Safety and Efficacy of Intracoronary Infusion of Allogeneic Human Cardiac Stem Cells in Patients With ST-Segment Elevation Myocardial Infarction and Left Ventricular Dysfunction.

RATIONALE: Allogeneic cardiac stem cells (AlloCSC-01) have shown protective, immunoregulatory, and regenerative properties with a robust safety profile in large animal models of heart disease. OBJECTIVE: To investigate the safety and feasibility of early administration of AlloCSC-01 in patients with ST-segment-elevation myocardial infarction. METHODS AND RESULTS: CAREMI (Safety and Efficacy of Intracoronary Infusion of Allogeneic Human Cardiac Stem Cells in Patients With STEMI and Left Ventricular Dysfunction) was a phase I/II multicenter, randomized, double-blind, placebo-controlled trial in patients with ST-segment-elevation myocardial infarction, left ventricular ejection fraction ≤45%, and infarct size ≥25% of left ventricular mass by cardiac magnetic resonance, who were randomized (2:1) to receive AlloCSC-01 or placebo through the intracoronary route at days 5 to 7. The primary end point was safety and included all-cause death and major adverse cardiac events at 30 days (all-cause death, reinfarction, hospitalization because of heart failure, sustained ventricular tachycardia, ventricular fibrillation, and stroke). Secondary safety end points included major adverse cardiac events at 6 and 12 months, adverse events, and immunologic surveillance. Secondary exploratory efficacy end points were changes in infarct size (percentage of left ventricular mass) and indices of ventricular remodeling by magnetic resonance at 12 months. Forty-nine patients were included (92% male, 55±11 years), 33 randomized to AlloCSC-01 and 16 to placebo. No deaths or major adverse cardiac events were reported at 12 months. One severe adverse events in each group was considered possibly related to study treatment (allergic dermatitis and rash). AlloCSC-01 elicited low levels of donor-specific antibodies in 2 patients. No immune-related adverse events were found, and no differences between groups were observed in magnetic resonance-based efficacy parameters at 12 months. The estimated treatment effect of AlloCSC-01 on the absolute change from baseline in infarct size was -2.3% (95% confidence interval, -6.5% to 1.9%). CONCLUSIONS: AlloCSC-01 can be safely administered in ST-segment-elevation myocardial infarction patients with left ventricular dysfunction early after revascularization. Low immunogenicity and absence of immune-mediated events will facilitate adequately powered studies to demonstrate their clinical efficacy in this setting. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov . Unique identifier: NCT02439398.

Rationale and Design of a Clinical Trial to Evaluate the Safety and Efficacy of Intracoronary Infusion of Allogeneic Human Cardiac Stem Cells in Patients With Acute Myocardial Infarction and Left Ventricular Dysfunction: The Randomized Multicenter Double-Blind Controlled CAREMI Trial (Cardiac Stem Cells in Patients With Acute Myocardial Infarction).

RATIONALE: Stem cell therapy has increased the therapeutic armamentarium in the fight against ischemic heart disease and heart failure. The administration of exogenous stem cells has been investigated in patients suffering an acute myocardial infarction, with the final aim of salvaging jeopardized myocardium and preventing left ventricular adverse remodeling and functional deterioration. However, phase I and II clinical trials with autologous and first-generation stem cells have yielded inconsistent benefits and mixed results. OBJECTIVE: In the search for new and more efficient cellular regenerative products, interesting cardioprotective, immunoregulatory, and cardioregenerative properties have been demonstrated for human cardiac stem cells. On the other hand, allogeneic cells show several advantages over autologous sources: they can be produced in large quantities, easily administered off-the-shelf early after an acute myocardial infarction, comply with stringent criteria for product homogeneity, potency, and quality control, and may exhibit a distinctive immunologic behavior. METHODS AND RESULTS: With a promising preclinical background, CAREMI (Cardiac Stem Cells in Patients With Acute Myocardial Infarction) has been designed as a double-blind, 2:1 randomized, controlled, and multicenter clinical trial that will evaluate the safety, feasibility, and efficacy of intracoronary delivery of allogeneic human cardiac stem cell in 55 patients with large acute myocardial infarction, left ventricular dysfunction, and at high risk of developing heart failure. CONCLUSIONS: This phase I/II clinical trial represents a novel experience in humans with allogeneic cardiac stem cell in a rigorously imaging-based selected group of acute myocardial infarction patients, with detailed safety immunologic assessments and magnetic resonance imaging-based efficacy end points. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02439398.

Non-invasive in vivo imaging of cardiac stem/progenitor cell biodistribution and retention after intracoronary and intramyocardial delivery in a swine model of chronic ischemia reperfusion injury.

BACKGROUND: The safety and efficacy of cardiac stem/progenitor cells (CSC) have been demonstrated in previous preclinical and clinical assays for heart failure. However, their optimal delivery route to the ischemic heart has not yet been assessed. This study was designed to determine by a non-invasive imaging technique (PET/CT) the biodistribution and acute retention of allogeneic pig CSC implanted by two different delivery routes, intracoronary (IC) and intramyocardial (IM), in a swine preclinical model of chronic ischemia-reperfusion. METHODS: Ischemia-reperfusion was induced in six Goettingen hybrid minipigs by 90 min coronary artery occlusion followed by reperfusion. Thirty days later, animals were allocated to receive IC (n = 3) or NOGA®-guided IM injection (n = 3) of 50 million of 18F-FDG/GFP-labeled allogeneic pig CSC. Acute retention was quantified by PET/CT 4 h after injection and cell engraftment assessed by immunohistochemical quantification of GFP+ cells three days post-injection. RESULTS: Biodistribution of 18F-FDG-labeled CSC was clearly visualized by PET/CT imaging and quantified. No statistical differences in acute cell retention (percentage of injected dose, %ID) were found in the heart when cells were administered by NOGA®-guided IM (13.4 ± 3.4%ID) or IC injections (17.4 ± 4.1%ID). Interestingly, engrafted CSC were histologically detected only after IM injection. CONCLUSION: PET/CT imaging of 18F-FDG-labeled CSC allows quantifying biodistribution and acute retention of implanted cells in a clinically relevant pig model of chronic myocardial infarction. Similar levels of acute retention are achieved when cells are IM or IC administered. However, acute cell retention does not correlate with cell engraftment, which is improved by IM injection.

Delayed administration of allogeneic cardiac stem cell therapy for acute myocardial infarction could ameliorate adverse remodeling: experimental study in swine.

BACKGROUND: The optimal timing of cardiac stem cells administration is still unclear. We assessed the safety of same-day and delayed (one week) delivery and the possible influence of the timing on the therapeutic outcomes of allogeneic porcine cardiac stem cells administration after acute myocardial infarction in a closed-chest ischemia-reperfusion model. METHODS: Female swine surviving 90 min occlusion of the mid left anterior descending coronary artery received an intracoronary injection of 25x10(6) porcine cardiac stem cells either two hours (n = 5, D0) or 7 days (n = 6, D7) after reperfusion. Controls received intracoronary injection of vehicle on day 7 (n = 6, CON). Safety was defined in terms of absence of major cardiac events, changes to the ECG during injection, post-administration coronary flow assessed using the TIMI scale and cardiac troponin I determination after the intervention. Cardiac Magnetic Resonance was performed for morphological and functional assessment prior to infarction, before injection (D7 and CON groups only), at one and 10 weeks. Samples were taken from the infarct and transition areas for pathological examination. RESULTS: No major adverse cardiac events were seen during injection in any group. Animals receiving the therapy on the same day of infarction (D0 group) showed mild transient ST changes during injection (n = 4) and, in one case, slightly compromised coronary flow (TIMI 2). Cardiac function parameters and infarct sizes were not significantly different between groups, with a trend towards higher ejection fraction in the treated groups. Ventricular volumes indexed to body surface area increased over time in control animals, and decreased by the end of the study in animals receiving the therapy, significantly so when comparing End Diastolic Volume between CON and D7 groups (CON: 121.70 ml/m(2) ± 26.09 ml/m(2), D7: 98.71 ml/m(2) ± 8.30 ml/m(2), p = 0.037). The treated groups showed less organization of the collagenous scar, and a significantly (p = 0.019) higher amount of larger, more mature vessels at the infarct border. CONCLUSIONS: The intracoronary injection of 25x10(6) allogeneic cardiac stem cells is generally safe, both early and 7 days after experimental infarction, and alleviates myocardial dysfunction, with a greater limitation of left ventricular remodeling when performed at one week.

Management of vagal paragangliomas: review of 17 patients.

Vagal paragangliomas are very rare benign vascular tumors of neuroendocrine nature, and are much less frequent than carotid and jugulo-tympanic tumors. The goal of this retrospective study is to review the clinical and genetic findings, surgical treatment, and complications of vagal paragangliomas, as well as to discuss the management options. During the period 1990-2013, 17 patients with vagal paragangliomas were referred to our institution. There were ten patients with isolated tumors, and seven with multicentric paragangliomas. There were nine women and eight men. Mean age of patients was 51.4 years. Five cases had a positive family history of paraganglioma (29.4 %). Germline mutations of SDH genes were found in six of our patients (35.3 %). Many options were considered in the management of vagal paragangliomas. Surgical treatment was performed in 11 young patients (64.7 %) using different approaches: in 4 patients the tumor was resected through a transcervical approach; in 3 through a transcervical-transmandibular approach; in 1 it was resected using a transcervical-transmastoid approach, and in 3 a type A infratemporal fossa approach was performed. In all operated cases, the removal of the tumor led to sacrificing of the vagus nerve. Postoperative hypoglossal nerve deficit was reported in 4 cases (36.3 %). In six elderly patients (35.3 %), we decided to "wait-and-scan" in order to avoid creating greater morbidity than that of the tumor itself. Many factors should be considered in the treatment of vagal paragangliomas: the age and general condition of the patient, the biological behavior of the tumor, tumor size, genetic results, bilaterality, multicentricity, lower cranial nerve function, and of course the potential morbidity of the surgical treatment itself. Rehabilitation and, possibly surgery, are necessary to treat postoperative lower cranial nerve deficits.

Intracoronary Administration of Microencapsulated HGF in a Reperfused Myocardial Infarction Swine Model.

Therapy microencapsulation allows minimally invasive, safe, and effective administration. Hepatocyte growth factor (HGF) has angiogenic, anti-inflammatory, anti-apoptotic, and anti-fibrotic properties. Our objective was to evaluate the cardiac safety and effectiveness of intracoronary (IC) administration of HGF-loaded extended release microspheres in an acute myocardial infarction (AMI) swine model. An IC infusion of 5 × 106 HGF-loaded microspheres (MS+HGF, n = 7), 5 × 106 placebo microspheres (MS, n = 7), or saline (SAL, n = 7) was performed two days after AMI. TIMI flow and Troponin I (TnI) values were assessed pre- and post-treatment. Cardiac function was evaluated with magnetic resonance imaging (cMR) before injection and at 10 weeks. Plasma cytokines were determined to evaluate the inflammatory profile and hearts were subjected to histopathological evaluation. Post-treatment coronary flow was impaired in five animals (MS+HGF and MS group) without significant increases in TnI. One animal (MS group) died during treatment. There were no significant differences between groups in cMR parameters at any time (p > 0.05). No statistically significant changes were found between groups neither in cytokines nor in histological analyses. The IC administration of 5 × 106 HGF-loaded-microspheres 48 h post-AMI did not improve cardiac function, nor did it decrease inflammation or cardiac fibrosis in this experimental setting.

Corrigendum to "Plasmatic Membrane Expression of Adhesion Molecules in Human Cardiac Progenitor/Stem Cells Might Explain Their Superior Cell Engraftment after Cell Transplantation".

[This corrects the article DOI: 10.1155/2020/8872009.].

Bioreactor-based 3D human myocardial ischemia/reperfusion in vitro model: a novel tool to unveil key paracrine factors upon acute myocardial infarction.

During acute myocardial infarction (AMI), Ischemia/Reperfusion (I/R) injury causes cardiomyocyte (CM) death and loss of tissue function, making AMI one of the major causes of death worldwide. Cell-based in vitro models of I/R injury have been increasingly used as a complementary approach to preclinical research. However, most approaches use murine cells in 2D culture setups, which are not able to recapitulate human cellular physiology, as well as nutrient and gas gradients occurring in the myocardium. In this work we established a novel human in vitro model of myocardial I/R injury using CMs derived from human induced pluripotent stem cells (hiPSC-CMs), which were cultured as 3D aggregates in stirred tank bioreactors. We were able to recapitulate important hallmarks of AMI, including loss of CM viability with disruption of cellular ultrastructure, increased angiogenic potential, and secretion of key proangiogenic and proinflammatory cytokines. Conditioned medium was further used to probe human cardiac progenitor cells (hCPCs) response to paracrine cues from injured hiPSC-CMs through quantitative whole proteome analysis (SWATH-MS). I/R injury hiPSC-CM conditioned media incubation caused upregulation of hCPC proteins associated with migration, proliferation, paracrine signaling, and stress response-related pathways, when compared to the control media incubation. Our results indicate that the model developed herein can serve as a novel tool to interrogate mechanisms of action of human cardiac populations upon AMI.

Plasmatic Membrane Expression of Adhesion Molecules in Human Cardiac Progenitor/Stem Cells Might Explain Their Superior Cell Engraftment after Cell Transplantation.

Human bone marrow mesenchymal stem cells (BM-MSCs) and cardiac progenitor/stem cells (CPCs) have been extensively studied as a potential therapeutic treatment for myocardial infarction (MI). Previous reports suggest that lower doses of CPCs are needed to improve cardiac function relative to their bone marrow counterparts. Here, we confirmed this observations and investigated the surface protein expression profile that might explain this effect. Myocardial infarction was performed in nude rats by permanent ligation of the left coronary artery. Cardiac function and infarct size before and after cell transplantation were evaluated by echocardiography and morphometry, respectively. The CPC and BM-MSC receptome were analyzed by proteomic analysis of biotin-labeled surface proteins. Rats transplanted with CPCs showed a greater improvement in cardiac function after MI than those transplanted with BM-MSCs, and this was associated with a smaller infarct size. Analysis of the receptome of CPCs and BM-MSCs showed that gene ontology biological processes and KEGG pathways associated with adhesion mechanisms were upregulated in CPCs compared with BM-MSCs. Moreover, the membrane protein interactome in CPCs showed a strong relationship with biological processes related to cell adhesion whereas the BM-MSCs interactome was more related to immune regulation processes. We conclude that the stronger capacity of CPCs over BM-MSCs to engraft in the infarcted area is likely linked to a more pronounced cell adhesion expression program.

Human adipose mesenchymal stem cells modulate myeloid cells toward an anti-inflammatory and reparative phenotype: role of IL-6 and PGE2.

BACKGROUND: Mesenchymal stem cells (MSCs) activate the endogenous immune regulatory system, inducing a therapeutic effect in recipients. MSCs have demonstrated the ability to modulate the differentiation of myeloid cells toward a phagocytic and anti-inflammatory profile. Allogeneic, adipose-derived MSCs (ASCs) have been investigated for the management of complex perianal fistula, with darvadstrocel being the first ASC therapy approved in Europe in March 2018. Additionally, ASCs are being explored as a potential treatment in other indications. Yet, despite these clinical advances, their mechanism of action is only partially understood. METHODS: Freshly isolated human monocytes from the peripheral blood were differentiated in vitro toward M0 non-polarized macrophages (Mphs), M1 pro-inflammatory Mphs, M2 anti-inflammatory Mphs, or mature dendritic cells (mDCs) in the presence or absence of ASCs, in non-contact conditions. The phenotype and function of the differentiated myeloid populations were determined by flow cytometry, and their secretome was analyzed by OLINK technology. We also investigated the capacity of ASCs to modulate the phenotype and function of terminally differentiated M1 Mphs. The role of soluble factors interleukin (IL)-6 and prostaglandin E2 (PGE2) on the ability of ASCs to modulate myeloid cells was assessed using neutralization assays, CRISPR/Cas9 knock-down of cyclooxygenase 2 (COX-2), and ASC-conditioned medium assays using pro-inflammatory stimulus. RESULTS: Co-culture of monocytes in the presence of ASCs resulted in the polarization of Mphs and mDCs toward an anti-inflammatory and phagocytic phenotype. This was characterized by an increase in phagocytic receptors on the cell surface of Mphs (M0, M1, and M2) and mDCs, as well as modulation of chemokine receptors and reduced expression of pro-inflammatory, co-stimulatory molecules. ASCs also modulated the secretome of Mphs and mDCs, demonstrated by reduced expression of pro-inflammatory factors and increased expression of anti-inflammatory and reparative factors. Chemical inhibition of PGE2 with indomethacin abolished this modulatory effect, whereas treatment with a neutralizing anti-IL-6 antibody resulted in a partial abolishment. The knock-down of COX-2 in ASCs and the use of IL-1ß-activated ASC-conditioned media confirmed the key role of PGE2 in ASC-mediated myeloid modulation. In our in vitro experimental settings, ASCs failed to modulate the phenotype and function of terminally polarized M1 Mphs. CONCLUSIONS: The results demonstrate that ASCs are able to modulate the in vitro differentiation of myeloid cells toward an anti-inflammatory and reparative profile. This modulatory effect was mediated mainly by PGE2 and, to a lesser extent, IL-6.

Microencapsulated Insulin-Like Growth Factor-1 therapy improves cardiac function and reduces fibrosis in a porcine acute myocardial infarction model.

Insulin-like growth factor-1 (IGF-1) has demonstrated beneficial effects after myocardial infarction (MI). Microencapsulation of IGF-1 could potentially improve results. We aimed to test the effect of an intracoronary (IC) infusion of microencapsulated IGF-1 in a swine acute MI model. For that purpose IC injection of a 10 ml solution of 5 × 106 IGF-1 loaded microspheres (MSPs) (n = 8, IGF-1 MSPs), 5 × 106 unloaded MSPs (n = 9; MSPs) or saline (n = 7; CON) was performed 48 hours post-MI. Left ventricular ejection fraction (LVEF), indexed ventricular volumes and infarct size (IS) were determined by cardiac magnetic resonance at pre-injection and 10 weeks. Animals were euthanized at 10 weeks, and myocardial fibrosis and vascular density were analysed. End-study LVEF was significantly greater in IGF-1 MSPs compared to MSPs and CON, while ventricular volumes exhibited no significant differences between groups. IS decreased over time in all groups. Collagen volume fraction on the infarct area was significantly reduced in IGF-1 MSPs compared to CON and MSPs. Vascular density analysis of infarct and border zones showed no significant differences between groups. In conclusion, the IC injection of 5 × 106 IGF-1 loaded MSPs in a porcine acute MI model successfully improves cardiac function and limits myocardial fibrosis, which could be clinically relevant.

Human cardiac progenitor cell activation and regeneration mechanisms: exploring a novel myocardial ischemia/reperfusion in vitro model.

BACKGROUND: Numerous studies from different labs around the world report human cardiac progenitor cells (hCPCs) as having a role in myocardial repair upon ischemia/reperfusion (I/R) injury, mainly through auto/paracrine signaling. Even though these cell populations are already being investigated in cell transplantation-based clinical trials, the mechanisms underlying their response are still poorly understood. METHODS: To further investigate hCPC regenerative process, we established the first in vitro human heterotypic model of myocardial I/R injury using hCPCs and human-induced pluripotent cell-derived cardiomyocytes (hiPSC-CMs). The co-culture model was established using transwell inserts and evaluated in both ischemia and reperfusion phases regarding secretion of key cytokines, hiPSC-CM viability, and hCPC proliferation. hCPC proteome in response to I/R was further characterized using advanced liquid chromatography mass spectrometry tools. RESULTS: This model recapitulates hallmarks of I/R, namely hiPSC-CM death upon insult, protective effect of hCPCs on hiPSC-CM viability (37.6% higher vs hiPSC-CM mono-culture), and hCPC proliferation (approximately threefold increase vs hCPCs mono-culture), emphasizing the importance of paracrine communication between these two populations. In particular, in co-culture supernatant upon injury, we report higher angiogenic functionality as well as a significant increase in the CXCL6 secretion rate, suggesting an important role of this chemokine in myocardial regeneration. hCPC whole proteome analysis allowed us to propose new pathways in the hCPC-mediated regenerative process, including cell cycle regulation, proliferation through EGF signaling, and reactive oxygen species detoxification. CONCLUSION: This work contributes with new insights into hCPC biology in response to I/R, and the model established constitutes an important tool to study the molecular mechanisms involved in the myocardial regenerative process.

Extracellular Vesicles Released by Allogeneic Human Cardiac Stem/Progenitor Cells as Part of Their Therapeutic Benefit.

The positive effects of therapeutic human allogeneic cardiac stem/progenitor cells (hCPC) in terms of cardiac repair/regeneration are very likely mediated by paracrine effects. Our previous studies revealed the advantageous immune interactions of allogeneic hCPC and proposed them as part of the positive paracrine effects occurring upon their application postmyocardial infarction (MI). Currently, extracellular vesicles/exosomes (EV/Exs) released by stem/progenitor cells are also proposed as major mediators of paracrine effects of therapeutic cells. Along this line, we evaluated contribution of EV/Exs released by therapeutic hCPC to the benefit of their successful allogeneic clinical application. Through tailored allogeneic in vitro human assay models mimicking the clinical setting, we demonstrate that hCPC-released EV/Exs were rapidly and efficiently up-taken by chief cellular actors of cardiac repair/regeneration. This promoted MAPK/Erk1/2 activation, migration, and proliferation of human leukocyte antigens (HLA)-mismatched hCPC, mimicking endogenous progenitor cells and cardiomyocytes, and enhanced endothelial cell migration, growth, and organization into tube-like structures through activation of several signaling pathways. EV/Exs also acted as pro-survival stimuli for HLA-mismatched monocytes tuning their phenotype toward an intermediate anti-inflammatory pro-angiogenic phenotype. Thus, while positively impacting the intrinsic regenerative and angiogenic programs, EV/Exs released by therapeutic allogeneic hCPC can also actively contribute to shaping MI-inflammatory environment, which could strengthen the benefits of hCPC allogeneic interactions. Collectively, our data might forecast the application of allogeneic hCPC followed by their cell-free EV/Exs as a strategy that will not only elicit the cell-contact mediated reparative/regenerative immune response but also have the desired long-lasting effects through the EV/Exs. Stem Cells Translational Medicine 2019;8:911&924.

Dissecting Allo-Sensitization After Local Administration of Human Allogeneic Adipose Mesenchymal Stem Cells in Perianal Fistulas of Crohn's Disease Patients.

Adipose mesenchymal stem cells (ASC) are considered minimally immunogenic. This is due to the low expression of human leukocyte antigens I (HLA-I), lack of HLA-II expression and low expression of co-stimulatory molecules such as CD40 and CD80. The low rate of observed immunological rejection as well as the immunomodulatory qualities, position ASC as a promising cell-based therapy for the treatment of a variety of inflammatory indications. Yet, few studies have addressed relevant aspects of immunogenicity such as ASC donor-to-patient HLA histocompatibility or assessment of immune response triggered by ASC administration, particularly in the cases of presensitization. The present study aims to assess allo-immune responses in a cohort of Crohn's disease patients administered with allogeneic ASC (darvadstrocel formerly Cx601) for the treatment of complex perianal fistulas. We identified donor-specific antibodies (DSA) generation in a proportion of patients and observed that patients showing preexisting immunity were prone to generating DSA after allogeneic therapy. Noteworthy, naïve patients generating DSA at week 12 (W12) showed a significant reduction in DSA titer at week 52 (W52), whereas DSA titer was reduced in pre-sensitized patients only with no specificities against the donor administered. Remarkably, we did not observe any correlation of DSA generation with ASC therapeutic efficacy. In vitro complement-dependent cytotoxicity (CDC) studies have revealed limited cytotoxic levels based upon HLA-I expression and binding capacity even in pro-inflammatory conditions. We sought to identify CDC coping mechanisms contributing to the limited cytotoxic killing observed in ASC in vitro. We found that ASC express membrane-bound complement regulatory proteins (mCRPs) CD55, CD46, and CD59 at basal levels, with CD46 more actively expressed in pro-inflammatory conditions. We demonstrated that CD46 is a main driver of CDC signaling; its depletion significantly enhances sensitivity of ASC to CDC. In summary, despite relatively high clearance, DSA generation may represent a major challenge for allogeneic cell therapy management. Sensitization may be a significant concern when evaluating re-treatment or multi-donor trials. It is still unknown whether DSA generation could potentially be the consequence of donor-to-patient interaction and, therefore, subsequently link to efficacy or biological activity. Lastly, we propose that CDC modulators such as CD46 could be used to ultimately link CDC specificity with allogeneic cell therapy efficacy.

Dose-dependent improvement of cardiac function in a swine model of acute myocardial infarction after intracoronary administration of allogeneic heart-derived cells.

BACKGROUND: Allogeneic cardiac-derived progenitor cells (CPC) without immunosuppression could provide an effective ancillary therapy to improve cardiac function in reperfused myocardial infarction. We set out to perform a comprehensive preclinical feasibility and safety evaluation of porcine CPC (pCPC) in the infarcted porcine model, analyzing biodistribution and mid-term efficacy, as well as safety in healthy non-infarcted swine. METHODS: The expression profile of several pCPC isolates was compared with humans using both FACS and RT-qPCR. ELISA was used to compare the functional secretome. One week after infarction, female swine received an intracoronary (IC) infusion of vehicle (CON), 25 × 106 pCPC (25 M), or 50 × 106 pCPC (50 M). Animals were followed up for 10 weeks using serial cardiac magnetic resonance imaging to assess functional and structural remodeling (left ventricular ejection fraction (LVEF), systolic and diastolic volumes, and myocardial salvage index). Statistical comparisons were performed using Kruskal-Wallis and Mann-Whitney U tests. Biodistribution analysis of 18F-FDG-labeled pCPC was also performed 4 h after infarction in a different subset of animals. RESULTS: Phenotypic and functional characterization of pCPC revealed a gene expression profile comparable to their human counterparts as well as preliminary functional equivalence. Left ventricular functional and structural remodeling showed significantly increased LVEF 10 weeks after IC administration of 50 M pCPC, associated to the recovery of left ventricular volumes that returned to pre-infarction values (LVEF at 10 weeks was 42.1 ± 10.0% in CON, 46.5 ± 7.4% in 25 M, and 50.2 ± 4.9% in 50 M, p < 0.05). Infarct remodeling was also improved following pCPC infusion with a significantly higher myocardial salvage index in both treated groups (0.35 ± 0.20 in CON; 0.61 ± 0.20, p = 0.04, in 25 M; and 0.63 ± 0.17, p = 0.01, in 50 M). Biodistribution studies demonstrated cardiac tropism 4 h after IC administration, with substantial myocardial retention of pCPC-associated tracer activity (18% of labeled cells in the heart), and no obstruction of coronary flow, indicating their suitability as a cell therapy product. CONCLUSIONS: IC administration of allogeneic pCPC at 1 week after acute myocardial infarction is feasible, safe, and associated with marked structural and functional benefit. The robust cardiac tropism of pCPC and the paracrine effects on left ventricle post-infarction remodeling established the preclinical bases for the CAREMI clinical trial (NCT02439398).

Definition of a cell surface signature for human cardiac progenitor cells after comprehensive comparative transcriptomic and proteomic characterization.

Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Assisted by complementary RNAseq analysis, we defined the fraction of the CPC proteome associable with specific functions by comparison with human bone marrow mesenchymal stem cells (MSC), the reference population for cell therapy, and human dermal fibroblasts (HDF), as a distant reference. Label-free proteomic analysis identified 526 proteins expressed differentially in CPC. iTRAQ analysis confirmed differential expression of a substantial proportion of those proteins in CPC relative to MSC, and systems biology analysis defined a clear overrepresentation of several categories related to enhanced angiogenic potential. The CPC plasma membrane compartment comprised 1,595 proteins, including a minimal signature of 167 proteins preferentially or exclusively expressed by CPC. CDH5 (VE-cadherin),  OX2G (OX-2 membrane glycoprotein; CD200), GPR4 (G protein-coupled receptor 4), CACNG7 (calcium voltage-gated channel auxiliary subunit gamma 7) and F11R (F11 receptor; junctional adhesion molecule A; JAM-A; CD321) were selected for validation. Their differential expression was confirmed both in expanded CPC batches and in early stages of isolation, particularly when compared against cardiac fibroblasts. Among them, GPR4 demonstrated the highest discrimination capacity between all cell lineages analyzed.

Human cardiac stem cells inhibit lymphocyte proliferation through paracrine mechanisms that correlate with indoleamine 2,3-dioxygenase induction and activity.

Transplantation of allogeneic human cardiac/stem progenitor cells (hCSCs) is currently being tested in several phase I/II clinical trials as a novel and promising therapy for restoration of myocardial tissue function in acute myocardial infarction (AMI) patients. Previous findings demonstrate that these cells have an immune suppressive profile interacting with different populations from the immune system, resulting in overall attenuation of myocardial inflammation. However, transplanted hCSCs are still recognized and cleared from the injured site, impairing long retention times in the tissue that could translate into a higher clinical benefit.In this work, through modeling allogeneic hCSC/T lymphocyte interaction in vitro by direct contact, transwell inserts, and hCSC conditioned medium, our results demonstrate that hCSCs exert an immune-suppressive effect on T lymphocyte proliferation not only through the previously described cell contact-dependent programmed cell death-1 (PD1)/programmed death ligand-1 (PDL-1) axis but also through a paracrine mechanism associated with indoleamine 2,3-dioxygenase (IDO) enzyme-mediated tryptophan metabolism. Such findings constitute a step forward in better understanding the mechanisms of action of transplanted hCSCs in allogeneic settings.

Pathogenic validation of unique germline intronic variants of RB1 in retinoblastoma patients using minigenes.

Precise identification of the pathogenic character of germline mutations in the retinoblastoma gene (RB1) is fundamental to provide genetic counselling to patients at risk of developing retinoblastoma. In contrast to bona fide oncogenic RB1 mutations like nonsense or frameshift mutations, and those affecting invariant dinucleotides at splice sites, intronic variants affecting less conserved splice motifs require additional analysis to ascertain whether splicing is altered. Although the frequency of these variations is low, their impact on genetic counselling is high, since they are usually associated with low penetrance phenotypes and unaffected carriers. In this work, we used minigene assays to study infrequent germline intronic variations for which functional data were not available. Using this approach, the aberrant splicing and the resulting oncogenic nature of three intronic RB1 mutations was established (c.501-15T>G, c.719-9C>G, c.2326-8T>A). Conversely, the intronic variant c.1961-12T>C was categorized by minigene assay as a very infrequent neutral polymorphism. To our knowledge this is the first report describing the use of minigene constructs to study the oncogenic character of intronic RB1 variants detected during mutational screening and show the utility of this approach to ascertain the oncogenic nature of unique RB1 intronic variants for which no previous functional and clinical data are available. Minigene assay can be especially useful when lymphocyte RNA is not available for study, or when aberrant mRNA can not be detected as a consequence of nonsense mediated decay. Since RB1 minigene are time-consuming assays, owing to the genomic organization of the RB1 gene, it should be welcome the design of new expression vectors that make this type of studies more straightforward.

Postoperative Pain in Adult Tonsillectomy: Is There Any Difference Between the Technique?

Tonsillectomy is one of the most common surgical procedures performed worldwide. Several techniques have been developed to reduce morbidity and enhance recovery after tonsillectomy. Our study was designed to compare post-operative pain with three different techniques: cold dissection (CD), monopolar-bipolar dissection (MBD) and coblation dissection (CBD). 103 adults were scheduled for elective tonsillectomy from September 2014 to December 2015, and were randomized to CD, MBD and CBD. Post-operative pain was assessed using visual analogue scale (VAS) and Lattinen Test (LT). We did not find significant differences between the groups in the VAS pain scores (p > 0.05), except for the first day, when CBD tonsillectomy showed a higher pain score (p < 0.05). The differences in LT scores between the three techniques were not statistically significant (p > 0.05). Comparison of analgesic consumption between CD, MBD and CBD did not found any significant differences irrespective of the technique used. When first and second week after surgery were compared, differences in analgesics requirements were statistically significant (p < 0.05). Seventeen cases (16.5%) of secondary haemorrhage were reported, but there were no statistical differences in the rate of postoperative bleeding between the three groups (p > 0.05). We conclude that in our study comparison of the three techniques, CD, MBD and CBD, did not show significant differences in the post-tonsillectomy pain scores and bleeding rate.

Human Cardiac-Derived Stem/Progenitor Cells Fine-Tune Monocyte-Derived Descendants Activities toward Cardiac Repair.

Cardiac repair following MI relies on a finely regulated immune response involving sequential recruitment of monocytes to the injured tissue. Monocyte-derived cells are also critical for tissue homeostasis and healing process. Our previous findings demonstrated the interaction of T and natural killer cells with allogeneic human cardiac-derived stem/progenitor cells (hCPC) and suggested their beneficial effect in the context of cardiac repair. Therefore, we investigated here whether monocytes and their descendants could be also modulated by allogeneic hCPC toward a repair/anti-inflammatory phenotype. Through experimental in vitro assays, we assessed the impact of allogeneic hCPC on the recruitment, functions and differentiation of monocytes. We found that allogeneic hCPC at steady state or under inflammatory conditions can incite CCL-2/CCR2-dependent recruitment of circulating CD14+CD16- monocytes and fine-tune their activation toward an anti-inflammatory profile. Allogeneic hCPC also promoted CD14+CD16- monocyte polarization into anti-inflammatory/immune-regulatory macrophages with high phagocytic capacity and IL10 secretion. Moreover, hCPC bended the differentiation of CD14+CD16- monocytes to dendritic cells (DCs) toward anti-inflammatory macrophage-like features and impaired their antigen-presenting function in favor of immune-modulation. Collectively, our results demonstrate that allogeneic hCPC could reshape monocytes, macrophages as well as DCs responses by favoring their anti-inflammatory/tolerogenic activation/polarization. Thereby, therapeutic allogeneic hCPC might also contribute to post-infarct myocardial healing by modeling the activities of monocytes and their derived descendants.