Cardiomyocyte Progenitor Cells Research Articles

Poor in vivo transduction of myocardium by lentiviral vectors is likely caused by a post-entry block of transduction

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Stichting LSH-TKI Introduction In cardiac gene therapy, two prototypic viral vectors are typically considered for long-term transgene expression, i.e. lentivirus (LV) and adeno-associated virus (AAV) vectors. Compared to AAV vectors, LV vectors have the advantages of a larger insert capacity and the ability to confer permanent transgene expression due to integration of the vector genome in the host cell’s chromosomal DNA . However, standard VSV G protein-pseudotyped LV vectors (VSV G-LV vectors) has shown suboptimal transduction of cardiac myocytes in vivo. Purpose In this study, we aimed to improve LV-mediated transduction of cardiomyocytes in vivo by evaluating LV vectors carrying various different envelopes and compared the most potent variants with AAV1 capsid-pseudotyped AAV2 vectors (AAV2/1 vectors). Methods We made a panel of LV vectors pseudotyped with envelope proteins from 76 different viruses carrying an expression cassette for enhanced green fluorescent protein (eGFP). Transduction efficiencies in neonatal rat ventricular myocytes (NRVMs), human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and cardiomyocyte progenitor cells (CMPCs) were determined by fluorescence-activated cell sorting (FACS) as percentage eGFP-positive cells. Male NOD -SCID mice aged 3-6 months were injected directly into the left ventricular myocardium with eGFP-encoding VSV G-LV and AAV2/1 vectors at doses of 1.4 x 10^7 infectious units and 2.0 x 10^10 genome copies, respectively, or PBS as a control. Mice were sacrificed one and four weeks after surgery. Per heart, left ventricular and eGFP-positive area was determined from 40 – 50 slides and the ratio was used as a measure of transduction efficiency. Results Of the panel of pseudotyped LVs none was able to consistently outperform VSV G-LV vectors, which reached transduction efficiencies in vitro of 58% in NRVMs to 96% in hiPSC-CMs and 100% in CMPCs . In AAV2/1 vector-injected hearts, the eGFP expressing area in the left ventricle increased from 1.07% in week 1 to 5.42% in week 4. In contrast, eGFP expression in LV-injected hearts was 0.31% and 0.16% after 1 week and 4 weeks, respectively (figure 1). Conclusion In vitro screening of a panel of pseudotyped LV vectors showed standard VSV G-LV vectors as top performer. Despite encouraging in vitro results and previously reported findings, VSV G-LV vectors was unable to provide for robust in vivo transduction. The observation that none of the 76 different envelopes yielded a meaningful improvement of lentiviral transduction rates together with the discrepancy between in vitro and in vivo transduction efficiencies, suggest that a post-entry block of transduction likely represents an important component of the poor in vivo performance.Figure 1in vivo transduction

Direct cellular reprogramming techniques for cardiovascular regenerative therapeutics.

Cardiovascular diseases remain a leading cause of hospitalization affecting approximately 38 million people worldwide. While pharmacological and revascularization techniques can improve the patient's survival and quality of life, they cannot help reversing myocardial infarction injury and heart failure. Direct reprogramming of somatic cells to cardiomyocyte and cardiac progenitor cells offers a new approach to cellular reprogramming and paves the way for translational regenerative medicine. Direct reprogramming can bypass the pluripotent stage with the potential advantage of non-immunogenic cell products, reduced carcinogenic risk, and no requirement for embryonic tissue. The process of directly reprogramming cardiac cells was first achieved through the overexpression of transcription factors such as GATA4, MEF2C, and TBX5. However, over the past decade, significant work has been focused on enhancing direct reprogramming using a mixture of transcription factors, microRNAs, and small molecules to achieve cardiac cell fate. This review discusses the evolution of direct reprogramming, recent progress in achieving efficient cardiac cell fate conversion, and describes the reprogramming mechanisms at a molecular level. We also explore various viral and non-viral delivery methods currently being used to aid in the delivery of reprogramming factors to improve efficiency. However, further studies will be needed to overcome molecular and epigenetic barriers to successfully achieve translational cardiac regenerative therapeutics.

Wnt9 directs zebrafish heart tube assembly via a combination of canonical and non-canonical pathway signaling.

During zebrafish heart formation, cardiac progenitor cells converge at the embryonic midline where they form the cardiac cone. Subsequently, this structure transforms into a heart tube. Only little is known about the molecular mechanisms that control these morphogenetic processes. Here, we use light sheet microscopy and combine genetic, molecular biological and pharmacological tools to show that the paralogous genes wnt9a/b are required for the assembly of the nascent heart tube. In wnt9a/b double mutants, cardiomyocyte progenitor cells are delayed in their convergence towards the embryonic midline, the formation of the heart cone is impaired and the transformation into an elongated heart tube fails. The same cardiac phenotype occurs when both canonical and non-canonical Wnt signaling pathways are simultaneously blocked by pharmacological inhibition. This demonstrates that Wnt9a/b and canonical and non-canonical Wnt signaling regulate the migration of cardiomyocyte progenitor cells and control the formation of the cardiac tube. This can be partly attributed to their regulation of the timing of cardiac progenitor cell differentiation. Our study demonstrates how these morphogens activate a combination of downstream pathways to direct cardiac morphogenesis.

Hey2 enhancer activity defines unipotent progenitors for left ventricular cardiomyocytes in juxta-cardiac field of early mouse embryo

The cardiac crescent is the first structure of the heart and contains progenitor cells of the first heart field, which primarily differentiate into left ventricular cardiomyocytes. The interface between the forming cardiac crescent and extraembryonic tissue is known as the juxta-cardiac field (JCF), and progenitor cells in this heart field contribute to the myocardium of the left ventricle and atrioventricular canal as well as the epicardium. However, it is unclear whether there are progenitor cells that differentiate specifically into left ventricular cardiomyocytes. We have previously demonstrated that an enhancer of the gene encoding the Hey2 bHLH transcriptional repressor is activated in the ventricular myocardium during mouse embryonic development. In this study, we aimed to investigate the characteristics of cardiomyocyte progenitor cells and their cell lineages by analyzing Hey2 enhancer activity at the earliest stages of heart formation. We found that the Hey2 enhancer initiated its activity prior to cardiomyocyte differentiation within the JCF. Hey2 enhancer-active cells were present rostrally to the Tbx5-expressing region at the early phase of cardiac crescent formation and differentiated exclusively into left ventricular cardiomyocytes in a lineage distinct from the Tbx5-positive lineage. By the late phase of cardiac crescent formation, Hey2 enhancer activity became significantly overlapped with Tbx5 expression in cells that contribute to the left ventricular myocardium. Our study reveals that a population of unipotent progenitor cells for left ventricular cardiomyocytes emerge in the JCF, providing further insight into the mode of cell type diversification during early cardiac development.

Deciphering the Cardiovascular Potential of Human CD34+ Stem Cells.

Ex vivo monitored human CD34+ stem cells (SCs) injected into myocardium scar tissue have shown real benefits for the recovery of patients with myocardial infarctions. They have been used previously in clinical trials with hopeful results and are expected to be promising for cardiac regenerative medicine following severe acute myocardial infarctions. However, some debates on their potential efficacy in cardiac regenerative therapies remain to be clarified. To elucidate the levels of CD34+ SC implication and contribution in cardiac regeneration, better identification of the main regulators, pathways, and genes involved in their potential cardiovascular differentiation and paracrine secretion needs to be determined. We first developed a protocol thought to commit human CD34+ SCs purified from cord blood toward an early cardiovascular lineage. Then, by using a microarray-based approach, we followed their gene expression during differentiation. We compared the transcriptome of undifferentiated CD34+ cells to those induced at two stages of differentiation (i.e., day three and day fourteen), with human cardiomyocyte progenitor cells (CMPCs), as well as cardiomyocytes as controls. Interestingly, in the treated cells, we observed an increase in the expressions of the main regulators usually present in cardiovascular cells. We identified cell surface markers of the cardiac mesoderm, such as kinase insert domain receptor (KDR) and the cardiogenic surface receptor Frizzled 4 (FZD4), induced in the differentiated cells in comparison to undifferentiated CD34+ cells. The Wnt and TGF-β pathways appeared to be involved in this activation. This study underlined the real capacity of effectively stimulated CD34+ SCs to express cardiac markers and, once induced, allowed the identification of markers that are known to be involved in vascular and early cardiogenesis, demonstrating their potential priming towards cardiovascular cells. These findings could complement their paracrine positive effects known in cell therapy for heart disease and may help improve the efficacy and safety of using ex vivo expanded CD34+ SCs.

A novel receptor-interacting protein-1 (RIP1) inhibitor (547) protects human cardiac cells from ischemia/reperfusion-triggered necroptotic cell death

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): M.M.C.P. is supported by a Netherlands Cardiovascular Research Initiative (CVON) grant (REMAIN 2014B27) and J.P.G.S is supported by Horizon2020 ERC-2016-COG-EVICARE [725229] and BRAVE (grant number 874827. Purpose Restoring perfusion, either by coronary angioplasty or bypass anastomosis, is performed around 2 million times per year throughout Europe to treat patients after myocardial infarction. Although this treatment is highly efficient to resolve myocardial ischemia, reperfusion is accompanied by damage through the release of reactive oxygen species (ROS). This ischemia/reperfusion (I/R) injury is manifested by cardiomyocyte death, caused to a certain extent by necroptosis. As a potential means of cardioprotection, we applied the small molecule compound 547 inhibiting RIP1-kinase, a central mediator of the necroptosis pathway, in an in vitro model of human cardiac I/R injury. Methods Human foetal cardiomyocyte progenitor cells (hfCPCs) were cultured and characterized for cell type defining markers before being subjected to simulated I/R damage using hydrogen peroxide (H2O2). Cardioprotective effects of 547 were assessed 24 hours after induction of damage and application of the compound. Cell viability and necroptosis pathway activation were assessed by flow cytometry, immunocytochemistry, and western blotting. Furthermore, mitochondrial damage was determined by JC-1 staining. Results hfCPCs expressed indicative markers SCA1, GATA4, PECAM-1, VEGF and C-KIT. Treatment with compound 547 significantly protected hfCPCs from H2O2 induced damage (viable cells: 84,3 ± 4,3% vs 1,6 ± 0,31%; P<0,0001). 547 decreased the necrotic cell population (DAPI+ AnnV-) (3,0 ± 1,1% vs 10,1 ± 1,5%, P<0,0001) and reduced phosphorylation of necrosome components RIP1 (1,3 ± 0,35 fold vs 3,7 ± 0,78 fold; P< 0,01), RIP3 (1,0 ± 0,46 fold vs 4,3 ± 1,5 fold; P< 0,05), and MLKL (1,1 ± 0,15 fold vs 5,7 ± 1,7 fold; P< 0,01) without decreasing unphosphorylated protein or mRNA levels. Interestingly, 547 treatment prevented oxidative stress induced loss of nuclear RIP1 and RIP3 as measured by immunocytochemistry and western blotting of cell fractions (nuclear/cytoplasmic ratio RIP1: 0,80 ± 0,0006 [Control] vs 1,5 ± 0,17 fold [547]; P< 0,01; nuclear/cytoplasmic ratio RIP3: 0,76 ± 0,10 [Control] vs 1,6 ± 0,18 fold [547]; P< 0,01). Remarkably, compound 547 also decreased H2O2–induced mitochondrial depolarization (JC-1 staining/membrane depolarization index: 4,5 ± 0,85% vs 1,2 ± 0,38%; P< 0,05)) and expression and activation of CAMKII (0,46 ± 0,13 fold; P<0,01), a protein mediating mitochondria-dependent cell death. Conclusions Administration of compound 547 led to inhibition of necroptosis, increased cell viability and reduced mitochondrial damage in an in vitro human cardiac I/R injury model, emphasizing the potential of necroptosis inhibition for cardioprotection. Assessing clinical applicability will require further studies in relevant in vivo models.

In Vivo Performance of a Cell and Factor Free Multifunctional Fiber Mesh Modulating Postinfarct Myocardial Remodeling

AbstractGuidance of postinfarct myocardial remodeling processes by an epicardial patch system may alleviate the consequences of ischemic heart disease. As macrophages are highly relevant in balancing immune response and regenerative processes their suitable instruction would ensure therapeutic success. A polymeric mesh capable of attracting and instructing monocytes by purely physical cues and accelerating implant degradation at the cell/implant interface is designed. In a murine model for myocardial infarction the meshes are compared to those either coated with extracellular matrix or loaded with induced cardiomyocyte progenitor cells. All implants promote macrophage infiltration and polarization in the epicardium, which is verified by in vitro experiments. 6 weeks post‐MI, especially the implantation of the mesh attenuates left ventricular adverse remodeling processes as shown by reduced infarct size (14.7% vs 28–32%) and increased wall thickness (854 µm vs 400–600 µm), enhanced angiogenesis/arteriogenesis (more than 50% increase compared to controls and other groups), and improved heart function (ejection fraction = 36.8% compared to 12.7–31.3%). Upscaling as well as process controls is comprehensively considered in the presented mesh fabrication scheme to warrant further progression from bench to bedside.

Characterisation of the single-cell human cardiomyocytes taken from the excess heart tissue of the right ventricular outlet in congenital heart disease.

Once resected, the heart tissues were transported at 37 °C, in Dulbecco's Modified Eagle's medium/ DMEM (4.5g.L-1, antibiotic-antimycotic 3x, PRP10% (v/v)), to reach the lab within 30min, weighted and grouped into less than 500mg and more than 1000mg (n = 4). Each sample was digested with 250 U.mL-1 Collagenase type V and 4U.mL-1 Proteinase XXIV in the MACS™ C-tube (Milltenyi, Germany), then dissociated using the MACS™ Octo Dissociator with Heater (Milltenyi, Germany) for 60min at 37 °C. All cells isolated were rod-shaped cells; viability was up to 90%. The cell density obtained from the 500mg group were 4,867 ± 899 cells.mg-1 tissue weight, significantly higher compared to the 1,000mg group; had 557 ± 490 cells.mg-1 tissue weight (mean of (n = 3) ± 95% C.l). The isolated cells were analyzed using FACs BD Flowcytometer, expressed cTnT + 13.38%, PECAM-1 + /VCAM-1- 32.25%, cKit + 7.85%, ICAM + 85.53%, indicating the cardiomyocyte progenitor cells. Cardiomyocytes taken from the wasted heart tissue might be a candidate of cardiomyocytes source to study interventions to the heart as it contained up to 13.38% cardiomyocytes, and 32.25% of cardiac progenitor cells. Moreover, perhaps when cardiac cell therapy needs autologous cardiomyocytes, less than 500mg tissue weight can be considered as sufficient.

Mechanism-Based Cardiac Regeneration Strategies in Mammals.

Heart failure in adults is a leading cause of morbidity and mortality worldwide. It can arise from a variety of diseases, with most resulting in a loss of cardiomyocytes that cannot be replaced due to their inability to replicate, as well as to a lack of resident cardiomyocyte progenitor cells in the adult heart. Identifying and exploiting mechanisms underlying loss of developmental cardiomyocyte replicative capacity has proved to be useful in developing therapeutics to effect adult cardiac regeneration. Of course, effective regeneration of myocardium after injury requires not just expansion of cardiomyocytes, but also neovascularization to allow appropriate perfusion and resolution of injury-induced inflammation and interstitial fibrosis, but also reversal of adverse left ventricular remodeling. In addition to overcoming these challenges, a regenerative therapy needs to be safe and easily translatable. Failure to address these critical issues will delay the translation of regenerative approaches. This review critically analyzes current regenerative approaches while also providing a framework for future experimental studies aimed at enhancing success in regenerating the injured heart.

Abstract P456: High Glucose Suppresses Cardiomyocyte Progenitor Cell Regenerative Capacity And The Role Of Mir-195/ezh2 Crosstalk In Gestational Diabetes Mellitus

Introduction: Gestational diabetes mellitus (GDM) is associated with a five-fold increase in congenital heart defects. It is critical to determine the biological effects of diabetes mellitus (DM) in vivo and high glucose in vitro on neonatal cardiomyocyte progenitor cells (nCPCs) to maximize their regenerative potential. In the present study we seek to investigate the roles of Mir-195 and its hypothesized target gene, enhancer of zeste homolog 2 (Ezh2), in GDM. Hypothesis: We predict that high glucose is associated with decreased cellular proliferation, viability and increased senescence through oxidative stress. We also hypothesize that expression of Mir-195 will be higher in DM-nCPCs and inhibit cell proliferation via Ezh2 silencing. Methods: We subjected nCPCs in vitro to increasing glucose concentrations; cellular proliferation, migration, reactive oxygen species generation, and apoptosis were assessed using cell counting kit-8, wound healing, dihydroethidium, and annexin assays respectively. Our in vivo experiments involved injecting four-week old female mice with streptozocin. After pairing diabetic mice with non-diabetic male mice, timed embryos at E14.5 were evaluated for viability, proliferation and characterization. mRNA expression levels of Mir-195 and Ezh2 protein levels were detected using RT-qPCR and western blot analysis respectively. Lipofectamine transfection, with siRNA inhibiting Mir-195, was performed on c-kit+ cardiac stem cells obtained from diabetic mothers. Results: We found that subjecting nCPCs in vitro to increased glucose concentration led to increased % cell death, decreased proliferation and expression of paracrine factors indicating poorer secretome quality from these cells. Our in vivo models showed that maternal diabetes impedes prenatal development as decreased expression of c-kit+/Lin- cells and ISL1+ cells and increased DHE positivity were seen in DM-nCPCs at E14.5. Expression of Mir-195 was higher in DM-nCPCs but Ezh2 mRNA and protein expression levels were significantly decreased. siRNA inhibition of Mir-195 revealed higher EZH2 expression in c-kit+ cells and concomitant increase in regenerative capacity. Conclusion: In conclusion, the viability of DM-nCPCs both in vivo and in vitro is decreased compared to NDM-nCPCs suggesting decreased postnatal regenerative capacity. Mir-195 is associated with increased apoptosis and decreased proliferation of nCPCs via abrogation of the protective effects of EZH2.

Generating dHAND homozygous knockout human embryonic stem cell line (WAe009-A-59) by episomal vector-based CRISPR/Cas9 system

In order to determine the function of dHAND in a specific subset of cardiomyocyte progenitor cells responsible for the heart and its function in the cellular mechanism of Hand2−/− mouse ventricular hypoplasia, we established an allelic knockout model of dHAND in human embryonic stem cells (hESCs-H9) by an episomal vector-based CRISPR/Cas9 system. This dHAND KO hESC line maintained normal karyotype and typical primed pluripotent human stem cell morphology, and maintained pluripotency, could differentiate into all three germ layers in vivo.

Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1.

Electronic pacemakers still face major shortcomings that are largely intrinsic to their hardware-based design. Radical improvements can potentially be generated by gene or cell therapy-based biological pacemakers. Our previous work identified adenoviral gene transfer of Hcn2 and SkM1, encoding a “funny current” and skeletal fast sodium current, respectively, as a potent combination to induce short-term biological pacing in dogs with atrioventricular block. To achieve long-term biological pacemaker activity, alternative delivery platforms need to be explored and optimized. The aim of the present study was therefore to investigate the functional delivery of Hcn2/SkM1 via human cardiomyocyte progenitor cells (CPCs). Nucleofection of Hcn2 and SkM1 in CPCs was optimized and gene transfer was determined for Hcn2 and SkM1 in vitro. The modified CPCs were analyzed using patch-clamp for validation and characterization of functional transgene expression. In addition, biophysical properties of Hcn2 and SkM1 were further investigated in lentivirally transduced CPCs by patch-clamp analysis. To compare both modification methods in vivo, CPCs were nucleofected or lentivirally transduced with GFP and injected in the left ventricle of male NOD-SCID mice. After 1 week, hearts were collected and analyzed for GFP expression and cell engraftment. Subsequent functional studies were carried out by computational modeling. Both nucleofection and lentiviral transduction of CPCs resulted in functional gene transfer of Hcn2 and SkM1 channels. However, lentiviral transduction was more efficient than nucleofection-mediated gene transfer and the virally transduced cells survived better in vivo. These data support future use of lentiviral transduction over nucleofection, concerning CPC-based cardiac gene delivery. Detailed patch-clamp studies revealed Hcn2 and Skm1 current kinetics within the range of previously reported values of other cell systems. Finally, computational modeling indicated that CPC-mediated delivery of Hcn2/SkM1 can generate stable pacemaker function in human ventricular myocytes. These modeling studies further illustrated that SkM1 plays an essential role in the final stage of diastolic depolarization, thereby enhancing biological pacemaker functioning delivered by Hcn2. Altogether these studies support further development of CPC-mediated delivery of Hcn2/SkM1 and functional testing in bradycardia models.

MiR-33a-5p targets NOMO1 to modulate human cardiomyocyte progenitor cells proliferation and differentiation and apoptosis

Purpose MicroRNA (miRNA) is known to be involved in the pathological process of congenital heart disease (CHD), and nodal modulator1 (NOMO1) is a critical determinant of heart formation. The present study aims to discover the effect of miR-33a-5p and NOMO1 on CHD. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect expressions of miR-33a-5p mimic or inhibitor and overexpressed NOMO1 plasmid orNOMO1 knockdown. Human cardiomyocyte progenitor cells (hCMPCs) proliferation was measured by cell counting kit-8 (CCK-8) at 24, 48 and 72 h. Flow cytometry was applied to investigate hCMPCs cell cycle progression and apoptosis. Expressions of cell apoptotic proteins Bax, Cleaved(C) caspase-3 and Bcl-2, and expressions of cardiomyocyte differentiation markers GATA4, troponin T (cTnT) and myocyte enhancer factor2C (MEF2C) in hCMPCs were identified by qRT-PCR and western blot. Target genes and potential binding sites of NOMO1 and miR-33a-5p were predicted with Targetscan 7.2, and was confirmed through dual-luciferase reporter assay. Results Up-regulation of miR-33a-5p inhibited hCMPCs proliferation, cell cycle G0/S transition but promoted hCMPCs apoptosis, which was partially mitigated by overexpressed NOMO1. NOMO1 was the target gene of miR-33a-5p. Expressions of Bax and C caspase-3 were enhanced but expressions of Bcl-2, GATA4, cTnT and MEF2C were reduced by up-regulation of miR-33a-5p, which was partially mitigated by overexpressed NOMO1. Conclusion Up-regulation of miR-33a-5p inhibited hCMPCs proliferation, cell cycle G0/S transition and differentiation into cardiomyocytes but promoted apoptosis via targeting NOMO1.

Phenyl Saligenin Phosphate Disrupts Cell Morphology and the Actin Cytoskeleton in Differentiating H9c2 Cardiomyoblasts and Human-Induced Pluripotent Stem-Cell-Derived Cardiomyocyte Progenitor Cells.

We have previously shown that phenyl saligenin phosphate (PSP), an organophosphorus compound which is classed as a weak inhibitor of acetylcholinesterase, triggered cytotoxicity in mitotic and differentiated H9c2 cardiomyoblasts. The aim of this study was to assess whether sublethal concentrations of PSP could disrupt the morphology of differentiating rat H9c2 cardiomyoblasts and human-induced pluripotent stem-cell-derived cardiomyocyte progenitor cells (hiPSC-CMs) and to assess the underlying cytoskeletal changes. PSP-induced changes in protein expression were monitored via Western blotting, immunocytochemistry, and proteomic analysis. PSP-mediated cytotoxicity was determined by measuring MTT reduction, LDH release, and caspase-3 activity. Sublethal exposure to PSP (3 μM) induced morphological changes in differentiating H9c2 cells (7, 9, and 13 days), reflected by reduced numbers of spindle-shaped cells. Moreover, this treatment (7 days) attenuated the expression of the cytoskeletal proteins cardiac troponin I, tropomyosin-1, and α-actin. Further proteomic analysis identified nine proteins (e.g., heat shock protein 90-β and calumenin) which were down-regulated by PSP exposure in H9c2 cells. To assess the cytotoxic effects of organophosphorus compounds in a human cell model, we determined their effects on human-induced pluripotent stem-cell-derived cardiomyocyte progenitor cells. Chlorpyrifos and diazinon-induced cytotoxicity (48 h) was evident only at concentrations >100 μM. By contrast, PSP exhibited cytotoxicity in hiPSC-CMs at a concentration of 25 μM following 48 h exposure. Finally, sublethal exposure to PSP (3 μM; 7 days) induced morphological changes and decreased the expression of cardiac troponin I, tropomyosin-1, and α-actin in hiPSC-CMs. In summary, our data suggest cardiomyocyte morphology is disrupted in both cell models by sublethal concentrations of PSP via modulation of cytoskeletal protein expression.

Cardiac differentiation of induced pluripotent stem cells derived from mesenchymal stem cells

Background & Aim Induced pluripotent stem cells (iPSCs) are commonly studied for clinical use due to its flexibility in differentiating into any desirable cellular products. iPSCs are commonly derived from fibroblasts or cells from peripheral blood due to its accessibility. Although not common, we have derived iPSCs from mesenchymal stem cells (MSC) for several purposes. We are developing an allogenic combinatory cellular therapy for heart disease using both MSCs with iPSC derived cardiomyocyte (CM) or cardiac progenitor cells (CPCs). We theorize, by utilizing iPSC derived from MSCs, it provides more compatibility when transplanting the two final cellular products from one donor into patients, thus yielding better therapeutic effects. Also, recent changes in donor qualification increase the financial burden when launching new cellular therapeutics due to the increase of required testing on donor and final cellular product. In this scenario, by reprogramming the MSCs into iPSCs then differentiate into CM, we eliminated the additional cost of qualifying the parent cells of iPSCs. Methods, Results & Conclusion Although cardiac differentiation of iPSC is well studied, the optimal differentiation condition needed to be tested for each cell line used. The initial evaluation must be performed to determine the optimal cell density, the concentration of CHIR99021, and the time point of switching to IWP2. After determining these factors, the cardiac differentiation was performed on both IMR90 (lung fibroblast) and MSC derived iPSCs in order to evaluate the efficiency of differentiation of the latter iPSC. The iPSCs were plated onto Vitronectin coated plates until the desired confluence reached using iPSC culture medium. At day 0 of the cardiac differentiation process, the iPSC culture medium was replaced with CHIR99021in differentiation medium (RPMI-1640 with B-27 without insulin). Two or three days later, IWP2 replaced CHIR99021 in the differentiation medium for another two days. After day 7 of the cardiac differentiation process, cells were maintained with CM culture medium (RPMI-1640 with B-27 with insulin) for another seven days. The medium was changed every three to four days. In summary, only the optimized combination of conditions yield beating CM culture at day 14 from both MSC and IMP90 derived iPSCs. CM derived from both iPSCs stained positive for Troponin T. At day 7 of the cardiac differentiation, the cultures were stained positive for GHRHR, which will be used as a marker for CPC selection. Induced pluripotent stem cells (iPSCs) are commonly studied for clinical use due to its flexibility in differentiating into any desirable cellular products. iPSCs are commonly derived from fibroblasts or cells from peripheral blood due to its accessibility. Although not common, we have derived iPSCs from mesenchymal stem cells (MSC) for several purposes. We are developing an allogenic combinatory cellular therapy for heart disease using both MSCs with iPSC derived cardiomyocyte (CM) or cardiac progenitor cells (CPCs). We theorize, by utilizing iPSC derived from MSCs, it provides more compatibility when transplanting the two final cellular products from one donor into patients, thus yielding better therapeutic effects. Also, recent changes in donor qualification increase the financial burden when launching new cellular therapeutics due to the increase of required testing on donor and final cellular product. In this scenario, by reprogramming the MSCs into iPSCs then differentiate into CM, we eliminated the additional cost of qualifying the parent cells of iPSCs. Although cardiac differentiation of iPSC is well studied, the optimal differentiation condition needed to be tested for each cell line used. The initial evaluation must be performed to determine the optimal cell density, the concentration of CHIR99021, and the time point of switching to IWP2. After determining these factors, the cardiac differentiation was performed on both IMR90 (lung fibroblast) and MSC derived iPSCs in order to evaluate the efficiency of differentiation of the latter iPSC. The iPSCs were plated onto Vitronectin coated plates until the desired confluence reached using iPSC culture medium. At day 0 of the cardiac differentiation process, the iPSC culture medium was replaced with CHIR99021in differentiation medium (RPMI-1640 with B-27 without insulin). Two or three days later, IWP2 replaced CHIR99021 in the differentiation medium for another two days. After day 7 of the cardiac differentiation process, cells were maintained with CM culture medium (RPMI-1640 with B-27 with insulin) for another seven days. The medium was changed every three to four days.

Telocytes in the Myocardium of Children with Congenital Heart Disease Tetralogy of Fallot.

Telocytes, a new type of interstitial stem cells with long thin processes that form a three-dimensional network around cardiomyocytes, vessels, and nerve fibers were described in the myocardium of children with tetralogy of Fallot. Two types of morphologically different telocytes, spindle-shaped and rounded, were identified. Contacts of telocytes with stem cells and interstitial macrophages were found. Telocytes were more common in the immature myocardium, where the assembly of myofibrils in cardiomyocytes was not completed and small Ki-67+ cardiomyocyte progenitor cells were present. Telocytes expressed immunohistochemical markers CD117, vimentin, CD34, and CD44. Localization and ultrastructural characteristics of telocytes suggested their participation in stem cell differentiation, coordination of neoangiogenesis, and paracrine regulation of all components of the interstitium.

Identification of Key Factors in Cardiomyocyte Development by Single‐Cell Transcriptome Analysis

BackgroundA promising intervention for heart diseases is the application of cardiomyocytes differentiated from pluripotent stem cells. However, the driving factors of the differentiation process still remain unclear. This study aims to map the transcriptional landscapes of directional differentiation of pluripotent stem cells at multiple stages, using single‐cell sequencing data, in order to identify the key drivers of the differentiation process.MethodSingle‐cell sequencing data from pluripotent stem cell‐derived cardiomyocytes at age day 0, day 2, day 5, day 15 and day 30 were included and normalized. UMAP algorithm was then used to reduce the dimensionality of the transcriptome profiles, followed by community detection to group the cells and cell type identification using marker genes. Graph‐autocorrelation analysis was implemented to identify differentially expressed genes among cell groups with Gene Ontology term enrichment. Next, we calculated the pseudotimes of each cardiomyocyte to construct the pseudotime trajectories. Finally, knowledge based dynamic changes in gene expression were analyzed to identify the key factors driving the cardiomyocyte development.ResultWe identified 9 different cell types in the process of cardiomyocyte development, including cardiomyocytes, induced pluripotent stem cells, cardiogenic mesoderm cells, cardiovascular progenitor cells, embryonic stem cells, endoderm cells, epithelial progenitor cells, cardiomyocyte progenitor cells and multipotent progenitor cells. Identification of cardiomyocyte was confirmed with specific gene expression profiles such as significant enrichment in the biological processes of cell adhesion and supramolecular fiber organization. The expression of NKX2‐5, which encodes a homeobox‐containing transcription factor, was found to be at a lower level in the early stages and continually increased as cardiomyocyte develop, suggesting that NKX2‐5 was one of the driving factors of cardiac development, acting through promoting myocardial maturation and development.ConclusionSingle cell sequencing data revealed the dynamic changes of gene expression during cardiomyocyte development differentiation. NKX2‐5 was identified as a key factor in cardiac development. Our finding contributes to the understanding of the mechanism behind the cardiomyocyte development, and may serve as the basis for stem cell based therapies.Support or Funding InformationThis work was supported by National Natural Science Foundation of China 81774152 (to RZ) and 81770571 (to LZ).

Potential of mesenchymal- and cardiac progenitor cells for therapeutic targeting of B-cells and antibody responses in end-stage heart failure.

Upon myocardial damage, the release of cardiac proteins induces a strong antibody-mediated immune response, which can lead to adverse cardiac remodeling and eventually heart failure (HF). Stem cell therapy using mesenchymal stromal cells (MSCs) or cardiomyocyte progenitor cells (CPCs) previously showed beneficial effects on cardiac function despite low engraftment in the heart. Paracrine mediators are likely of great importance, where, for example, MSC-derived extracellular vesicles (EVs) also show immunosuppressive properties in vitro. However, the limited capacity of MSCs to differentiate into cardiac cells and the sufficient scaling of MSC-derived EVs remain a challenge to clinical translation. Therefore, we investigated the immunosuppressive actions of endogenous CPCs and CPC-derived EVs on antibody production in vitro, using both healthy controls and end-stage HF patients. Both MSCs and CPCs strongly inhibit lymphocyte proliferation and antibody production in vitro. Furthermore, CPC-derived EVs significantly lowered the levels of IgG1, IgG4, and IgM, especially when administered for longer duration. In line with previous findings, plasma cells of end-stage HF patients showed high production of IgG3, which can be inhibited by MSCs in vitro. MSCs and CPCs inhibit in vitro antibody production of both healthy and end-stage HF-derived immune cells. CPC-derived paracrine factors, such as EVs, show similar effects, but do not provide the complete immunosuppressive capacity of CPCs. The strongest immunosuppressive effects were observed using MSCs, suggesting that MSCs might be the best candidates for therapeutic targeting of B-cell responses in HF.

Biofabrication of Cell-Derived Nanovesicles: A Potential Alternative to Extracellular Vesicles for Regenerative Medicine.

Extracellular vesicles (EVs) are mediators of intercellular communication by transferring functional biomolecules from their originating cells to recipient cells. This intrinsic ability has gained EVs increased scientific interest in their use as a direct therapeutic in the field of regenerative medicine or as vehicles for drug delivery. EVs derived from stem cells or progenitor cells can act as paracrine mediators to promote repair and regeneration of damaged tissues. Despite substantial research efforts into EVs for various applications, their use remains limited by the lack of highly efficient and scalable production methods. Here, we present the biofabrication of cell-derived nanovesicles (NVs) as a scalable, efficient, and cost-effective production alternative to EVs. We demonstrate that NVs have a comparable size and morphology as EVs, but lack standard EV (surface) markers. Additionally, in vitro uptake experiments show that human fetal cardiac fibroblast, endothelial cells, and cardiomyocyte progenitor cells internalize NVs. We observed that cardiac progenitor cell-derived NVs and EVs are capable of activating mitogen-activated protein kinase 1/2 (MAPK1/2)-extracellular signal-regulated kinase, and that both NVs and EVs derived from A431 and HEK293 cells can functionally deliver Cre-recombinase mRNA or protein to other cells. These observations indicate that NVs may have similar functional properties as EVs. Therefore, NVs have the potential to be applied for therapeutic delivery and regenerative medicine purposes.

BREATHTAKING DIAGNOSIS: ATRIAL MYXOMA

SESSION TITLE: Monday Abstract Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: 10/21/2019 02:30 PM - 03:15 PM INTRODUCTION: Primary cardiac tumors are one of the least investigated subjects in oncological medicine. Among these primary cardiac tumors, atrial myxoma is one of the most common primary cardiac tumors mostly arising in the left atrium.We present a unique case of atrial myxoma that will highlight the broad importance of differential diagnosis in inpatient and outpatient setting. CASE PRESENTATION: A 57-year-old female presented for outpatient CT scan of the chest to rule out pulmonary embolism due to continued dyspnea at rest and orthopnea following upper respiratory tract infection. A CT scan of the chest was negative for pulmonary embolism; however, it revealed a 4.9 x 4.8 cm low-density mass in the left atrium (Panel A, arrow). A transesophageal echocardiogram showed a large heterogeneous left atrial mass attached to the interatrial septum with characteristic "plop” across the mitral valve causing functional mitral stenosis (Panel B, arrow). Cardiac catheterization revealed non-obstructive left anterior descending and right coronary arteries; however, left circumflex coronary artery was not visualized despite aortic root angiography indicating congenital absence (Panel C, arrows [LAD and RCA denote left anterior descending and right coronary arteries, respectively]). The patient underwent surgical resection of the mass (Panel D). Biopsy of the mass revealed tumor cells with elongated and fusiform stellate with bland nuclei and eosinophilic cytoplasm with dense lymphocytic aggregation consistent with myxoma (Panel E and F). The postoperative course was unremarkable. DISCUSSION: Myxomas are the most common benign primary cardiac tumors and occur predominantly in the left atrium and in women. Patients typically present with symptoms of heart failure secondary to mitral inflow obstruction; however, one-third of the patients, especially men present with the embolic phenomenon. Cardiac myxoma disproportionately affects the female population with 65% of myxomas occurring in them. Secondary of myxomas being familial in 4.5 to 10% of the cases it is recommended to screen first degree relatives [2]. Multiple theories have been proposed regards to the origin of these tumors. The initial thought process was to classify them as organized thrombotic structure but histological and molecular investigations reported the expression of different cardiac myocytes and transcription factors lead to propose that these lesions might have mesenchymal cardiomyocyte progenitor cells [3]. Cardiac myxomas even though classified as benign tumors have a tendency to seed in other locations causing inflammation, embolism, or intra-cardiac obstruction leading to mortality and significant morbidity. CONCLUSIONS: Prompt diagnosis is crucial since the results of surgical resection are excellent with dramatic symptoms improvement and rapid postoperative recovery. Reference #1: Lam, K. Y., Dickens, P., & Chan, A. C. (1993). Tumors of the heart. A 20-year experience with a review of 12,485 consecutive autopsies. Archives of pathology & laboratory medicine, 117(10), 1027-1031 Reference #2: Ekmektzoglou, K. A., Samelis, G. F., & Xanthos, T. (2008). Heart and tumors: location, metastasis, clinical manifestations, diagnostic approaches and therapeutic considerations. Journal of Cardiovascular Medicine, 9(8), 769-777. Reference #3: Amano J, Kono T, Wada Y, Zhang T, Koide N, Fujimori M, Ito K. Cardiac myxoma: its origin and tumor characteristics. Ann Thorac Cardiovasc Surg. 2003;9(4):215–221 DISCLOSURES: No relevant relationships by Pranjal Boruah, source=Web Response No relevant relationships by Gaurav Patel, source=Web Response No relevant relationships by Muhammad Siddique Pir, source=Web Response No relevant relationships by najam saqib, source=Web Response