Explant-derived Cells Research Articles

Inactivation of the NLRP3 inflammasome mediates exosome-based prevention of atrial fibrillation.

Rationale: Extracellular vesicles (EVs) from human explant-derived cells injected directly into the atria wall muscle at the time of open chest surgery reduce atrial fibrosis, atrial inflammation, and atrial fibrillation (AF) in a rat model of sterile pericarditis. Albeit a promising solution to prevent postoperative AF, the mechanism(s) underlying this effect are unknown and it is not clear if this benefit is dependent on EV dose. Methods: To determine the dose-efficacy relationship of EVs from human explant-derived cells in a rat model of sterile pericarditis. Increasing doses of EVs (106, 107, 108 or 109) or vehicle control were injected into the atria of middle-age male Sprague-Dawley rats at the time of talc application. A sham control group was included to demonstrate background inducibility. Three days after surgery, all rats underwent invasive electrophysiological testing prior to sacrifice. Results: Pericarditis increased the likelihood of inducing AF (p<0.05 vs. sham). All doses decreased the probability of inducing AF with maximal effects seen after treatment with the highest dose (109, p<0.05 vs. vehicle). Pericarditis increased atrial fibrosis while EV treatment limited the effect of pericarditis on atrial fibrosis with maximal effects seen after treatment with 108 or 109 EVs. Increasing EV dose was associated with progressive decreases in pro-inflammatory cytokine content, inflammatory cell infiltration, and oxidative stress. EVs decreased NLRP3 (NACHT, LRR, and PYD domains-containing protein-3) inflammasome activation though a direct effect on resident atrial fibroblasts and macrophages. This suppressive effect was exclusive to EVs produced by heart-derived cells as application of EVs from bone marrow or umbilical cords did not alter NLRP3 activity. Conclusions: Intramyocardial injection of incremental doses of EVs at the time of open chest surgery led to progressive reductions in atrial fibrosis and inflammatory markers. These effects combined to render atria resistant to the pro-arrhythmic effects of pericarditis which is mechanistically related to suppression of the NLRP3 inflammasome.

Prevention of atrial fibrillation after open chest surgery with extracellular vesicle therapy.

Almost half of patients recovering from open chest surgery experience atrial fibrillation (AF) that results principally from inflammation in the pericardial space surrounding the heart. Given that post-operative AF is associated with increased mortality, effective measures to prevent AF after open-chest surgery are highly desirable. In this study, we tested the concept that extracellular vesicles (EVs) isolated from human atrial explant-derived cells can prevent post-operative AF. Middle-aged female and male rats were randomized to undergo sham operation or induction of sterile pericarditis followed by trans-epicardial injection of human EVs or vehicle into the atrial tissue. Pericarditis increased the probability of inducing AF while EV treatment abrogated this effect in a sex independent manner. EV treatment reduced infiltration of inflammatory cells and production of pro-inflammatory cytokines. Atrial fibrosis and hypertrophy seen after pericarditis was markedly attenuated by EV pre-treatment; an effect attributable to suppression of fibroblast proliferation by EVs. Our study demonstrates that injection of extracellular vesicles at the time of open-chest surgery shows prominent anti-inflammatory effects and prevents AF due to sterile pericarditis. Translation of this finding to patients might provide an effective new strategy to prevent post-operative AF by reducing atrial inflammation and fibrosis.

Human heart explant-derived cell extracellular vesicles inhibit NLRP3 inflammasome activation in macrophages

Background: Abnormal activation of NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome plays a crucial role in promoting myocardial inflammation and adverse remodeling. Extracellular vesicles (EVs) secreted by heart explant-derived cells (HDCs) have been shown to reduce inflammation and confer cardioprotection in animal models of myocardial infarction. These salutary effects are suggested to be mediated by antiinflammatory microRNA (miRNA) cargo found within the EVs. However, whether HDC EVs can modulate NLRP3 inflammasome activation is not known. Hypothesis &amp; Study Objective: Given their antiinflammatory effects and abundantly enriched miRNA transcripts, we hypothesized that HDC EVs attenuate NLRP3 inflammasome activation. Therefore, the objective of this study was to assess the inhibitory effect of HDC EVs on NLRP3 inflammasome activation in macrophages-a major proinflammatory cell type recruited to myocardium after an ischemic insult. Methods: Human HDCs were cultured from cardiac biopsies in a clinical grade cell manufacturing facility. EVs were isolated from conditioned media (ultracentrifugation) and characterized for their identity (Nanosight &amp; antibody array). Monocytes (THP-1) were differentiated into macrophages with phorbol-myristate acetate (PMA; 3 days) and treated with EVs (4E+10 EVs/mL; 20 hours) before priming (lipopolysaccharide; 4 hours) and activating (nigericin; 1 hour) NLRP3 inflammasome. Caspase-1 activity, IL-1β and IL-18 levels in the culture supernatants were measured using a Caspase-Glo® 1 assay and ELISA respectively. EV miRNA cargo was profiled using nCounter ® human miRNA expression assay. Functional enrichment analysis and miRNA target prediction were performed using bioinformatic tools (Tam 2.0 &amp; miRWalk v3). Results: EV size (160±2 nm) and markers (ICAM, ALIX, CD81, CD63, EPCAM, ANXAS, TSG101, FLOT-1) confirmed EV identity. EV treatment significantly inhibited NLRP3 inflammasome induced caspase-1 activity (67% lower vs. no EV treated, n=4-6, p&lt;0.0001). Further, EV treated macrophages showed a significant reduction in IL-18 (623±8 vs.1462±43 pg/mL, n=3, p=0.0025) and a trend towards reduction in IL-1β levels (1292±115 vs. 2535±115 pg/mL, n=3, p=0.08). MicroRNA set analysis revealed that EVs are enriched with 22 anti-inflammatory miRNAs and importantly 3 of the top 10 significantly enriched functional terms (overrepresentation, p&lt;0.05) are found to be related to immunity and inflammation. Interestingly, EVs are enriched with 4 miRNAs (miR-100, miR-181a, miR-21, miR-22) predicted to inhibit NLRP3 inflammasome. Conclusion: Our findings show that human heart explant-derived cell EVs inhibit activation of NLRP3 inflammasome in macrophages and this effect in part is possibly mediated via transfer of their anti-inflammatory miRNA cargo. These preliminary findings encourage a preclinical investigation to assess EV modulatory effect on NLRP3 inflammasome in models of ischemic heart disease. Canadian Institutes of Health Research Project Grant 410103 (DRD) and University of Ottawa Heart Institute Strategic Research Endowed Fellowship (RV) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

EXTRACELLULAR VESICLES FROM HUMAN HEART EXPLANT-DERIVED CELLS ATTENUATE ACTIVATION OF THE NLRP3 INFLAMMASOME IN MACROPHAGES

Intramyocardial injection of heart explant-derived cells (EDCs) improves cardiac function in preclinical models of ischemic cardiomyopathy. This therapeutic benefit is partially attributable to the anti-inflammatory cargo (micro RNAs and proteins) enriched within the extracellular vesicles (EVs) released by EDCs. Recent work has shown that activation of the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome in both immune and non-immune cells plays a critical role in promoting cardiac inflammation and adverse remodeling.

PO-617-04 A DOSE ESCALATION STUDY USING EXTRACELLULAR VESICLES TO REDUCE ATRIAL FIBROSIS, INFLAMMATION, AND FIBRILLATION

Extracellular vesicles (EVs) from human Explant-Derived Cells delivered directly to the atria at the time of open chest surgery reduce atrial fibrosis/inflammation and AF in a rat model of sterile pericarditis. But the minimally effective dose is not known, nor is it clear if this benefit is dependent on EV dose.

PO-676-07 A DIRECT COMPARISON OF TWO ANTI-INFLAMMATORY STRATEGIES TO PREVENT POST-OPERATIVE ATRIAL FIBRILLATION REVEALS SUPERIOR ANTI-FIBROTIC AND ANTIARRHYTHMIC EFFICACY WITH EXTRACELLULAR VESICLES

Extracellular vesicles (EVs) from human Explant-Derived Cells delivered directly to the atria at the time of open chest surgery reduce atrial fibrosis/inflammation and AF in a rat model of sterile pericarditis. But the minimally effective dose is not known, nor is it clear if this benefit is dependent on EV dose.

Abstract 6970: Extracellular Vesicles Prevent Post-Operative Atrial Fibrillation

Half of patients recovering from cardiac surgery experience a malignant form of atrial fibrillation that results from inflammation in the pericardial space surrounding the heart. Given that post-operative atrial fibrillation (POAF) increases mortality, effective measures to prevent or treat POAF are highly desirable. In this study, we built on previous work to see if extracellular vesicles (EVs) isolated from human atrial explant-derived cell conditioned media can prevent POAF. Hypothesis: Human EVs will reduce AF inducibility in a rat model of sterile pericarditis by reducing inflammation and the malignant pro-fibrillatory transformation of atrial fibroblasts. Methods and Results: Middle aged female and male Sprague-Dawley rats (6 months old) were randomized to sham operation (n=24; 12 females, 12 males) or induction of sterile pericarditis followed by intra-atrial injection of 10 8 human EVs (n=34; 17 females, 17 males) or vehicle (n=34; 17 females, 17 males). Three days later, all rats underwent invasive electrophysiological testing prior to sacrifice. As expected, pericarditis increased the probability of inducing AF (Figure Panel A). EV treatment reduced the probability of AF. Recipient sex did not alter this effect. Analysis of atrial tissue histology (H&amp;E staining) and lysate (Luminex assays for IL-2, Il-18, MCP-1 and PDGF-AB) revealed that EV treatment abrogated the pro-inflammatory effects of pericarditis. As shown in Panel B, pericarditis increased both atrial fibrosis and mass while EV treatment limited the effect of pericarditis on atrial fibrosis and mass. These effects were attributable to the anti-fibrotic effect of EV treatment on atrial fibroblast proliferation profiled using in-vitro cultures exposed to pro-fibrillatory stimuli (i.e., IL-6 and TGFβ1). Conclusion: Intramyocardial injection of EVs at the time of open chest surgery may provide a facile acellular strategy to prevent POAF by reducing inflammation and fibrosis.

Electrophysiological engineering of heart-derived cells with calcium-dependent potassium channels improves cell therapy efficacy for cardioprotection

We have shown that calcium-activated potassium (KCa)-channels regulate fundamental progenitor-cell functions, including proliferation, but their contribution to cell-therapy effectiveness is unknown. Here, we test the participation of KCa-channels in human heart explant-derived cell (EDC) physiology and therapeutic potential. TRAM34-sensitive KCa3.1-channels, encoded by the KCNN4 gene, are exclusively expressed in therapeutically bioactive EDC subfractions and maintain a strongly polarized resting potential; whereas therapeutically inert EDCs lack KCa3.1 channels and exhibit depolarized resting potentials. Somatic gene transfer of KCNN4 results in membrane hyperpolarization and increases intracellular [Ca2+], which boosts cell-proliferation and the production of pro-healing cytokines/nanoparticles. Intramyocardial injection of EDCs after KCNN4-gene overexpression markedly increases the salutary effects of EDCs on cardiac function, viable myocardium and peri-infarct neovascularization in a well-established murine model of ischemic cardiomyopathy. Thus, electrophysiological engineering provides a potentially valuable strategy to improve the therapeutic value of progenitor cells for cardioprotection and possibly other indications.

Mybl2 rejuvenates heart explant-derived cells from aged donors after myocardial infarction.

While cell therapy is emerging as a promising option for patients with ischemic cardiomyopathy (ICM), the influence of advanced donor age and a history of ischemic injury on the reparative performance of these cells are not well defined. As such, intrinsic changes that result from advanced donor age and ischemia are explored in hopes of identifying a molecular candidate capable of restoring the lost reparative potency of heart explant‐derived cells (EDCs) used in cell therapy. EDCs were cultured from myocardial biopsies obtained from young or old mice 4 weeks after randomization to experimental myocardial infarction or no intervention. Advanced donor age reduces cell yield while increasing cell senescence and the secretion of senescence‐associated cytokines. A history of ischemic injury magnifies these effects as cells are more senescent and have lower antioxidant reserves. Consistent with these effects, intramyocardial injection of EDCs from aged ischemic donors provided less cell‐mediated cardiac repair. A transcriptome comparison of ICM EDCs shows aging modifies many of the pathways responsible for effective cell cycle control and DNA damage/repair. Over‐expression of the barely explored antisenescent transcription factor, Mybl2, in EDCs from aged ICM donors reduces cell senescence while conferring salutary effects on antioxidant activity and paracrine production. In vivo, we observed an increase in cell retention and vasculogenesis after treatment with Mybl2‐over‐expressing EDCs which improved heart function in infarcted recipient hearts. In conclusion, Mybl2 over‐expression rejuvenates senescent EDCs sourced from aged ICM donors to confer cell‐mediated effects comparable to cells from young nonischemic donors.

Deterministic paracrine repair of injured myocardium using microfluidic-based cocooning of heart explant-derived cells

While encapsulation of cells within protective nanoporous gel cocoons increases cell retention and pro-survival integrin signaling, the influence of cocoon size and intra-capsular cell-cell interactions on therapeutic repair are unknown. Here, we employ a microfluidic platform to dissect the impact of cocoon size and intracapsular cell number on the regenerative potential of transplanted heart explant-derived cells. Deterministic increases in cocoon size boosted the proportion of multicellular aggregates within cocoons, reduced vascular clearance of transplanted cells and enhanced stimulation of endogenous repair. The latter being attributable to cell-cell stimulation of cytokine and extracellular vesicle production while also broadening of the miRNA cargo within extracellular vesicles. Thus, by tuning cocoon size and cell occupancy, the paracrine signature and retention of transplanted cells can be enhanced to promote paracrine stimulation of endogenous tissue repair.

Substance P Administered after Myocardial Infarction Upregulates Microphthalmia-Associated Transcription Factor, GATA4, and the Expansion of c-Kit+ Cells.

Microphthalmia-associated transcription factor (MITF), a basic helix-loop-helix leucine zipper transcription factor, can govern gene expression by binding to E box elements in the promoter region of its target gene. Although high levels of MITF have been observed in cardiomyocytes and the heart, the role of MITF after myocardial infarction (MI) remains unclear. We investigated the association between substance P (SP)/neurokinin-1 receptor (NK1R) signaling and MITF expression after MI. Male Sprague-Dawley rats (8 weeks) were randomly divided in two groups: ischemia/reperfusion injury (I/R) and SP injection (5 nmol/kg, SP+I/R). At the end of 7 days, the left ventricle (LV; LV7daysI/R, LV7daysSP+I/R) and infarct-related areas (IA; IA7daysI/R, IA7daysSP+I/R) from the hearts were collected. Immunofluorescence staining demonstrated that the LV7daysSP+I/R had a larger population of c-Kit+ GATA4high cells, which markedly upregulated MITF, c-Kit, and GATA4. c-Kit+ cells in the explant-derived cells (EDCs) derived from IA7daysSP+I/R migrated more widely than EDCs IA7daysI/R. Immunofluorescence staining, western blot analysis, and qRT-PCR assay showed that SP-treated c-Kit+ cells exhibited a high expression of c-Kit, GATA4, and MITF. FTY720 (a MITF inhibitor), RP67580 (NK1R inhibitor), or both inhibited the migration and proliferation of c-Kit+ cells increased by SP and blocked the upregulation of c-Kit, GATA4, and MITF. Overall, we suggest that MITF might be a potential regulator in SP-mediated c-Kit+ cell expansion post-MI via c-Kit and GATA4.

GMP-Grade Methods for Cardiac Progenitor Cells: Cell Bank Production and Quality Control.

Cardiac explant-derived cells (cEDC), also referred as cardiac progenitors cells (CPC) (Barile et al., Cardiovasc Res 103(4):530-541, 2014; Barile et al., Cardiovasc Res 114(7):992-1005, 2018), represent promising candidates for the development of cell-based therapies, a novel and interesting treatment for cardioprotective strategy in heart failure (Kreke et al., Expert Rev Cardiovasc Ther 10(9):1185-1194, 2012). CPC have been tested in a preclinical setting for direct cell transplantation and tissue engineering or as a source for production of extracellular vesicles (EV) (Oh et al., J Cardiol 68(5):361-367, 2016; Barile et al., Eur Heart J 38(18):1372-1379, 2017; Rosen et al., J Am Coll Cardiol 64(9):922-937, 2014). CPC cultured as cardiospheres derived cells went through favorable Phase 1 and 2 studies demonstrating safety and possible efficacy (Makkar et al., Lancet 379(9819):895-904, 2012; Ishigami et al., Circ Res 120(7):1162-1173, 2017; Ishigami et al., Circ Res 116 (4):653-664, 2015; Tarui et al., J Thorac Cardiovasc Surg 150(5):1198-1207, 1208 e1191-1192, 2015). In this context and in view of clinical applications, cells have to be prepared and released according to Good Manufacturing Practices (GMP) (EudraLex-volume 4-good manufacturing practice (GMP) guidelines-Part I-basic requirements for medicinal products. http://ec.europa.eu/health/documents/eudralex/vol-4 ; EudraLex-volume 4-good manufacturing practice (GMP) guidelines-Part IV-guidelines on good manufacturing practices specific to advanced therapy medicinal products. http://ec.europa.eu/health/documents/eudralex/vol-4 ). This chapter describes GMP-grade methods for production and testing of a CPC Master Cell Bank (MCB), consisting of frozen aliquots of cells that may be used either as a therapeutic product or as source for the manufacturing of Exo for clinical trials.The MCB production method has been designed to isolate and expand CPC from human cardiac tissue in xeno-free conditions (Andriolo et al., Front Physiol 9:1169, 2018). The quality control (QC) methods have been implemented to assess the safety (sterility, endotoxin, mycoplasma, cell senescence, tumorigenicity) and identity/potency/purity (cell count and viability, RT-PCR, immunophenotype) of the cells (Andriolo et al., Front Physiol 9:1169, 2018).

Physiologic expansion of human heart-derived cells enhances therapeutic repair of injured myocardium

BackgroundSerum-free xenogen-free defined media and continuous controlled physiological cell culture conditions have been developed for stem cell therapeutics, but the effect of these conditions on the relative potency of the cell product is unknown. As such, we conducted a head-to-head comparison of cell culture conditions on human heart explant-derived cells using established in vitro measures of cell potency and in vivo functional repair.MethodsHeart explant-derived cells cultured from human atrial or ventricular biopsies within a serum-free xenogen-free media and a continuous physiological culture environment were compared to cells cultured under traditional (high serum) cell culture conditions in a standard clean room facility.ResultsTransitioning from traditional high serum cell culture conditions to serum-free xenogen-free conditions had no effect on cell culture yields but provided a smaller, more homogenous, cell product with only minor antigenic changes. Culture within continuous physiologic conditions markedly boosted cell proliferation while increasing the expression of stem cell-related antigens and ability of cells to stimulate angiogenesis. Intramyocardial injection of physiologic cultured cells into immunodeficient mice 1 week after coronary ligation translated into improved cardiac function and reduced scar burden which was attributable to increased production of pro-healing cytokines, extracellular vesicles, and microRNAs.ConclusionsContinuous physiological cell culture increased cell growth, paracrine output, and treatment outcomes to provide the greatest functional benefit after experimental myocardial infarction.

Abstract 404: Deterministic Paracrine Repair of Injured Myocardium using Microfluidic Cocooning of Heart Explant-Derived Cells

Previous work has shown that cocooning heart explant-derived cells (EDCs) within protective nanoporous gel capsules before intra-myocardial injection increases the retention of transplanted cells and the paracrine production of nanoparticles to improve post infarct cardiac function. In this study, we investigated the influence of cocoon size and intracapsular cell number on cell-treatment outcomes using a newly developed microfluidic-based (MF) cellular cocooning platform. Methods/Results: Traditional vortex-based encapsulation (Vx) inherently provides cocoons of varying diameters (30-100 μm; 68±5 μm). By altering the flow pressure ratios and the nozzle diameters within the MF chip, we encapsulated human EDCs within small (51±1 μm, MF50) and large (90±1 μm, MF90) diameter nanoporous gel cocoons for comparison with standard Vx-defined capsules (71±1 μm, MF70). MF cocooning mirrored the expected Poisson distribution with smaller cocoons having a greater proportion of single cells while larger diameter cocoons contained greater proportions of multicellular aggregates. Immunodeficient mice underwent left coronary artery ligation 1 week before randomization to echocardiographic guided intra-myocardial injection of EDCs (suspended or variable diameter cocoons) or vehicle. Increasing cocoon diameter stimulated progressive salutary effects on post-infarct function (ejection fraction), scar burden and newly generated peri-infarct blood vessels (isolectin B4+) and cardiomyocytes (BrdU+/TNT+) 4 weeks after treatment. Bioluminescent imaging of luciferase tagged cells revealed increasing cocoon diameter reduced the rate of cell clearance from injured tissues. Disrupting cell-cell contact within the capsules (using a custom antibody cocktail to block E/P-selectin and N-cadherin) reduced the amount and profile of pro-healing cytokines + nanoparticles delivered to injured myocardium. Conclusions: Increasing cocoon diameter and cell occupancy within protective nanoporous gel cocoons boosts paracrine-mediated repair of damaged myocardium by slowing clearance of cells from injured tissues and the number of cytokines + nanoparticles secreted by micro-encapsulated cells.

Insulin-like growth factor binding protein-7: A marker of conjunctivalization in an animal model of limbal stem cell deficiency

PurposeLimbal stem cell deficiency (LSCD) is characterized by the loss of limbal epithelial stem cells, resulting in a pathological process termed ‘conjunctivalization’ which compromises corneal transparency, leading to blindness. Current diagnosis for LSCD is limited because reliable conjunctiva-specific biomarkers are lacking. This study sought to address this shortcoming through the serendipitous discovery of insulin-like growth factor binding protein (IGFBP)-7. MethodsIGFBP-7 expression was determined in normal (n=83) and conjunctivalized (n=52) mouse corneas with experimentally-induced LSCD, and in cadaveric normal human corneas (n=7) and human pterygia (n=15); a disease characterized by the invasion of a conjunctivalized, fibrovascular pannus. Clinical assessments including slit-lamp microscopy, fluorescein staining and impression cytology, and biochemical, molecular and immunological assays were also conducted. ResultsMass spectrometry of conditioned media from mouse limbal explant-derived cells revealed the presence of IGFBP-7. This factor was expressed in normal limbal and conjunctival epithelium and conjunctivalized corneas from mice with LSCD, and in human pterygium epithelium but not in normal mouse or human corneal epithelium. Four weeks after inducing LSCD, IGFBP-7 staining was increased by 2.9-fold in mouse corneas compared to steady-state, and by 1.6-fold in impression cytology specimens derived from the same mice. Notably, IGFBP-7 was detected approximately 2-weeks earlier than Muc5AC. ConclusionsThis study provides novel insights into the specificity of IGFBP-7 for the mammalian conjunctival epithelium in health and disease. A point-of-care test for IGFBP-7 could be developed to assist clinicians in early diagnosis, and in monitoring disease progression, severity and therapeutic outcomes in patients with LSCD.

Glyoxalase 1 Prevents Chronic Hyperglycemia Induced Heart-Explant Derived Cell Dysfunction

Decades of work have shown that diabetes increases the risk of heart disease and worsens clinical outcomes after myocardial infarction. Because diabetes is an absolute contraindication to heart transplant, cell therapy is increasingly being explored as a means of improving heart function for these patients with very few other options. Given that hyperglycemia promotes the generation of toxic metabolites, the influence of the key detoxification enzyme glyoxalase 1 (Glo1) on chronic hyperglycemia induced heart explant-derived cell (EDC) dysfunction was investigated.Methods: EDCs were cultured from wild type C57Bl/6 or Glo1 over-expressing transgenic mice 2 months after treatment with the pancreatic beta cell toxin streptozotocin or vehicle. The effects of Glo1 overexpression was evaluated using in vitro and in vivo models of myocardial ischemia.Results: Chronic hyperglycemia reduced overall culture yields and increased the reactive dicarbonyl cell burden within EDCs. These intrinsic cell changes reduced the angiogenic potential and production of pro-healing exosomes while promoting senescence and slowing proliferation. Compared to intra-myocardial injection of normoglycemic cells, chronic hyperglycemia attenuated cell-mediated improvements in myocardial function and reduced the ability of transplanted cells to promote new blood vessel and cardiomyocyte growth. In contrast, Glo1 overexpression decreased oxidative damage while restoring both cell culture yields and EDC-mediated repair of ischemic myocardium. The latter was associated with enhanced production of pro-healing extracellular vesicles by Glo1 cells without altering the pro-healing microRNA cargo within.Conclusions: Chronic hyperglycemia decreases the regenerative performance of EDCs. Overexpression of Glo1 reduces dicarbonyl stress and prevents chronic hyperglycemia-induced dysfunction by rejuvenating the production of pro-healing extracellular vesicles.

Comparing the invivo and invitro effects of hypoxia (3% O2) on directly derived cells from murine cardiac explants versus murine cardiosphere derived cells

Coronary heart disease (CHD) is still one of the main causes of death in the world, despite significant advances in clinical treatments. Stem cell transplantation methods have the potential to improve cardiac function and patients' outcome following heart attack, but optimal cell types, cell preparation methods and cell delivery routes are yet to be developed. Mammalian hearts contain a small fraction of progenitor cells which, in culture, migrate out of the cardiac explants, known as explant-derived cell (EDCs) and contribute to spheroids known as cardiospheres (Csphs). Following further culture and cell passaging, Csphs give rise to cardiosphere-derived cells (CDCs). EDCs, Csphs and CDCs show in vitro and in vivo angiogenesis and tissue regeneration in myocardial ischemia. However, CDC and Csph formation is time consuming, expensive and not always successful. Therefore, this study aims to compare EDCs with CDCs and assess the effect of hypoxic preconditioning on their pro-angiogenic potential. The data showed that preconditioning EDCs in hypoxic cell culture enhances cell growth, viability and expression of stem cell and pro-angiogenic markers more than CDCs. In vivo experiments using a sub-dermal matrigel plug assay showed that EDCs and CDCs alone have limited pro-angiogenic potential; however, hypoxic preconditioning of EDCs and CDCs significantly enhances this process. Further research will increase our understanding of cardiac stem cell mediated angiogenesis and improve clinical therapies for myocardial infarction (MI) patients.

Dose-dependency and reversibility of radiation-induced injury in cardiac explant-derived cells of mice

We evaluated the dose-dependency and reversibility of radiation-induced injury in cardiac explant-derived cells (CDCs), a mixed cell population grown from heart tissues. Adult C57BL/6 mice were exposed to 0, 10, 50 and 250 mGy γ-rays for 7 days and atrial tissues were collected for experiments 24 hours after last exposure. The number of CDCs was significantly decreased by daily exposure to over 250 mGy. Interestingly, daily exposure to over 50 mGy significantly decreased the c-kit expression and telomerase activity, increased 53BP1 foci in the nuclei of CDCs. However, CD90 expression and growth factors production in CDCs were not significantly changed even after daily exposure to 250 mGy. We further evaluated the reversibility of radiation-induced injury in CDCs at 1 week and 3 weeks after a single exposure to 3 Gy γ-rays. The number and growth factors production of CDCs were soon recovered at 1 week. However, the increased expression of CD90 were retained at 1 week, but recovered at 3 weeks. Moreover, the decreased expression of c-kit, impaired telomerase activity, and increased 53BP1 foci were poorly recovered even at 3 weeks. These data may help us to find the most sensitive and reliable bio-parameter(s) for evaluating radiation-induced injury in CDCs.

The impact of patient co-morbidities on the regenerative capacity of cardiac explant-derived stem cells

BackgroundAlthough patient-sourced cardiac stem cells repair damaged myocardium, the extent to which medical co-morbidities influence cardiac-derived cell products is uncertain. Therefore, we investigated the influence of atherosclerotic risk factors on the regenerative performance of human cardiac explant-derived cells (EDCs).MethodsIn this study, the Long Term Stratification for survivors of acute coronary syndromes model was used to quantify the burden of cardiovascular risk factors within a group of patients with established atherosclerosis. EDCs were cultured from human atrial appendages and injected into immunodeficient mice 7 days post-left coronary ligation. Cytokine arrays and enzyme linked immunoassays were used to determine the release of cytokines by EDCs in vitro, and echocardiography was used to determine regenerative capabilities in vivo.ResultsEDCs sourced from patients with more cardiovascular risk factors demonstrated a negative correlation with production of pro-healing cytokines (such as stromal cell derived factor 1α) and exosomes which had negative effects on the promotion of angiogenesis and chemotaxis. Reductions in exosomes and pro-healing cytokines with accumulating medical co-morbidities were associated with increases in production of the pro-inflammatory cytokine interleukin-6 (IL-6) by EDCs. Increased patient co-morbidities were also correlated with significant attenuation in improvements of left ventricular ejection fraction.ConclusionsThe regenerative performance of the earliest precursor cell population cultured from human explant tissue declines with accumulating medical co-morbidities. This effect is associated with diminished production of pro-cardiogenic cytokines and exosomes while IL-6 is markedly increased. Predictors of cardiac events demonstrated a lower capacity to support angiogenesis and repair injured myocardium in a mouse model of myocardial infarction.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0321-4) contains supplementary material, which is available to authorized users.

Label-Free Imaging of Umbilical Cord Tissue Morphology and Explant-Derived Cells.

In situ detection of MSCs remains difficult and warrants additional methods to aid with their characterization in vivo. Two-photon confocal laser scanning microscopy (TPM) and second harmonic generation (SHG) could fill this gap. Both techniques enable the detection of cells and extracellular structures, based on intrinsic properties of the specific tissue and intracellular molecules under optical irradiation. TPM imaging and SHG imaging have been used for label-free monitoring of stem cells differentiation, assessment of their behavior in biocompatible scaffolds, and even cell tracking in vivo. In this study, we show that TPM and SHG can accurately depict the umbilical cord architecture and visualize individual cells both in situ and during culture initiation, without the use of exogenously applied labels. In combination with nuclear DNA staining, we observed a variance in fluorescent intensity in the vessel walls. In addition, antibody staining showed differences in Oct4, αSMA, vimentin, and ALDH1A1 expression in situ, indicating functional differences among the umbilical cord cell populations. In future research, marker-free imaging can be of great added value to the current antigen-based staining methods for describing tissue structures and for the identification of progenitor cells in their tissue of origin.