Advances in the Generation of Constructed Cardiac Tissue Derived from Induced Pluripotent Stem Cells for Disease Modeling and Therapeutic Discovery

The human heart lacks significant regenerative capacity; thus, the solution to heart failure (HF) remains organ donation, requiring surgery and immunosuppression. The demand for constructed cardiac tissues (CCTs) to model and treat disease continues to grow. Recent advances in induced pluripotent stem cell (iPSC) manipulation, CRISPR gene editing, and 3D tissue culture have enabled a boom in iPSC-derived CCTs (iPSC-CCTs) with diverse cell types and architecture. Compared with 2D-cultured cells, iPSC-CCTs better recapitulate heart biology, demonstrating the potential to advance organ modeling, drug discovery, and regenerative medicine, though iPSC-CCTs could benefit from better methods to faithfully mimic heart physiology and electrophysiology. Here, we summarize advances in iPSC-CCTs and future developments in the vascularization, immunization, and maturation of iPSC-CCTs for study and therapy.

Figure 2. Constructed Cardiac Tissues: (A).iPSC-CCTs are 3D tissues that produce more physiologically relevant, life-like cardiac tissue than 2D tissue cultures, which can be used to study physiology, test therapies, and be developed for transplantation.These emerging platforms can broadly be arranged by emphasis on high production volumes, that is, high-throughput, or on high physiological relevance.(B).Top-down strategies enable batch-scales, often leveraging iPSC self-assembly to produce tissues with diverse cell types using minimal intervention during culture.This is particularly useful for drug discovery and personalized screenings.Bottom-up strategies involve the modular assembly of cells and components, usually into scaffolded tissue, which enable the finetuning of cardiac tissues.This is particularly useful in organ modeling, including modeling healthy versus diseased tissue.Combining middle-out production methods and top-down self-assembly is promising for regenerative medicine, which benefits from biological complexity but also scalable production.2D, two-dimensional; 3D, three-dimensional; BoC, body-on-a-chip; CCT, constructed cardiac tissue; CO, cardiac organoid; CS, cardiac spheroid; EHT, engineered heart tissue; HoC, heart-on-achip; iPSC-CCT, iPSC-derived CCT; iPSC, induced pluripotent stem cell. .iPSC-CCTs are 3D tissues that produce more physiologically relevant, life-like cardiac tissue than 2D tissue cultures, which can be used to study physiology, test therapies, and be developed for transplantation.These emerging platforms can broadly be arranged by emphasis on high production volumes, that is, high-throughput, or on high physiological relevance.(B).Top-down strategies enable batch-scales, often leveraging iPSC self-assembly to produce tissues with diverse cell types using minimal intervention during culture.This is particularly useful for drug discovery and personalized screenings.Bottom-up strategies involve the modular assembly of cells and components, usually into scaffolded tissue, which enable the finetuning of cardiac tissues.This is particularly useful in organ modeling, including modeling healthy versus diseased tissue.Combining middle-out production methods and top-down self-assembly is promising for regenerative medicine, which benefits from biological complexity but also scalable production.2D, two-dimensional; 3D, three-dimensional; BoC, body-on-a-chip; CCT, constructed cardiac tissue; CO, cardiac organoid; CS, cardiac spheroid; EHT, engineered heart tissue; HoC, heart-on-a-chip; iPSC-CCT, iPSC-derived CCT; iPSC, induced pluripotent stem cell.

Cardiac Organoids (COs)
Spheroids are an important tissue engineering milestone, giving rise to robust platforms such as iPSC spheroids and cardiac spheroids (CSs).Unlike organoids, spheroids are generally simple aggregates of one or more cell lines, often terminally differentiated [9,61,75,76,83,84].CSs may be added to existing organoids to introduce new cell types and structures, even mimicking the fusion of the proepicardial organ with the developing heart [56].This "CCT building block" technique is used for coculture with MSCs, macrophages (MFs), and heart layer tissue to produce more complete systems [7,36,43,56,61].Spheroids have the advantage of well-defined ratios of cell populations [9,14].While spheroids are commonly used as tumor models given their hypoxic core [48,75,85], this hypoxic gradient can be useful in modeling cardiac ischemia [4,14,16,46].
Ventricular-and chamber-forming COs have been developed via biphasic Wnt modulation [56,86].All COs receive exogenous bFGF and BMP-4 to produce CMs and some ECs, though the further addition of VEGF-A produces larger EC and CF populations in distinct layers.EpiC CS aggregates are added and partially envelope the COs [56].Harnessing crosstalk between embryonic germ layers, both Silva et al. and Branco et al. achieved epicardium and myocardium induction through mesodermalendodermal co-emergence [52,71], while Drakhlis et al. used mesodermal-endodermal foregut co-emergence to establish the endocardium and myocardium [51,52], establishing the beginnings of an EC vascular network through para-and juxtracrine interactions with developing foregut tissue [51,71].Lewis-Israeli et al. achieved multiple, interconnected chambers in COs possessing all three heart layers from a single mesodermal lineage using triphasic Wnt modulation, boasting several populations of important supporting cells like ECs, CFs, and EpiCs, together resulting in microvasculature [45,47].While Lewis-Israeli et al. did not report staining for mural markers, Silva et al. also achieved significant epicardial populations and reported microvasculature stabilized by PCs and CF-like VSMCs.These studies suggest that substantial EpiC and epicardial-derived cell populations (e.g., CFs and mural cells like VSMCs/PCs) hold promise in developing mature vasculature through vessel stabilization and smooth muscle reinforcement [10,45,47,50,65,71] (Figure 4).CO technology continues to evolve, boasting cell-type diversity and a tendency toward vascularization [20,23,28,32,38,46,47,52,57,63,67,[70][71][72].
HoCs are capable of capturing several features of physiological tissue, but contributions are particularly innovative in studying and producing vascularized CCTs [3,24,30,34,39,46,58,75,76].Fluid driving with peristaltic, acoustic, and pressure-driven pumps offers the direct emulation of vascular perfusion.Modeling vascular perfusion promises to provide key insights into vascular development, perhaps even quantitatively, especially for flow-mechanosensitive ECs and associated Notch signaling [3,34,45,61,76].Obtaining vascular perfusion in vitro remains challenging, but HoCs can replicate perfusion by directing flow across specific cell populations [24,30,34,37,45,46,58,61,65,75,76] (Figure 4), which can also extend the lifetime of in vitro tissues [43,46].Producing gas gradients in HoCs may offer insight into therapeutic mechanisms.Several gasses are studied in heart medicine, and HoCs offer a platform for their integration with in vitro tissues.Nitric oxide/NO is a cardioprotective, vasodilatory gas implicated in angiogenesis and released during vascular perfusion in ECs [65].Reduced nitric oxide synthase levels are detected in CCTs after damaging inflammatory stimuli [65] and nitric oxide-releasing hydrogels show promise in treating MI [11].Interestingly, common toxic gasses in small quantities like carbon monoxide and hydrogen sulfide can have cardioprotective effects.The release of these gasses from implanted hydrogels can combat inflammation and apoptosis in MI [11].Perhaps such gasses could be directly introduced into cultures to simulate mechanotransduction and/or cardioprotective cues, for instance, in modeling vascular perfusion via fluid flow and nitric oxide exposure.
A powerful example of iPSC-CCTs in drug discovery comes from a series of Mills et al. publications in 2017, 2019, and 2021 using COs for HTDS.Their platform uses iPSC-derived COs, the coupling of tissue to physical stretchers, and the supplementation of culture media with BSA-FAs for metabolic maturation.This multifaceted platform reflects field-wide advancements, using biological, chemical, and physical methods to produce life-like tissues.After the platform was developed in 2017, researchers screened >100 cardio-regenerative compounds in 2019, successfully identifying drugs that induce the proliferation of otherwise quiescent CMs.Here, drug screening also contributed to our mechanistic knowledge, implicating the mevalonate signaling pathway in CM proliferation.Demonstrating adaptability and reproducibility, Mills et al. applied their established CO-HTDS platform to the COVID-19 pandemic, screening promising therapeutic compounds through 2021 from FDA drug libraries.Bromodomain and extraterminal family inhibitors (BETis) were discovered to rescue cardiac function from inflammation-induced dysfunction, specifically following infectious cytokine storms.Such screenings advance our understanding of pathogenesis, especially the role of inflammation in CVD.This platform synthesizes recent iPSC-CCT innovations to produce life-like tissue at a high enough throughput and scale to accommodate HTDS.Even in its infancy, CO-HTDS is capable of screening potentially hundreds of compounds.As HTDS is further developed, we can expect iPSC-CCTs to play an increasingly influential and productive role in drug discovery [17,18,44].

Regenerative Medicine
Cells, factors, and stimuli that induce specific tissue growth and development in CCTs could be applied as therapies to regenerate that same tissue in patients.iPSC technology offers such tools for manipulating cell fate, which are useful for regenerative medicine [76].Cardiac regenerative medicine seeks to replenish poorly proliferative CMs, regenerate supporting tissue, and re-establish vasculature to improve heart function [76].Pro-regeneration signals can be delivered through iPSC-CCTs via cellular and acellular constructs using various degrees of scaffold usage versus self-assembly [7,8,15,35,36] (Figure 3).Hydrogel CCTs frequently use controlled degradation with loaded cells and/or factors, sometimes spatially organized, to facilitate the recovery of damaged tissue [2,11,15].Several ongoing clinical trials deliver iPSC-CMs or iPSC-CCTs via injection, intravenous administration, and transplantation [1,8].Even so, conventional cell therapies struggle in clinical translation because of rapid cell death upon transplantation [10,30,34,35].
Transplanting tissues with diverse cell types, especially self-assembled CCTs like COs/organoids, has promise in overcoming low cell viability and enhancing host integration compared with cell solutions and hydrogels [1,7,30,36,38,48,83]. Cell-seeded hydrogels struggle to obtain physiological cell density, but these cell-cell interactions are recapitulated when iPSCs are guided to assemble into cohesive tissues with multiple cell types.The result is the improved viability of transplanted tissue and cellular communication with the host.Compared with monoculture transplants, cellular diversity demonstrates improved transplant viability and repair of IR injuries in several MI models [7,23,30,36,38].Though CCTs and iPSC-CCTs in preclinical studies are primarily transplanted into immunodeficient mice, rats, and pigs, transplanting autologous iPSCs holds promise in reducing immunogenic risk in humans without systemic immunosuppression [1,7,23,30,38,39,48,67,83].Hypoimmunogenic iPSC-CCTs have been developed using PD-L1 overexpression or MHC-II underexpression [34].Similar to the suppression of MHC-encoding HLA-I and -II genes, HLA-E overexpression has also been suggested [1].Excitingly, MHC-matched allogenic iPSC-CM injections survive for up to 12 weeks and improve MI recovery in cynomolgus monkeys.While non-fatal arrhythmia occurrence peaked 14 days after transplantation, integration with host CMs was observed, and this rate fell with time, possibly as a result of in vivo transplant maturation [27].A key success, host vascular integration is achieved using "biologically talkative" CSs or COs, whose robust paracrine signaling owes in part to crosstalk between cell types as the tissue develops [23,38,50,61,67].This has contributed to burgeoning clinical trials for iPSC-CCTs [1,8], including patches for treating ischemic myopathy [1,23], engineered myocardium for ventricular assistance [1], and CSs for treating HF [1,39].These methods represent significant advancements in the practicality and efficacy of cell therapy.
Acellular alternatives have also been explored to circumvent these conventional difficulties of cellular regenerative medicine.Stem cells were once thought to seed new tissue but are now thought to function primarily through paracrine signaling via secreting extracellular vesicles, particularly exosomes.Exosomes are protein-modified lipid vesicles containing growth factors, cytokines, miRNAs, and other compounds with potent regenerative, anti-inflammatory, and pro-angiogenic effects on damaged tissue [3,8,33,35,91].Exosomes secreted by explanted cardiac stem cells have been used [33,91,92], but recently, exosome therapy (ExT) derived from iPSC cardiac tissue exosomes has demonstrated therapeutic promise in studies and ongoing clinical trials [3,35].Conditioning cardiac stem cells and iPSC-CCTs with culture conditions can even produce exosomes tailored to heart healing [4,33,35,91,92].ExT boasts less invasive administration compared with surgical implantation, instead offering the intravenous and catheter-guided injection of heart-and injury-homing exosomes [3,10,33,35,78].ExT circumvents many challenges of cell therapy like viability, immunogenicity, and transplant arrhythmia or teratoma [10] while also retaining similar comprehensive benefits [8,35].These cell-derived therapies have seen tentative success as many exosome-loaded cardiac patches undergo clinical trials [3], with others in development [93].

Figure 1 .
Figure 1.Heart Composition: (A) The adult human heart is divided via the septum into left and right halves.Each side has an atrium, ventricle, and OFT, with the muscular left ventricle pumping blood through the aorta out to the body.The heart has three layers with an external sac, the pericardium.(B) Adult human heart tissue contains three layers: the endocardium, composed of ECs and EndCs; the myocardium, composed of CMs and CFs with vessels composed of ECs, VSMCs, and PCs (microvessels and capillaries have ECs and PCs; larger macro-vessels like arterioles and venules have ECs and VSMCs); and the epicardium, composed of MSCs and EpiCs.The EMT produces mesenchymal tissue below the EpiCs.(C).The embryonic heart emerges from the gastrula mesoderm as a fused vessel known as the heart tube, which contains the SHF, producing the RV, and the FHF, producing the LV.As cardiogenesis progresses, the heart tube loops, folds, and develops a septum, eventually producing the four heart chambers.CF, cardiac fibroblast; CM, cardiomyocyte; PC, pericyte; MSC, mesenchymal stem cell; EC, endothelial cell; EndC, endocardial cell; EMT, epithelial-to-mesenchymal transition; EpiC, epicardial cell; FHF, first heart field; OFT, outflow tract; L, left; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; R, right; SHF, second heart field; VSMC, vascular smooth muscle cell.

Figure 1 .
Figure 1.Heart Composition: (A) The adult human heart is divided via the septum into left and right halves.Each side has an atrium, ventricle, and OFT, with the muscular left ventricle pumping blood through the aorta out to the body.The heart has three layers with an external sac, the pericardium.(B) Adult human heart tissue contains three layers: the endocardium, composed of ECs and EndCs; the myocardium, composed of CMs and CFs with vessels composed of ECs, VSMCs, and PCs (microvessels and capillaries have ECs and PCs; larger macro-vessels like arterioles and venules have ECs and VSMCs); and the epicardium, composed of MSCs and EpiCs.The EMT produces mesenchymal tissue below the EpiCs.(C).The embryonic heart emerges from the gastrula mesoderm as a fused vessel known as the heart tube, which contains the SHF, producing the RV, and the FHF, producing the LV.As cardiogenesis progresses, the heart tube loops, folds, and develops a septum, eventually producing the four heart chambers.CF, cardiac fibroblast; CM, cardiomyocyte; PC, pericyte; MSC, mesenchymal stem cell; EC, endothelial cell; EndC, endocardial cell; EMT, epithelialto-mesenchymal transition; EpiC, epicardial cell; FHF, first heart field; OFT, outflow tract; L, left; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; R, right; SHF, second heart field; VSMC, vascular smooth muscle cell.

Figure 2 .
Figure2.Constructed Cardiac Tissues: (A).iPSC-CCTs are 3D tissues that produce more physiologically relevant, life-like cardiac tissue than 2D tissue cultures, which can be used to study physiology, test therapies, and be developed for transplantation.These emerging platforms can broadly be arranged by emphasis on high production volumes, that is, high-throughput, or on high physiological relevance.(B).Top-down strategies enable batch-scales, often leveraging iPSC self-assembly to produce tissues with diverse cell types using minimal intervention during culture.This is particularly useful for drug discovery and personalized screenings.Bottom-up strategies involve the modular assembly of cells and components, usually into scaffolded tissue, which enable the finetuning of cardiac tissues.This is particularly useful in organ modeling, including modeling healthy versus diseased tissue.Combining middle-out production methods and top-down self-assembly is promising for regenerative medicine, which benefits from biological complexity but also scalable production.2D, two-dimensional; 3D, three-dimensional; BoC, body-on-a-chip; CCT, constructed cardiac tissue; CO, cardiac organoid; CS, cardiac spheroid; EHT, engineered heart tissue; HoC, heart-on-a-chip; iPSC-CCT, iPSC-derived CCT; iPSC, induced pluripotent stem cell.

Figure 4 .
Figure 4. Vascularization Methods: (A).Vascularizing iPSC-CCTs is essential to improving their size and complexity through increased oxygen and nutrient availability.Native vessels come in various sizes and structures: arteries are large and possess external connective tissue layers; arterioles are small and also have an endothelium reinforced with smooth muscle; and capillaries are very small, with an endothelium supported by a basement membrane and PCs.(B).Factor addition is useful for vascularization.Inductive factors critical to respective cell types are as follows: BMP-4 for CMs and ECs; VEGF-A for ECs; PDGF-BB for VSMCs/PCs and MSCs; and bFGF for CFs and MSCs, while IGF-1, HGF-1, and nitric oxide/NO support CMs and EC-VSMC crosstalk.(C).Engineering approaches to vascularization include sacrificial bioinks (SWIFT) that dissolve away to leave channels for flow, branching vascular-like micropatterning, and the direct emulation of perfusion in a microfluidic chip.bFGF, basic fibroblast growth factor, synonymous with FGF-2; BMP-4, bone morphogenic protein four; CCT, constructed cardiac tissue, CF, cardiac fibroblast; CM, cardiomyocyte; EC, endothelial cell; HGF-1, hepatocyte growth factor one; iPSC-CCT, iPSC-derived CCT; iPSC, induced pluripotent stem cell; IGF-1, insulin-like growth factor one; MSC, mesenchymal stem cell; NO, nitric oxide; PDGF-BB, platelet-derived growth factor B form dimer; PC, pericyte; SWIFT, sacrificial writing into functional tissue; VEGF-A, vascular endothelial growth factor form A; VSMC, vascular smooth muscle cell.

Figure 4 .
Figure 4. Vascularization Methods: (A).Vascularizing iPSC-CCTs is essential to improving their size and complexity through increased oxygen and nutrient availability.Native vessels come in various sizes and structures: arteries are large and possess external connective tissue layers; arterioles are small and also have an endothelium reinforced with smooth muscle; and capillaries are very small, with an endothelium supported by a basement membrane and PCs.(B).Factor addition is useful for vascularization.Inductive factors critical to respective cell types are as follows: BMP-4 for CMs and ECs; VEGF-A for ECs; PDGF-BB for VSMCs/PCs and MSCs; and bFGF for CFs and MSCs, while IGF-1, HGF-1, and nitric oxide/NO support CMs and EC-VSMC crosstalk.(C).Engineering approaches to vascularization include sacrificial bioinks (SWIFT) that dissolve away to leave channels for flow, branching vascular-like micropatterning, and the direct emulation of perfusion in a microfluidic chip.bFGF, basic fibroblast growth factor, synonymous with FGF-2; BMP-4, bone morphogenic protein four; CCT, constructed cardiac tissue, CF, cardiac fibroblast; CM, cardiomyocyte; EC, endothelial cell; HGF-1, hepatocyte growth factor one; iPSC-CCT, iPSC-derived CCT; iPSC, induced pluripotent stem cell; IGF-1, insulin-like growth factor one; MSC, mesenchymal stem cell; NO, nitric oxide; PDGF-BB, platelet-derived growth factor B form dimer; PC, pericyte; SWIFT, sacrificial writing into functional tissue; VEGF-A, vascular endothelial growth factor form A; VSMC, vascular smooth muscle cell.

Cells 2024 , 25 Figure 5 .Figure 5 .
Figure5.Maturation Methods: iPSC-CM maturity is a critical feature of iPSC-CCT use in adult disease models, drug discovery, and the electromechanical integration of regenerative transplants into the host myocardium.CMs lose their proliferative ability with phenotypic maturity.Mature CMs have elongated morphology, aligned sarcomeres, binucleation, physiological hypertrophy, enlarged mitochondria performing primarily FAO, T-tubule networks for synchronized contraction, and proteins for calcium handling, resulting in longer APs with a positive contractile force-frequency relationship.Maturation is induced through various techniques: 1. mechanical stretching to 110-120% tissue length (length is often ramped up over time to increase CM length) at 1-3 Hz; 2. electrical cardiac stimulation with 3-5 V/cm at 2-6 Hz (frequency is often ramped up over time); 3. chemical supplementation with T3, Dex, other glucocorticoids, IGF-1, sometimes β-adrenergic agonists like adrenaline, and BSA-bound fatty acids to facilitate a switch from glucose to FAO metabo-Figure 5. Maturation Methods: iPSC-CM maturity is a critical feature of iPSC-CCT use in adult disease models, drug discovery, and the electromechanical integration of regenerative transplants CMs lose their proliferative ability with phenotypic maturity.Mature CMs have elongated morphology, aligned sarcomeres, binucleation, physiological hypertrophy, enlarged mitochondria performing primarily FAO, T-tubule networks for synchronized contraction, and proteins for calcium handling, resulting in longer APs with a positive contractile force-frequency relationship.Maturation is induced through various techniques: 1. mechanical stretching to 110-120% tissue length (length is often ramped up over time to increase CM length) at 1-3 Hz; 2. electrical cardiac stimulation with 3-5 V/cm at 2-6 Hz (frequency is often ramped up over time); 3. chemical supplementation with T3, Dex, other glucocorticoids, IGF-1, sometimes β-adrenergic agonists like adrenaline, and BSA-bound fatty acids to facilitate a switch from glucose to FAO metabolism; and 4. co-culture with diverse cell types, which increases iPSC-CM maturity and β-adrenergic/cAMP signaling.AP, action potential; BSA, bovine serum albumin; cAMP, cyclic adenosine monophosphate; CF, cardiac fibroblast; CM, cardiomyocyte; Dex, dexamethasone; EHT, engineered heart tissue; EC, endothelial cell; FAO, fatty acid oxidation; iPSC-CM, iPSC-derived CM; iPSC, induced pluripotent stem cell; IGF-1, insulin-like growth factor one; MSC, mesenchymal stem cell; MF, macrophage; PC, pericyte; T3, triiodothyronine; VSMC, vascular smooth muscle cell.

Table 1 .
Representative examples of iPSC-CCT platforms.Pros, cons, and features of platforms.Listed are notable iPSC-CCT publications and the first author, followed by that respective platform's advantages; disadvantages; a description of the cardiac tissue architecture; vascularization methods or method of modeling vascular interactions; and maturation methods of improving the phenotypic maturity of CM electrophysiology, metabolism, and morphology.