Cardiac Spheroids Research Articles

Cell number per spheroid and electrical conductivity of nanowires influence the function of silicon nanowired human cardiac spheroids.

Cardiovascular disease is the leading cause of death and disability worldwide. Due to the limited regenerative capacity of adult human hearts, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have received significant attention because they provide a patient specific cell source to regenerate damaged hearts. Despite the progress, current human hiPSC-CMs retain an immature phenotype that leads to difficulties for integration with adult myocardium after transplantation. To address this, we recently utilized electrically conductive silicon nanowires (e-SiNWs) to facilitate self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac spheroids. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. In this manuscript, we examined the effects of two important factors on the functions of nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. The results from these studies will allow for the development of suitable nanowired hiPSC cardiac spheroids to effectively deliver hiPSC-CMs for heart repair.

330 - Doxorubicin-Mediated Toxic Effects Are Mediated Via NO/eNOS in a Novel 3D in Vitro Model of the Human Heart

Background Nitric oxide (NO●) and endothelial nitric oxide synthase (eNOS) play a dual role in the human heart by both preventing and mediating the toxic effects of chemotherapeutics such as doxorubicin (DOX). Treatment of cancer patients with DOX can result in severe late-onset dilated cardiomyopathy more than one year after completion of treatment. This cardiotoxicity has been linked to DOX-mediated upregulation of eNOS, peroxynitrous acid generation and caspase 3 activation. However, currently available models used to study this signalling pathway have limited potential to identify the cellular and molecular targets of DOX and the mechanism of toxicity in humans. Aims to use a novel in vitro 3D co-culture model, the “cardiac spheroid” (CS), generated from human endothelial cells (ECs), cardiac myocytes (CMs) and fibroblasts (CFs) to recapitulate biochemical and morphological features typical of the human heart and examine the mechanisms of DOX toxicity. Results DOX-mediated dose dependent toxic effects on CSs are shown to be dependent on NO● synthesis. Inhibition of NO● formation using L-NIO reduced DOX-mediated toxic effects in CSs at DOX concentrations detected in DOX-treated patients. Analysis at the cellular level showed that EC-derived NO● plays a protective role, whereas most of the toxic effects of DOX/NO was derived from CFs. Silencing of eNOS with lentiviral NOS3 shRNA particles in ECs confirmed a dual role of NO in mediating DOX-derived toxic effects in CSs., Genetic eNOS inhibition in CFs supported the toxic role played by these cells. Conclusions This study provides the first direct evidence for a dual role of NO. in mediating both toxic and protective DOX-mediated effects in the human heart, and cellular crosstalk responsible for this. Determining the molecular and cellular targets of novel drugs in CSs has the potential to prevent unexpected toxic effects.

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.

Abstract 310: Vascular Endothelial Growth Factor and Endothelial Nitric Oxide Synthase-mediated Regulation of Cardiomyocyte Proliferation During Development in Humans and Mice

Our previous studies in early mouse embryonic development (E8.2) showing that vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) are expressed in embryonic endothelial cells, but not in embryonic cardiomyocytes, together with the findings by others indicating that NO is required for cardiomyocyte proliferation in mice, led us to investigate the relevancy of the VEGF/eNOS signaling pathway during cardiovascular development. First, wild type and NOS3 knockout mouse embryos between E8.0 and E17.0 were stained with antibodies against smooth muscle α-actin, phospho-histone H3 (PH3), VEGFR2 and PECAM, markers of cardiomyocytes, proliferating, progenitor and endothelial cells, respectively. Our confocal analysis showed hearts of E8.0 NOS3 nulls develop normally. However, E8.5 and E9.5 NOS3 nulls have reduced cardiomyocyte proliferation and impaired heart development. As consequence, hearts of E17 NOS3 nulls were approximately 20% smaller compared to wildtype hearts. To translate our findings to humans, we stained human heart specimens with antibodies against VEGFR2, eNOS, PH3 and sarcomeric α-actinin. Confocal analyses showed for the first time that VEGFR2 is highly expressed in the perinuclear region of human cardiomyocytes of a young donor. They also showed a correlation between eNOS expression and cardiomyocyte proliferation in humans. Consequently, we developed an in vitro three-dimensional co-culture model of human endothelial cells, cardiomyocytes and fibroblasts: “human cardiac tissue spheroids” (HCTSs). Our data showed that laminin and collagen type IV synthesis is increased in VEGF-treated HCTSs generated using CMs from an older donor compared to untreated cultures, suggesting a role for VEGF and eNOS in postnatal human heart development. In conclusion, our data showed that VEGF and eNOS play a similar role in mediating cardiomyocytes proliferation and heart regeneration in both mice and humans. Current studies are focusing on evaluating molecular targets of the VEGF/eNOS signalling pathway in human proliferating cardiomyocytes, which may have significant therapeutical impact for stem cell differentiation, and prevention of cardiovascular complications such as myocardial infarction.

Development of a three-dimensional pre-vascularized scaffold-free contractile cardiac patch for treating heart disease

The aim of our study was to develop a completely scaffold-free, viable, contractile cardiac tissue capable of being grafted into the damaged native heart. Our technology is based on the fundamental characteristics of the self-assembling nature of cells. We created contractile cardiac spheroids by plating a mixture of rat neonatal ventricular cardiomyocytes, human dermal fibroblasts, and human coronary microartery endothelial cells in ultralow attachment plates. First, the optimal cell ratios for the 3 cell sources were determined. Next, approximately 1 × 10(4) optimal spheroids were fused into a patch-like construct, and the morphologic characteristics and mechanical functions of these patches were evaluated. Finally, the cardiac patches were grafted into the hearts of F344 nude rats, and histologic studies were performed after transplantation. Synchronous beating of the cardiac patch was confirmed electrophysiologically and mechanically. A micronetwork of endothelial cells was also demonstrated in the construct, and the histologic study performed 5 days after transplantation showed the grafts to be viable, with functioning microvascular structures inside the graft tissue. We consider the application of our scaffold-free 3-dimensional tissue engineering technology to cardiac regeneration therapy is feasible and expect that this technology will become a promising tool for the treatment of end-stage heart failure.

Silicon nanowire-induced maturation of cardiomyocytes derived from human induced pluripotent stem cells.

The current inability to derive mature cardiomyocytes from human pluripotent stem cells has been the limiting step for transitioning this powerful technology into clinical therapies. To address this, scaffold-based tissue engineering approaches have been utilized to mimic heart development in vitro and promote maturation of cardiomyocytes derived from human pluripotent stem cells. While scaffolds can provide 3D microenvironments, current scaffolds lack the matched physical/chemical/biological properties of native extracellular environments. On the other hand, scaffold-free, 3D cardiac spheroids (i.e., spherical-shaped microtissues) prepared by seeding cardiomyocytes into agarose microwells were shown to improve cardiac functions. However, cardiomyocytes within the spheroids could not assemble in a controlled manner and led to compromised, unsynchronized contractions. Here, we show, for the first time, that incorporation of a trace amount (i.e., ∼0.004% w/v) of electrically conductive silicon nanowires (e-SiNWs) in otherwise scaffold-free cardiac spheroids can form an electrically conductive network, leading to synchronized and significantly enhanced contraction (i.e., >55% increase in average contraction amplitude), resulting in significantly more advanced cellular structural and contractile maturation.

Correction for Fares et al., Matrix Metalloproteinases 2 and 9 Are Differentially Expressed in Patients with Indeterminate and Cardiac Clinical Forms of Chagas Disease

Volume 81, no. 10, p. [3600–3608][1], 2013. Page 3605: [Figure 4][2] should appear as shown below. ![FIG 4][3] FIG 4 Evaluation of matrix protein expression in cardiac spheroids. (A) Representative bands of matrix protein expression: collagen type I, fibronectin, laminin, MMP-2, and MMP-9

Matrix Metalloproteinases 2 and 9 Are Differentially Expressed in Patients with Indeterminate and Cardiac Clinical Forms of Chagas Disease

Dilated chronic cardiomyopathy (DCC) from Chagas disease is associated with myocardial remodeling and interstitial fibrosis, resulting in extracellular matrix (ECM) changes. In this study, we characterized for the first time the serum matrix metalloproteinase 2 (MMP-2) and MMP-9 levels, as well as their main cell sources in peripheral blood mononuclear cells from patients presenting with the indeterminate (IND) or cardiac (CARD) clinical form of Chagas disease. Our results showed that serum levels of MMP-9 are associated with the severity of Chagas disease. The analysis of MMP production by T lymphocytes showed that CD8(+) T cells are the main mononuclear leukocyte source of both MMP-2 and MMP-9 molecules. Using a new 3-dimensional model of fibrosis, we observed that sera from patients with Chagas disease induced an increase in the extracellular matrix components in cardiac spheroids. Furthermore, MMP-2 and MMP-9 showed different correlations with matrix proteins and inflammatory cytokines in patients with Chagas disease. Our results suggest that MMP-2 and MMP-9 show distinct activities in Chagas disease pathogenesis. While MMP-9 seems to be involved in the inflammation and cardiac remodeling of Chagas disease, MMP-2 does not correlate with inflammatory molecules.

Development of 3-Dimensional Prevascularized Scaffold-Free Contractile Cardiac Patch for Heart Regeneration

Purpose The aim of our study is to develop completely scaffold-free, viable, contractile cardiac tissue which can be grafted to the damaged native heart. Methods and Materials Our technology is based on the fundamental characteristics of self-assembling nature of cells. We called these self-assembled spherical cell aggregates as “spheroids”. We created contractile cardiac spheroids by plating a mixture of 800 cells of rat neonate ventricular cardiomyocytes (RNVCM), 100 cells of human normal dermal fibroblasts (HNDFB), and 100 cells of human coronary micro-artery endothelial cells (HCMEC) in ultra-low-attachment plate. After 24 hr cultivation, prevascularized contractile cardiac spheroids were fabricated. Next, 10000 spheroids were fused into patch like construct with a maximum diameter of 1cm with 200μm in thickness. We evaluated morphological characteristics, and electro- and mechanical function. We also grafted the cardiac patch onto the heart of F344 nude rats, and performed histological study 3 weeks after transplantation. Results We confirmed synchronous beating of the cardiac patch electro-physiologically, and mechanically (video-motion analysis). Expression of connexin 43 (a component of gap junction) and micro network of endothelial cell in the construct were demonstrated. Histological study performed 3 weeks after transplantation showed that the grafts were viable with functioning micro-vascular structure inside the graft. [ figure 1 ] Conclusions We consider that applying our scaffold-free 3-Dimensinal tissue engineering technology to cardiac regeneration therapy is feasible. We expect that this technology will become a promising tool to treat end-stage heart failure.

Fibrosis and Hypertrophy Induced byTrypanosoma cruziin a Three‐Dimensional Cardiomyocyte‐Culture System

Cardiac damages caused by in vivo infection with Trypanosoma cruzi are still not fully clarified. Here we describe for the first time an in vitro model of fibrosis, hypertrophy, and remodeling induced by T. cruzi in cardiomyocyte spheroids (cardiac microtissues). In this new 3-dimensional system, cardiac spheroids showed spontaneous contractility, with typical cardiac morphology and production of extracellular matrix components. There were 4- and 6-fold increases, respectively, in the area and the volume of T. cruzi-infected cardiomyocytes and whole microtissues, together with a 50% reduction of the cell population. Immunofluorescence showed increased expression of fibronectin, collagen IV, and laminin in the microtissues 144 h after infection. T. cruzi infection induced an increase in both the cellular area and the extracellular matrix components in cardiac spheroids, which contributed to an increase in total microtissue volume, making this a powerful 3-dimensional in vitro model for the study of cardiac-tissue hypertrophy, fibrosis, and remodeling.