Spheroids of cardiomyocytes derived from human-induced pluripotent stem cells improve recovery from myocardial injury in mice

Abstract

The microenvironment of native heart tissue may be better replicated when cardiomyocytes are cultured in three-dimensional clusters (i.e., spheroids) than in monolayers or as individual cells. Thus, we differentiated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs) and allowed them to form spheroids and spheroid fusions that were characterized in vitro and evaluated in mice after experimentally induced myocardial infarction (MI). Synchronized contractions were observed within 24 h of spheroid formation, and optical mapping experiments confirmed the presence of both Ca²⁺ transients and propagating action potentials. In spheroid fusions, the intraspheroid conduction velocity was 7.0 ± 3.8 cm/s on days 1–2 after formation, whereas the conduction velocity between spheroids increased significantly (P = 0.003) from 0.8 ± 1.1 cm/s on days 1–2 to 3.3 ± 1.4 cm/s on day 7. For the murine MI model, five-spheroid fusions (200,000 hiPSC-CMs/spheroid) were embedded in a fibrin patch and the patch was transplanted over the site of infarction. Later (4 wk), echocardiographic measurements of left ventricular ejection fraction and fractional shortening were significantly greater in patch-treated animals than in animals that recovered without the patch, and the engraftment rate was 25.6% or 30% when evaluated histologically or via bioluminescence imaging, respectively. The exosomes released from the spheroid patch seemed to increase cardiac function. In conclusion, our results established the feasibility of using hiPSC-CM spheroids and spheroid fusions for cardiac tissue engineering, and, when fibrin patches containing hiPSC-CM spheroid fusions were evaluated in a murine MI model, the engraftment rate was much higher than the rates we have achieved via the direct intramyocardial injection.

NEW & NOTEWORTHY Spheroids fuse in culture to produce structures with uniformly distributed cells. Furthermore, human cardiac lineage-induced pluripotent stem cells in cardiomyocytes in adjacent fused spheroids became electromechanically coupled as the fusions matured in vitro, and when the spheroids were combined with a biological matrix and administered as a patch over the infarcted region of mouse hearts, the engraftment rate exceeded 25%, and the treatment was associated with significant improvements in cardiac function via a paracrine mechanism, where exosomes released from the spheroid patch.

INTRODUCTION

Human-induced pluripotent stem cells (hiPSCs) have enormous potential for cell-based therapeutic applications because they can be used to generate an unlimited number of terminally differentiated cell types for use in tissue engineering, drug development, and disease modeling. However, despite efforts to develop clinically effective treatments, only very small proportions of transplanted cells are retained and continue to survive at the administration site, and this low engraftment rate is believed to be one of the primary barriers to successful therapy (1, 16). The engraftment rate is typically higher when cells are administered in engineered tissues rather than injected directly in the myocardium, and a wide variety of biomaterials and fabrication techniques have been investigated over the last decade (3). Researchers are also developing methods to facilitate the vascularization of engineered myocardial tissues, which may also increase engraftment.

During the early stages of embryonic development, cellular differentiation is linked to the formation of aggregates of individual cells. Cultured cells also self-associate, and the resulting interactions between adjacent cells have an important role in the maturation of both cells and tissues. When cultured in low-attachment round-bottomed wells, cardiomyocytes (CMs) cluster into spheroids, which are fundamentally different from two-dimensional monolayers and may be more efficient for the development of three-dimensional (3-D) tissues, such as cardiac muscle (14). Spheroids can also be used as building blocks during tissue fabrication and could be combined with other cellular aggregates to form structures with complex architectures that may more accurately reproduce the properties of native tissues, thereby enhancing their effectiveness in clinical applications, drug testing, and disease modeling.

Here, we present the results from a series of experiments with spheroids of CMs that had been differentiated from hiPSCs. Our findings indicate no significant necrotic core in large spheroid (~800-µm diameter), which beats synchronously in dish. The spheroids fuse in culture to produce structures with uniformly distributed cells and electrical properties such as action potential (AP) and Ca²⁺ transient (CaT) propagation in engineered myocardial tissue. Furthermore, when the spheroids were fused together within the fibrin matrix and administered as a patch over the infarcted region of mouse hearts, the spheroid patch was rapidly vascularized, the engraftment rate for the administered cells at week 4 posttransplantation was remarkably high (>25%), and the treatment was associated with significant improvements in infarct size and cardiac function.

METHODS

Generation and characterization of hiPSC-CMs.

The hiPSCs (GRiPS) were generated by transfecting human male cardiac fibroblasts with Sendai viruses coding for transgenic human octamer-binding transcription factor 4, sex-determining region Y box 2, Kruppel-like factor 4, and c-Myc (18). The plasmid pCDH-MSCV-firefly luciferase (Luc) 2-EF1-green fluorescent protein (GFP)-T2A-Puro was a gift from Dr. Wu (Stanford University). To make the lentivirus, pCDH-MSCV-Luc2-EF1-GFP-T2A-Puro was cotransfected with packaging plasmids pVSVg (no. 8454, AddGene), pRSV-Rev (no. 12253, AddGene), and pMDLg/pRRE (no. 12251, AddGene) into human embryonic kidney (HEK)-293(F)T cells. The virus-containing culture medium was harvested 48 h after transfection and centrifuged at 3,000 rpm at 4°C for 10 min to remove cell debris; next, the supernatant was filtered through a 0.45-µm low-protein-binding membrane (Millipore) and used immediately for hiPSC transduction. After transduction, hiPSCs were cultured with puromycin (5 µg/ml) on Matrigel-coated dishes for 4 days, puromycin-resistant colonies were selected and expanded in culture, and GFP and Luc expression was verified via fluorescence microscopy and bioluminescent imaging (BLI). BLI imaging was performed in the University of Alabama at Birmingham small animal imaging core facility with a Xenogen IVIS 100 in vivo Imaging System and Living Image software (Caliper Life Sciences); hiPSC- and CM-derived spheroids were imaged in six-well culture plates after the addition of d-luciferin (150 µg/ml).

Stable GFP/Luc transfectants were then subjected to CM differentiation protocol that involved culturing the cells with CHIR99021 in RPMI basal medium with B27 “minus insulin” (B27⁻) supplement at the first step. Later (24 h), cells were recovered and cultured with RPMI basal medium and B27⁻ supplement for 2 days, and cells were then cultured in RPMI basal medium with B27⁻ and the Wnt signaling inhibitor IWR1 for 48 h followed by a 48-h culture in RPMI/B27⁻ alone and the addition of 5 µM insulin. Spontaneously contracting hiPSC-CMs typically appeared 8 days after differentiation was initiated, and cells were enriched via metabolic selection (12). The procedure began on day 10 with changing the CM differentiation medium to selection medium, which consists of glucose-free RPMI medium supplemented with 4 mM lactate and B27 supplement. On day 12, the selection medium was changed back to the differentiation medium for 1 day. This cycle of differentiation/selection medium change was repeated on days 13−16. On day 16, monolayers were trypsinized, and cells were subjected to the spheroid formation. CMs were characterized via immunohistochemical analyses for the expression of cardiac troponin T (cTnT), connexin43, ventricular myosin light chain 2 (MLC2v), and myosin light chain 2A (MLC2A).

Generation of hiPSC-CM spheroids and spheroid fusions.

hiPSC-CMs were suspended in RPMI medium supplemented with B27 (RPMI/B27⁺), plated in ultralow attachment 96 U-well plates (product no. 7007, Corning) at varying concentrations (2.5 × 10³−3 × 10⁵ cells/well), and incubated in a humidified atmosphere at 37°C and 5% CO₂; the culture medium was changed every 2 days. Cells plated at concentrations below 2,500 cells/well-formed loosely associated clusters but not spheroids. Spheroid fusions were formed by transferring 50 spheroids of ~200-µm diameter or 2 spheroids of ~800-µm diameter in 2-ml round-bottomed test tubes containing maintenance medium; spontaneous beating was typically observed within 1 day of fusion and the fusions contracted into small compact tissue-like structures by day 7.

Transmembrane voltage and Ca²⁺ optical mapping.

Spheroids were stained with a low-affinity Ca²⁺-sensitive dye (Cal-520FF, 10 μM) (7) for 1 h, transferred to a perfusion chamber mounted on an inverted microscope, and held in place with two nylon meshes (280-μm pore size): one mesh submerged in the perfusion solution (HBSS at 36–37°C) and one mesh mounted on a micromanipulator; next, the spheroids were stained with a transmembrane voltage-sensitive dye (RH-237, 2.5 μM) for 5 min. Fluorescence was excited with a 200-W Hg/Xe arc lamp, and the optical signal was recorded with a 16 × 16 photodiode array (Hamamatsu) at a spatial resolution of 110 µm/diode as previously described (11). RH-237 fluorescence was excited at 560/55 nm and measured at >650 nm, and Cal-520FF fluorescence was excited at 480/40 nm and measured at 535/50 nm. Motion artifacts caused by spheroid contractions were eliminated by supplementing the perfusion solution with 10 μM of blebbistatin, and the optical signal was digitally filtered to increase the signal-to-noise ratio. Spheroids were stimulated with 2-ms rectangular pulses delivered to the edge of the spheroid from the tip of a bipolar electrode. The electrode consisted of a glass pipette filled with HBSS and a silver wire coiled around the pipette tip, and the current was delivered at 1.5 times the excitation threshold. Activation times were measured at 50% of the maximum AP amplitude and used to construct isochronal maps of activation spread. Conduction velocity was calculated at each recording site from local activation times and averaged across the whole map using the custom-designed data-analysis software. AP duration (APD) and CaT duration were measured at 50% (APD₅₀ and CaT₅₀, respectively) and 80% (APD₈₀ and CaT₈₀, respectively) recovery.

The murine model of myocardial infarction and treatment administration.

All experimental protocols that involved the use of animals were approved by the Institutional Animal Care and Use Committee of the Alabama at Birmingham and performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23). Experiments were performed in NOD/SCID Gamma mice (stock no. 005557, The Jackson Laboratory). Animals were anesthetized with inhaled isoflurane (1.5–2%), intubated, and ventilated; next, a left thoracotomy was performed, and the left anterior descending coronary artery (LAD) was ligated with an 8-0 nonabsorbable suture. After myocardial infarction (MI) induction, the chest muscles and skin were closed, and mice received intraperitoneal injections of buprenorphine (0.1 mg/kg) every 12 h for up to 3 days and intraperitoneal injections of carprofen (5 mg/kg) every 12 h for up to 1 day after surgery.

The experimental treatment consisted of five hiPSC-CM spheroids (2 $\times$10⁵ cells/spheroid) suspended in a fibrin matrix containing 1 ml fibrinogen solution (25 mg/ml) and 1 ml thrombin (80 NIH U/ml, Sigma-Aldrich) supplemented with 2 µl of 400 mM CaCl₂ and 200 mM ε-aminocaproic acid (Sigma-Aldrich), which was positioned over the site of infarction in animals from the MI + spheroid group (n = 6) immediately after LAD ligation and held in place with a film of sodium hyaluronate-carboxymethylcellulose that was sutured to the heart and prevented adhesion to the serous pericardium. The patch was withheld from animals in the MI group (n = 6), and animals in the sham group (n = 8) underwent all surgical procedures for MI induction except for the ligation step.

Echocardiography.

Echocardiographic imaging was performed as previously described (17). Briefly, mice were lightly anesthetized with 1.5–2% isoflurane until the heart rate stabilized at 400–500 beats/min; next, parasternal long-axis and two-dimensional short-axis images were obtained with a high-resolution Micro-Ultrasound system (Vevo 2100, VisualSonics). Images were analyzed with a Vevo 2100 system, and Vevo Analysis software was used to calculate the left ventricular (LV) ejection fraction (EF), fractional shortening (FS), end-diastolic volume (EDV), and end-systolic volume (ESV).

Engraftment rate.

Because the cells used in this study were of human origin and stably expressed GFP and the Luc reporter gene, the engraftment rate was determined both histologically and via in vivo BLI of luciferase activity. Histological assessments were performed in cryostained sections with the corresponding primary rabbit anti-Turbo-GFP (Evrogen), mouse anti-human nuclear antigen (HNA; Abcam), rabbit anti-human (h)cTnT (Abcam), and secondary FITC-donkey anti-rabbit, Cy3-donkey anti-mouse, and Cy5-donkey anti-mouse (Jackson ImmunoResearch Laboratory) antibodies. Each mouse heart was fixed and subjected to whole organ 10-µm serial sectioning. Every 10th section was stained with hcTnT/HNA antibodies and counterstained with DAPI. The number of hcTnT/HNA-positive cells was counted over the whole section and normalized by the number of DAPI-positive cells. This number was multiplied by 10 to obtain the number of cells in a 10-section block of tissue. A total of 20 blocks were analyzed in each heart in this fashion. The total number of cells from these 20 blocks was divided by the number of transplanted cells to determine the engraftment rate. BLI was performed at the University of Alabama at Birmingham Small Animal Imaging Shared Facility with a Xenogen IVIS-100 system. Briefly, varying numbers of hiPSC-CM spheroids (0, 1, 2, 3, 4, 5, 6, 7, and 8) containing known quantities of cells were transferred to a 48-well plate and imaged, and the data were used to plot a standard curve of the relationship between BLI intensity and number of cells. Animals were anesthetized with isoflurane and intraperitoneally injected with d-luciferin (375 mg/kg body wt); bioluminescence images were obtained 10 min later, and the BLI signal intensity was compared with the standard curve to determine the number of engrafted cells.

Infarct size.

Infarct size was evaluated as previously described (8, 13). Briefly, hearts were frozen, and coronal plane sections were cut from the apex to base at 1.0-mm intervals; next, sections were fixed in Bouin’s solution and stained with Sirius red to identify the scarred tissue and with fast green to identify noninfarcted myocardial tissues. Digital photographs were taken and analyzed with NIH ImageJ 1.36b software, and infarct size was calculated according to the following formula: infarct size = (scar area)/(total LV area) × 100%.

Immunostaining.

Murine hearts were cut into halves from the middle of the infarct, with one of the halves being frozen for cryosectioning and the other half stored in 10% formalin for paraffin-embedded sectioning. Cryosections were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 for 15 min, and blocked with 5% donkey serum for 30 min. Primary antibodies (Table 1) were added to the blocking solution, and sections were incubated at 4°C overnight; next, sections were incubated with secondary antibodies for 1 h at room temperature and stained with DAPI. Paraffin-embedded tissues were cut into 7-μm sections, and sections were then fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized in 0.1% Triton X-100 at 4°C for 10 min, and blocked with UltraV block (Thermo Scientific) for 7 min. The primary antibodies rabbit anti-Turbo-GFP (Evrogen), mouse anti-CD31 (CD31, Dako), rabbit anti-HNA (Abcam), and rabbit anti-cTnT (Abcam) were added to the UltraV block buffer, and sections were incubated at 4°C overnight; next, sections were incubated with the fluorescence-conjugated secondary antibodies FITC-donkey anti-mouse, FITC-donkey anti-rabbit, Cy3-donkey anti-mouse, Cy3-donkey anti-rabbit, and Cy5-donkey anti-mouse (Jackson ImmunoResearch Laboratory) for 1 h at room temperature, stained with DAPI, washed, and examined under a fluorescence microscope (Olympus XI).

Table 1.

Antibodies

Antigen	Vendor	Catalog No.	Type
Human cardiac troponin T	Abcam	ab91605	Rabbit monoclonal
NKX2.5	ThermoFisher	PA5-47322	Goat polyclonal
Anti-human nuclear antigen antibody	Abcam	ab191181	Mouse monoclonal
Anti-cardiac troponin I	Abcam	ab47003	Rabbit polyclonal
Anti-CopGFP	Evrogen	AB501	Rabbit polyclonal
VEGF	R&D Systems	AB-293-NA	Mouse monoclonal
CD63	Santa Cruz Biotechnology	sc-5275	Mouse monoclonal
Anti-CD31	Abcam	ab28364	Rabbit polyclonal
Connexin43	Abcam	Ab11370	Rabbit polyclonal
MLC2 atrial	Abcam	ab68086	Mouse polyclonal
MLC2 ventricular	Proteintech	10906-1-AP	Rabbit polyclonal
FITC-donkey anti-mouse	Jackson ImmunoResearch Laboratory	715-095-150	Polyclonal
Cy3-donkey anti-rabbit	Jackson ImmunoResearch Laboratory	711-165-152	Polyclonal
Cy5-donkey anti-mouse	Jackson ImmunoResearch Laboratory	715-175-150	Polyclonal

NKX2.5, NK2 homeobox 5; GFP, green fluorescent protein; VEGF, vascular endothelial growth factor; MLC2, myosin light chain 2.

Apoptosis analysis.

Cryosections of spheroids and embedded mouse hearts were TUNEL stained using an in situ Cell Death Detection Kit (Roche Applied Science) as directed by the manufacturer’s instructions and viewed at ×40 magnification, as previously reported (18). The number of TUNEL-positive cells and the total number of cells were counted in 5 fields/slide, 5–6 slides/animal.

Exosome isolation and detection.

A volume of 15 ml of fresh exosome-depleted culture medium was used for culturing of either 2 × 10⁵ hiPSC-derived cardiac cells grown in monolayers on round glass coverslips inside 24-well plates or spheroids generated in low-adhesion round-bottomed plates from an equivalent number (2 × 10⁵) of CMs. The culture medium from each type of CM culture was collected on days 1 and 7 and centrifuged at 1,500 rpm for 10 min. The resulting supernatants were passed through 0.22-μm filters to remove cell debris, and exosomes were then extracted from the medium using an ExoQuick precipitation solution kit (System Biosciences), resuspended in 50 µl PBS (pH 7.4), and stored at −80°C. Exosome identification nanoparticle tracking analysis was performed with a NanoSight LM10 (Malvern Instruments, Worcestershire, UK), and protein content in each sample was measured via a MicroBCA protein assay (ThermoFisher Scientific). Incorporation of VEGF was determined by Western blot analysis, as previously described (4). Exosome ultrastructure was analyzed by transmission electron microscopy and negative staining for background identification.

Enzyme-linked immunosorbent assays.

ELISAs were performed by using a VEGF Quantikine ELISA kit purchased from R&D (catalog no. DVE00). In brief, 15 ml of fresh exosome-depleted culture medium were used for culturing of either 2 × 10⁵ hiPSC-derived cardiac cells and were grown in monolayers on round glass coverslips inside 24-well plates or spheroids generated in low-adhesion round-bottomed plates from an equivalent number (2 × 10⁵) of CMs. The cell culture supernatants of monolayers and spheroids were collected before the experiment and stored at −20°C before use. First, 50 µl of assay diluent, contained in the ELISA kit, were added to each well followed by 200 µl of standard, the sample of a monolayer, or spheroid. Plates were incubated for 2 h at room temperature on a horizontal shaker. Plates were then washed four times with 400 µl washing buffer/well for each wash. The antibody conjugates were added to each well in a 200-µl volume. The plate was then incubated at room temperature for 1 h. Plates were again washed four times with washing buffer. The enzymatic color reaction was carried out by adding 200 µl of substrate solution to each well and incubation of the plate at room temperature for 20 min. Finally, 50 µl of stop solution were added to each well, and the color change of the solution in each well from blue to yellow was monitored visually. Plates were analyzed by the BioTek Synergy Hybrid Plate Reader at 450-, 540-, and 570-nm visible wavelengths and the concentration of the protein was calculated based on the background-normalized absorbance.

Statistical analysis.

Data are presented as means ± SE. Significance (P < 0.05) was evaluated via the Student’s t-test for comparisons between two values and via ANOVA or repeated ANOVA. All statistical analyses were performed with SPSS software (version 13.0, SPSS).

RESULTS

Generation and characterization of hiPSC-derived CMs.

hiPSCs were derived from human cardiac fibroblasts as previously described (17) and transduced with lentiviral vectors coding for GFP and Luc, which would enable cells differentiated from hiPSCs to be detected after administration to mouse hearts via immunohistochemistry (GFP) and bioluminescent imaging (Luc); transduction was confirmed via fluorescence and immunofluorescence (Fig. 1Ai) analysis of GFP expression. hiPSCs were cultured in monolayers for three passages and then differentiated into CMs (hiPSC-CMs) as previously described (17). Spontaneously contracting cells typically appeared 8–10 days after differentiation was initiated, GFP expression in the differentiated cells was confirmed via fluorescence and immunofluorescence [Fig. 1Aii and Supplemental Video S1 (Supplemental Material for this article is available at the American Journal of Physiology-Heart and Circulatory Physiology website)], and CM identity was confirmed via immunofluorescent assessments of the expression of the CM markers MLC2v, hcTnT, and connexin43 (Fig. 1D, ii and iii).

Fig. 1.

Generation and characterization of human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs). A: hiPSCs were differentiated into CMs for 16 days and then transferred in varying concentrations to round-bottomed wells; images were obtained via light microscopy at the indicated time points after plating. B: spheroid sizes were determined 24 after plating and plotted against the number of plated cells. C: spheroid-derived CMs were harvested on day 7 after spheroid plating and were stained for NK2 homeobox 5 (NKX2.5), phalloidin, and cardiac troponin I (cTnI) expression, and nuclei were counterstained with DAPI. D: CMs were also stained for human cardiac troponin T (hcTnT) expression (i) and for expression of the CM marker proteins ventricular myosin light chain 2 (MLC2v) (ii), myosin light chain 2A (MLC2a) (iii), and connexin43 (iv); nuclei were counterstained with DAPI. E: cryosections of a single spheroid were stained with hematoxylin and eosin (H&E; i) for expression of the necrosis marker phosphorylated mixed-lineage kinase domain-like (p-MLKL) protein (ii). Nuclei were counterstained with DAPI. F: conduction velocities and Ca²⁺ transients were measured in individual large (>800-µm diameter; i) day 7 spheroids to generate optical maps of action potential propagation (ii) and traces of individual action potentials (blue) and Ca²⁺ transients (red; iii). The number of each trace in iii corresponds to the location identified in the map shown in ii.

Generation and characterization of hiPSC-CM spheroids.

Spheroid formation was evaluated by transferring varying amounts of hiPSC-CMs into ultra-low-attachment, round-bottomed, 96-well plates (Fig. 1A). When seeded at a density of <2,500 cells/well, hiPSC-CMs formed loose clusters rather than spheroids (data not shown), but, at higher densities, cells quickly associated and occupied most of the free space. Spheroid sizes were proportional to the number of transferred cells after 24 h (Fig. 1B), and analyses of hiPSC-CMs identity were confirmed via immunofluorescent assessments of the expression of the cardiomyocyte-specific markers NK2 homeobox 5 (NKX2.5), cardiac troponin I (cTnI), and phalloidin. For MLC2v and MLC2A staining procedures, CMs were harvested on day 7 after spheroid plating. CMs showed expression of both MLC2v and MLC2A, as revealed by immunofluorescence staining with the corresponding antibodies [Fig. 1D, ii (MLC2V) and iii (MLC2A)]. Cells exhibited striation, which indicates that CMs matured in spheroids (Fig. 1C). Cells were uniformly distributed throughout the spheroids (Fig. 1Ei), and, although a small number of cells located in the center of the spheroids expressed the necrosis-specific marker phosphorylated mixed-lineage kinase domain-like (Fig. 1Eii), spheroids were composed almost entirely of live cells and displayed ample evidence of hcTnT and GFP expression (Fig. 2, A and C).

Fig. 2.

Generation and characterization of spheroid fusions. A: ∼50 small (200-µm-diameter), i.e., type 1, fusion and 2 big (800-µm-diameter), i.e., type 2, fusion human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs) were cultured in low-attachment round-bottomed wells; images were obtained 1 and 7 days after plating via light microscopy. A and B: fusions of types 1 and 2 cultured for 7 days. C–E: conduction velocities and Ca²⁺ transients were measured in the fusions of two large (>800-µm diameter) spheroids (i.e., type 2 fusions) after 1 (C) and 7 (D) days of culture (i) to generate optical maps of action potential propagation (ii) and traces of individual action potentials (blue) and Ca²⁺ transients (red; iii); the number of each trace in iii corresponds to the location identified in the map shown in ii. iv, Activation time (AT) for each trace was plotted against the map location. E: type 2 fused 7-day spheroids were stained with calcein-AM for live cells (green) and ethidium homodimer-1 for dead cells (red). F and G: durations of action potentials (F) and Ca²⁺ transients (G) until 50% (i.e., APD₅₀ and CaT₅₀, respectively; i) and 80% (i.e., APD₈₀ and CaT₈₀, respectively; ii) recovery were determined for a single large spheroid (red) and for fusions of 2 large spheroids after 1 (blue) and 7 (green) days in culture.*P < 0.05 [single spheroids (n = 16), spheroid fusion days 1–2 (n = 8), and spheroid fusion day 7 (n = 7), all individual measurements were obtained for each spheroid/fusion].

Optical mapping experiments of individual hiPSC-CM spheroids (Fig. 1Fi) indicated that the AP spread uniformly (Fig. 1Fii) with an average conduction velocity of 9.4 ± 5.7 cm/s (n = 16) and a prominent plateau phase (Fig. 1Fiii) similar to that observed in the adult human ventricular myocardium. However, unlike adult human myocardial tissue, CaTs of hiPSC-CM spheroids had a slow rising phase, increasing from 10% to 90% of peak amplitude over 59 ± 25 ms (n = 5). APD₅₀ (138 ± 45 ms) and CaT₅₀ (170 ± 61 ms) as well as APD₈₀ (161 ± 50 ms) and CaT₈₀ (62 ± 85 ms) were somewhat shorter than those observed in a normal human myocardium of 350 ms. The differences in CaTs may be partially attributable to our use of a low-affinity Ca²⁺-sensitive dye (Cal-520FF).

Generation and characterization of the hiPSC-CM spheroid fusions.

The fusion of multiple spheroids into an engineered cardiac tissue-like aggregate was evaluated by combining ~50 small (~200 µm) or 2–5 large (~800 µm) 1-day-old spheroids (i.e., type 1 or type 2 fusions, respectively) in 2-ml round-bottomed tubes. Spontaneous contractions were observed in the majority of fusions just 1 day later (Supplemental Videos S2 and S4), but the spheroids remained loosely associated, with individual spheroids easily distinguishable in each fusion (Fig. 2, A and B). By day 7, all of the spheroids within the fusion beat synchronously (Supplemental Videos S3 and S5), which suggests that the cells within each spheroid and in adjacent spheroids were electronically coupled. Day 7 fusions were also more compact and resembled tissue-like structures; however, type 1 fusions contained gaps that were likely caused by incomplete association of individual spheroids, rather than necrosis, because the fused regions displayed ample evidence of cTnT and GFP expression (Fig. 2C). Gaps were not present in the fusions of larger spheroids, and, consequently, type 2 fusions were used for all subsequent electrophysiological assessments and in vivo experiments.

Optical mapping of two-spheroid type 2 fusions indicated that the adjacent spheroids were poorly coupled after 1–2 days of culture (Fig. 2C): the average conduction velocity was 7.0 ± 3.8 cm/s within each spheroid but just 0.8 ± 1.1 cm/s (n = 8, P = 0.002 vs. intraspheroid) at the interface, and the AP upstrokes at the interface were biphasic, which is typically observed in regions where conduction is delayed. By day 7 (Fig. 2D), the interspheroid conduction velocity had increased significantly (to 3.3 ± 1.4 cm/s, n = 7, P = 0.003 vs. days 1–2) but remained somewhat slower than intraspheroid conduction (6.0 ± 2.1 cm/s, P = 0.017 vs. interspheroid), and the biphasic nature of APs at the spheroid interface was less pronounced. Most of the cells were positive for calcein-AM staining, indicating that cells were alive after 7 days of fusion and very few cells were positive for dead staining such as ethidium homodimer 1 (Fig. 2E). CaTs were consistent with AP traces at days 1–2 and 7, whereas both APD₅₀ and APD₈₀ as well as CaT₅₀ and CaT₈₀ in day 7 type 2 fusions were similar to the durations in individual spheroids and significantly longer than in day 1–2 type 2 fusions (Fig. 2, F and G). Collectively, these observations indicate that spheroid fusions became progressively more mature and electronically coupled during the 7-day culture period.

Patches composed of hiPSC-CM spheroids in a fibrin matrix improve cardiac performance in a murine model of MI.

The cardioprotective/regenerative capacity of hiPSC-CM spheroid fusions was evaluated in a murine model of MI. MI was surgically induced by permanently ligating the coronary artery, as previously described (20), and animals in the MI + spheroid group were treated with a single five-spheroid fusion containing a total of 1 million hiPSC-CMs. The spheroids were suspended in a fibrin patch positioned over the site of infarction and held in place with a film of sodium hyaluronate carboxymethylcellulose that was sutured to the heart preventing adhesion to the serous pericardium (Fig. 3A). The spheroids-patch and film were withheld from animals in the MI group and animals in the sham group underwent all surgical procedures for MI induction except for the ligation step. After MI and spheroid administration (4 wk), echocardiographic measurements of LV EF (Fig. 3C), FS (Fig. 3D), ESV (Fig. 3E), and EDV (Fig. 3F) as well as histological assessments of infarct size (Fig. 3, G and H) and the ratios of heart weight to body weight (Fig. 3B) were significantly better in animals from the MI + spheroid group than in the MI group.

Fig. 3.

Patch-mediated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CM) transplantation improves recovery from mice with myocardial infarction (MI). MI was surgically induced in mice. A: animals in the MI + spheroid group were treated with a single 5-spheroid fusion containing a total of 1 × 10⁶ cells/animal. The total number of hiPSC-CMs cells administered was 1 million. Spheroids were suspended in a fibrin/thrombin patch positioned over the site of infarction. The experimental treatment was withheld from animals in the MI group; fused spheroids of the whole patch and animals in the sham group underwent all surgical procedures for MI induction except the ligation step. B: heart weight-to-body weight ratios (HW/BW) were determined for animals euthanized at week 4. *P < 0.05. C–F: echocardiographic assessments of left ventricular (LV) ejection fractions (EF; C), LV fractional shortening (FS; D), end-systolic volume (ESV; E), and end-diastolic volume (EDV; F) were performed before MI induction or sham surgery (baseline) and 4 wk afterward. *P < 0.05. G and H: cryosections of LVs of animals euthanized at week 4 were stained with Sirius red/fast green to visualize regions of fibrotic (red) and nonfibrotic (green) tissue (G), and infarct size was then quantified as the percentage of the total LV surface that was stained red (H). *P < 0.05.

Patch-mediated hiPSC-CM spheroid transplantation is associated with high rates of engraftment.

Transplanted hiPSC-CMs were identified at the injury site via histological assessments of the expression of GFP, hcTnT, and/or HNA (Fig. 4A), and the engraftment rate was determined histologically, by counting the number of cells that expressed both hcTnT and HNA, and via BLI assessments in living mice (Fig. 4B); both methods indicated that the engraftment rate exceeded 25% (hcTnT/HNA expression: 25.57 ± 3.10% and BLI: 30.99 ± 2.5%; Fig. 4C).

Fig. 4.

Patch-mediated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CM) spheroid transplantation leads to a high rate of engraftment. A: transplanted hiPSC-CMs were identified at the site of administration in myocardial infarction (MI) + spheroid animals by staining for the expression of green fluorescent protein (GFP), human cardiac troponin T (hcTnT), and/or human nuclear antigen (HNA); native-like and transplanted CMs were visualized by staining for cTnT, and nuclei were counterstained with DAPI. B: bioluminescence assessments were performed in living animals before euthanization at week 4. i, Known quantities of cultured hiPSC-CM derived spheroids were imaged, and the data were used to plot a standard curve of the relationship between bioluminescent imaging (BLI) intensity and cell number (ii). iii, Bioluminescence images were obtained 10 min after mice were injected with d-luciferin; next, the BLI signal intensity was compared with the standard curve to determine the number of engrafted cells. C: engraftment rate was calculated by dividing the number of hcTnT/human nuclear antigen (HNA) double-positive cells (histology) or the number of cells determined via BLI by the total number of cells administered and expressed as a percentage.

Patches containing hiPSC-CM spheroid are readily vascularized by the native circulatory system.

Macroscopic observations indicated that the transplanted spheroid-containing patches were well vascularized (Fig. 5A), and this observation was confirmed in immunostained sections of heart tissue. Regions composed of predominantly native-like tissue structures were identified via cTnI and α-actinin expression, which occurs primarily in mature CMs, whereas the patch region was identified by the presence of a large number of transplanted (i.e., HNA and/or hcTnT positive) cells, and blood vessels were identified via expression of the endothelial marker CD31 (Fig. 5B); CD31 was abundantly expressed in regions populated by transplanted cells. CD31-expressing vascular structures were also significantly more common (Fig. 5C), whereas the proportion of apoptotic (i.e., TUNEL-positive) cells was significantly lower (Fig. 5D) at the border zone of the infarct in animals from the MI + spheroid group than from the corresponding region of hearts in the MI group. Our in vitro experiments showed that exosomes secreted by spheroids on days 1 and 7 contained a higher load of VEGF than exosomes from CM monolayer cultures (Fig. 6, A and B). The concentration of free VEGF, estimated by the ELISA method, was also higher in spheroid culture medium compared with that in the medium from CMs cultured in monolayers (Fig. 6C). The data support the concept of a paracrine mechanism of cell therapy (2, 10, 15, 19) that is associated with the VEGF released from grafts of hiPSC-CM spheroids (Fig. 6D).

Fig. 5.

Patches containing human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CM) spheroids are well vascularized 4 wk after transplantation. A: sections from the border of the infarcted region were stained for CD31 expression, and nuclei were counterstained with DAPI; next, vascular density was quantified as the number of CD31-positive structures/unit area. B: vascularity of the hiPSC-CM spheroid patch was visible in macroscopic images. C: cryosections from the region of treatment administration in myocardial infarction (MI) + spheroid animals were viewed via bright-field microscopy and stained for the expression of cardiac troponin I (cTnI), α-actinin (to identify mature native like CMs), human nuclear antigen (HNA), and/or hcTnT (to identify transplanted hiPSC-CMs) and CD31 (to identify endothelial cells on the vasculature); nuclei were counterstained with DAPI. D: sections from the infarct border zone were TUNEL stained, and nuclei were counterstained with DAPI; next, apoptosis was quantified as the percentage of cells that were TUNEL positive. *P < 0.05.

Fig. 6.

Characteristics and cytoprotective effects of spheroid-secreted exosomes compared with monolayers. Exosomes were isolated from the monolayers and spheroid culture medium at days 1 and 7. A: exosome morphology was evaluated via electron microscopy (bar = 100 nm) (i), and exosome size was evaluated via nanoparticle tracking analysis for monolayers (ii and iv) and spheroids (iii and v). B: VEGF protein expression Western blot of exosomes derived from monolayers and spheroids culture media at days 1 and 7. C: VEGF protein concentration measured via ELISA. D: cryosections from the region of treatment administration in myocardial infarction (MI) + spheroid animals were viewed via flouresence microscopy and stained for the expression of CD63 (to identify exosomes on the transplanted spheroid patches), hcTnT(to identify transplanted hiPSC-CMs), and VEGF; nuclei were counterstained with DAPI.

DISCUSSION

The important findings are that those hiPSC-CM spheroid fusions became more compact and mature over time and large (~800 µm diameter). The spheroids could be fused to form more complex necrosis-free structures. Furthermore, although electronic signal propagation was initially impaired at the interface between two fused spheroids, conduction velocity increased significantly as the fusion matured over a 7-day culture period, and contractile activity gradually became more synchronous, which suggests that CMs in each spheroid were electronically coupled to each other and to CMs in the adjacent spheroid. The transplantation of large spheroids was also associated with significantly better graft size that was accompanied by improvements in cardiac function, infarct size, and remodeling when suspended in a fibrin scaffold and transplanted over the site of myocardial injury in infarcted mouse hearts.

Cellular aggregation is an important event during embryonic development because it facilitates interactions between cells that are crucial for tissue differentiation and maturation. Cells also aggregate in culture, forming clusters that may replicate the interactions present in 3-D tissues such as cardiac muscle. Here, we show that when cultured in low-attachment round-bottomed wells, hiPSC-CMs form spheroids of cells that begin to contract synchronously within 24 h of formation and that the size of the spheroid is proportional to the available number of cells.

A number of studies have suggested that cardiac functional parameters improve when cells are administered via direct intramyocardial injection, (1, 16) or as a patch of engineered tissue (5, 17); however, the effectiveness of these approaches is limited by a variety of factors (6, 14, 21), including, perhaps most importantly, the exceptionally small proportion of cells that are engrafted by the native-like tissues at the site of administration. When tested in a murine MI model, we and others have previously carried out several CM implantation studies using either CM patch (5, 9) or CM suspension (20). It should be noted that in all previous studies performed both in our laboratory and by others, the engraftment rate was consistently and significantly lower than in the present study involving spheroids (less than ~10% vs. more than ~25%, respectively). Importantly, all of the studies performed in our laboratory (20) were performed under the same experimental conditions as the spheroid studies. These conditions included the same animal model, infarction procedure, surgical personnel, and source of CMs, CM differentiation and culturing methods, and engraftment rate assessment time point and methods. Two different methods (Luc quantification based and histological cell staining and counting based) were used in all of these studies. The treatment was associated with improvements in cardiac function and tissue vascularity. Notably, the in vivo experiments reported here were conducted in the same animal model used by Gao et al. (5), and the engraftment rate (25–30%) was more than twofold higher at the same time point (4 wk after patch administration). The underlying mechanism of the observed functional benefits is likely related to a cytokine effect, such as VEGF within the exosome that are released from the grafted spheroids (2, 10, 15, 19). These data indicate that the survival of transplanted CM spheroids is associated with a robust increase of angiogenesis, which, in turn, improved myocardial perfusion and cardiac function of the recipient heart. We performed in vitro experiments to show that exosomes secreted by spheroids on days 1 and 7 contain higher load of VEGF than exosomes from CM monolayer cultures (Fig. 6, A and B). The concentration of free VEGF, estimated by the ELISA method, was also higher in spheroid culture medium compared with that in the medium from CMs cultured in monolayers (Fig. 6C). The data support the concept of a paracrine mechanism of cell therapy that is associated with the VEGF released from grafts of hiPSC-CM spheroids (Fig. 6D). The engraftment rate of >25% found at 4 wk posttransplantation was a surprise and a prominent finding that was examined and validated in parallel by two independent methods (BLI and histological cell staining/counting). Because the low engraftment rate in cardiac cell therapy is one of the major obstacles in the field, this novel finding is highly significant.

Conclusions

The results presented here demonstrate that cultured hiPSC-CMs form spheroids, that the size of the spheroids can be controlled by varying the number of hiPSC-CMs available, and that large (~800-µm diameter) spheroids fuse in culture to produce structures with uniformly distributed cells. Furthermore, hiPSC-CMs in adjacent fused spheroids became electromechanically coupled as the fusions matured in vitro, and, when the spheroids were combined with a biological matrix and administered as a patch over the infarcted region of mouse hearts, the engraftment rate exceeded 25%, and the treatment was associated with significant improvements in cardiac function via a paracrine mechanism, where exosomes released from the spheroid patch.

GRANTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants RO1-HL-95077, HL-114120, HL-131017, and UO1-HL-134764 (to J. Zhang).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.M., W.Z., V.G.F., L.G., C.W., and R.K. performed experiments; S.M., W.Z., and V.G.F. analyzed data; S.M., W.Z., and V.G.F. interpreted results of experiments; S.M., L.G., C.W., R.K., and A.V.B. prepared figures; S.M., W.Z., V.G.F., A.V.B., and J.Z. drafted manuscript; S.M., V.G.F., A.V.B., and J.Z. edited and revised manuscript; J.Z. conceived and designed research; J.Z. approved final version of manuscript.