Nanowired human cardiac organoid transplantation enables highly efficient and effective recovery of infarcted hearts.

Human cardiac organoids hold remarkable potential for cardiovascular disease modeling and human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) transplantation. Here, we show cardiac organoids engineered with electrically conductive silicon nanowires (e-SiNWs) significantly enhance the therapeutic efficacy of hPSC-CMs to treat infarcted hearts. We first demonstrated the biocompatibility of e-SiNWs and their capacity to improve cardiac microtissue engraftment in healthy rat myocardium. Nanowired human cardiac organoids were then engineered with hPSC-CMs, nonmyocyte supporting cells, and e-SiNWs. Nonmyocyte supporting cells promoted greater ischemia tolerance of cardiac organoids, and e-SiNWs significantly improved electrical pacing capacity. After transplantation into ischemia/reperfusion-injured rat hearts, nanowired cardiac organoids significantly improved contractile development of engrafted hPSC-CMs, induced potent cardiac functional recovery, and reduced maladaptive left ventricular remodeling. Compared to contemporary studies with an identical injury model, greater functional recovery was achieved with a 20-fold lower dose of hPSC-CMs, revealing therapeutic synergy between conductive nanomaterials and human cardiac organoids for efficient heart repair.

Targeting HIF-α for robust prevascularization of human cardiac organoids.

Prevascularized 3D microtissues have been shown to be an effective cell delivery vehicle for cardiac repair. To this end, our lab has explored the development of self-organizing, prevascularized human cardiac organoids by co-seeding human cardiomyocytes with cardiac fibroblasts, endothelial cells, and stromal cells into agarose microwells. We hypothesized that this prevascularization process is facilitated by the endogenous upregulation of hypoxia-inducible factor (HIF) pathway in the avascular 3D microtissues. In this study, we used Molidustat, a selective PHD (prolyl hydroxylase domain enzymes) inhibitor that stabilizes HIF-α, to treat human cardiac organoids, which resulted in 150 ± 61% improvement in endothelial expression (CD31) and 220 ± 20% improvement in the number of lumens per organoids. We hypothesized that the improved endothelial expression seen in Molidustat treated human cardiac organoids was dependent upon upregulation of VEGF, a well-known downstream target of HIF pathway. Through the use of immunofluorescent staining and ELISA assays, we determined that Molidustat treatment improved VEGF expression of non-endothelial cells and resulted in improved co-localization of supporting cell types and endothelial structures. We further demonstrated that Molidustat treated human cardiac organoids maintain cardiac functionality. Lastly, we showed that Molidustat treatment improves survival of cardiac organoids when exposed to both hypoxic and ischemic conditions in vitro. For the first time, we demonstrate that targeted HIF-α stabilization provides a robust strategy to improve endothelial expression and lumen formation in cardiac microtissues, which will provide a powerful framework for prevascularization of various microtissues in developing successful cell transplantation therapies.

Engineering a Chemically Defined Hydrogel Bioink for Direct Bioprinting of Microvasculature.

Vascularizing printed tissues is a critical challenge in bioprinting. While protein-based hydrogel bioinks have been successfully used to bioprint microvasculature, their compositions are ill-defined and subject to batch variation. Few studies have focused on engineering proangiogenic bioinks with defined properties to direct endogenous microvascular network formation after printing. Here, a peptide-functionalized alginate hydrogel bioink with defined mechanical, rheological, and biochemical properties is developed for direct bioprinting of microvascularized tissues. An integrin-binding peptide (RGD) and a vascular endothelial growth factor-mimetic peptide with a protease-sensitive linker are conjugated onto a biodegradable alginate to synergistically promote vascular morphogenesis and capillary-scale endothelial tube formation. Partial ionic crosslinking before printing converts the otherwise unprintable hydrogel into a viscoelastic bioink with excellent printability and cytocompatibility. We use the bioink to fabricate a compartmentalized vascularized tissue construct, wherein we observe pericyte-endothelial cell colocalization and angiogenic sprouting across a tissue interface, accompanied by deposition of fibronectin and collagen in vascular and tissue components, respectively. This study provides a tunable and translational "off-the-shelf" hydrogel bioink with defined composition for vascularized bioprinting.

Evolutionarily conserved sequence motif analysis guides development of chemically defined hydrogels for therapeutic vascularization.

Biologically active ligands (e.g., RGDS from fibronectin) play critical roles in the development of chemically defined biomaterials. However, recent decades have shown only limited progress in discovering novel extracellular matrix-protein-derived ligands for translational applications. Through motif analysis of evolutionarily conserved RGD-containing regions in laminin (LM) and peptide-functionalized hydrogel microarray screening, we identified a peptide (a1) that showed superior supports for endothelial cell (EC) functions. Mechanistic studies attributed the results to the capacity of a1 engaging both LM- and Fn-binding integrins. RNA sequencing of ECs in a1-functionalized hydrogels showed ~60% similarities with Matrigel in "vasculature development" gene ontology terms. Vasculogenesis assays revealed the capacity of a1-formulated hydrogels to improve EC network formation. Injectable alginates functionalized with a1 and MMPQK (a vascular endothelial growth factor-mimetic peptide with a matrix metalloproteinase-degradable linker) increased blood perfusion and functional recovery over decellularized extracellular matrix and (RGDS + MMPQK)-functionalized hydrogels in an ischemic hindlimb model, illustrating the power of this approach.

Human cardiac organoids for the modelling of myocardial infarction and drug cardiotoxicity.

Environmental factors are the largest contributors to cardiovascular disease. Here we show that cardiac organoids that incorporate an oxygen-diffusion gradient and that are stimulated with the neurotransmitter noradrenaline model the structure of the human heart after myocardial infarction (by mimicking the infarcted, border and remote zones), and recapitulate hallmarks of myocardial infarction (in particular, pathological metabolic shifts, fibrosis and calcium handling) at the transcriptomic, structural and functional levels. We also show that the organoids can model hypoxia-enhanced doxorubicin cardiotoxicity. Human organoids that model diseases with non-genetic pathological factors could help with drug screening and development.

Transcriptomic analysis of short-term 17α-ethynylestradiol exposure in two Californian sentinel fish species sardine (Sardinops sagax) and mackerel (Scomber japonicus).

Endocrine disrupting chemicals (EDCs) are substances which disrupt normal functioning of the endocrine system by interfering with hormone regulated physiological pathways. Aquatic environments provide the ultimate reservoir for many EDCs as they enter rivers and the ocean via effluent discharges and accumulate in sediments. One EDC widely dispersed in municipal wastewater effluent discharges is 17α-ethynylestradiol (EE2), which is one of the most widely prescribed medicines. EE2 is a bio-active estrogen employed in the majority of oral contraceptive pill formulations. As evidence of the health risks posed by EDCs mount, there is an urgent need to improve diagnostic tools for monitoring the effects of pollutants. As the cost of high throughput sequencing (HTS) diminishes, transcriptional profiling of an organism in response to EDC perturbation presents a cost-effective way of screening a wide range of endocrine responses. Coastal pelagic filter feeding fish species analyzed using HTS provide an excellent tool for EDC risk assessment in the marine environment. Unfortunately, there are limited genome sequence data and annotation for many of these species including Pacific sardine (Sardinops sagax) and chub mackerel (Scomber japonicus), which limits the utility of molecular tools such as HTS to interrogate the effects of endocrine disruption. In this study, we carried out RNA sequencing (RNAseq) of liver RNA harvested from wild sardine and mackerel exposed for 5 h under laboratory conditions to a concentration of 12.5 pM EE2 in the tank water. We developed an analytical framework for transcriptomic analyses of species with limited genomic information. EE2 exposure altered expression patterns of key genes involved in important metabolic and physiological processes. The systems approach presented here provides a powerful tool for obtaining a comprehensive picture of endocrine disruption in aquatic organisms.

De Novo Hepatic Transcriptome Assembly and Systems Level Analysis of Three Species of Dietary Fish, Sardinops sagax, Scomber japonicus, and Pleuronichthys verticalis.

The monitoring of marine species as sentinels for ecosystem health has long been a valuable tool worldwide, providing insight into how both anthropogenic pollution and naturally occurring phenomena (i.e., harmful algal blooms) may lead to human and animal dietary concerns. The marine environments contain many contaminants of anthropogenic origin that have sufficient similarities to steroid and thyroid hormones, to potentially disrupt normal endocrine physiology in humans, fish, and other animals. An appropriate understanding of the effects of these endocrine disrupting chemicals (EDCs) on forage fish (e.g., sardine, anchovy, mackerel) can lead to significant insight into how these contaminants may affect local ecosystems in addition to their potential impacts on human health. With advancements in molecular tools (e.g., high-throughput sequencing, HTS), a genomics approach offers a robust toolkit to discover putative genetic biomarkers in fish exposed to these chemicals. However, the lack of available sequence information for non-model species has limited the development of these genomic toolkits. Using HTS and de novo assembly technology, the present study aimed to establish, for the first time for Sardinops sagax (Pacific sardine), Scomber japonicas (Pacific chub mackerel) and Pleuronichthys verticalis (hornyhead turbot), a de novo global transcriptome database of the liver, the primary organ involved in detoxification. The assembled transcriptomes provide a foundation for further downstream validation, comparative genomic analysis and biomarker development for future applications in ecotoxicogenomic studies, as well as environmental evaluation (e.g., climate change) and public health safety (e.g., dietary screening).

Inspiration from heart development: Biomimetic development of functional human cardiac organoids.

Recent progress in human organoids has provided 3D tissue systems to model human development, diseases, as well as develop cell delivery systems for regenerative therapies. While direct differentiation of human embryoid bodies holds great promise for cardiac organoid production, intramyocardial cell organization during heart development provides biological foundation to fabricate human cardiac organoids with defined cell types. Inspired by the intramyocardial organization events in coronary vasculogenesis, where a diverse, yet defined, mixture of cardiac cell types self-organizes into functional myocardium in the absence of blood flow, we have developed a defined method to produce scaffold-free human cardiac organoids that structurally and functionally resembled the lumenized vascular network in the developing myocardium, supported hiPSC-CM development and possessed fundamental cardiac tissue-level functions. In particular, this development-driven strategy offers a robust, tunable system to examine the contributions of individual cell types, matrix materials and additional factors for developmental insight, biomimetic matrix composition to advance biomaterial design, tissue/organ-level drug screening, and cell therapy for heart repair.

Development of peptide-functionalized synthetic hydrogel microarrays for stem cell and tissue engineering applications.

Synthetic polymer microarray technology holds remarkable promise to rapidly identify suitable biomaterials for stem cell and tissue engineering applications. However, most of previous microarrayed synthetic polymers do not possess biological ligands (e.g., peptides) to directly engage cell surface receptors. Here, we report the development of peptide-functionalized hydrogel microarrays based on light-assisted copolymerization of poly(ethylene glycol) diacrylates (PEGDA) and methacrylated-peptides. Using solid-phase peptide/organic synthesis, we developed an efficient route to synthesize methacrylated-peptides. In parallel, we identified PEG hydrogels that effectively inhibit non-specific cell adhesion by using PEGDA-700 (M. W.=700) as a monomer. The combined use of these chemistries enables the development of a powerful platform to prepare peptide-functionalized PEG hydrogel microarrays. Additionally, we identified a linker composed of 4 glycines to ensure sufficient exposure of the peptide moieties from hydrogel surfaces. Further, we used this system to directly compare cell adhesion abilities of several related RGD peptides: RGD, RGDS, RGDSG and RGDSP. Finally, we combined the peptide-functionalized hydrogel technology with bioinformatics to construct a library composed of 12 different RGD peptides, including 6 unexplored RGD peptides, to develop culture substrates for hiPSC-derived cardiomyocytes (hiPSC-CMs), a cell type known for poor adhesion to synthetic substrates. 2 out of 6 unexplored RGD peptides showed substantial activities to support hiPSC-CMs. Among them, PMQKMRGDVFSP from laminin ß4 subunit was found to support the highest adhesion and sarcomere formation of hiPSC-CMs. With bioinformatics, the peptide-functionalized hydrogel microarrays accelerate the discovery of novel biological ligands to develop biomaterials for stem cell and tissue engineering applications. STATEMENT OF SIGNIFICANCE: In this manuscript, we described the development of a robust approach to prepare peptide-functionalized synthetic hydrogel microarrays. Combined with bioinformatics, this technology enables us to rapidly identify novel biological ligands for the development of the next generation of functional biomaterials for stem cell and tissue engineering applications.

Nanowires and Electrical Stimulation Synergistically Improve Functions of hiPSC Cardiac Spheroids.

The advancement of human induced pluripotent stem-cell-derived cardiomyocyte (hiPSC-CM) technology has shown promising potential to provide a patient-specific, regenerative cell therapy strategy to treat cardiovascular disease. Despite the progress, the unspecific, underdeveloped phenotype of hiPSC-CMs has shown arrhythmogenic risk and limited functional improvements after transplantation. To address this, tissue engineering strategies have utilized both exogenous and endogenous stimuli to accelerate the development of hiPSC-CMs. Exogenous electrical stimulation provides a biomimetic pacemaker-like stimuli that has been shown to advance the electrical properties of tissue engineered cardiac constructs. Recently, we demonstrated that the incorporation of electrically conductive silicon nanowires to hiPSC cardiac spheroids led to advanced structural and functional development of hiPSC-CMs by improving the endogenous electrical microenvironment. Here, we reasoned that the enhanced endogenous electrical microenvironment of nanowired hiPSC cardiac spheroids would synergize with exogenous electrical stimulation to further advance the functional development of nanowired hiPSC cardiac spheroids. For the first time, we report that the combination of nanowires and electrical stimulation enhanced cell-cell junction formation, improved development of contractile machinery, and led to a significant decrease in the spontaneous beat rate of hiPSC cardiac spheroids. The advancements made here address critical challenges for the use of hiPSC-CMs in cardiac developmental and translational research and provide an advanced cell delivery vehicle for the next generation of cardiac repair.

Engineering alginate as bioink for bioprinting.

Recent advances in three-dimensional (3-D) printing offer an excellent opportunity to address critical challenges faced by current tissue engineering approaches. Alginate hydrogels have been used extensively as bioinks for 3-D bioprinting. However, most previous research has focused on native alginates with limited degradation. The application of oxidized alginates with controlled degradation in bioprinting has not been explored. Here, a collection of 30 different alginate hydrogels with varied oxidation percentages and concentrations was prepared to develop a bioink platform that can be applied to a multitude of tissue engineering applications. The authors systematically investigated the effects of two key material properties (i.e. viscosity and density) of alginate solutions on their printabilities to identify a suitable range of material properties of alginates to be applied to bioprinting. Further, four alginate solutions with varied biodegradability were printed with human adipose-derived stem cells (hADSCs) into lattice-structured, cell-laden hydrogels with high accuracy. Notably, these alginate-based bioinks were shown to be capable of modulating proliferation and spreading of hADSCs without affecting the structure integrity of the lattice structures (except the highly degradable one) after 8days in culture. This research lays a foundation for the development of alginate-based bioink for tissue-specific tissue engineering applications.

3D printing facilitated scaffold-free tissue unit fabrication.

Tissue spheroids hold great potential in tissue engineering as building blocks to assemble into functional tissues. To date, agarose molds have been extensively used to facilitate fusion process of tissue spheroids. As a molding material, agarose typically requires low temperature plates for gelation and/or heated dispenser units. Here, we proposed and developed an alginate-based, direct 3D mold-printing technology: 3D printing microdroplets of alginate solution into biocompatible, bio-inert alginate hydrogel molds for the fabrication of scaffold-free tissue engineering constructs. Specifically, we developed a 3D printing technology to deposit microdroplets of alginate solution on calcium containing substrates in a layer-by-layer fashion to prepare ring-shaped 3D hydrogel molds. Tissue spheroids composed of 50% endothelial cells and 50% smooth muscle cells were robotically placed into the 3D printed alginate molds using a 3D printer, and were found to rapidly fuse into toroid-shaped tissue units. Histological and immunofluorescence analysis indicated that the cells secreted collagen type I playing a critical role in promoting cell-cell adhesion, tissue formation and maturation.