ABSTRACT
Cardiomyocyte (CM) proliferation and maturation are highly linked processes, however, the extent to which these processes are controlled by a single signaling axis is unclear. Here, we show the previously undescribed role of Hedgehog (HH)-GLI2-CKS1B cascade in regulation of the toggle between CM proliferation and maturation. Here we show downregulation of GLI-signaling in adult human CM, adult murine CM, and in late-stage hiPSC-CM leading to their maturation. In early-stage hiPSC-CM, inhibition of HH- or GLI-proteins enhanced CM maturation with increased maturation indices, increased calcium handling, and transcriptome. Mechanistically, we identified CKS1B, as a new effector of GLI2 in CMs. GLI2 binds the CKS1B promoter to regulate its expression. CKS1B overexpression in late-stage hiPSC-CMs led to increased proliferation with loss of maturation in CMs. Next, analysis of datasets of patients with heart disease showed a significant enrichment of GLI2-signaling in patients with ischemic heart failure (HF) or dilated-cardiomyopathy (DCM) disease, indicating operational GLI2-signaling in the stressed heart. Thus, the Hh-GLI2-CKS1B axis regulates the proliferation-maturation transition and provides targets to enhance cardiac tissue engineering and regenerative therapies.
Subject(s)
Cell Proliferation , Myocytes, Cardiac , Zinc Finger Protein Gli2 , Myocytes, Cardiac/metabolism , Humans , Zinc Finger Protein Gli2/metabolism , Zinc Finger Protein Gli2/genetics , Animals , Mice , Hedgehog Proteins/metabolism , Signal Transduction , Induced Pluripotent Stem Cells/metabolism , Cell DifferentiationABSTRACT
Cell-cell fusion is a normal physiological mechanism that requires a well-orchestrated regulation of intracellular and extracellular factors. Dysregulation of this process could lead to diseases such as osteoporosis, malformation of muscles, difficulties in pregnancy, and cancer. Extensive literature demonstrates that fusion occurs between cancer cells and other cell types to potentially promote cancer progression and metastasis. However, the mechanisms governing this process in cancer initiation, promotion, and progression are less well-studied. Fusogens involved in normal physiological processes such as syncytins and associated factors such as phosphatidylserine and annexins have been observed to be critical in cancer cell fusion as well. Some of the extracellular factors associated with cancer cell fusion include chronic inflammation and inflammatory cytokines, hypoxia, and viral infection. The interaction between these extracellular factors and cell's intrinsic factors potentially modulates actin dynamics to drive the fusion of cancer cells. In this review, we have discussed the different mechanisms that have been identified or postulated to drive cancer cell fusion.
Subject(s)
Neoplasms , Humans , Cell Fusion , Neoplasms/pathologyABSTRACT
While the field of tissue engineering has progressed rapidly with the advent of 3D bioprinting and human induced pluripotent stem cells (hiPSCs), impact is limited by a lack of functional, thick tissues. One way around this limitation is to 3D bioprint tissues laden with hiPSCs. In this way, the iPSCs can proliferate to populate the thick tissue mass prior to parenchymal cell specification. Here we design a perfusion bioreactor for an hiPSC-laden, 3D-bioprinted chamber with the goal of proliferating the hiPSCs throughout the structure prior to differentiation to generate a thick tissue model. The bioreactor, fabricated with digital light projection, was optimized to perfuse the interior of the hydrogel chamber without leaks and to provide fluid flow around the exterior as well, maximizing nutrient delivery throughout the chamber wall. After 7 days of culture, we found that intermittent perfusion (15 s every 15 min) at 3 ml min-1provides a 1.9-fold increase in the density of stem cell colonies in the engineered tissue relative to analogous chambers cultured under static conditions. We also observed a more uniform distribution of colonies within the tissue wall of perfused structures relative to static controls, reflecting a homogeneous distribution of nutrients from the culture media. hiPSCs remained pluripotent and proliferative with application of fluid flow, which generated wall shear stresses averaging â¼1.0 dyn cm-2. Overall, these promising outcomes following perfusion of a stem cell-laden hydrogel support the production of multiple tissue types with improved thickness, and therefore increased function and utility.
Subject(s)
Induced Pluripotent Stem Cells , Pluripotent Stem Cells , Humans , Tissue Scaffolds/chemistry , Tissue Engineering , Perfusion , Cell Differentiation , Hydrogels , BioreactorsABSTRACT
Automaticity involves Ca2+ handling at the cell membrane and sarcoplasmic reticulum (SR). Abnormal or acquired automaticity is thought to initiate ventricular arrhythmias associated with myocardial ischemia. Ca2+ flux from mitochondria can influence automaticity, and lysosomes also release Ca2+. Therefore, we tested whether lysosomal Ca2+ flux could influence automaticity. We studied ventricular human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), hiPSC 3D engineered heart tissues (EHTs), and ventricular cardiomyocytes isolated from infarcted mice. Preventing lysosomal Ca2+ cycling reduced automaticity in hiPSC-CMs. Consistent with a lysosomal role in automaticity, activating the transient receptor potential mucolipin channel (TRPML1) enhanced automaticity, and two channel antagonists reduced spontaneous activity. Activation or inhibition of lysosomal transcription factor EB (TFEB) increased or decreased total lysosomes and automaticity, respectively. In adult ischemic cardiomyocytes and hiPSC 3D EHTs, reducing lysosomal Ca2+ release also inhibited automaticity. Finally, TRPML1 was up-regulated in cardiomyopathic patients with ventricular tachycardia (VT) compared with those without VT. In summary, lysosomal Ca2+ handling modulates abnormal automaticity, and reducing lysosomal Ca2+ release may be a clinical strategy for preventing ventricular arrhythmias.
ABSTRACT
Introduction: Fibroblasts are mesenchymal cells that predominantly produce and maintain the extracellular matrix (ECM) and are critical mediators of injury response. In the heart, valve interstitial cells (VICs) are a population of fibroblasts responsible for maintaining the structure and function of heart valves. These cells are regionally distinct from myocardial fibroblasts, including left ventricular cardiac fibroblasts (LVCFBs), which are located in the myocardium in close vicinity to cardiomyocytes. Here, we hypothesize these subpopulations of fibroblasts are transcriptionally and functionally distinct. Methods: To compare these fibroblast subtypes, we collected patient-matched samples of human primary VICs and LVCFBs and performed bulk RNA sequencing, extracellular matrix profiling, and functional contraction and calcification assays. Results: Here, we identified combined expression of SUSD2 on a protein-level, and MEOX2, EBF2 and RHOU at a transcript-level to be differentially expressed in VICs compared to LVCFBs and demonstrated that expression of these genes can be used to distinguish between the two subpopulations. We found both VICs and LVCFBs expressed similar activation and contraction potential in vitro, but VICs showed an increase in ALP activity when activated and higher expression in matricellular proteins, including cartilage oligomeric protein and alpha 2-Heremans-Schmid glycoprotein, both of which are reported to be linked to calcification, compared to LVCFBs. Conclusion: These comparative transcriptomic, proteomic, and functional studies shed novel insight into the similarities and differences between valve interstitial cells and left ventricular cardiac fibroblasts and will aid in understanding region-specific cardiac pathologies, distinguishing between primary subpopulations of fibroblasts, and generating region-specific stem-cell derived cardiac fibroblasts.
ABSTRACT
During cardiac development and morphogenesis, cardiac progenitor cells differentiate into cardiomyocytes that expand in number and size to generate the fully formed heart. Much is known about the factors that regulate initial differentiation of cardiomyocytes, and there is ongoing research to identify how these fetal and immature cardiomyocytes develop into fully functioning, mature cells. Accumulating evidence indicates that maturation limits proliferation and conversely proliferation occurs rarely in cardiomyocytes of the adult myocardium. We term this oppositional interplay the proliferation-maturation dichotomy. Here we review the factors that are involved in this interplay and discuss how a better understanding of the proliferation-maturation dichotomy could advance the utility of human induced pluripotent stem cell-derived cardiomyocytes for modeling in 3-dimensional engineered cardiac tissues to obtain truly adult-level function.
Subject(s)
Induced Pluripotent Stem Cells , Tissue Engineering , Humans , Tissue Engineering/methods , Myocytes, Cardiac/physiology , Myocardium , Cell Differentiation/physiology , Cell ProliferationABSTRACT
The intervertebral disc (IVD) aids in motion and acts to absorb energy transmitted to the spine. With little inherent regenerative capacity, degeneration of the intervertebral disc results in intervertebral disc disease, which contributes to low back pain and significant disability in many individuals. Increasing evidence suggests that IVD degeneration is a disease of the whole joint that is associated with significant inflammation. Moreover, studies show elevated macrophage accumulation within the IVD with increasing levels of disease severity; however, we still need to understand the roles, be they causative or consequential, of macrophages during the degenerative process. In this narrative review, we discuss hallmarks of IVD degeneration, showcase evidence of macrophage involvement during disc degeneration, and explore burgeoning research aimed at understanding the molecular pathways regulating macrophage functions during intervertebral disc degeneration.
Subject(s)
Intervertebral Disc Degeneration , Intervertebral Disc Displacement , Intervertebral Disc , Humans , Intervertebral Disc Degeneration/metabolism , Intervertebral Disc/metabolism , Inflammation/metabolism , Intervertebral Disc Displacement/metabolism , Macrophages/metabolismABSTRACT
Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that mesenchymal signaling cells (MSCs) promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. In this manuscript, we tested the hypothesis that MSCs produce trophic factors that alter macrophage subsets. To this end, we collected conditioned medium from human, bone marrow-derived STRO3+ MSCs. We then cultured human bone marrow-derived macrophages in MSC conditioned medium (CM) and performed single cell RNA-sequencing. Comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. To determine if factors from MSC CM simulated effects of the anti-inflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-ß signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-ß neutralization partially limited the effects of MSC CM. In conclusion, our study identified a unique macrophage subset induced by MSCs within hypoxic conditions and supports that MSCs alter macrophage phenotypes through TGF-ß-dependent mechanisms.
ABSTRACT
Bionic-engineered tissues have been proposed for testing the performance of cardiovascular medical devices and predicting clinical outcomes ex vivo. Progress has been made in the development of compliant electronics that are capable of monitoring treatment parameters and being coupled to engineered tissues; however, the scale of most engineered tissues is too small to accommodate the size of clinical-grade medical devices. Here, we show substantial progress toward bionic tissues for evaluating cardiac ablation tools by generating a centimeter-scale human cardiac disk and coupling it to a hydrogel-based soft-pressure sensor. The cardiac tissue with contiguous electromechanical function was made possible by our recently established method to 3D bioprint human pluripotent stem cells in an extracellular matrix-based bioink that allows for in situ cell expansion prior to cardiac differentiation. The pressure sensor described here utilized electrical impedance tomography to enable the real-time spatiotemporal mapping of pressure distribution. A cryoablation tip catheter was applied to the composite bionic tissues with varied pressure. We found a close correlation between the cell response to ablation and the applied pressure. Under some conditions, cardiomyocytes could survive in the ablated region with more rounded morphology compared to the unablated controls, and connectivity was disrupted. This is the first known functional characterization of living human cardiomyocytes following an ablation procedure that suggests several mechanisms by which arrhythmia might redevelop following an ablation. Thus, bionic-engineered testbeds of this type can be indicators of tissue health and function and provide unique insight into human cell responses to ablative interventions.
Subject(s)
Bionics , Catheter Ablation , Humans , Catheter Ablation/methods , Myocytes, Cardiac/metabolism , Tissue Engineering/methods , Arrhythmias, Cardiac/metabolismABSTRACT
Predicting rupture risk in intracranial aneurysms is among one of the most critical questions in vascular surgery. The processes that govern an aneurysm growth are multifaceted and complex, but may be summarized into three components: hemodynamics, biology, and mechanics. We review and connect the literature in the three disciplines, identifying considerable strides in recent history and current gaps in research. Taken together, the findings from each field elucidate how and why certain aneurysms rupture, whereas others remain stable. These parameters could eventually inform a translatable predictive model that optimizes risk evaluation and physician's decision-making in treatment options for aneurysms.
Subject(s)
Aneurysm, Ruptured , Intracranial Aneurysm , Aneurysm, Ruptured/surgery , Biology , Hemodynamics , Humans , Intracranial Aneurysm/surgeryABSTRACT
More than 60% of hypertrophic cardiomyopathy (HCM)-causing mutations are found in the gene loci encoding cardiac myosin-associated proteins including myosin heavy chain (MHC) and myosin binding protein C (MyBP-C). Moreover, patients with more than one independent HCM mutation may be at increased risk for more severe disease expression and adverse outcomes. However detailed mechanistic understanding, especially at early stages of disease progression, is limited. To identify early-stage HCM triggers, we generated single (MYH7 c.2167C > T [R723C] with a known pathogenic significance in the MHC converter domain) and double (MYH7 c.2167C > T [R723C]; MYH6 c.2173C > T [R725C] with unknown significance) myosin gene mutations in human induced pluripotent stem cells (hiPSCs) using a base-editing strategy. Cardiomyocytes (CMs) derived from hiPSCs with either single or double mutation exhibited phenotypic characteristics consistent with later-stage HCM including hypertrophy, multinucleation, altered calcium handling, metabolism, and arrhythmia. We then probed mutant CMs at time points prior to the detection of known HCM characteristics. We found MYH7/MYH6 dual mutation dysregulated extracellular matrix (ECM) remodeling, altered integrin expression, and interrupted cell-ECM adhesion by limiting the formation of focal adhesions. These results point to a new phenotypic feature of early-stage HCM and reveal novel therapeutic avenues aimed to delay or prohibit disease onset.
ABSTRACT
The extracellular matrix (ECM) provides essential cues to promote endothelial specification during tissue development in vivo; correspondingly, ECM is considered essential for endothelial differentiation outside of the body. However, systematic studies to assess the precise contribution of individual ECM proteins to endothelial differentiation have not been conducted. Further, the multi-component nature of differentiation protocols makes it challenging to study the underlying mechanisms by which the ECM contributes to cell fate. In this study, we determined that Laminin 411 alone increases endothelial differentiation of induced pluripotent stem cells over collagen I or Matrigel. The effect of ECM was shown to be independent of vascular endothelial growth factor (VEGF) binding capacity. We also show that ECM-guided endothelial differentiation is dependent on activation of focal adhesion kinase (FAK), integrin-linked kinase (ILK), Notch, and ß-catenin pathways. Our results indicate that ECM contributes to endothelial differentiation through multiple avenues, which converge at the expression of active ß-catenin.
Subject(s)
Laminin , beta Catenin , Extracellular Matrix/metabolism , Laminin/metabolism , Signal Transduction/physiology , Vascular Endothelial Growth Factor A/metabolism , beta Catenin/metabolismABSTRACT
Each heartbeat that pumps blood throughout the body is initiated by an electrical impulse generated in the sinoatrial node (SAN). However, a number of disease conditions can hamper the ability of the SAN's pacemaker cells to generate consistent action potentials and maintain an orderly conduction path, leading to arrhythmias. For symptomatic patients, current treatments rely on implantation of an electronic pacing device. However, complications inherent to the indwelling hardware give pause to categorical use of device therapy for a subset of populations, including pediatric patients or those with temporary pacing needs. Cellular-based biological pacemakers, derived in vitro or in situ, could function as a therapeutic alternative to current electronic pacemakers. Understanding how biological pacemakers measure up to the SAN would facilitate defining and demonstrating its advantages over current treatments. In this review, we discuss recent approaches to creating biological pacemakers and delineate design criteria to guide future progress based on insights from basic biology of the SAN. We emphasize the need for long-term efficacy in vivo via maintenance of relevant proteins, source-sink balance, a niche reflective of the native SAN microenvironment, and chronotropic competence. With a focus on such criteria, combined with delivery methods tailored for disease indications, clinical implementation will be attainable.
Subject(s)
Arrhythmias, Cardiac/therapy , Biological Clocks , Sinoatrial Node/physiopathology , Action Potentials/physiology , Arrhythmias, Cardiac/physiopathology , Humans , Prosthesis DesignABSTRACT
Differentiation of pluripotent stem cells to cardiomyocytes is influenced by culture conditions including the extracellular matrices or similar synthetic scaffolds on which they are grown. However, the molecular mechanisms that link the scaffold with differentiation outcomes are not fully known. Here, we determined by immunofluorescence staining and mass spectrometry approaches that extracellular matrix (ECM) engagement by mouse pluripotent stem cells activates critical components of canonical wingless/integrated (Wnt) signaling pathways via kinases of the focal adhesion to drive cardiomyogenesis. These kinases were found to be differentially activated depending on type of ECM engaged. These outcomes begin to explain how varied ECM composition of in vivo tissues with development and in vitro model systems gives rise to different mature cell types, having broad practical applicability for the design of engineered tissues.
Subject(s)
Cell Differentiation , Extracellular Matrix/metabolism , Focal Adhesions/metabolism , Models, Cardiovascular , Myocytes, Cardiac/metabolism , Pluripotent Stem Cells/metabolism , Wnt Signaling Pathway , Animals , MiceABSTRACT
Cardiac fibroblasts (CFBs) support heart function by secreting extracellular matrix (ECM) and paracrine factors, respond to stress associated with injury and disease, and therefore are an increasingly important therapeutic target. We describe how developmental lineage of human pluripotent stem cell-derived CFBs, epicardial (EpiC-FB), and second heart field (SHF-FB) impacts transcriptional and functional properties. Both EpiC-FBs and SHF-FBs exhibited CFB transcriptional programs and improved calcium handling in human pluripotent stem cell-derived cardiac tissues. We identified differences including in composition of ECM synthesized, secretion of growth and differentiation factors, and myofibroblast activation potential, with EpiC-FBs exhibiting higher stress-induced activation potential akin to myofibroblasts and SHF-FBs demonstrating higher calcification and mineralization potential. These phenotypic differences suggest that EpiC-FBs have utility in modeling fibrotic diseases while SHF-FBs are a promising source of cells for regenerative therapies. This work directly contrasts regional and developmental specificity of CFBs and informs CFB in vitro model selection.
Subject(s)
Cell Lineage/physiology , Myofibroblasts/physiology , Pluripotent Stem Cells/physiology , Cell Differentiation/physiology , Cells, Cultured , Extracellular Matrix/physiology , Humans , Myocardium/pathology , Myocytes, Cardiac/physiology , Phenotype , Transcription, Genetic/physiologyABSTRACT
RATIONALE: One goal of cardiac tissue engineering is the generation of a living, human pump in vitro that could replace animal models and eventually serve as an in vivo therapeutic. Models that replicate the geometrically complex structure of the heart, harboring chambers and large vessels with soft biomaterials, can be achieved using 3-dimensional bioprinting. Yet, inclusion of contiguous, living muscle to support pump function has not been achieved. This is largely due to the challenge of attaining high densities of cardiomyocytes-a notoriously nonproliferative cell type. An alternative strategy is to print with human induced pluripotent stem cells, which can proliferate to high densities and fill tissue spaces, and subsequently differentiate them into cardiomyocytes in situ. OBJECTIVE: To develop a bioink capable of promoting human induced pluripotent stem cell proliferation and cardiomyocyte differentiation to 3-dimensionally print electromechanically functional, chambered organoids composed of contiguous cardiac muscle. METHODS AND RESULTS: We optimized a photo-crosslinkable formulation of native ECM (extracellular matrix) proteins and used this bioink to 3-dimensionally print human induced pluripotent stem cell-laden structures with 2 chambers and a vessel inlet and outlet. After human induced pluripotent stem cells proliferated to a sufficient density, we differentiated the cells within the structure and demonstrated function of the resultant human chambered muscle pump. Human chambered muscle pumps demonstrated macroscale beating and continuous action potential propagation with responsiveness to drugs and pacing. The connected chambers allowed for perfusion and enabled replication of pressure/volume relationships fundamental to the study of heart function and remodeling with health and disease. CONCLUSIONS: This advance represents a critical step toward generating macroscale tissues, akin to aggregate-based organoids, but with the critical advantage of harboring geometric structures essential to the pump function of cardiac muscle. Looking forward, human chambered organoids of this type might also serve as a test bed for cardiac medical devices and eventually lead to therapeutic tissue grafting.
Subject(s)
Bioprinting/methods , Cell Differentiation , Myocytes, Cardiac/physiology , Organoids/physiology , Tissue Engineering/methods , Action Potentials , Cell Proliferation , Cells, Cultured , Extracellular Matrix Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/physiology , Myocardial Contraction , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Organoids/cytology , Organoids/metabolismABSTRACT
Fusion between cells of different organisms (i.e., xenogeneic hybrids) can occur, and for humans this may occur in the course of tissue transplantation, animal handling, and food production. Previous work shows that conferred advantages are rare in xenogeneic hybrids, whereas risks of cellular dysregulation are high. Here, we explore the transcriptome of individual xenogeneic hybrids of human mesenchymal stem cells and murine cardiomyocytes soon after fusion and ask whether the process is stochastic or involves conserved pathway activation. Toward this end, single-cell RNA sequencing was used to analyze the transcriptomes of hybrid cells with respect to the human and mouse genomes. Consistent with previous work, hybrids possessed a unique transcriptome distinct from either fusion partner but were dominated by the cardiomyocyte transcriptome. New in this work is the documentation that a few genes that were latent in both fusion partners were consistently expressed in hybrids. Specifically, human growth hormone 1, murine ribosomal protein S27, and murine ATP synthase H+ transporting, mitochondrial Fo complex subunit C2 were expressed in nearly all hybrids. The consistent activation of latent genes between hybrids suggests conserved signaling mechanisms that either cause or are the consequence of fusion of these 2 cell types and might serve as a target for limiting unwanted xenogeneic fusion in the future.-Yuan, C., Freeman, B. T., McArdle, T. J., Jung, J. P., Ogle, B. M. Conserved pathway activation following xenogeneic, heterotypic fusion.
Subject(s)
Cell Fusion , Human Growth Hormone/metabolism , Hybrid Cells/metabolism , Mesenchymal Stem Cells/metabolism , Myocytes, Cardiac/metabolism , Transcriptome , Animals , Cells, Cultured , Coculture Techniques , High-Throughput Nucleotide Sequencing , Human Growth Hormone/genetics , Humans , Hybrid Cells/cytology , Mesenchymal Stem Cells/cytology , Mice , Myocytes, Cardiac/cytologyABSTRACT
On March 1 and 2, 2018, the National Institutes of Health 2018 Progenitor Cell Translational Consortium, Cardiovascular Bioengineering Symposium, was held at the University of Alabama at Birmingham. Convergence of life sciences and engineering to advance the understanding and treatment of heart failure was the theme of the meeting. Over 150 attendees were present, and >40 scientists presented their latest work on engineering human functional myocardium for disease modeling, drug development, and heart failure research. The scientists, engineers, and physicians in the field of cardiovascular sciences met and discussed the most recent advances in their work and proposed future strategies for overcoming the major roadblocks of cardiovascular bioengineering and therapy. Particular emphasis was given for manipulation and using of stem/progenitor cells, biomaterials, and methods to provide molecular, chemical, and mechanical cues to cells to influence their identity and fate in vitro and in vivo. Collectively, these works are profoundly impacting and progressing toward deciphering the mechanisms and developing novel treatments for left ventricular dysfunction of failing hearts. Here, we present some important perspectives that emerged from this meeting.
Subject(s)
Biological Science Disciplines , Biomedical Engineering , Biomedical Research , Heart Failure , Interdisciplinary Communication , Animals , Cooperative Behavior , Diffusion of Innovation , Heart/physiopathology , Heart Failure/metabolism , Heart Failure/pathology , Heart Failure/physiopathology , Heart Failure/therapy , Humans , Myocardium/metabolism , Myocardium/pathology , Recovery of Function , RegenerationABSTRACT
Recessive dystrophic Epidermolysis Bullosa (RDEB) is caused by mutations in collagen-type VII gene critical for the dermoepidermal junction (DEJ) formation. Neither tissues of animal models nor currently available in vitro models are amenable to the quantitative assessment of mechanical adhesion between dermal and epidermal layers. Here, we created a 3D in vitro DEJ model using extracellular matrix (ECM) proteins of the DEJ anchored to a poly(ethylene glycol)-based slab (termed ECM composites) and seeded with human keratinocytes and dermal fibroblasts. Keratinocytes and fibroblasts of healthy individuals were well maintained in the ECM composite and showed the expression of collagen type VII over a 2-week period. The ECM composites with healthy keratinocytes and fibroblasts exhibited yield stress associated with the separation of the model DEJ at 0.268 ± 0.057 kPa. When we benchmarked this measure of adhesive strength with that of the model DEJ fabricated with cells of individuals with RDEB, the yield stress was significantly lower (0.153 ± 0.064 kPa) consistent with our current mechanistic understanding of RDEB. In summary, a 3D in vitro model DEJ was developed for quantification of mechanical adhesion between epidermal- and dermal-mimicking layers, which can be utilized for assessment of mechanical adhesion of the model DEJ applicable for Epidermolysis Bullosa-associated therapeutics. © 2018 The Authors. Journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3231-3238, 2018.
Subject(s)
Epidermolysis Bullosa Dystrophica/pathology , Extracellular Matrix Proteins/chemistry , Fibroblasts/pathology , Keratinocytes/pathology , Polyethylene Glycols/chemistry , Tissue Scaffolds/chemistry , Biomechanical Phenomena , Cells, Cultured , Coculture Techniques , Collagen Type VII/analysis , Dermis/pathology , Humans , Immobilized Proteins/chemistry , Male , Stress, MechanicalABSTRACT
Cardiovascular disease is the global leading cause of death. One route to address this problem is using biomedical imaging to measure the molecules and structures that surround cardiac cells. This cellular microenvironment, known as the cardiac extracellular matrix, changes in composition and organization during most cardiac diseases and in response to many cardiac treatments. Measuring these changes with biomedical imaging can aid in understanding, diagnosing, and treating heart disease. This chapter supports those efforts by reviewing representative methods for imaging the cardiac extracellular matrix. It first describes the major biological targets of ECM imaging, including the primary imaging target of fibrillar collagen. Then it discusses the imaging methods, describing their current capabilities and limitations. It categorizes the imaging methods into two main categories: organ-scale noninvasive methods and cellular-scale invasive methods. Noninvasive methods can be used on patients, but only a few are clinically available, and others require further development to be used in the clinic. Invasive methods are the most established and can measure a variety of properties, but they cannot be used on live patients. Finally, the chapter concludes with a perspective on future directions and applications of biomedical imaging technologies.