scholarly journals Interindividual heterogeneity affects the outcome of human cardiac tissue decellularization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miguel F. Tenreiro ◽  
Henrique V. Almeida ◽  
Tomás Calmeiro ◽  
Elvira Fortunato ◽  
Lino Ferreira ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Sodium Dodecyl ◽  
Cell Alignment ◽  
Tissue Samples ◽  
Cardiac Tissues ◽  
Human Cardiac Tissue ◽  
Induced Pluripotent ◽  
Ecm Architecture ◽  
Triton X ◽  
Extended Exposure

AbstractThe extracellular matrix (ECM) of engineered human cardiac tissues corresponds to simplistic biomaterials that allow tissue assembly, or animal derived off-the-shelf non-cardiac specific matrices. Decellularized ECM from human cardiac tissue could provide a means to improve the mimicry of engineered human cardiac tissues. Decellularization of cardiac tissue samples using immersion-based methods can produce acceptable cardiac ECM scaffolds; however, these protocols are mostly described for animal tissue preparations. We have tested four methods to decellularize human cardiac tissue and evaluated their efficiency in terms of cell removal and preservation of key ECM components, such as collagens and sulfated glycosaminoglycans. Extended exposure to decellularization agents, namely sodium dodecyl sulfate and Triton-X-100, was needed to significantly remove DNA content by approximately 93% in all human donors. However, the biochemical composition of decellularized tissue is affected, and the preservation of ECM architecture is donor dependent. Our results indicate that standardization of decellularization protocols for human tissue is likely unfeasible, and a compromise between cell removal and ECM preservation must be established in accordance with the scaffold’s intended application. Notwithstanding, decellularized human cardiac ECM supported human induced pluripotent-derived cardiomyocyte (hiPSC-CM) attachment and retention for up to 2 weeks of culture, and promoted cell alignment and contraction, providing evidence it could be a valuable tool for cardiac tissue engineering.

Abstract 366: Engineered Human Cardiac Tissue from Skeletal Muscle Derived Cells and Induced Pluripotent Stem Cell Derived Cardiomyocytes

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Kimimasa Tobita ◽  
Jason S Tchao ◽  
Jong Kim ◽  
Bo Lin ◽  
Johnny Huard ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Cardiac Tissue ◽  
Troponin I ◽  
Troponin T ◽  
Induced Pluripotent Stem Cell ◽  
Human Cardiac Tissue ◽  
Induced Pluripotent ◽  
Immature Myocardium

We have previously shown that rat skeletal muscle derived stem cells differentiate into an immature cardiomyocyte (CM) phenotype within a 3-dimensional collagen gel engineered cardiac tissue (ECT). Here, we investigated whether human skeletal muscle derived progenitor cells (skMDCs) can differentiate into a CM phenotype within ECT similar to rat skeletal muscle stem cells and compared the human skMDC-ECT properties with ECT from human induced pluripotent stem cell (iPSc) derived CMs. SkMDCs differentiated into a cardiac muscle phenotype within ECT and exhibited spontaneous beating activity as early as culture day 4 and maintained their activity for more than 2 weeks. SkMDC-ECTs stained positive for cardiac specific troponin-T and troponin-I, and were co-localized with fast skeletal muscle myosin heavy chain (sk-fMHC) with a striated muscle pattern similar to fetal myocardium. The iPS-CM-ECTs maintained spontaneous beating activity for more than 2 weeks from ECT construction. iPS-CM stained positive for both cardiac troponin-T and troponin-I, and were also co-localized with sk-fMHC while the striated expression pattern of sk-fMHC was lost similar to post-natal immature myocardium. Connexin-43 protein was expressed in both engineered tissue types, and the expression pattern was similar to immature myocardium. The skMDC-ECT significantly upregulated expression of cardiac-specific genes compared to conventional 2D culture. SkMDC-ECT displayed cardiac muscle like intracellular calcium ion transients. The contractile force measurements demonstrated functional properties of fetal type myocardium in both ECTs. Our results suggest that engineered human cardiac tissue from skeletal muscle progenitor cells mimics developing fetal myocardium while the engineered cardiac tissue from inducible pluripotent stem cell-derived cardiomyocytes mimics post-natal immature myocardium.

Abstract 30: In vitro Fabrication of Human Cardiac Tissues With Porcine Perfusable Blood Vessels

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Akitoshi Inui ◽  
Hidekazu Sekine ◽  
Kazunori Sano ◽  
Izumi Dobashi ◽  
Azumi Yoshida ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Cardiac Tissue ◽  
Severe Heart Failure ◽  
Perfusion Culture ◽  
Cardiac Cell ◽  
Cell Sheets ◽  
Cardiac Tissues ◽  
Bioreactor System ◽  
Human Cardiac Tissue

The definitive treatment of severe heart failure is heart transplantation; however the number of heart transplantation procedures performed in Japan per year ranges from 30-40 due to donor shortage. Therefore, recently other treatments such as ventricular assist device or regenerative therapy by human cardiac tissue engineering have been developed and are considered as appropriate alternatives. We have developed an original technology, which was named cell-sheet based tissue engineering to fabricate functional three-dimensional tissue by layering cell sheets. The utilization of this technique allowed us to successfully engineer thick rat cardiac tissue with perfusable blood vessels in vitro. Here, we demonstrate a technique to engineer human cardiac tissue with perfusable blood vessels using cardiac cell sheets derived from human induced pluripotent stem cells, and porcine small intestine as a vascular bed for perfusion culture. The small intestine was harvested from with a branch of the superior mesenteric artery and vein and underwent mucosal resection after harvested tissue was cut open. To engineer cardiac tissue with perfusable blood vessels, cardiac cell sheets co-cultured with endothelial cells, were triple-layered and then was overlaid on the vascular bed in the bioreactor system. One day after perfusion culture, overlaid cardiac tissues pulsated spontaneously and were synchronized. The cardiac tissue construct was viable tissue without any observable necrosis. Furthermore we examined the possibility of transplantation of the in vitro engineered human cardiac tissue with the connectable host artery and vein. Engineered cardiac tissue was removed from the bioreactor system after 4-day perfusion, and transplanted to another pig heart. The branch of the superior mesenteric artery and vein of the graft were then reconnected to the host internal thoracic artery and vein. When the cardiac tissue reperfused, it began to beat spontaneously after a few minutes. We believe that this method is useful to fabricate functional cardiac tissue and may become an appropriate treatment for severe heart failure.

scholarly journals Virome Sequencing in Patients With Myocarditis

2020 ◽  
Vol 13 (7) ◽  
Author(s):  
Bettina Heidecker ◽  
Simon H. Williams ◽  
Komal Jain ◽  
Alexandra Oleynik ◽  
Dimitri Patriki ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Cardiac Tissue ◽  
Mononuclear Cells ◽  
Endogenous Retrovirus ◽  
Fulminant Myocarditis ◽  
Human Endogenous Retrovirus ◽  
Peripheral Blood Mononuclear ◽  
Tissue Samples ◽  
Cardiac Tissues ◽  
Barr Virus

Background: Polymerase chain reaction analyses of cardiac tissues have detected viral sequences in up to 67% of cases of myocarditis. However, viruses have not been implicated in giant cell myocarditis (GCM). Furthermore, efforts to detect viruses implicated in myocarditis have been unsuccessful in more accessible samples such as peripheral blood. Methods: We used Virome Capture Sequencing for Vertbrate Viruses (VirCapSeq-VERT), a method that simultaneously screens for all known vertebrate viruses, to investigate viruses in 33 patients with myocarditis. We investigated peripheral blood mononuclear cells (n=24), plasma (n=27), endomyocardial biopsies (n=2), and cardiac tissue samples from explanted hearts (n=13). Results: Nine patients (27%) had GCM and 4 patients (13%) had fulminant myocarditis. We found the following viruses in the blood of patients with myocarditis: Epstein Barr virus (n=11, 41%), human pegivirus (n=1, 4%), human endogenous retrovirus K (n=27, 100%), and anellovirus (n=15, 56%). All tissue samples from fulminant myocarditis (n=2) and GCM (n=13) contained human endogenous retrovirus K. Conclusions: No nucleic acids from viruses previously implicated in myocarditis or other human illnesses were detected in relevant amounts in cardiac tissue samples from GCM or in blood samples from other types of myocarditis. These findings do not exclude a role for viral infection in GCM but do suggest that if viruses are implicated, the mechanism is likely to be indirect rather than due to cytotoxic infection of myocardium.

Human induced pluripotent stem cell-derived cardiac tissue on a thin collagen membrane with natural microstructures

2016 ◽  
Vol 4 (11) ◽  
pp. 1655-1662 ◽  
Author(s):  
Li Wang ◽  
Xiaoqing Zhang ◽  
Cong Xu ◽  
Hui Liu ◽  
Jianhua Qin
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Cardiac Tissue ◽  
Induced Pluripotent Stem Cell ◽  
Collagen Membrane ◽  
Cardiac Tissues ◽  
New Strategy ◽  
Induced Pluripotent

We present a new strategy to produce a thin collagen membrane from porcine tendons and engineered cardiac tissues using hiPSC-derived cardiomyocytes.

scholarly journals Quantification of Cardiomyocyte Alignment from Three-Dimensional (3D) Confocal Microscopy of Engineered Tissue

2017 ◽  
Vol 23 (4) ◽  
pp. 826-842 ◽  
Author(s):  
William J. Kowalski ◽  
Fangping Yuan ◽  
Takeichiro Nakane ◽  
Hidetoshi Masumoto ◽  
Marc Dwenger ◽  
...  
Keyword(s):  
Multiple Scales ◽  
Orientational Order ◽  
Three Dimensional ◽  
Biological Tissues ◽  
Cardiac Cells ◽  
Cell Alignment ◽  
Cardiac Tissues ◽  
Automated Method ◽  
Synthetic Images ◽  
Induced Pluripotent

AbstractBiological tissues have complex, three-dimensional (3D) organizations of cells and matrix factors that provide the architecture necessary to meet morphogenic and functional demands. Disordered cell alignment is associated with congenital heart disease, cardiomyopathy, and neurodegenerative diseases and repairing or replacing these tissues using engineered constructs may improve regenerative capacity. However, optimizing cell alignment within engineered tissues requires quantitative 3D data on cell orientations and both efficient and validated processing algorithms. We developed an automated method to measure local 3D orientations based on structure tensor analysis and incorporated an adaptive subregion size to account for multiple scales. Our method calculates the statistical concentration parameter,κ, to quantify alignment, as well as the traditional orientational order parameter. We validated our method using synthetic images and accurately measured principal axis and concentration. We then applied our method to confocal stacks of cleared, whole-mount engineered cardiac tissues generated from human-induced pluripotent stem cells or embryonic chick cardiac cells and quantified cardiomyocyte alignment. We found significant differences in alignment based on cellular composition and tissue geometry. These results from our synthetic images and confocal data demonstrate the efficiency and accuracy of our method to measure alignment in 3D tissues.

scholarly journals Chronic optical pacing conditioning of h-iPSC engineered cardiac tissues

2019 ◽  
Vol 10 ◽  
pp. 204173141984174 ◽  
Author(s):  
Marc Dwenger ◽  
William J Kowalski ◽  
Fei Ye ◽  
Fangping Yuan ◽  
Joseph P Tinney ◽  
...  
Keyword(s):  
Stem Cell ◽  
Electrical Stimulation ◽  
Pluripotent Stem Cell ◽  
Cardiac Tissue ◽  
Induced Pluripotent Stem Cell ◽  
Mechanical Stretch ◽  
In Vitro Models ◽  
Cardiac Tissues ◽  
Induced Pluripotent

The immaturity of human induced pluripotent stem cell derived engineered cardiac tissues limits their ability to regenerate damaged myocardium and to serve as robust in vitro models for human disease and drug toxicity studies. Several chronic biomimetic conditioning protocols, including mechanical stretch, perfusion, and/or electrical stimulation promote engineered cardiac tissue maturation but have significant technical limitations. Non-contacting chronic optical stimulation using heterologously expressed channelrhodopsin light-gated ion channels, termed optogenetics, may be an advantageous alternative to chronic invasive electrical stimulation for engineered cardiac tissue conditioning. We designed proof-of-principle experiments to successfully transfect human induced pluripotent stem cell derived engineered cardiac tissues with a desensitization resistant, chimeric channelrhodopsin protein, and then optically paced engineered cardiac tissues to accelerate maturation. We transfected human induced pluripotent stem cell engineered cardiac tissues using an adeno-associated virus packaged chimeric channelrhodopsin and then verified optically paced by whole cell patch clamp. Engineered cardiac tissues were then chronically optically paced above their intrinsic beat rates in vitro from day 7 to 14. Chronically optically paced resulted in improved engineered cardiac tissue electrophysiological properties and subtle changes in the expression of some cardiac relevant genes, though active force generation and histology were unchanged. These results validate the feasibility of a novel chronically optically paced paradigm to explore non-invasive and scalable optically paced–induced engineered cardiac tissue maturation strategies.

scholarly journals SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes

2020 ◽  
Author(s):  
Denisa Bojkova ◽  
Julian Wagner ◽  
Mariana Shumliakivska ◽  
Galip Aslan ◽  
Umber Saleem ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Cardiac Injury ◽  
Transcriptional Response ◽  
Human Cardiomyocytes ◽  
Global Pandemic ◽  
Virus Rna ◽  
Human Cardiac Tissue ◽  
Induced Pluripotent ◽  
Apoptotic Effects

Background: The coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality in COVID-19 patients. It is unclear whether cardiac injury may have been caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here we investigate whether human cardiomyocytes are permissive for SARS-CoV-2 infection. Methods: Infection was induced by two strains of SARS-CoV-2 (FFM1 and FFM2) in human induced pluripotent stem cells-derived cardiomyocytes (hiPS-CM) and in two models of human cardiac tissue. Results: We show that SARS-CoV-2 infects hiPS-CM as demonstrated by detection of intracellular double strand viral RNA and viral spike glycoprotein protein expression. Increasing concentrations of virus RNA are detected in supernatants of infected cardiomyocytes, which induced infections in CaCo-2 cell lines documenting productive infections. SARS-COV-2 infection induced cytotoxic and pro-apoptotic effects and abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signaling, apoptosis and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a iPS-derived human 3D cardiosphere tissue models. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. Conclusions: The demonstration that cardiomyocytes are permissive for SARS-CoV-2 infection in vitro warrants the further in depth monitoring of cardiotoxic effects in COVID-19 patients.

scholarly journals Single‐Cell Transcriptomics of Engineered Cardiac Tissues From Patient‐Specific Induced Pluripotent Stem Cell–Derived Cardiomyocytes Reveals Abnormal Developmental Trajectory and Intrinsic Contractile Defects in Hypoplastic Right Heart Syndrome

2020 ◽  
Vol 9 (20) ◽  
Author(s):  
Yin‐Yu Lam ◽  
Wendy Keung ◽  
Chun‐Ho Chan ◽  
Lin Geng ◽  
Nicodemus Wong ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Pluripotent Stem Cell ◽  
Cardiac Tissue ◽  
Induced Pluripotent Stem Cell ◽  
Developmental Trajectory ◽  
Single Cell Rna Sequencing ◽  
Human Cardiac Tissue ◽  
Induced Pluripotent

Background To understand the intrinsic cardiac developmental and functional abnormalities in pulmonary atresia with intact ventricular septum (PAIVS) free from effects secondary to anatomic defects, we performed and compared single‐cell transcriptomic and phenotypic analyses of patient‐ and healthy subject–derived human‐induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs) and engineered tissue models. Methods and Results We derived hiPSC lines from 3 patients with PAIVS and 3 healthy subjects and differentiated them into hiPSC‐CMs, which were then bioengineered into the human cardiac anisotropic sheet and human cardiac tissue strip custom‐designed for electrophysiological and contractile assessments, respectively. Single‐cell RNA sequencing (scRNA‐seq) of hiPSC‐CMs, human cardiac anisotropic sheet, and human cardiac tissue strip was performed to examine the transcriptomic basis for any phenotypic abnormalities using pseudotime and differential expression analyses. Through pseudotime analysis, we demonstrated that bioengineered tissue constructs provide pro‐maturational cues to hiPSC‐CMs, although the maturation and development were attenuated in PAIVS hiPSC‐CMs. Furthermore, reduced contractility and prolonged contractile kinetics were observed with PAIVS human cardiac tissue strips. Consistently, single‐cell RNA sequencing of PAIVS human cardiac tissue strips and hiPSC‐CMs exhibited diminished expression of cardiac contractile apparatus genes. By contrast, electrophysiological aberrancies were absent in PAIVS human cardiac anisotropic sheets. Conclusions Our findings were the first to reveal intrinsic abnormalities of cardiomyocyte development and function in PAIVS free from secondary effects. We conclude that hiPSC‐derived engineered tissues offer a unique method for studying primary cardiac abnormalities and uncovering pathogenic mechanisms that underlie sporadic congenital heart diseases.

Periodontal Bacterial DNA and Their Link to Human Cardiac Tissue: Findings of a Pilot Study

2015 ◽  
Vol 66 (01) ◽  
pp. 083-090 ◽  
Author(s):  
Dirk Ziebolz ◽  
Christoph Rost ◽  
Julia Schmidt ◽  
Regina Waldmann-Beushausen ◽  
Friedrich Schöndube ◽  
...  
Keyword(s):  
Pilot Study ◽  
Aortic Valve ◽  
Cardiac Tissue ◽  
Binding Protein ◽  
Myocardial Revascularization ◽  
Tissue Samples ◽  
Bacterial Dna ◽  
Protocol Biopsies ◽  
Human Cardiac Tissue ◽  
Lps Binding

Background The aim of this pilot study was to detect correlations of microbiological DNA, inflammatory proteins, and infection parameters in patients with periodontal disease (PD) and valvular heart disease (VHD). Methods A perioperative comprehensive dental examination for the investigation of periodontal status, including sampling of specific subgingival bacteria, was performed in 10 patients with indication for surgery of aortic valve stenosis with or without concomitant myocardial revascularization. Standard protocol biopsies were taken from right atrium (A), left septal myocardium (M), and aortic valve (V). Eleven periodontal pathogens DNA in oral and cardiac tissue samples (A/M/V) were analyzed using polymerase chain reaction. For cardiac tissue samples, Western blot analysis of LPS-binding protein (LBP), immunohistochemical (IHC) detection of LBP-big42, LPS-binding protein receptor (CD14), and macrophages (CD68), as well as inflammation scoring measurement were performed. Results Periodontitis was present in all patients with severe intensity in 7, moderate in 2 and mild in one patient. Same bacterial DNA was detected in A, M, and V in different distribution, and detection was more often in atrium than in myocardium or valve tissue. Morphological investigation revealed increased extracellular inflammatory cell migration. In IHC markers of LBP, CD68 and CD14 showed positive findings for all patients in atrium and myocardium. Conclusion Our results demonstrate the presence of oral bacterial DNA in human cardiac tissue, as well as inflammatory markers potentially indicating connection of PD and VHD. Further investigation is necessary to confirm these preliminary data.

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