontology analysis was performed through DAVID.26 To determine cardiomyocyte and fibroblast transcriptomes the following algorithm was applied: (1) counts (>10) of purified PSC-derived cardiomyocytes (HES2, iCELL, hiPS-G1; n=3 from each line) and fibroblasts from 3 different sources (heart, skin, gingiva; n=3 from each source) were pooled and the differentially expressed genes (P<0.05, corrected by Benjamini-Hochberg method for multiple testing27) between cardiomyocyte and fibroblast pools determined using edgeR28; (2) log2 changes of differentially expressed genes were calculated and genes omitted with a log2 difference lower than mean log2 of all cardiomyocyte genes; (3) resulting cardiomyocyte- and fibroblast-enriched genes were screened for RPKM values in adult healthy heart and all genes with RPKM <1 in adult heart were omitted.

3D Printing of Flexible Holders for EHM Patches

Flexible holders for the EHM patch construction were printed on a Connex350 (Stratasys) 3D printer using the biocompatible MED610 polymer as stiff base and TangoBlack polymer for the flexible poles. Support material was sprayed off using a Balco Powerblast waterjet. Holders were incubated for 15 minutes in isopropanol to dissolve traces of support material, sprayed again, rinsed, and soaked in water for at least 5 days to bleed out leftovers from the polymerization process. Holders were then sterilized by plasma cleaning for 30 s (Harrick Plasma).

Imaging of EHM Patch Function

For each measurement, plates were recorded inside a 37°C climate chamber for at least 2 minutes at 50 frames per second resolution using a Basler acA2000 8 bit monochrome camera with a Kowa 35-mm lens from ≈45-cm distance. Back light was set to bleach background pixels and facilitate video analysis by a custom-made Matlab code. The region of interest was manually adjusted to patch size, nonbackground pixels were selected by an intensity cutoff, and the Matlab imdilate and imfill commands were used to close gaps and fill holes in the tissue leaving pixels belonging to patch and poles white (1) and everything else black (0). The number of white pixels represents the surface area sa at time t and was converted to fractional area change (FAC) by division through the maximum sa of a contraction cycle. Contraction peaks, ie, the time points of maximal FAC, were identified automatically; FAC and beating frequency, determined from peak-to-peak intervals, were averaged over 2 minutes.

Implantation of Human Patches

EHM patches were epicardially implanted into immunosuppressed athymic (nude) rats (Charles River) as described previously.17

Flow Cytometry and Immunofluorescence Staining

Quantitative and qualitative analyses were performed after antibody labeling (online-only Data Supplement Table II) as described previously.29

Statistical Analyses

Data are presented as mean±standard error of the mean. Statistical differences between 2 groups were tested by 2-sided unpaired or paired Student t tests. In case of ≥3 groups, 1-way or 2-way unrepeated or repeated-measures ANOVA with appropriate post hoc testing was performed. The performed tests are specified in the respective figure legends. Statistical testing was performed with GraphPad Prism 6.

Additional methods are described in the online-only Data Supplement Material.

Results

Definition of Cell Composition in EHM

EHM formation comprises 2 macroscopically distinguishable phases (Figure 1A): (1) EHM consolidation in casting molds with an onset of spontaneous beating in variably sized areas within 24 hours, and (2) EHM maturation with coordinated and rhythmic contractions of the whole tissue after 3 days and onward (online-only Data Supplement Movie I). In pilot experiments, we defined 1.5×106 ESC (H9.2 and HES3)–derived cardiomyocytes suspended in 500-µL collagen type I/Matrigel hydrogels as the optimal condition for the construction of force-generating EHM online-only Data Supplement Table I).

Figure 1.

Figure 1. Defining human EHM. A, EHM generation is characterized by 2 phases: EHM consolidation for 3 days (Left, casting mold with 4 EHMs; Inset, magnification of EHM in mold) and EHM maturation for at least 7 days under mechanical load (Right, EHM on flexible PDMS holders). Bars: 5 mm (Left), 1 mm (Right). B, Force of contraction (FOC; normalized to maximal FOC) in relation to output cardiomyocyte percentage (actinin+ cells) of EHM made from HES2-RFP, HES2, and hiPS-BJ lines. Blue square indicates an EHM sample constructed from SIRPA2A-selected30 cardiomyocytes. Gray area indicates optimal cardiomyocyte percentage across indicated lines (mean±SD). C, Purification of cardiomyocytes for defined EHM generation. Quantification of cardiomyocyte purity (actinin+ cells) before and after enrichment by metabolic selection; n=8, P<0.05 by 2-tailed, paired Student t test. D, Macroscopic appearance of EHM with >92% CM (CM EHM) and EHM with >92% CM supplemented with HFF (70:30% CM+HFF EHM). Immunostaining for actinin (green), f-actin (red), and nuclei (blue) in CM EHM (Middle) and CM+HFF EHM (Right). Bars: 5 mm (Left), 50 µm (Middle and Right). E, Titration of the optimal CM:HFF ratio. Output CM percentage and force per CM in 2-week-old EHM made with indicated input cell ratios of purified CMs and HFFs. Colors indicate the input CM:HFF ratio of respective EHMs (each circle represents one individual EHM with an additional empty circle indicating the mean±SEM of the respective groups). F, Force of contraction (FOC) recorded under increasing calcium concentrations and electric stimulation at 1.5 Hz in 4-week EHMs constructed according to the undefined Starting Protocol (n=19; Table) and defined, Serum-free Protocol (n=59; Table); pooled data from EHM generated from different ESC and iPSC lines (please refer also to online-only Data Supplement Figure IV for detailed information); *P<0.05 by 2-way ANOVA with the Tukey multiple comparisons post hoc test. ANOVA indicates analysis of variance; CM, cardiomyocyte; EHM, engineered human myocardium; ESC, embryonic stem cell; HFF, human foreskin fibroblast; iPSC, induced pluripotent stem cell; and SEM, standard error of the mean.

Using HES2 and hiPSC-BJ cardiac differentiation cultures with different cardiomyocyte content, we found that EHM containing a cardiomyocyte:nonmyocyte composition of 1:1 at the time of force assessment developed maximal contractile forces (Figure 1B). This was in agreement with recent reports15 and our own experience in rodent models31 on the critical role of nonmyocytes for the engineering of force-generating myocardium. We next formally tested the effect of the cardiomyocyte:nonmyocyte ratio by using metabolic selection23 for cardiomyocyte purification (Figure 1C) and HFFs; EHM constructed directly from enriched cardiomyocyte populations did not condense and contained mostly rounded cardiomyocytes (Figure 1D; online-only Data Supplement Movie II). The addition of HFFs at a 70%/30% cardiomyocyte/fibroblast input ratio was optimal for the construction of force-generating EHM loops with a cardiomyocyte:fibroblast output ratio of ≈1:1 (Figure 1E), confirming our initial findings (Figure 1B).

By defining the nonmyocyte input, we observed advanced cardiomyocyte maturation with reduced variability in the functional maturation of EHM (online-only Data Supplement Figure IA) and a higher mean actinin fluorescence intensity per cell, indicating higher sarcomeric protein content per individual cardiomyocyte (online-only Data Supplement Figure IB). Furthermore, the classical inotropic and lusitropic (relaxation) responses to isoprenaline were enhanced in defined EHM (online-only Data Supplement Figure IC). The number of immature ventricular cardiomyocytes (defined by simultaneous expression of MLC2A and MLC2V) was greatly reduced by EHM culture with more pronounced ventricular maturation in defined EHM (online-only Data Supplement Figure ID and IE). Defining the nonmyocyte population in EHM not only reduced intraline (online-only Data Supplement Figure IA), but also interline variability (online-only Data Supplement Figure IF). Moreover, expression of pluripotency-associated genes and cell cycle activity in cardiomyocytes and nonmyocytes were markedly reduced in defined EHM (online-only Data Supplement Figure II). Taken together, we conclude that defining the nonmyocyte cell fraction increases the robustness of the EHM protocol also with respect to its organotypic contractile function and ventricular fate.

Development of a Defined, Serum-Free EHM Construction Protocol Toward Current Good Manufacturing Practice

The EHM Starting Protocol, which was devised from our original rodent tissue engineering protocol,29 included a variety of undefined matrix (Matrigel) and serum (horse serum, fetal calf serum, chick embryo extract) components (Table). We first defined the matrix components and observed that EHM could be constructed from medical-grade bovine collagen without Matrigel (Matrix Protocol), without any reduction in functionality online-only Data Supplement Figure IIIA and IIIB). The addition of laminin (5 µg/EHM) or fibronectin (5 µg/EHM) to the Matrix Protocol did not further improve EHM function (online-only Data Supplement Figure IIIC). Factorial screens, including the assessment of the B27 supplement, were performed next with the aim to replace all animal culture medium components. To expedite the initial screens, we used simple HES2-cardiomyocyte aggregate cultures (online-only Data Supplement Figure IIID) and subsequently tested putative cardio-instructive factors in EHM. We first selected a particular B27 medium supplementation (4% with insulin) based on cell viability. We subsequently selected growth factors (FGF-2, insulin-like growth factor 1 [IGF-1], transforming growth factor-β1 [TGF-β1], vascular endothelial growth factor 165 [VEGF165]) for EHM testing according to the following criteria: (1) neutral or enhanced cell viability, and (2) enhanced cardiomyocyte actinin content or cardiomyocyte size. Last, we confirmed that the combination of FGF-2, IGF-1, TGF-β1, and VEGF165 was maximally effective in supporting the formation of force-generating EHMs (online-only Data Supplement Figure IIIE). In agreement with the important role of extracellular matrix remodeling in early EHM cultures,29 we found that TGF-β1 treatment in the consolidation phase (day 0–3) was necessary for enhanced EHM function (online-only Data Supplement Figure IIIE). It is interesting to note that we observed that antioxidants were not critical for EHM function and that omitting insulin (B27 minus insulin) enhanced EHM function in comparison with insulin-containing B27 (online-only Data Supplement Figure IIIF). This led to the definition of a minimal Serum-free Protocol containing 4% B27 without insulin plus TGF-β1, IGF-1, FGF-2, VEGF165 (Table). Last, testing of the basal media identified calcium supplementation to physiological concentrations (1.2 mmol/L) as a critical parameter for optimal outcome (online-only Data Supplement Figure IIIG). Collectively, these experiments established a defined, Serum-free Protocol with markedly enhanced contractile performance in comparison with the undefined Starting Protocol (Figure 1F;online-only Data Supplement Movie III) and applicability to various ESC- and iPSC-EHM models (online-only Data Supplement Figure IV).

Evidence for Structural and Functional Maturation of EHMs

We next investigated whether the defined, Serum-free Protocol supports EHM maturation. Enzymatic dispersion of EHMs revealed cardiomyocytes with an elongated phenotype with sarcomeres in registry (Figure 2A, online-only Data Supplement Figure VA). In comparison with serum-containing EHM cultures and in line with the functional outcome (Figure 1F), intact rod-shaped cardiomyocytes from EHMs constructed according to the Serum-free Protocol presented with a larger volume (12 101±1240 versus 5649±1410 µm3), but similar aspect ratio (7.6±0.4 versus 6.7±0.9; n=28/10). In comparison with 2D monolayer cardiomyocyte cultures and EHM constructed according to the Starting Protocol, sarcomere size was larger in EHM constructed according to the defined, Serum-free Protocol (1.93±0.01 versus 1.81±0.01 versus 1.84±0.01 µm; >120 sarcomeres were analysed in 12/8/10 cardiomyocytes in the respective groups. The low cardiomyocyte volume (20 000–35 000 µm3 reported in adult human cardiomyocytes32) was mainly attributable to a smaller cell width in EHM (width: 13±0.5 versus 20–35 µm; length: 92±4 versus 60–150 µm in adult human cardiomyocytes32,33). Note that cardiomyocytes in EHM exhibited a similar width as observed in 6-week-old infant heart (4–12 µm33). Ultrastructural analyses revealed that cardiomyocytes in EHM displayed a remarkable degree of sarcomere organization with clearly distinguishable Z, I, A, H, and M bands (Figure 2B, online-only Data Supplement Figure IVB). Consistent with earlier reports on largely absent M bands, even in extended stem cell–derived cardiomyocyte monolayer cultures34 and tissue-engineered models,1113,15 we found little organization of M bands in monolayer cardiomyocytes, but a high degree of organization in EHM-derived cardiomyocytes (online-only Data Supplement Figure IVC). Also, enhanced MLC2V organization and presence of n-cadherin+ intercalated disk-like structures were observed in EHM cardiomyocytes (online-only Data Supplement Figure IVB and IVC).

Figure 2

Figure 2 . Morphological and functional maturation of EHM.A, Immunostaining of isolated cardiomyocyte from 4-week EHM (hiPS-G1). Top, myosin heavy chain (green); Middle, brightfield image with nucleus labeled with Hoechst (blue; Bottom, overlay; bar: 20 µm). B,Electron micrographs of 4-week EHMs (hiPS-G1), low-power (Left, bar: 2.5 µm) and high-power magnification (Right, characteristic sarcomere structures are labeled; Mito, mitchondria; bar: 1 µm). C,FOC per cross-sectional area (CSA) of serum-free EHM from HES2 and hiPS-G1 at the indicated time points in culture; n=12/14/8 for weeks 2/4/6 in HES2 EHM and n=7/10/8 for weeks 2/4/8 in hiPS-G1 EHM *P<0.05 by 2-way ANOVA with the Tukey multiple comparison post hoc test. D, Force-frequency response of hiPS-G1-EHM (at 4 weeks in culture) generated according to the Starting Protocol (red; n=8) and defined, Serum-free Protocol (black; n=21). §P<0.05 versus 1 Hz of the respective group by 2-way ANOVA with the Tukey multiple comparison post hoc test; *P<0.05 by 2-way repeated measures ANOVA followed by the Sidak multiple comparison test. E, Representative force traces recorded from hiPS-G1-EHM (at 4 weeks in culture) at 1.5-Hz stimulation with an intermittent stimulation pause (10 s); enhanced FOC at the reintroduction of electric stimulation, ie, postrest potentiation, is characteristic for cardiomyocytes with mature intracellular calcium storage and release (the dotted line marks prepause baseline maximal FOC. F, Representative action potentials recorded by impaling electrode measurements in EHM developed under the Starting Protocol (HES3) and the Serum-free Protocol (HES2); the table summarizes data recorded from together 51 independent action potential recordings; values in parentheses indicate maximally negative RMP and fastest dV/dtmax recorded in the respective groups. ANOVA indicates analysis of variance; APA, action potential amplitude; APD, action potential duration; EHM, engineered human myocardium; MP, membrane potential; and RMP, resting membrane potential.

Functional maturation was a continuous process with enhanced inotropic responses to calcium in older EHM (Figure 2C); this was despite similar cardiomyocyte content (online-only Data Supplement Figure VIA). Because EHM cross-sectional area decreased over time in culture (online-only Data Supplement Figure VIB), we opted to correct FOC by cross-sectional area to allow a direct comparison of the different models (HES and iPSC) and their developmental stages (Figure 2C); uncorrected FOC is displayed in online-only Data Supplement Figure VIC). The average maximal FOC developed by EHM after 8 weeks in culture (6.2±0.8 mN/mm2 at 1.5 Hz; n=8) exceeded the reported FOC (≈1 mN/mm2 at 1 Hz) in papillary muscle from human infants (3–14 months after birth)35 markedly, but remained lower than the FOC recorded in adult nonfailing myocardium (≈25 mN/mm2 at ≈1.5 Hz).36 It is interesting to note that a positive force-frequency behavior (Bowditch phenomenon), which is absent in newborns and present in infants,35 was clearly developed in defined, serum-free EHM (+19±5% at 2 Hz, +22±6% at 3 Hz versus 1 Hz, studied at 4 weeks) in contrast to EHM constructed according to the undefined Starting Protocol (Figure 2D). In agreement with this finding, postrest potentiation (enhanced FOC by +9±1% [n=7] in the first electrically stimulated beat after a stimulation pause) was observed, providing evidence for intracellular calcium storage and release by the sarcoplasmic reticulum (Figure 2E).

Electrophysiological studies revealed that EHMs comprised mainly working myocardium-like cells without pronounced spontaneous phase 4 depolarization (Figure 2F). This suggests that the spontaneous contractions of EHM are under the control of a small portion of pacemaker cells in EHM (<10% of all cells analyzed).

Molecular Maturation of EHM

We next sought to use an unbiased approach to determine whether signs of molecular maturation could be identified inline with the observed structural and functional maturation of EHM. Hence, we first determined the differential transcriptome in high purity (94±2% ACTN2+ by flow cytometry, n=9) PSC-derived cardiomyocytes (n=777 transcripts; HES2, hiPS-G1, hiPS-CDI; each n=3) and fibroblasts (n=200 transcripts; skin-, gingiva-, heart-derived fibroblasts; each n=3) by RNA sequencing (Figure 3A). As anticipated cardiomyocyte, the cardiomyocyte (CM) transcriptome was highly enriched for sarcomeric transcripts, and the fibroblast transcriptome was enriched for transcripts encoding for extracellular matrix–associated proteins and proteins mediating cell-cell or cell-matrix interactions (Figure 3B, refer to online-only Data Supplement Tables III and IV for a full list of the identified CM and fibroblast transcriptomes including a comparison with fetal and adult heart expression levels).

Figure 3.

Figure 3. Molecular maturation of serum-free EHM.A,Strategy to determine cardiomyocyte and fibroblast transcriptomes from RNAseq data obtained from purified pluripotent stem cell–derived (PSC) cardiomyocytes (n=3 hES2 RFP, n=3 iCell CM, n=3 hiPS-G1) and primary fibroblasts (n=3 HFF, n=3 human cardiac fibroblasts, n=3 human gingiva fibroblasts). B,RPKM values of the 29 most abundantly expressed transcripts in PSC-derived cardiomyocytes and primary fibroblasts. C, Heatmap of cardiomyocyte transcripts in 22-day-old cardiomyocyte monolayer cultures (2D D22), 60-day-old cardiomyocyte monolayer cultures (2D D60), 6-week-old EHMs (note that cardiomyocyte age in these EHMs was similar to 2D D60 cultures), fetal heart, and adult heart. Boxed areas indicate cardiomyocyte maturation genes; adult, increasing expression with development (upper box), and embryonic, decreasing expression with development (lower box). D, Histogram of cardiomyocyte gene expression level (RPKM) in comparison with fetal heart as reference. Comparison of 22-day-old cardiomyocyte monolayer cultures (2D, gray box) as starting point, 60-day-old cardiomyocyte monolayer cultures (2D, blue box), and 6-week EHM cultures (red box). E, Venn diagram and corresponding list of differentially expressed cardiomyocyte maturation genes with specific regulation in EHM, 60-day-old cardiomyocyte monolayer cultures (2D), or both (overlap in Venn diagram; P<0.05 corrected for multiple testing by Benjamini-Hochberg method). CM indicates cardiomyocyte; EHM, engineered human myocardium; FC, fold change; HFF, human foreskin fibroblast; and RPKM, reads per kilobase of transcript per million.

We next used the CM transcriptome to establish a temporal gene expression profile from embryonic to adult heart, taking embryonic CMs (22-day-old monolayer HES2 CM, n=3), fetal heart (n=3), and adult heart (n=4) as reference time points. We classified 3 gene clusters: (1) genes with continuous increase (adult CM genes; n=218; Figure 3C, upper box), (2) genes with continuous decrease (embryonic CM genes; n=128; Figure 3C, lower box), and (3) genes without clear trajectory (n=431) in expression (refer to online-only Data Supplement Table V for a full list of genes in each of the 3 clusters). Transcriptional profiling revealed that transcription of 174 and 110 of the adult CM genes was enhanced and transcription of 94 and 72 of the embryonic CM genes was reduced in parallel EHM (6 weeks) and 2D (60 days) cultures, respectively. Direct comparison of the CM maturation gene transcripts (embryonic plus adult CM genes; n=346) showed higher frequencies of fetal-adult gene expression levels in EHM, indicating enhanced molecular maturation in EHM versus 2D (Figure 3D). In comparison with 2D cultures, but also with undefined EHM cultures, a significant upregulation of pivotal CM genes involved in “ventricular cardiac muscle tissue morphogenesis” (gene ontology:0055010) further confirmed the cardiotypic development and a high degree of maturation in EHM constructed according to the defined, Serum-free Protocol (Figure 3E, online-only Data Supplement Figure VII).

Simulation of Heart Failure in EHM by Neurohumoral Overstimulation

The sympathetic nerve system controls heart function via the release of catecholamines and subsequent adrenoceptor activation. Experimentally, acute addition of isoprenaline (β1- and β2-adrenoceptor agonist) is used to simulate organotypic responses to catecholamine stimulation, including enhanced force development (inotropy), beating frequency (chronotropy), and relaxation (lusitropy); chronic application of norephinephrin (NE; α1-, α2-, β1- > β2-adrenoceptor agonist) is classically used to induce pathological CM hypertrophy.

Although effects on chronotropy have been well established in human PSC-derived CMs, so far, there is little evidence for regular inotropic responses,11,13,15 suggesting functional immaturity of the β-adrenergic signaling cascade. Transcriptome analyses revealed lower transcript abundance for most adrenergic receptors, including, in particular, the β1(ADRB1)- and β2(ADRB2)-adrenoceptors, in EHM versus adult myocardium (online-only Data Supplement Figure VIIIA). Irrespective of the transcript levels, we observed a robust inotropic response of EHM to isoprenaline, which was significantly enhanced in serum-free versus serum-containing cultures (online-only Data Supplement Figure VIIIB and VIIIC). It is interesting to note that EHM displayed a similar sensitivity (EC50: 10±1 nmol/L; online-only Data Supplement Figure VIIID) to isoprenaline as that reported for nonfailing myocardium.37 Classical pharmacological β1- and β2-adrenoceptor blocking experiments with CGP-20712A and ICI-118551, respectively, revealed that 32±6% of the acute inotropic effect in EHM were mediated via ADRB1 (online-only Data Supplement Figure VIIIE).

Chronic catecholamine overstimulation (serum levels in patients with heart failure: 1–10 nmol/L NE) contributes to heart failure development and progression.38 In iPSC models, results have been variable with recent reports demonstrating the need for defined media to elicit CM hypertrophy.39 We asked whether EHMs would exhibit the clinically observed heart failure phenotype, including β-adrenergic desensitization, CM hypertrophy, and the release of biomarkers (such as brain natriuretic peptide40). To recapitulate sympathetic overstimulation, we exposed EHM to NE at clinically relevant concentrations (0.001–1 µmol/L) for 7 days. We also included a group of EHMs exposed to endothelin-1 (0.01 µmol/L), a well-established inducer of CM hypertrophy via the alternative Gq-protein transduction pathway.41 Similarly as observed in patients, chronic NE stimulation induced contractile dysfunction in a concentration-dependent manner (Figure 4A) with desensitization to acute β-adrenergic stimulation (Figure 4B), which, according to its underlying mechanism, only occurred under NE and not endothelin-1. To enable a cell type–specific analysis of cell size and cell composition, we developed a color-coded EHM model comprising RFP+-CMs and GFP+-fibroblasts amenable to flow cytometry analyses (Figure 4C, online-only Data Supplement Movie IV). This allowed us to confirm enhanced CM hypertrophy (Figure 4D, online-only Data Supplement Figure IX) and death (Figure 4E) in response to increasing NE concentrations. We also found the clinically relevant biomarker N-terminal pro B-type natriuretic peptide released in a concentration-dependent manner (Figure 4F) and a blunted force-frequency response in serum-free, but not serum-containing EHM (online-only Data Supplement Figure XA). A consistent observation was that the pathological phenotype was, in general, more pronounced in serum-free EHM (summarized in online-only Data Supplement Figure XB) with a significantly reduced hypertrophic response in serum-containing EHM. This finding is consistent with earlier data on the hypertrophy-masking effects of serum in human PSC-derived CMs.39 It is notable that the pathological phenotype could be partially or fully prevented by β1-adrenoreceptor and α1-adrenoreceptor blockade with metoprolol and phenoxybenzamine, respectively, demonstrating the applicability of EHM in the in vitro simulation of heart failure and its prevention by pharmacological means (Figure 4G).

Figure 4.

Figure 4. Modeling heart failure in color-coded EHM.A, Effect of 7-day treatment with indicated concentrations (in µmol/L) of norepinephrine (NE) or endothelin-1 (ET-1) on FOC of EHM; *P<0.05 versus Control by 2-way ANOVA with the Tukey multiple comparison post hoc test, n=8 to 10 per Control and NE groups, n=4 for ET-1 group. B, Inotropic response to acute isoprenaline (ISO) stimulation in EHM previously exposed to 7-day NE or ET-1 at the indicated concentrations (same EHM as in A); *P<0.05 versus Control (Ctr) by 1-way ANOVA with the Tukey multiple comparison post hoc test. C, Left, Macroscopic view of color-coded EHM (RFP+-CM: red, GFP+-Fib: green); scale bar: 1 cm. Middle, Cross section of color-coded EHM (red: actinin+-CM, green: GFP+-Fib); scale bar: 500 µm; Inset, magnification, scale bar: 50 µm. Right, Flow cytometry of RFP+-CM and GFP+-Fib after enzymatic dispersion of color-coded EHM. D, CM size measured by determination of RFP median fluorescence intensity (MFI, please refer to online-only Supplement Figure IX for experimental details); *P<0.05 versus Ctr by 1-way ANOVA with the Tukey multiple comparison post hoc test. E, Cell type distribution in color-coded EHM assessed by total cell quantification after enzymatic dispersion and subsequent flow cytometry for the separation of RFP+-CM and GFP+-Fib (from same EHM as in A); *P<0.05 for cardiomyocyte number versus Ctr by 1-way ANOVA with the Tukey multiple comparison post hoc test. F, NT-proBNP secretion per CM into the culture medium (n=3/group). G, Maximal FOC, response to ISO, CM viability, and CM size in comparison with control (dashed line) in EHM treated with 1 µmol/L NE with and without preincubation with 5 µmol/L metoprolol (Met) or 5 µmol/L phenoxybenzamine (Phen); *P<0.05 versus Ctr by 1-way ANOVA with the Tukey multiple comparison post hoc test (n=4–10/group). ANOVA indicates analysis of variance; CM, cardiomyocyte; EHM, engineered human myocardium; Fib, fibroblast; FOC, force of contraction; and NT-proBNP, N-terminal pro B-type natriuretic peptide.

Scaling of EHM for Heart Repair

Remuscularization of myocardial scar tissue in the failing heart will require sizable muscle surrogates. Accordingly, we tested whether large EHM can be engineered under the defined, Serum-free EHM Protocol. We also reasoned that casting patches rather than loops would facilitate scaling toward clinical needs. Accordingly, we developed stamps with flexible tips by 3D printing for the penetration of EHM mixtures cast into a size-adapted mold (Figure 5A). This allowed us to scale EHM patches variably, reaching sizes for clinical translation (15×17 mm and 35×34 mm containing 10×106 and 40×106 CMs respectively; thickness: 0.5±0.1 mm, n=5; Figure 5B and 5C). Cells in EHM patches were homogenously distributed and structurally organized along traction force lines (Figure 5C). It is important to note that EHM patches and loops contracted similarly (online-only Data Supplement Movie V). Because nondisruptive measurements will finally be essential to document EHM patch quality, we developed an optical force assessment strategy by correlating FOC recorded in individual EHM loops with FAC in EHM patches from the same production runs. This analysis revealed a correlation of FOC and FAC recorded in EHM loops and patches, respectively (online-only Data Supplement Figure XI); further refinement of this measure will be required to account for homogeneity, shape, and force distribution of the different culture formats.

Figure 5.

Figure 5. Scaling of EHM for heart repair.A, Technical drawings of the EHM patch manufacturing devices: Top left, 3D-printed patch holder with flexible poles; Top right, inverted patch holder positioned in hexagonal casting mold; Bottom, top view on patch holder for small and large EHM patch with dimensions in millimeters. B, Display of different EHM designs (from left to right): small (1.5×106 cells/500 µL) and big (2.5×106 cells/900 µL) loops, fusion of 5 big loops according to technology reported earlier for rat,42 small (10×106 cells/2 mL) and clinical-sized large (40×106 cells/8 mL) patch. C, Overview and 90° projections of an immunostained (f-actin in green) small EHM patch (image stitched together from 24× 850×850 µm tiles); boxed areas magnified on right for a demonstration of cell orientation. Bars: 5 mm (overview) and 1 mm (magnifications). D, Explanted rat heart 4 weeks after epicardial implantation of an EHM patch in a RNU rat; bar: 1 cm. E, Overview of human EHM on rat heart, immunostaining of human MYH7 (red), dashed line outlines the human EHM; bar: 500 µm. F, Immunostaining of human EHM 107 days after implantation, cardiac troponin T (red), sarcomeric actinin (green), nuclei (blue); bar: 100 µm. G, Immunostaining of CD31 (white) and human specific β1-integrin (red); bar: 500 µm. EHM indicates engineered human myocardium.

In continuation of a recently completed experimental series for the assessment of feasibility and safety of EHM grafting,17 we now tested whether EHM patches would be retained after engraftment. In line with our recent study with EHM loops, we could demonstrate that EHM patches formed sizable and structurally highly developed grafts in RNU rats (Figure 5D through 5F), which were progressively vascularized (Figure 5G).

Discussion

Our study demonstrates that differentiated, force-generating human heart muscle can be generated in vitro under defined, serum-free conditions for applications in heart failure modeling and tissue-engineered heart repair. While the definition of cell composition and culture conditions reduced variability and procedural complexity, it also supported CM structural and functional maturation beyond the current state-of-the-art. The reported protocol is adaptable to current good manufacturing practice and thus serves as the basis for highly standardized in vitro assay development and clinical translation of tissue-engineered heart repair.

A number of factors have been previously identified to support maturation of human CMs in tissue-engineered heart muscle, such as mechanical stimulation13 and electric stimulation,12 and the coculture of CMs and fibroblasts, as well.15 In this study, we systematically screened culture conditions and identified the minimal requirements for EHM formations under highly defined conditions (Table: Serum-free Protocol). So far unrecognized were the need for an adaptation of extracellular calcium to physiological levels (1.2 mmol/L) and supplementation of TGFβ-1 during EHM consolidation. The requirement for calcium adaptations was identified serendipitously while testing different basal media with normal and reduced (RPMI 0.42 mmol/L) calcium. This observation is in agreement with the previously reported essential role of calcium for myofibrillogenesis in the mouse.43 The mode of action of TGFβ-1 during EHM consolidation appears to be enhanced fibroblast-mediated extracellular matrix remodeling, which was found earlier to be crucial in rodent EHM models.29 Last, addition of IGF-1, FGF-2, VEGF165, and B27 without insulin were sufficient to replace all serum supplements. The use of clinical grade bovine collagen instead of the widely used Matrigel supplemented hydrogels44 further assisted in defining culture conditions.

Using our highly defined EHM protocol, we observed advanced structural, functional, and molecular maturation of CMs. In fact, to our knowledge, the following maturation characteristics have not been reported so far: (1) structural maturation with a rod-shaped CM morphology and sarcomers with distinguishable M bands; both parameters are rarely observed even in extended (1-year) monolayer cultures45; (2) dominant ventricular structural and functional maturation evidenced by abundant Myl2 (MLC2V) positivity and characteristic action potential kinetics; and (3) functional maturation with contractile forces and physiological responses such as a positive force-frequency behavior observed only in postnatal myocardium.35,46 Although functional β1-adrenergic signaling is minute in immature PSC-derived CMs,47 defined EHM displayed a robust β1-mediated inotropic response. The cardiotoxic effect of elevated norepinephrine levels further argues for relevant adrenergic signaling to model disease mechanisms of heart failure. Consistent with recent work, the biomechanical stimulation of EHM may accelerate β-adrenergic maturation in comparison with monolayer CMs.47 Spontaneous contractions of EHM require specialized pacemaker cells. Random impalements with sharp electrodes for AP recordings did not identify bona fide pacemaker cells in defined, serum-free EHM. Optical imaging after loading with voltage-sensitive dyes or the use of genetically encoded voltage sensors48 may help to better localize regions with pacemaker activity and guide detailed electrophysiological studies to define the underlying mechanisms of EHM automaticity.

Transcriptional profiling in 6-week EHM was in agreement with the structural and functional data, confirming an advanced degree of maturation in comparison with parallel monolayer cultures. However, reaching a fully adult phenotype remains a challenging task. In fact, unbiased global transcriptome profiling suggested that EHMs are, at large, similar to fetal human heart at 13 weeks of gestation, despite some morphological (M bands) and functional (Bowditch phenomenon) properties that develop postnatally. This suggests, on the one hand, that our defined, serum-free EHM protocol supports bona fide heart development in the dish to a notable extent, and, on the other hand, introduces an unbiased approach for the benchmarking of tissue-engineered myocardium.

Taken together, we conclude that the serum-free EHM protocol can serve as the foundation for the definition of specific biological, pharmacological, or biophysical interventions controlling heart development. Whether in vitro interventions will finally enable the speeding up of heart development in a dish beyond the pace of natural cardiomyogenesis remains to be elucidated. The principle propensity for advanced maturation was further supported by long-term in vitro culture and in vivo implantation studies. We consider this an important prerequisite for applications of EHM in disease modeling, drug screens, and tissue-engineered heart repair.

Acknowledgments

The authors thank M. Hoch, I. Quentin, D. Reher, A. Schraut, and K. Sharkova for excellent technical assistance. The authors thank D. Ziebolz for providing gingiva samples, C. Rogge for preparing EHM during early phases of this study, and S. Lutz for sharing cardiac fibroblast cultures and antibodies. The authors also acknowledge B. Downie, T. Lingner, and G. Salinas from the Transcriptome and Genome Analysis Laboratory, University Medical Center Göttingen, for their support.

Footnotes

The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.116.024145/-/DC1.

Circulation is available at http://circ.ahajournals.org.

Correspondence to: Wolfram-Hubertus Zimmermann, MD, Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Georg-August-University, Robert-Koch-Str 40, 37075 Göttingen, Germany. E-mail

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Clinical Perspective

What Is New?

  • Proof-of-concept for the engineering of scalable force-generating human myocardium from a variety of human pluripotent stem cells and biopsy-derived fibroblasts under defined, serum-free conditions.

  • Evidence for morphological, molecular, and functional maturation beyond the present state-of-the-art is demonstrated (eg, positive force-frequency response, sarcomere assembly with robust M-band formation).

  • Simulation of a human heart failure phenotype in the dish with (1) contractile dysfunction, (2) loss of a positive force-frequency response, (3) adrenergic signal desensitization, (4) cardiomyocyte hypertrophy, and (5) biomarker release (N-terminal pro B-type natriuretic peptide) by chronic catecholamine stimulation.

  • Implantability of scalable engineered human myocardium patches is demonstrated.

What Are the Clinical Implications?

  • Robustness and readiness of defined, serum-free engineered human myocardium for applications in translational studies is demonstrated.

  • Advanced morphological, molecular, and functional maturation, and organotypic responses to physiological (positive force-frequency response) and pathological (norepinephrine-induced heart failure) stimuli, as well, are key for the utility of engineered human myocardium in heart failure modeling.

  • Simulated heart failure in engineered human myocardium may be exploited for the development of novel heart failure therapeutics.

  • The reported defined, serum-free protocol will facilitate the engineering of human myocardium according to current good manufacturing practice for applications in tissue-engineered heart repair.

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