Abstract 13381: hiPSC of CIITA and B2M KO Derived Cardiac Cells Engineered 3D Spheroid Transplantation Induce Tachycardia in Myocardial Infarction in Swine

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Saidulu Mattapally ◽  
Jianyi Zhang
Keyword(s):  
Cardiac Fibroblasts ◽  
Quantitative Polymerase Chain Reaction ◽  
Hla Class I ◽  
Cardiac Cells ◽  
Border Zone ◽  
Large Animal ◽  
Myocardial Repair ◽  
Large Animal Models ◽  
Organ Systems ◽  
Significant Difference

Introduction: Human induced pluripotent stem cells (hiPSCs) are among one the most significant discoveries in life sciences. As a promising —biological drug for cell therapy, multiple lineages of iPSC-derived cardiac cells have been administered in human clinical trials in multiple important organ systems. The potential immunogenicity of hiPSC-derived cardiac cells continues to be one of the concerns in large animal models. Methods: In the present study, WT hiPSCs were generated by transfecting male human cardiac fibroblasts with Sendai viruses coding for OCT4, SOX2, KLF4, and C-MYC.Then hiPSCs carrying knockout mutations for both HLA Class I and Class II (HLAI/II-KOhiPSC) were generated via CRISPR/Cas9 gene-editing technology (Mattapally et al, 2018). hiPSCs were differentiated into cardiomyocytes (CM) endothelial cells (ECs) and spheroid cultured was performed as previously described (Mattapally et al, 2018). We evaluated spheroid transplantation and its potency for myocardial repair in the Swine. Programmed stimulation was used to determine the arrhythmogenic outcome. Results: To determine the engraftment efficacy of HLAI/II KO compared to WT Spheroid, a swine study was performed. After LAD ligation in swine, 800μm spheroid was injected into the border zone of the left ventricle. After transplantation, cell engraftment was monitored by Q-PCR. At week 4, there was a significant difference between the 2 groups. Animal groups included: MI hearts treated with 500 WT Spheroid injection (MI+WT Spheroid, n=5), MI hearts treated with 500 KO Spheroid injection (n=6), MI only hearts (n=6); the fourth group of animals underwent sham surgery (Sham, n=6). Arrhythmia was studied by programmed electrical stimulations (PES) and conduction velocities measured with electrode mapping, and the engraftment rate by calculation of quantitative polymerase chain reaction measurements of expression of the human Y chromosome. Engraftment of iPSC-CMs was found in both treatment groups; however, a significantly higher engraftment rate was found in KO Spheroid. The spheroid treatment is associated with significant changes in arrhythmogenicity. Conclusion: Our study established the improved graft but associated with arrhythmogenicity.

scholarly journals Complex electrophysiological remodeling in postinfarction ischemic heart failure

2018 ◽  
Vol 115 (13) ◽  
pp. E3036-E3044 ◽  
Author(s):  
Bence Hegyi ◽  
Julie Bossuyt ◽  
Leigh G. Griffiths ◽  
Rafael Shimkunas ◽  
Zana Coulibaly ◽  
...  
Keyword(s):  
Heart Failure ◽  
Single Channel ◽  
Ionic Currents ◽  
Border Zone ◽  
Delayed Rectifier ◽  
Large Animal ◽  
Current Profile ◽  
Large Animal Models ◽  
Remote Zone ◽  
Arrhythmogenic Substrate

Heart failure (HF) following myocardial infarction (MI) is associated with high incidence of cardiac arrhythmias. Development of therapeutic strategy requires detailed understanding of electrophysiological remodeling. However, changes of ionic currents in ischemic HF remain incompletely understood, especially in translational large-animal models. Here, we systematically measure the major ionic currents in ventricular myocytes from the infarct border and remote zones in a porcine model of post-MI HF. We recorded eight ionic currents during the cell’s action potential (AP) under physiologically relevant conditions using selfAP-clamp sequential dissection. Compared with healthy controls, HF-remote zone myocytes exhibited increased late Na+ current, Ca2+-activated K+ current, Ca2+-activated Cl− current, decreased rapid delayed rectifier K+ current, and altered Na+/Ca2+ exchange current profile. In HF-border zone myocytes, the above changes also occurred but with additional decrease of L-type Ca2+ current, decrease of inward rectifier K+ current, and Ca2+ release-dependent delayed after-depolarizations. Our data reveal that the changes in any individual current are relatively small, but the integrated impacts shift the balance between the inward and outward currents to shorten AP in the border zone but prolong AP in the remote zone. This differential remodeling in post-MI HF increases the inhomogeneity of AP repolarization, which may enhance the arrhythmogenic substrate. Our comprehensive findings provide a mechanistic framework for understanding why single-channel blockers may fail to suppress arrhythmias, and highlight the need to consider the rich tableau and integration of many ionic currents in designing therapeutic strategies for treating arrhythmias in HF.

scholarly journals Natural history study of glycan accumulation in large animal models of GM2 gangliosidoses

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243006
Author(s):  
Catlyn Cavender ◽  
Linley Mangini ◽  
Jeremy L. Van Vleet ◽  
Carley Corado ◽  
Emma McCullagh ◽  
...  
Keyword(s):  
Animal Models ◽  
Natural History ◽  
Biochemical Study ◽  
Sandhoff Disease ◽  
Large Animal ◽  
Natural History Study ◽  
Large Animal Models ◽  
Significant Difference ◽  
Feline Model ◽  
Gm2 Gangliosidoses

β-hexosaminidase is an enzyme responsible for the degradation of gangliosides, glycans, and other glycoconjugates containing β-linked hexosamines that enter the lysosome. GM2 gangliosidoses, such as Tay-Sachs and Sandhoff, are lysosomal storage disorders characterized by β-hexosaminidase deficiency and subsequent lysosomal accumulation of its substrate metabolites. These two diseases result in neurodegeneration and early mortality in children. A significant difference between these two disorders is the accumulation in Sandhoff disease of soluble oligosaccharide metabolites that derive from N- and O-linked glycans. In this paper we describe our results from a longitudinal biochemical study of a feline model of Sandhoff disease and an ovine model of Tay-Sachs disease to investigate the accumulation of GM2/GA2 gangliosides, a secondary biomarker for phospholipidosis, bis-(monoacylglycero)-phosphate, and soluble glycan metabolites in both tissue and fluid samples from both animal models. While both Sandhoff cats and Tay-Sachs sheep accumulated significant amounts of GM2 and GA2 gangliosides compared to age-matched unaffected controls, the Sandhoff cats having the more severe disease, accumulated larger amounts of gangliosides compared to Tay-Sachs sheep in their occipital lobes. For monitoring glycan metabolites, we developed a quantitative LC/MS assay for one of these free glycans in order to perform longitudinal analysis. The Sandhoff cats showed significant disease-related increases in this glycan in brain and in other matrices including urine which may provide a useful clinical tool for measuring disease severity and therapeutic efficacy. Finally, we observed age-dependent increasing accumulation for a number of analytes, especially in Sandhoff cats where glycosphingolipid, phospholipid, and glycan levels showed incremental increases at later time points without signs of peaking. This large animal natural history study for Sandhoff and Tay-Sachs is the first of its kind, providing insight into disease progression at the biochemical level. This report may help in the development and testing of new therapies to treat these disorders.

Abstract 16: Enhancing Myocardial Repair With CardioChimeras

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Pearl Quijada ◽  
Hazel T Salunga ◽  
Nirmala Hariharan ◽  
Jonathan Cubillo ◽  
Farid El-Sayed ◽  
...  
Keyword(s):  
Infarct Size ◽  
Troponin T ◽  
Capillary Density ◽  
Anterior Wall ◽  
Left Ventricular ◽  
Parent Cell ◽  
Large Animal ◽  
Myocardial Repair ◽  
Post Injury ◽  
Large Animal Models

Dual cell transplantation of cardiac progenitor cells (CPCs) and mesenchymal stem cells (MSCs) after infarction enhances myocardial repair and performance in large animal models relative to delivery of either cell population individually. However, a single stem cell to support both direct and indirect mechanisms of myocardial repair has yet to be identified. CardioChimeras (CCs), a progenitor cell formed by fusion between CPCs and MSCs were analysed for reparative potential after myocardial infarction (MI) relative to individual parents cell or combined parent cell delivery. Two representative CCs, CardioChimera 1 (CC1) and CardioChimera 2 (CC2) were used for this study. CC1 and CC2 improved left ventricular anterior wall thickness (AWT) at 4 weeks, but only CC1 treatment preserved AWT at 18 weeks relative to no cell treatment (PBS). Ejection fraction was enhanced at 6 weeks post injury in CC1 and CC2 groups, which was maintained in CC1, CC2 and CPC + MSC combined groups up to 18 weeks. Infarct size was decreased by 5% in CC1 and CC2 hearts, whereas CPC + MSC and CPC parent groups remained unchanged when comparing 4 to 12 week change in scar size. MSC and PBS groups displayed marked increases in infarct size (10-15%). CC1 and CC2 showed enhanced engraftment potential by 3-fold relative to CPC + MSC and CPC hearts. In contrast, MSCs were detected at low levels (0.04%). CC1 and CC2 discovered within the myocardium expressed early commitment marker cardiac troponin T relative to controls. CC1 and CC2 treatment increased capillary density within the infarct, indicating that cell persistence facilitates paracrine mediated vasculature stabilization and/or formation. CCs merge the application of distinct cells into a single entity for cellular therapeutic intervention in the progression of heart failure. CCs represent a tractable cellular system that improves upon combinatorial cell therapy approaches and supports myocardial regeneration.

Abstract 12083: Lymphatic Vasculature Mediates Cardiac Progenitor Cell Trafficking After Myocardial Infarction

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Maria Cimini ◽  
Luca Troncone ◽  
Antonio Di Rocco ◽  
Sergio Signore ◽  
Antonio Cannata ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Endothelial Cells ◽  
Cardiac Cells ◽  
Border Zone ◽  
Integrin Subunit ◽  
Cell Trafficking ◽  
Myocardial Repair ◽  
Inhibitory Peptide ◽  
Direct Role

Myocardial infarction (MI) and post-MI heart failure are major causes of morbidity and mortality. Cardiac progenitor cells (CPC) regenerate injured myocardium, due to their ability to migrate and engraft in the wounded area. Yet, the mechanisms governing CPC trafficking in the diseased myocardium are largely unknown. We examined a hitherto unaddressed role of cardiac lymphatic microvessels (LMV) in CPC mobilization to the site of infarction. We detected that in acute and chronic MI, mouse CPC accumulated in the infarct border zone in proximity of LMV and traversed their wall. We showed that human CPC actively intravasated into the lumen of three-dimensional LMV formed by human lymphatic endothelial cells (LEC) in vitro. The binding of CPC to LEC was hindered in the presence of N-cadherin inhibitory peptide or E-selectin neutralizing antibody. CPC displayed unique pattern of interactions with LEC. Specifically, the propensity of CPC to adhere to LEC at basal conditions and after pre-stimulation with an inflammatory mediator TNFα was higher comparing to human cardiac microvascular endothelial cells (CMEC). Trans-migration of CPC through LEC but not CMEC monolayer was enhanced following treatment with TNFα. Interference with the SDF-1/CXCR4 pathway diminished CPC intravasation via LEC but did not affect the CPC ability to traverse CMEC. Intriguingly, we found that the bioactive lipid S1P, secreted by endothelial cells, had a significant impact on CPC migration. S1P binds to 5 different S1P receptors (S1P1-5). We established that the expression of S1P3 was enriched in CPC. Although exposure to S1P mitigated CPC motility, an S1P3-selective agonist evoked strong chemotaxis of CPC, whereas down-regulation of S1P3 blocked CPC translocation. Accordingly, CPC activation with S1P3 agonist reduced their contacts with the extracellular matrix, while S1P ligand induced the organization of adhesion complexes containing β1-integrin subunit. The direct role of S1P3 in CPC trafficking in vivo is supported by our observations that S1P3 expression was increased in the cardiac cells associated with LMV after MI. In conclusion, we provide evidence that specific interactions with LMV may promote CPC trafficking in the infarcted heart facilitating myocardial repair.

scholarly journals Differentiation and Application of Human Pluripotent Stem Cells Derived Cardiovascular Cells for Treatment of Heart Diseases: Promises and Challenges

2021 ◽  
Vol 9 ◽  
Author(s):  
Yu Gao ◽  
Jun Pu
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Heart Diseases ◽  
Embryonic Stem ◽  
Human Pluripotent Stem Cells ◽  
Cardiac Cells ◽  
Human Embryos ◽  
Large Animal ◽  
Large Animal Models ◽  
Cardiovascular Cells

Human pluripotent stem cells (hPSCs) are derived from human embryos (human embryonic stem cells) or reprogrammed from human somatic cells (human induced pluripotent stem cells). They can differentiate into cardiovascular cells, which have great potential as exogenous cell resources for restoring cardiac structure and function in patients with heart disease or heart failure. A variety of protocols have been developed to generate and expand cardiovascular cells derived from hPSCs in vitro. Precisely and spatiotemporally activating or inhibiting various pathways in hPSCs is required to obtain cardiovascular lineages with high differentiation efficiency. In this concise review, we summarize the protocols of differentiating hPSCs into cardiovascular cells, highlight their therapeutic application for treatment of cardiac diseases in large animal models, and discuss the challenges and limitations in the use of cardiac cells generated from hPSCs for a better clinical application of hPSC-based cardiac cell therapy.

Abstract 16635: Enhanced Cardiac Delivery of a Small Soluble Antifibrotic Peptide With Liposomal Encapsulation

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Swati D Sonkawade ◽  
Shirley Xu ◽  
Saraswati Pokharel ◽  
Umesh C Sharma
Keyword(s):  
Membrane Trafficking ◽  
Cardiac Fibrosis ◽  
Cardiac Fibroblasts ◽  
Target Cells ◽  
Large Animal ◽  
Inhibitory Peptide ◽  
Free Peptide ◽  
Large Animal Models ◽  
Distribution Studies

Introduction: N-acetyl-ser-asp-lys-pro (Ac-SDKP) is an enodogenous anti-fibrotic peptide with a great potential for use as a “ replacement therapy ” in fibrotic myocardial remodeling. However, its therapeutic use is limited by its short half-life (4.5 mins) and a negligible bioavailibity. Hypothesis: We hypothesize that liposomal encapsulation of Ac-SDKP (L-Ac-SDKP) enhances its stability, and improves delivery into the myocardial target cells at a higher efficiency than free Ac-SDKP. This will maximize retention time, allowing intermittent dosing and minimizing off-target effects. Methods: We synthesized L-Ac-SDKP using zwitterionic lipid formulation for better cell membrane penetration. We used Fluorophore (FITC)-conjugated Ac-SDKP as a tracer, and scrambled peptides and blank liposomes as controls. After confirming an enhanced uptake efficiency of L-Ac-SDKP into the adult cardiac fibroblasts, we performed in vivo L-Ac-SDKP uptake and distribution studies in a model of cardiac fibrosis induced by 30Gy of thoracic ionizing radiation exposure. Results: Confocal microscopy of the cultured cardiac fibroblasts showed L-Ac-SDKP distribution localized to perinuclear areas corroborating its known effects on cell-cycle and nuclear membrane trafficking compared to the free peptide. Along with flow cytometry analysis L-Ac-SDKP uptake was seen in 98% of cells as opposed to 7% when treated with free Ac-SDKP. In vivo, the mouse model of myocardial fibrosis showed significantly increased L-Ac-SDKP uptake (33±3 fold), compared to normal controls (14±3 fold) after 5 daily injections. Quantification of Ac-SDKP in the cardiac homogenate reached a maximum therapeutic concentration of 1.4 pg/mg after 2 doses in irradiated mice, whereas even higher concentration could be achieved after 5 cumulative doses. Conclusion: We conclude that liposomal-conjugation is a highly effective therapeutic approach for targeting cardiac fibrosis using a potent fibroblast inhibitory peptide, Ac-SDKP as a therapeutic agent. Translational studies in large animal models are currently in progress for further testing its therapeutic utility.

Abstract 135: Reprogramming of Pig Cardiac Fibroblasts to Cardiomyocyte Fate: Implications for Gene Therapy to Treat Myocardial Infarction

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Vivek P Singh ◽  
Megumi Mathison ◽  
Xueping Xu ◽  
Vivek K Patel ◽  
Narasimhaswamy S Belaguli ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Troponin T ◽  
Cardiac Fibroblasts ◽  
Human Fibroblasts ◽  
Large Animal ◽  
Large Animal Models ◽  
New Therapeutic Approaches ◽  
Cardiovascular Regeneration ◽  
Treat Heart Failure

There is an urgent clinical need to develop new therapeutic approaches to treat heart failure, but the biology of cardiovascular regeneration is complex. A new generation of reprogramming technology involves trans-differentiating one adult somatic cell type directly into another. We reported previously that administration of gene transfer vectors encoding Gata4 (G), Mef 2c (M) and Tbx5 (T), reprograms rat cardiac fibroblasts into induced cardiomyocytes (iCMs) in vitro and improves cardiac function in myocardial infarction models. Previous cardiac reprogramming studies were restricted to rodent and human fibroblasts. However studies in large animal models relevant for pre-clinical studies is lacking. The Aim of the present study was to determine the optimal combination of factors necessary for direct reprogramming of porcine fibroblasts towards a cardiomyocyte lineage for the first time. Here we have used human lentiviral system to express various cardiomyocyte enriched transcriptional regulators such as GMT, Hand2, myocardin, and two microRNAs, miR-590 and mir-199, in porcine cardiac fibroblasts. Reprogramming of fibroblasts into iCMs was determined 4 weeks post-virus transduction by FACS analysis for the activation of endogenous cardiac troponin T (cTnT). GMT alone was not sufficient to reprogram porcine fibroblasts although this combination was shown previously to be sufficient to convert rodent fibroblasts into iCMs. Addition of mir-199 to GMT was also not sufficient to promote reprogramming. However, adding miR-590 or Hand2, and myocardin (HM) to GMT resulted in reprogramming, although the efficiency remained low. Together, our results show that combination of 5 distinct transcription regulators (GMTHM) or GMT plus mir-590 is necessary for reprogramming porcine fibroblast. These findings demonstrate that pig fibroblasts can be directly reprogrammed toward the cardiomyocyte lineage, and represent a step toward possible therapeutic application of this reprogramming approach in a pre-clinical setting.

scholarly journals Assessment of the Growth and Reproductive Performance of Cloned Pietrain Boars

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 2053
Author(s):  
Junsong Shi ◽  
Baohua Tan ◽  
Lvhua Luo ◽  
Zicong Li ◽  
Linjun Hong ◽  
...  
Keyword(s):  
Growth Rate ◽  
Body Size ◽  
Growth Performance ◽  
Reproductive Performance ◽  
Semen Quality ◽  
Small Body ◽  
Economic Benefits ◽  
Large Animal ◽  
Significant Difference ◽  
F1 Progeny

How to maximize the use of the genetic merits of the high-ranking boars (also called superior ones) is a considerable question in the pig breeding industry, considering the money and time spent on selection. Somatic cell nuclear transfer (SCNT) is one of the potential ways to answer the question, which can be applied to produce clones with genetic resources of superior boar for the production of commercial pigs. For practical application, it is essential to investigate whether the clones and their progeny keep behaving better than the “normal boars”, considering that in vitro culture and transfer manipulation would cause a series of harmful effects to the development of clones. In this study, 59,061 cloned embryos were transferred into 250 recipient sows to produce the clones of superior Pietrain boars. The growth performance of 12 clones and 36 non-clones and the semen quality of 19 clones and 28 non-clones were compared. The reproductive performance of 21 clones and 25 non-clones were also tested. Furthermore, we made a comparison in the growth performance between 466 progeny of the clones and 822 progeny of the non-clones. Our results showed that no significant difference in semen quality and reproductive performance was observed between the clones and the non-clones, although the clones grew slower and exhibited smaller body size than the non-clones. The F1 progeny of the clones showed a greater growth rate than the non-clones. Our results demonstrated through the large animal population showed that SCNT manipulation resulted in a low growth rate and small body size, but the clones could normally produce F1 progeny with excellent growth traits to bring more economic benefits. Therefore, SCNT could be effective in enlarging the merit genetics of the superior boars and increasing the economic benefits in pig reproduction and breeding.

Small RNA Sequencing to Discover Circulating MicroRNA Biomarkers of Testicular Toxicity in Dogs

2020 ◽  
pp. 109158182096151
Author(s):  
Jennifer C. Shing ◽  
Kai Schaefer ◽  
Shaun E. Grosskurth ◽  
Andy H. Vo ◽  
Tatiana Sharapova ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Exploratory Study ◽  
Quantitative Polymerase Chain Reaction ◽  
Small Rna Sequencing ◽  
Circulating Microrna ◽  
Testicular Toxicity ◽  
Potential Candidate ◽  
Drug Induced ◽  
Organ Systems

Predictive indicators of testicular toxicity could improve drug development by allowing early in-life screening for this adverse effect before it becomes severe. We hypothesized that circulating microRNAs (miRNAs) could serve as testicular toxicity biomarkers in dogs. Herein, we describe the results of an exploratory study conducted to discover biomarkers of drug-induced testicular injury. Following a dose-selection study using the testicular toxicant ethylene glycol monomethyl ether (EGME), we chose a dose of 50 mg/kg/d EGME to avoid systemic toxicity and treated 2 groups of dogs (castrated, non-castrated) for 14 to 28 days. Castrated animals were used as negative controls to identify biomarkers specific for testicular toxicity because EGME can cause toxicity to organ systems in addition to the testis. Blood was collected daily during the dosing period, followed by recovery for 29 to 43 days with less frequent sampling. Dosing was well tolerated, resulting in mild-to-moderate degeneration in testes and epididymides. Global profiling of serum miRNAs at selected dosing and recovery time points was completed by small RNA sequencing. Bioinformatics data analysis using linear modeling demonstrated several circulating miRNAs that were differentially abundant during the dosing period compared with baseline and/or castrated control samples. Confirmatory reverse transcription quantitative polymerase chain reaction data in these animals was unable to detect sustained alterations of miRNAs in serum, except for 1 potential candidate cfa-miR-146b. Taken together, we report the results of a comprehensive exploratory study and suggest future directions for follow-up research to address the challenge of developing diagnostic biomarkers of testicular toxicity.

Sign in / Sign up

Export Citation Format

Share Document