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Therapeutic potential of clinical-grade human induced pluripotent stem cell-derived cardiac tissues

Objectives To establish a protocol to prepare and transplant clinical-grade human induced pluripotent stem cell (hiPSC)-derived cardiac tissues (HiCTs) and to evaluate the therapeutic potential in an animal myocardial infarction (MI) model. Methods We simultaneously differentiated clinical-grade hiPSCs into cardiovascular cell lineages with or without the administration of canonical Wnt inhibitors, generated 5- layer cell sheets with insertion of gelatin hydrogel microspheres (GHMs) (HiCTs), and transplanted them onto an athymic rat MI model. Cardiac function was evaluated by echocardiography and cardiac magnetic resonance imaging and compared with that in animals with sham and transplantation of 5-layer cell sheets without GHMs. Graft survival, ventricular remodeling, and neovascularization were evaluated histopathologically. Results The administration of Wnt inhibitors significantly promoted cardiomyocyte (CM) (P < .0001) and vascular endothelial cell (EC) (P = .006) induction, which resulted in cellular components of 52.0 ± 6.1% CMs and 9.9 ± 3.0% ECs. Functional analyses revealed the significantly lowest left ventricular end-diastolic volume and highest ejection fraction in the HiCT group. Histopathologic evaluation revealed that the HiCT group had a significantly larger median engrafted area (4 weeks, GHM(-) vs HiCT: 0.4 [range, 0.2-0.7] mm2 vs 2.2 [range, 1.8-3.1] mm2; P = .005; 12 weeks, 0 [range, 0-0.2] mm2 vs 1.9 [range, 0.1-3.2] mm2; P = .026), accompanied by the smallest scar area and highest vascular density at the MI border zone. Conclusions Transplantation of HiCTs generated from clinical-grade hiPSCs exhibited a prominent therapeutic potential in a rat MI model and may provide a promising therapeutic strategy in cardiac regenerative medicine.

HiCTs generated from clinical-grade hiPSCs show prominent therapeutic potential.

CENTRAL MESSAGE
Transplantation of clinical-grade human induced pluripotent stem cell-derived cardiac tissues resulted in functional recovery in an animal myocardial infarction model and may be a promising therapeutic strategy in cardiac regenerative medicine. PERSPECTIVE We have established a method to generate and transplant clinical-grade human induced pluripotent stem cell-derived cardiac tissues and validated their therapeutic potential in myocardial regeneration accompanied by vascular networks in an animal myocardial infarction model.The preclinical proof-of-concept of the efficacy of this therapeutic strategy provides further support for this therapeutic modality in patients with severe heart failure.

See Commentaries on pages 375 and 377.
Video clip is available online.
Stem cell products manufactured from various stem cell populations are being increasingly applied for cardiac regenerative therapy [1][2][3] Among the stem cell populations tested in basic research, pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have demonstrated a robust potential for cardiac function restoration owing to their ability to produce cardiovascular cells for supplementation to an injured heart. 4 Pluripotent stem cell-based cardiac regenerative therapy may be an optimal therapeutic strategy for severe heart failure, considering the shortage of donor hearts for organ transplantation worldwide. 5,6 The benefit of iPSCs over ESCs is the availability of patients' own autologous cells for treatment. When iPSC products are generated autologously, better engraftment free from the risk of immune rejection after transplantation can be theoretically anticipated. Despite the advantage of autologous iPSCs, disadvantages in the setting of autologous iPSC transplantation exist, including the cost and time required for quality control for each individual. 7 Furthermore, autologous cell products from patients with cardiomyopathy attributed to genetic mutations may take over its disease phenotype, which would hamper the therapeutic effects of the products. Clinical-grade human iPSCs (hiPSCs) established from human leukocyte antigen (HLA)-homozygous healthy volunteers are currently being considered as a quality-controlled cell source available for allogeneic transplantation. This cell stock or bank has been established in which good manufacturing protocol (GMP)-grade cell-processing facilities provide qualitycontrolled hiPSCs for clinical use. [8][9][10][11] We have been investigating the systematic induction of various cardiovascular cells-cardiomyocytes (CMs), vascular endothelial cells (ECs), and vascular mural cells (MCs)-from ESCs and iPSCs and preclinical validation of the therapeutic potential of induced cardiovascular cell-engineered 3-dimensional (3D) transplantable grafts using animal disease models. [12][13][14][15][16][17][18] In the present study, we aimed to evaluate the differentiation efficacy of clinical-grade hiPSCs into cardiovascular lineages as a cellular candidate for future clinical use and to validate the preclinical functional efficacy of transplantation surgery using hiPSC-derived cardiac tissue (HiCT) bioengineered by clinical-grade hiPSCderived cardiac cell sheets and gelatin hydrogel microspheres (GHMs) 19,20 in a rat myocardial infarction (MI) model.

METHODS
The experimental materials and methods are described in more detail in Appendix 1.

Differentiation of Clinical-Grade hiPSCs Into Cardiovascular Cell Lineages
We used clinical-grade hiPSC lines (FfI01s04 and QHJI01s04) that are peripheral monocyte-derived HLA-homozygous hiPSCs established at the Facility for iPS Cell Therapy Center for iPS Cell Research and Application, Kyoto University, Japan. Clinical-grade hiPSCs were simultaneously differentiated into cardiovascular cell lineages with (Wiþ) or without (Wi-) canonical Wnt inhibitors XAV939 and IWP4 at differentiation days 5 to 7 (Figure 1, A). At 13 to 15 days after differentiation, cardiovascular cells were dissociated and subjected to flow cytometry analysis. Cells from the Wi þ group were seeded onto 12-well temperature-responsive culture plates. At 2 to 4 days after seeding, self-pulsating cell sheets were collected and then subjected to flow cytometry analysis.

Preparation of HiCTs
We generated 5 layers of cell sheets with insertion of GHMs (HiCTs) and also prepared 5 layers of stacked sheets without GHMs [GHM(-)] for transplantation experiments.

Abbreviations and Acronyms
3D

Animals
Male athymic nude rats (F344/NJcl-rnu/rnu, 8-12 weeks old) were purchased from CLEA Japan (Tokyo, Japan), and housed in a controlled environment. All animal experiment protocols were approved by the Animal Experimentation Committee of Kyoto University (#Med Kyo 19540). All animal experiments were performed according to the Guidelines for Animal Experiments of Kyoto University, which conform to Japanese law and the US National Research Council's Guide for the Care and Use of Laboratory Animals.
Induction of MI, HiCT Transplantation, and Follow-up MI was induced by permanent ligation of the left anterior descending coronary artery as reported previously. 15 Rats with left ventricular (LV) fractional shortening (FS) <30% by echocardiography on day 7 after ligation were enrolled in further experiments. Each enrolled rat was assigned at random to 1 of the 3 groups: 4-week observation, MI operation without transplantation group (sham; n ¼ 18), 5-layer cell sheets without GHM transplantation group [GHM(-); n ¼ 12], and the HiCT transplantation group (HiCT; n ¼ 12). Parts of each group [sham, n ¼ 8; GHM(-), n ¼ 6; HiCT, n ¼ 5] were followed up to 12 weeks post-transplantation. Echocardiography and cardiac magnetic resonance imaging (MRI) were conducted and compared among the groups.
At the end of the observation period, the animals were sacrificed, and graft survival, ventricular remodeling, and neovascularization after MI were histopathologically evaluated and compared among the groups. As a 3D evaluation of the vascular network formation among engrafted HiCTs, we performed light sheet fluorescence microscopy (LSFM) on transplanted rat hearts after tissue clearing by the clear, unobstructed brain imaging cocktails and computational analysis sample (CUBIC) method. 21,22

Statistical Analysis
Nonnormally distributed data are presented as median (interquartile range [IQR]), and normally distributed data are presented as mean AE standard deviation (SD). All data analyses were performed using JMP Pro 15.1 (SAS Institute, Cary, NC). P values for comparisons between 2 groups were obtained using the Wilcoxon/Kruskal-Wallis test. Comparisons between >2 groups were also performed using the Wilcoxon/Kruskal-Wallis test and post hoc comparisons between groups were performed with the Steel-Dwass multiple-comparison test. P < .05 was considered statistically significant.

Cardiovascular Cell Induction and Transplantable Cardiac Tissue Formation From Clinical-Grade hiPSCs
We collected cells on differentiation day 15 and subjected to flow cytometry for analysis of cellular components that resulted in significantly higher cardiac isoform of troponin T (cTnT) þ CM components (Wi-vs Wiþ, 34.5 AE 8.9% vs 52.0 AE 6.1%; P < .0001) and VE-cadherin þ EC components (Wi-vs Wiþ, 7.1 AE 3.1% vs 9.9 AE 3.0%; . We layered the collected cell sheets using GHMs to generate HiCTs ( Figure 1, E). The HiCT diameter was approximately 10 mm, and the thickness was approximately 800 mm.

Transplantation of HiCTs and Functional Recovery in a Rat MI Model
There were no significant differences among the 3 groups in echocardiographic parameters before transplantation (after MI induction). Echocardiography at 4 weeks posttransplantation revealed significantly lower LV diastolic dimension (LVDd) and LV end-diastolic volume (LVEDV) and higher LV ejection fraction (LVEF) and LV FS in the

Engraftment of HiCTs and Vascular Network Formation After Transplantation
We next histologically evaluated the transplanted grafts. All surviving rats during the observation period were free from tumor formation both macroscopically and histologically. We      (Figure 3, E and F), indicating that functional recovery after HiCT transplantation was mediated by the attenuation of LV remodeling by virtue of the HiCT transplantation.

Neovascularization After HiCT Transplantation and Vascular Network Formation in Engrafted HiCT
We evaluated the vascular density of the MI border zone and found that HiCT transplantation significantly increased the number of vWF þ capillaries in close proximity to the graft compared with the sham and GHM(-) groups at 4 and 12 weeks after transplantation [sham vs GHM(-) vs HiCT, 4 weeks: 2.5 AE 0.8 mm 2 vs 4.5 AE 0.3 mm 2 vs 9.6 AE 1.1 mm 2 , P ¼ .0008; 12 weeks: 2.1 AE 1.2 mm 2 vs 2.9 AE 0.6 mm 2 vs 7.6 AE 1.6/mm 2 , P ¼ .006] (Figure 4, A and B) LSFM analysis revealed that although vascular structure in the grafts was not clearly observed at 3 days posttransplantation, marked vascular network formation in the grafts was observed at 2 weeks post-transplantation (Figure 4, C; Video 3). Histologic evaluation of vascular structures in the grafts at 12 weeks post-transplantation revealed that the vascular structure consisted of both human cells (Ku80 þ ) and nonhuman rat cells (Ku80 -/hematoxylin þ ), indicating that the formation of chimeric vascular networks originated from both hosts and grafts (Figure 4, D).

DISCUSSION
In this study, we validated the efficacy of the inhibition of canonical Wnt/b-catenin signaling pathway in the differentiation of clinical-grade hiPSCs into cardiovascular lineage cells. The transplantation of HiCTs onto a rat MI model exhibited greater cell survival and recovery of cardiac function in accordance with attenuated LV remodeling, possibly mediated by neovascularization ( Figure 5). This work provides evidence supporting the possible clinical use of iPSCs in cardiovascular medicine and cardiac regenerative therapy, as originally established by Shinya Yamanaka. 23 We first attempted to further improve our previously reported cell differentiation method 16 with modifications in which we evaluated the effect of inhibiting the canonical Wnt/b-catenin signaling pathway at the mesoderm stage of differentiation, which is reported to promote cardiac cell differentiation from pluripotent stem cells. [24][25][26] The Wnt/ b-catenin signaling pathway is involved in a multitude of developmental processes and the maintenance of adult tissue homeostasis, including cell differentiation, proliferation, organogenesis, tissue regeneration, and tumorigenesis. 25 Although up-regulation of the Wnt/b-catenin signal is required for mesoderm differentiation, the cardiac specification process requires its inhibition, indicating the biphasic effect of the Wnt/b-catenin signal. 24 In the canonical Wnt/b-catenin signaling cascade, XAV939 inhibits tankyrase activity and increases the protein levels of the axin-GSK3b complex, which promotes b-catenin degradation by stabilizing axin. 27 IWP4 prevents palmitylation of Wnt proteins, which inhibits Wnt production. 25 Administering these inhibitors of the Wnt/b-catenin signaling cascade during the cardiac specification process in our protocol at differentiation day 5 to 7 (mesoderm state) effectively increased CM (by 1.5-fold) and EC (by 1.4-fold) components. The validation of enhanced cardiovascular cell differentiation from clinical-grade hiPSCs through timedependent regulation of canonical Wnt/b-catenin signal activity would be advantageous in promoting cardiac regenerative medicine based on hiPSCs. This is our first report of an effective modification of the method of induction of clinical-grade human iPS cell lines prepared for actual clinical use.
To further promote stem cell therapy, a suitable transplantation format that enables efficient cell engraftment is indispensable, considering the relatively poor efficiency of engraftment after transcoronary infusion or direct cell   injection into the myocardium owing to mechanical loss and the low survival rate of transplanted cells. 28,29 There is a known limitation of layering cell sheets to deliver a larger number of cells in vivo, as layering more than 3 cell sheets (>80 mm thickness) does not provide a thicker construct because of hypoxic cell damage inside of the construct, leading to central necrosis. 30 To overcome the problem associated with cell sheet layering, we have established a bioengineered technology to enhance the viability of thick-layered mouse ESC-derived cardiac tissue sheets (>1 mm; 15 layers) using GHMs, 19,20 a biomaterial supporting oxygen and nutrient supply, which attenuates hypoxic cell death and apoptosis in vitro and augments engrafted volume and period in vivo. 15 In this study, we demonstrate that the application of GHMs with the layering of clinical-grade HiCTs boosts the temporospacial potential of engraftment of human tissue in rat hearts, as well as functional recovery by virtue of the enhanced engraftment ( Figure 6). Histologic evaluation revealed that HiCT transplantation attenuated LV remodeling, which is commonly mediated by paracrine effects, such as neovascularization. 16,31 Tachibana and colleagues 32 reported that iPSC-derived CM transplantation conferred functional recovery in a mouse MI model with paracrine effects mediated by various cytokines with antiapoptosis, proangiogenesis, or pro-cell migration effects. We previously reported that VEGF serves mainly as an angiogenic paracrine factor in mouse ESC-derived cardiac tissue sheet transplantation. 16 The therapeutic effect in this study might be attributed to the augmentation of antiremodeling, angiogenic paracrine effects mediated by the engrafted tissue.
HiCT transplantation exhibited greater and longer engraftment of transplanted cells compared with GHM(-) transplantation. The engrafted tissue was supported by a vascular network consisting of chimeric vasculature with human and nonhuman vascular cells, which would have contributed to longer survival of the engrafted tissue. Although this requires further investigation, we presume that the engrafted tissue served as a "biological drug reservoir" that can provide preferential cytokines for the repair of damaged heart tissue through the circulatory connection between host and graft, and the therapeutic effects as a "reservoir" sustained by the longer survival of the engrafted tissue with a vascular network. The survival of transplanted grafts depends on an adequate nutrient and oxygen supply, which would be provided by direct diffusion from the surrounding tissue until the vascular network is formed. It may be possible that transplanted cardiac tissues supplemented with GHMs could survive only by direct diffusion until the development of tissue-equipped vascular networks that allow the long-term engraftment of HiCTs.
In this study, we validated the therapeutic potential of HiCTs using clinical-grade materials. The preclinical proof-of-concept of therapeutic efficacy with clinicalgrade products has great significance for the realization of cardiac regenerative medicine using hiPSCs. We also need to recognize the areas remaining to be resolved for the clinical application of this strategy, including investigations of tumorigenicity 8,33 and arrhythmogenicity. 34 In particular, the risk of tumorigenicity should be studied before clinical implementation of this cell-based therapy. At least in the present study with its 4-and 12-week observation periods, we did not detect any neoplasm formation. In general, activation of the Wnt signaling pathway may be related to cancer formation owing to accelerated cell proliferation, according to previous  studies. 25 On the other hand, we performed Wnt inhibition (not activation) in the present study, which might explain why we did not observe tumor formation. Nonetheless, we recognize the importance of further investigations of tumorigenicity with longer observation periods. In addition, further validation of therapeutic effects and mechanisms is needed in more severe pathologic conditions such as chronic ischemic cardiomyopathy and idiopathic dilated cardiomyopathy, the main disorders associated with heart transplantation and regenerative therapy. This, along with further refinements in the manufacturing and quality control of clinical-grade products, could provide a fundamental technological basis for hiPSC-based cardiac regenerative therapy as a standard therapeutic option in the future.
This study has several limitations. First, there is an interspecies difference between rats and humans, which raises the possibility that the immune reaction in rats might not be relevant to that in humans. To avoid interspecies bias, allogeneic transplantation experiments will be indispensable in our future studies. 34,35 Second, because MI in this study model was not atherosclerotic as in human clinical settings, but rather was induced by experimental coronary ligation, some of the biological effects of HiCT transplantation in our rat MI model might be inconsistent with those in human treatment. This possibility should be evaluated using such medical modalities as myocardial perfusion scintigraphy in clinical trials.

CONCLUSIONS
HiCT generated from clinical-grade hiPSCs is a feasible cell product for treating MI, as validated by our rat MI model experiments. Clinical-grade hiPSCs are a potentially reasonable cell source for cardiac regenerative medicine with foreseeable clinical applications.

Conflict of Interest Statement
Dr Yamashita is a founder, equity holder, and scientific adviser of iHeart Japan Corporation. Drs Yamashita and Masumoto are co-inventors listed on multiple pluripotent stem cell-related patents. All other authors reported no conflicts of interest.
The Journal policy requires editors and reviewers to disclose conflicts of interest and to decline handling or reviewing manuscripts for which they may have a conflict of interest. The editors and reviewers of this article have no conflicts of interest.
We thank Kenji Kakuta (iHeart Japan Corp) for providing the clinical-grade HiCT; Yasuyo Matsubara and Naoko Tada

APPENDIX 1. METHODS Differentiation of Clinical-Grade hiPSCs into Cardiovascular Cell Lineages
We used clinical-grade hiPSC lines (FfI01s04 and QHJI01s04) that are peripheral monocyte-derived HLA-homozygous hiPSCs established at the Facility for iPS Cell Therapy Center for iPS Cell Research and Application, Kyoto University. For cardiovascular cell differentiation, we dissociated hiPSCs using 30.5 TrypLE Select (Thermo Fisher Scientific, Waltham, Mass) diluted with 0.5 mM EDTA and plated them onto Matrigel-coated plates (growth factor reduced, 1:60 dilution; Corning Inc, Corning, NY) at a density of approximately 150,000 to 160,000 cells/cm 2 in AK02N or AK03N medium (Ajinomoto Pharma, Tokyo, Japan) with 10 mM Y-27632 (FUJIFILM Wako Pure Chemical, Osaka, Japan) 2 days before induction. Cells were covered with Matrigel (1:60 dilution) and AK02N or AK03N medium 1 day before induction. The medium was replaced with RPMI þ B27 medium (RPMI 1640 plus 2 mM L-glutamine or Glutamax plus B27 supplement without insulin) (Thermo Fisher Scientific, Waltham, Mass) supplemented with 100 ng/mL Activin A (R&D Systems, Minneapolis, Minn) for 1 day, followed by 10 ng/mL bone morphogenetic protein 4 (BMP4; R&D Systems) and 10 ng/mL basic fibroblast growth factor (bFGF; FUJIFILM Wako Pure Chemical) for 4 days without culture medium change.
At differentiation day 5, the culture medium was replaced with RPMI þ B27 medium with 50 ng/mL recombinant human vascular endothelial growth factor 165 amino-acid isoform (VEGF 165 ; FUJIFILM Wako Pure Chemicals) and Wnt inhibitors (5 mM XAV939 [Merck Millipore, Burlington, Mass] and 2.5 mM IWP4 [Stemgent, Cambridge, Mass]). Culture medium was refreshed every other day with RPMI þ B27 medium supplemented with 50 ng/mL VEGF 165 . All reagents were prepared with clinical-grade materials for the culture of QHJI01s04 iPS cells.
At 13 to 15 days after differentiation, cardiovascular cells were dissociated by incubation with Accumax (Innovative Cell Technologies, San Diego, Calif) or Versene (Thermo Fisher Scientific) plus 0.05% Trypsin solution with EDTA (Thermo Fisher Scientific) and seeded on fetal bovine serum (FBS)-coated 12-well temperatureresponsive culture plates (UpCell; CellSeed, Tokyo, Japan) E1,E2 with a cell density of 1.5 3 10 6 cells/well with 3 mL of alpha minimum essential medium (aMEM; Thermo Fisher Scientific) or RPMI 1640 supplemented with 10% FBS or B27, 50 to 100 ng/mL VEGF 165 , and 20 mM Y-27632 or, alternatively, 5.5 mmol/L 2mercaptoethanol, 50,000 U/L penicillin, and 50 mg/L streptomycin, at 37 C under 5% CO 2 . At 2 to 4 days after seeding, the culture plates were moved to room temperature for detachment of cell sheets. Cell sheets detached spontaneously within 15 to 30 minutes,.

Preparation of HiCTs
Each detached cell sheet was gently aspirated into a 10-mL pipette and transferred onto a noncoated or Matrigel-coated 60-mm culture dish. Once the cell sheet was spread without folds by aspirating the medium, GHMs dissolved with 10 mL of phosphate-buffered saline (50 mg/mL) were spread on the surface of the cell sheet. The dishes were incubated at 37 C for 45 minutes to allow the cell sheet to adhere to the culture dish. Then the second cell sheet was placed on the top of the first sheet and attached without folds by aspirating the media. Similarly, stacked constructs with 5 layers (HiCTs) were prepared. We also prepared 5 layers of stacked sheet without GHMs as GHM(-). Transplantation was performed 1 day after stacking. HiCTs and GHM(-) generated with GMPgrade materials were prepared and provided by iHeart Japan Corporation (Kyoto, Japan). GHMs were prepared by dehydrothermal cross-linking of gelatin microspheres prepared in a water in oil emulsion state according to a previously reported method. E1,E3 GMP-grade GHMs were provided by Shibuya-Kogyo Industries (Kanazawa, Japan).

Flow Cytometry Analysis
For flow cytometry analysis, differentiated cells were stained with combinations of allophycocyanin-conjugated CD90, phycoerythrin (PE)-conjugated PDGFR-b (BD Biosciences, San Jose, Calif), PE-conjugated vascular endothelial cadherin (VE-cadherin), and PE-conjugated TRA-1-60 (BD Biosciences). Cells were stained with the LIVE/DEAD Fixable Aqua Dead Cell Staining Kit (Thermo Fisher Scientific) to eliminate dead cells. For intracellular proteins, cells were fixed with 4% paraformaldehyde (PFA) and stained with combinations of cardiac isoform of troponin T (cTnT; Thermo Fisher Scientific) labeled with Alexa Fluor 488 using Zenon technology (Thermo Fisher Scientific), then subjected to analysis with a FACSAria II (BD Biosciences) and FACS-Diva software version 8.0 (BD Biosciences).

Animals
Male athymic nude rats (F344/NJcl-rnu/rnu, 8-12 weeks old) were purchased from CLEA Japan (Tokyo, Japan) and housed in a controlled environment. All animal experimental protocols were approved by the Animal Experimentation Committee of Kyoto University (#Med Kyo 19540). All animal experiments were performed according to the Guidelines for Animal Experiments of Kyoto University, which conform to Japanese law and the US National Research Council's Guide for the Care and Use of Laboratory Animals.

MI Induction and HiCT Transplantation
The animals were intubated with a 16-gauge angiocath (Terumo, Tokyo, Japan) and mechanically ventilated under