The only treatment available for end stage liver diseases is orthotopic liver transplantation. Although there is a big donor scarcity, many donor livers are discarded as they do not qualify for ...transplantation. Alternatively, decellularization of discarded livers can potentially render them transplantable upon recellularization and functional testing. The success of this approach will heavily depend on the quality of decellularized scaffolds which might show variability due to factors including age. Here we assessed the age-dependent differences in liver extracellular matrix (ECM) using rat and human livers. We show that the liver matrix has higher collagen and glycosaminoglycan content and a lower growth factor content with age. Importantly, these changes lead to deterioration in primary hepatocyte function potentially due to ECM stiffening and integrin-dependent signal transduction. Overall, we show that ECM changes with age and these changes significantly affect cell function thus donor age should be considered as an important factor for bioengineering liver substitutes.
The heart wall tissue, or the myocardium, is one of the main targets in cardiovascular
disease prevention and treatment. Animal models have not been sufficient in mimicking the
human myocardium as ...evident by the very low clinical translation rates of cardiovascular
drugs. Additionally, current in vitro models of the human myocardium
possess several shortcomings such as lack of physiologically relevant co-culture of
myocardial cells, lack of a 3D biomimetic environment, and the use of non-human
cells. In
this study, we address these shortcomings through the design and manufacture of a
myocardium-on-chip (MOC) using 3D cell-laden hydrogel constructs and human induced pluripotent stem
cell
(hiPSC) derived myocardial cells. The MOC utilizes 3D spatially controlled co-culture of hiPSC
derived cardiomyocytes (iCMs) and hiPSC derived endothelial cells (iECs) integrated
among iCMs as well as in capillary-like side channels, to better mimic the
microvasculature seen in native myocardium. We first fully characterized iCMs using
immunostaining, genetic, and electrochemical analysis and iECs through immunostaining and
alignment analysis to ensure their functionality, and then seeded these cells sequentially into
the MOC device. We
showed that iECs could be cultured within the microfluidic device without losing their
phenotypic lineage commitment, and align with the flow upon physiological level shear
stresses. We were able to incorporate iCMs within the device in a spatially
controlled manner with the help of photocrosslinkable polymers. The iCMs were shown to be
viable and functional within the device up to 7 days, and were integrated with the iECs. The iCMs and
iECs in this study were derived from the same hiPSC cell line, essentially
mimicking the myocardium of an individual human patient. Such devices are essential for
personalized medicine studies where the individual drug response of patients with
different genetic backgrounds can be tested in a physiologically relevant manner.
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Studying heart tissue is critical for understanding and developing treatments for cardiovascular diseases. In this work, we fabricated precisely controlled and biomimetic engineered ...model tissues to study how cell-cell and cell-matrix interactions influence myocardial cell survival upon exposure to pathological level oxidative stress. Specifically, the interactions of endothelial cells (ECs) and cardiomyocytes (CMs), and the role of hypoxia inducible factor-1α (HIF-1α), with its novel alternative regulator, HIF-1α antisense RNA1 (HIF1A-AS1), in these interactions were investigated. We encapsulated CMs in photo-crosslinkable, biomimetic hydrogels with or without ECs, then exposed to oxidative stress followed by normoxia. With precisely controlled microenvironment provided by the model tissues, cell-cell interactions were restricted to be solely through the secreted factors. CM survival after oxidative stress was significantly improved, in the presence of ECs, when cells were in the model tissues that were functionalized with cell attachment motifs. Importantly, the cardioprotective effect of ECs was reduced when HIF-1α expression was knocked down suggesting that HIF-1α is involved in cardioprotection from oxidative damage, provided through secreted factors conferred by the ECs. Using model tissues, we showed that cell survival increased with increased cell-cell communication and enhanced cell-matrix interactions. In addition, whole genome transcriptome analysis showed, for the first time to our knowledge, a possible role for HIF1A-AS1 in oxidative regulation of HIF-1α. We showed that although HIF1A-AS1 knockdown helps CM survival, its effect is overridden by CM-EC bidirectional interactions as we showed that the conditioned media taken from the CM-EC co-cultures improved CM survival, regardless of HIF1A-AS1 expression.
Cardiovascular diseases, most of which are associated with oxidative stress, is the most common cause of death worldwide. Thus, understanding the molecular events as well as the role of intercellular communication under oxidative stress is upmost importance in its prevention. In this study we used 3D engineered tissue models to investigate the role of HIF-1α and its regulation in EC-mediated cardioprotection. We showed that EC-mediated protection is only possible when there is a bidirectional crosstalk between ECs and CMs even without physical cell-cell contact. In addition, this protective effect is at least partially related to cell-ECM interactions and HIF-1α, which is regulated by HIF1A-AS1 under oxidative stress.
End-stage liver diseases lead to mortality of millions of patients, as the only treatment available is liver transplantation and donor scarcity means that patients have to wait long periods before ...receiving a new liver. In order to minimize donor organ scarcity, a promising bioengineering approach is to decellularize livers that do not qualify for transplantation. Through decellularization, these organs can be used as scaffolds for developing new functional organs. In this process, the original cells of the organ are removed and ideally should be replaced by patient-specific cells to eliminate the risk of immune rejection. Induced pluripotent stem cells (iPSCs) are ideal candidates for developing patient-specific organs, yet the maturity and functionality of iPSC-derived cells do not match those of primary cells. In this study, we introduced iPSCs into decellularized rat liver scaffolds prior to the start of differentiation into hepatic lineages to maximize the exposure of iPSCs to native liver matrices. Through exposure to the unique composition and native 3D organization of the liver microenvironment, as well as the more efficient perfusion culture throughout the differentiation process, iPSC differentiation into hepatocyte-like cells was enhanced. The resulting cells showed significantly higher expression of mature hepatocyte markers, including important CYP450 enzymes, along with lower expression of fetal markers, such as AFP. Importantly, the gene expression profile throughout the different stages of differentiation was more similar to native development. Our study shows that the native 3D liver microenvironment has a pivotal role to play in the development of human-origin hepatocyte-like cells with more mature characteristics.
Modeling human disease as precisely as possible is of upmost importance in understanding the underlying pathology and discovering effective therapies. Therefore, disease models that are highly ...controlled and composed of human-origin cells that present the disease phenotype are crucial. The human induced pluripotent stem cell (hiPSC)-based tissue model we present in this study is an important example of human-origin tissue model with controlled gene expression. Through CRISPR/Cas9 editing of hypoxia inducible factor 1α in hiPSCs, we developed tissue models that show the age and disease-dependent endothelial deterioration. This model holds promise for various biomedical applications as more realistic disease phenotypes can be created using fully human-origin platforms.
The mortality rate of many complex multicellular organisms increases with age, which suggests that net ageing damage is accumulative, despite remodelling processes. But how exactly do these little ...mishaps in the cellular level accumulate and spread to become a systemic catastrophe? To address this question we present experiments with synthetic tissues, an analytical model consistent with experiments, and a number of implications that follow the analytical model. Our theoretical framework describes how shape, curvature and density influences the propagation of failure in a tissue subjected to oxidative damage. We propose that ageing is an emergent property governed by interaction between cells, and that intercellular processes play a role that is at least as important as intracellular ones.
Purpose:
The decellularization of Vascularized Composite Allotransplantation (VCA) represents a first step in circumventing the need for immunosuppression by depleting donor cells from VCAs. Here we ...present a model of decellularization of VCAs in rats.
Methods:
Rat hind limbs based upon the common femoral artery were harvested and perfused through the vascular pedicle at 1ml/min through with 1% sodium dodecyl sulfate (SDS) (group 1) or 0.2% SDS (group 2) or were immersed in SDS 1% (group 3), followed by perfusion (group 1 and 2) or immersion (group 3) in 1% Triton X-100. The degree of decellularization was assessed quantitatively by determining DNA content and qualitatively by histology in all groups at the same locations. The group yielding best decellularization outcome was selected for further assessment for the preservation of ECM content and vasculature and was transplanted orthotopically to assess the ability to reperfuse these decellularized limbs in vivo. RESULTS - Gross examination and DNA quantification of less than 50 ng/mg tissue demonstrated successful decellularization only in group 2 while preserving tissue matrix architecture, including vessels. The arterial network was found to be perfusable and intact during in vivo orthotopic transplantation of the decellularized limb. No outflow was observed, however, after 115 minutes of arterial reperfusion.
Conclusions:
We describe a successful decellularization protocol for rat hindlimb VCAs by perfusion ex-vivo of 1% SDS. During its in vivo transplantation, decellularization alone allows for a perfusable arterial network and with further optimization, we aim to achieve venous perfusion as well.
Studying heart tissue is critical for understanding and developing treatments for cardiovascular diseases. In this work, we fabricated precisely controlled and biomimetic engineered model tissues to ...study how cell-cell and cell-matrix interactions influence myocardial cell survival upon exposure to pathological level oxidative stress. Specifically, the interactions of endothelial cells (ECs) and cardiomyocytes (CMs), and the role of hypoxia inducible factor-1α (HIF-1α), with its novel alternative regulator, HIF-1α antisense RNA1 (HIF1A-AS1), in these interactions were investigated. We encapsulated CMs in photo-crosslinkable, biomimetic hydrogels with or without ECs, then exposed to oxidative stress followed by normoxia. With precisely controlled microenvironment provided by the model tissues, cell-cell interactions were restricted to be solely through the secreted factors. CM survival after oxidative stress was significantly improved, in the presence of ECs, when cells were in the model tissues that were functionalized with cell attachment motifs. Importantly, the cardioprotective effect of ECs was reduced when HIF-1α expression was knocked down suggesting that HIF-1α is involved in cardioprotection from oxidative damage, provided through secreted factors conferred by the ECs. Using model tissues, we showed that cell survival increased with increased cell-cell communication and enhanced cell-matrix interactions. In addition, whole genome transcriptome analysis showed, for the first time to our knowledge, a possible role for HIF1A-AS1 in oxidative regulation of HIF-1α. We showed that although HIF1A-AS1 knockdown helps CM survival, its effect is overridden by CM-EC bidirectional interactions as we showed that the conditioned media taken from the CM-EC co-cultures improved CM survival, regardless of HIF1A-AS1 expression.
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Myocardial infarction (MI) is one of the most common among cardiovascular diseases. Endothelial cells (ECs) are considered to have protective effects on cardiomyocytes (CMs) under ...stress conditions such as MI; however, the paracrine CM-EC crosstalk and the resulting endogenous cellular responses that could contribute to this protective effect are not thoroughly investigated. Here we created biomimetic synthetic tissues containing CMs and human induced pluripotent stem cell (hiPSC)-derived ECs (iECs), which showed improved cell survival compared to single cultures under conditions mimicking the aftermath of MI, and performed high-throughput RNA-sequencing to identify target pathways that could govern CM-iEC crosstalk and the resulting improvement in cell viability. Our results showed that single cultured CMs had different gene expression profiles compared to CMs co-cultured with iECs. More importantly, this gene expression profile was preserved in response to oxidative stress in co-cultured CMs while single cultured CMs showed a significantly different gene expression pattern under stress, suggesting a stabilizing effect of iECs on CMs under oxidative stress conditions. Furthermore, we have validated the in vivo relevance of our engineered model tissues by comparing the changes in the expression levels of several key genes of the encapsulated CMs and iECs with in vivo rat MI model data and clinical data, respectively. We conclude that iECs have protective effects on CMs under oxidative stress through stabilizing mitochondrial complexes, suppressing oxidative phosphorylation pathway and activating pathways such as the drug metabolism-cytochrome P450 pathway, Rap1 signaling pathway, and adrenergic signaling in cardiomyocytes pathway.
Heart diseases are the leading cause of death worldwide. Oxidative stress is a common unwanted outcome that especially occurs due to the reperfusion following heart attack or heart surgery. Standard methods of in vivo analysis do not allow dissecting various intermingled parameters, while regular 2D cell culture approaches often fail to provide a biomimetic environment for the physiologically relevant cellular phenotypes. In this research, a systematic genome-wide transcriptome profiling was performed on myocardial cells in a biomimetic 3D hydrogel-based synthetic model tissue, for identifying possible target genes and pathways as protecting regulators against oxidative stress. Identification of such pathways would be very valuable for new strategies during heart disease treatment by reducing the cellular damage due to reperfusion injury.
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Deaths attributed to ischemic heart disease increased by 41.7% from 1990 to 2013. This is primarily due to an increase in the aged population, however, research on cardiovascular ...disease (CVD) has been overlooking aging, a well-documented contributor to CVD. The use of young animals is heavily preferred due to lower costs and ready availability, despite the prominent differences between young and aged heart structure and function. Here we present the first human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (iCM)-based, in vitro aged myocardial tissue model as an alternative research platform. Within 4 months, iCMs go through accelerated senescence and show cellular characteristics of aging. Furthermore, the model tissues fabricated using aged iCMs, with stiffness resembling that of aged human heart, show functional and pharmacological deterioration specific to aged myocardium. Our novel tissue model with age-appropriate physiology and pathology presents a promising new platform for investigating CVD or other age-related diseases.
In vitro and in vivo models of cardiovascular disease are aimed to provide crucial insight on the pathology and treatment of these diseases. However, the contribution of age-dependent cardiovascular changes is greatly underestimated through the use of young animals and premature cardiomyocytes. Here, we developed in vitro aged cardiac tissue models that mimic the aged heart tissue microenvironment and cellular phenotype and present the first evidence that age-appropriate in vitro disease models can be developed to gain more physiologically-relevant insight on development, progression, and amelioration of cardiovascular diseases.
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