Engineered platforms for mimicking cardiac development and drug screening.

Congenital heart defects are associated with significant health challenges, demanding a deep understanding of the underlying biological mechanisms and, thus, better devices or platforms that can recapitulate human cardiac development. The discovery of human pluripotent stem cells has substantially reduced the dependence on animal models. Recent advances in stem cell biology, genetic editing, omics, microfluidics, and sensor technologies have further enabled remarkable progress in the development of in vitro platforms with increased fidelity and efficiency. In this review, we provide an overview of advancements in in vitro cardiac development platforms, with a particular focus on technological innovation. We categorize these platforms into four areas: two-dimensional solid substrate cultures, engineered substrate architectures that enhance cellular functions, cardiac organoids, and embryos/explants-on-chip models. We conclude by addressing current limitations and presenting future perspectives.

Electroactive Biomaterials and Systems for Cell Fate Determination and Tissue Regeneration: Design and Applications.

During natural tissue regeneration, tissue microenvironment and stem cell niche including cell-cell interaction, soluble factors, and extracellular matrix (ECM) provide a train of biochemical and biophysical cues for modulation of cell behaviors and tissue functions. Design of functional biomaterials to mimic the tissue/cell microenvironment have great potentials for tissue regeneration applications. Recently, electroactive biomaterials have drawn increasing attentions not only as scaffolds for cell adhesion and structural support, but also as modulators to regulate cell/tissue behaviors and function, especially for electrically excitable cells and tissues. More importantly, electrostimulation can further modulate a myriad of biological processes, from cell cycle, migration, proliferation and differentiation to neural conduction, muscle contraction, embryogenesis, and tissue regeneration. In this review, endogenous bioelectricity and piezoelectricity are introduced. Then, design rationale of electroactive biomaterials is discussed for imitating dynamic cell microenvironment, as well as their mediated electrostimulation and the applying pathways. Recent advances in electroactive biomaterials are systematically overviewed for modulation of stem cell fate and tissue regeneration, mainly including nerve regeneration, bone tissue engineering, and cardiac tissue engineering. Finally, the significance for simulating the native tissue microenvironment is emphasized and the open challenges and future perspectives of electroactive biomaterials are concluded.

Direct Cardiac Reprogramming with Engineered miRNA Scaffolds.

Ischemic heart disease is a predominant cause of death worldwide. The loss or death of cardiomyocytes due to restricted blood flow often results in a cardiac injury. Myofibroblasts replace these injured cardiomyocytes to preserve structural integrity. However, the depleted cardiomyocytes lead to cardiac dysfunction such as pathological cardiac dilation, reduced cardiac contraction, and fibrosis. Repair and regeneration of myocardium are the best possible therapy for end-stage heart failure patients because the current cardiomyocytes restoration therapies are limited to heart transplantation only. The emergence of interests to directly reprogram a mammalian heart with minimal regenerative capacity holds a promising future in the field of cardiovascular regenerative medicine. Repair and regeneration become the two crucial factors in the field of cardiovascular regenerative medicine since heart muscles have no substitutes, like heart valves or blood vessels. Cardiac regeneration includes strategies to reprogram with diverse factors like small molecules, genetic and epigenetic regulators. However, there are some constraints like low efficacy, immunogenic problems, and unsafe delivery systems that pose a daunting challenge in human trial translations. Hence, there is a need for a holistic nanoscale approach in regulating cell fate effectively and efficiently with a safer delivery and a suitable microenvironment that mimics the extracellular matrix. In this review, we have discussed the current state-of-the-art techniques, challenges in direct reprogramming of fibroblasts to cardiac muscle, and prospects of biomaterials in miRNA delivery and cardiac regeneration predominantly during the past decade (2008-2019).

Engineered heart tissue models from hiPSC-derived cardiomyocytes and cardiac ECM for disease modeling and drug testing applications.

Cardiac tissue engineering provides unique opportunities for cardiovascular disease modeling, drug testing, and regenerative medicine applications. To recapitulate human heart tissue, we combined human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a chitosan-enhanced extracellular-matrix (ECM) hydrogel, derived from decellularized pig hearts. Ultrastructural characterization of the ECM-derived engineered heart tissues (ECM-EHTs) revealed an anisotropic muscle structure, with embedded cardiomyocytes showing more mature properties than 2D-cultured hiPSC-CMs. Force measurements confirmed typical force-length relationships, sensitivity to extracellular calcium, and adequate ionotropic responses to contractility modulators. By combining genetically-encoded calcium and voltage indicators with laser-confocal microscopy and optical mapping, the electrophysiological and calcium-handling properties of the ECM-EHTs could be studied at the cellular and tissue resolutions. This allowed to detect drug-induced changes in contraction rate (isoproterenol, carbamylcholine), optical signal morphology (E-4031, ATX2, isoproterenol, ouabin and quinidine), cellular arrhythmogenicity (E-4031 and ouabin) and alterations in tissue conduction properties (lidocaine, carbenoxolone and quinidine). Similar assays in ECM-EHTs derived from patient-specific hiPSC-CMs recapitulated the abnormal phenotype of the long QT syndrome and catecholaminergic polymorphic ventricular tachycardia. Finally, programmed electrical stimulation and drug-induced pro-arrhythmia led to the development of reentrant arrhythmias in the ECM-EHTs. In conclusion, a novel ECM-EHT model was established, which can be subjected to high-resolution long-term serial functional phenotyping, with important implications for cardiac disease modeling, drug testing and precision medicine. STATEMENT OF SIGNIFICANCE: One of the main objectives of cardiac tissue engineering is to create an in-vitro muscle tissue surrogate of human heart tissue. To this end, we combined a chitosan-enforced cardiac-specific ECM hydrogel derived from decellularized pig hearts with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from healthy-controls and patients with inherited cardiac disorders. We then utilized genetically-encoded calcium and voltage fluorescent indicators coupled with unique optical imaging techniques and force-measurements to study the functional properties of the generated engineered heart tissues (EHTs). These studies demonstrate the unique potential of the new model for physiological and pathophysiological studies (assessing contractility, conduction and reentrant arrhythmias), novel disease modeling strategies ("disease-in-a-dish" approach) for studying inherited arrhythmogenic disorders, and for drug testing applications (safety pharmacology).

Flexible nanofilms coated with aligned piezoelectric microfibers preserve the contractility of cardiomyocytes.

The use of engineered cardiac tissue for high-throughput drug screening/toxicology assessment remains largely unexplored. Here we propose a scaffold that mimics aspects of cardiac extracellular matrix while preserving the contractility of cardiomyocytes. The scaffold is based on a poly(caprolactone) (PCL) nanofilm with magnetic properties (MNF, standing for magnetic nanofilm) coated with a layer of piezoelectric (PIEZO) microfibers of poly(vinylidene fluoride-trifluoroethylene) (MNF+PIEZO). The nanofilm creates a flexible support for cell contraction and the aligned PIEZO microfibers deposited on top of the nanofilm creates conditions for cell alignment and electrical stimulation of the seeded cells. Our results indicate that MNF+PIEZO scaffold promotes rat and human cardiac cell attachment and alignment, maintains the ratio of cell populations overtime, promotes cell-cell communication and metabolic maturation, and preserves cardiomyocyte (CM) contractility for at least 12 days. The engineered cardiac construct showed high toxicity against doxorubicin, a cardiotoxic molecule, and responded to compounds that modulate CM contraction such as epinephrine, propranolol and heptanol.

Bioprinting 3D microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip.

Engineering cardiac tissues and organ models remains a great challenge due to the hierarchical structure of the native myocardium. The need of integrating blood vessels brings additional complexity, limiting the available approaches that are suitable to produce integrated cardiovascular organoids. In this work we propose a novel hybrid strategy based on 3D bioprinting, to fabricate endothelialized myocardium. Enabled by the use of our composite bioink, endothelial cells directly bioprinted within microfibrous hydrogel scaffolds gradually migrated towards the peripheries of the microfibers to form a layer of confluent endothelium. Together with controlled anisotropy, this 3D endothelial bed was then seeded with cardiomyocytes to generate aligned myocardium capable of spontaneous and synchronous contraction. We further embedded the organoids into a specially designed microfluidic perfusion bioreactor to complete the endothelialized-myocardium-on-a-chip platform for cardiovascular toxicity evaluation. Finally, we demonstrated that such a technique could be translated to human cardiomyocytes derived from induced pluripotent stem cells to construct endothelialized human myocardium. We believe that our method for generation of endothelialized organoids fabricated through an innovative 3D bioprinting technology may find widespread applications in regenerative medicine, drug screening, and potentially disease modeling.

Electrical stimulation of cardiac adipose tissue-derived progenitor cells modulates cell phenotype and genetic machinery.

A major challenge of cardiac tissue engineering is directing cells to establish the physiological structure and function of the myocardium being replaced. Our aim was to examine the effect of electrical stimulation on the cardiodifferentiation potential of cardiac adipose tissue-derived progenitor cells (cardiac ATDPCs). Three different electrical stimulation protocols were tested; the selected protocol consisted of 2 ms monophasic square-wave pulses of 50 mV/cm at 1 Hz over 14 days. Cardiac and subcutaneous ATDPCs were grown on biocompatible patterned surfaces. Cardiomyogenic differentiation was examined by real-time PCR and immunocytofluorescence. In cardiac ATDPCs, MEF2A and GATA-4 were significantly upregulated at day 14 after stimulation, while subcutaneous ATDPCs only exhibited increased Cx43 expression. In response to electrical stimulation, cardiac ATDPCs elongated, and both cardiac and subcutaneous ATDPCs became aligned following the linear surface pattern of the construct. Cardiac ATDPC length increased by 11.3%, while subcutaneous ATDPC length diminished by 11.2% (p = 0.013 and p = 0.030 vs unstimulated controls, respectively). Compared to controls, electrostimulated cells became aligned better to the patterned surfaces when the pattern was perpendicular to the electric field (89.71 ± 28.47º for cardiac ATDPCs and 92.15 ± 15.21º for subcutaneous ATDPCs). Electrical stimulation of cardiac ATDPCs caused changes in cell phenotype and genetic machinery, making them more suitable for cardiac regeneration approaches. Thus, it seems advisable to use electrical cell training before delivery as a cell suspension or within engineered tissue.