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There are no definitive therapies for patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Therefore, new therapeutic strategies are needed to improve clinical outcomes, particularly in patients with severe disease. This case series explores the safety and effectiveness of intravenous allogeneic cardiosphere-derived cells (CDCs), formulated as CAP-1002, in critically ill patients with confirmed coronavirus disease 2019 (COVID-19). Adverse reactions to CAP-1002, clinical status on the World Health Organization (WHO) ordinal scale, and changes in pro-inflammatory biomarkers and leukocyte counts were analyzed. All patients (n = 6; age range 19-75 years, 1 female) required ventilatory support (invasive mechanical ventilation, n = 5) with PaO 2 /FiO 2 ranging from 69 to 198. No adverse events related to CAP-1002 administration were observed. Four patients (67%) were weaned from respiratory support and discharged from the hospital. One patient remains mechanically ventilated as of April 28th, 2020; all survive. A contemporaneous control group of critically ill COVID-19 patients (n = 34) at our institution showed 18% overall mortality at a similar stage of hospitalization. Ferritin was elevated in all patients at baseline (range of all patients 605.43-2991.52 ng/ml) and decreased in 5/6 patients (range of all patients 252.89-1029.90 ng/ml). Absolute lymphocyte counts were low in 5/6 patients at baseline (range 0.26-0.82 × 10 3 /µl) but had increased in three of these five patients at last follow-up (range 0.23-1.02 × 10 3 /µl). In this series of six critically ill COVID-19 patients, intravenous infusion of CAP-1002 was well tolerated and associated with resolution of critical illness in 4 patients. This series demonstrates the apparent safety of CAP-1002 in COVID-19. While this initial experience is promising, efficacy will need to be further assessed in a randomized controlled trial.

Single-vessel, intracoronary infusion of stem cells under stop-flow conditions has proven safe but achieves only limited myocardial coverage. Continuous flow intracoronary delivery to one or more coronary vessels may achieve broader coverage for treating cardiomyopathy, but has not been investigated. Using nonocclusive coronary guiding catheters, we infused allogeneic cardiosphere-derived cells (CDCs) either in a single vessel or sequentially in all three coronary arteries in porcine ischemic cardiomyopathy and used magnetic resonance imaging (MRI) to assess structural and physiological outcomes. Vehicle-infused animals served as controls. Single-vessel stop-flow and continuous-flow intracoronary infusion revealed equivalent effects on scar size and function. Sequential infusion into each of the three major coronary vessels under stop-flow or continuous-flow conditions revealed equal efficacy, but less elevation of necrotic biomarkers with continuous-flow delivery. In addition, multi-vessel delivery resulted in enhanced global and regional tissue function compared to a triple-vessel placebo-treated group. The functional benefits after global cell infusion were accompanied histologically by minimal inflammatory cellular infiltration, attenuated regional fibrosis and enhanced vessel density in the heart. Sequential multi-vessel non-occlusive delivery of CDCs is safe and provides enhanced preservation of left ventricular function and structure. The current findings provide preclinical validation of the delivery method currently undergoing clinical testing in the Dilated cardiomYopathy iNtervention With Allogeneic MyocardIally-regenerative Cells (DYNAMIC) trial of CDCs in heart failure patients.

Stem cell transplantation is a promising strategy for therapeutic cardiac regeneration, but current therapies are limited by inefficient interaction between potentially beneficial cells (either exogenously transplanted or endogenously recruited) and the injured tissue. Here we apply targeted nanomedicine to achieve in vivo cell-mediated tissue repair, imaging and localized enrichment without cellular transplantation. Iron nanoparticles are conjugated with two types of antibodies (one against antigens on therapeutic cells and the other directed at injured cells) to produce magnetic bifunctional cell engager (MagBICE). The antibodies link the therapeutic cells to the injured cells, whereas the iron core of MagBICE enables physical enrichment and imaging. We treat acute myocardial infarction by targeting exogenous bone marrow-derived stem cells (expressing CD45) or endogenous CD34-positive cells to injured cardiomyocytes (expressing myosin light chain. Targeting can be further enhanced by magnetic attraction, leading to augmented functional benefits. MagBICE represents a generalizable platform technology for regenerative medicine.

Over the past 2 decades, there have been numerous stem cell studies focused on cardiac diseases, ranging from proof-of-concept to phase 2 trials. This series of papers focuses on the legacy of these studies and the outlook for future treatment of cardiac diseases with stem cell therapies. The first section by Drs. Rosen and Myerburg is an independent review that analyzes the basic science and translational strategies supporting the rapid advance of stem cell technology to the clinic, the philosophies behind them, trial designs, and means for going forward that may impact favorably on progress. The second and third sections were collected as responses to the initial section of this review. The commentary by Drs. Francis and Cole discusses the review by Drs. Rosen and Myerburg and details how trial outcomes can be affected by noise, poor trial design (particularly the absence of blinding), and normal human tendencies toward optimism and denial. The final, independent paper by Dr. Marbán takes a different perspective concerning the potential for positive impact of stem cell research applied to heart disease and future prospects for its clinical application. (Compiled by the JACC editors)., (Copyright © 2014 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Controversy surrounds the identity, origin, and physiologic role of endogenous cardiomyocyte progenitors in adult mammals. Using an inducible genetic labeling approach to identify small non-myocyte cells expressing cardiac markers, we find that activated endogenous cardioblasts are rarely evident in the normal adult mouse heart. However, myocardial infarction results in significant cardioblast activation at the site of injury. Genetically labeled isolated cardioblasts express cardiac transcription factors and sarcomeric proteins, exhibit spontaneous contractions, and form mature cardiomyocytes in vivo after injection into unlabeled recipient hearts. The activated cardioblasts do not arise from hematogenous seeding, cardiomyocyte dedifferentiation, or mere expansion of a preformed progenitor pool. Cell therapy with cardiosphere-derived cells amplifies innate cardioblast-mediated tissue regeneration, in part through the secretion of stromal cell-derived factor 1 by transplanted cells. Thus, stimulation of endogenous cardioblasts by exogenous cells mediates therapeutic regeneration of injured myocardium., (© 2014 The Authors. Published under the terms of the CC BY 4.0 license.)

Objectives: This study sought to compare the regenerative potency of cells derived from healthy and diseased human hearts., Background: Results from pre-clinical studies and the CADUCEUS (CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction) trial support the notion that cardiosphere-derived cells (CDCs) from normal and recently infarcted hearts are capable of regenerating healthy heart tissue after myocardial infarction (MI). It is unknown whether CDCs derived from advanced heart failure (HF) patients retain the same regenerative potency., Methods: In a mouse model of acute MI, we compared the regenerative potential and functional benefits of CDCs derived from 3 groups: 1) non-failing (NF) donor: healthy donor hearts post-transplantation; 2) MI: patients who had an MI 9 to 35 days before biopsy; and 3) HF: advanced cardiomyopathy tissue explanted at cardiac transplantation., Results: Cell growth and phenotype were identical in all 3 groups. Injection of HF CDCs led to the greatest therapeutic benefit in mice, with the highest left ventricular ejection fraction, thickest infarct wall, most viable tissue, and least scar 3 weeks after treatment. In vitro assays revealed that HF CDCs secreted higher levels of stromal cell-derived factor (SDF)-1, which may contribute to the cells' augmented resistance to oxidative stress, enhanced angiogenesis, and improved myocyte survival. Histological analysis indicated that HF CDCs engrafted better, recruited more endogenous stem cells, and induced greater angiogenesis and cardiomyocyte cell-cycle re-entry. CDC-secreted SDF-1 levels correlated with decreases in scar mass over time in CADUCEUS patients treated with autologous CDCs., Conclusions: CDCs from advanced HF patients exhibit augmented potency in ameliorating ventricular dysfunction post-MI, possibly through SDF-1–mediated mechanisms.

Self-assembling heart-derived stem cell clusters named cardiospheres (CSps) improve function and attenuate remodeling in rodent models of acute myocardial infarction. The effects of CSps in chronically remodeled myocardium post-MI, and the underlying mechanisms, remain unknown. One month after permanent coronary ligation, rats were randomly assigned to injection of vehicle (controls) or CSps in the peri-infarct area. One month post-injection, CSps increased left ventricular function, reduced scar mass and collagen density, and enhanced vascularity within the infarct zone compared to controls. Immunoblots revealed Tgfβ-1/smad cascade downregulation and an increase in soluble endoglin post-CSp injection. Six months post-transplantation, left ventricular function further improved and cardiomyocyte hypertrophy was attenuated in the CSp-treated group. In vitro, co-culture of CSps with fibroblasts recapitulated the suppression of the Tgf-β1/smad pathway changes, responses which were blunted by neutralizing antibody against endoglin. Thus, cardiosphere transplantation enhances angiogenesis and reduces fibrosis in chronically infarcted myocardium, leading to partial reversal of cardiac dysfunction. The underlying mechanism involves inhibition of Tgf-β1/smad signaling by CSp-secreted soluble endoglin.

Cardiosphere-derived cells (CDCs) have been shown to regenerate infarcted myocardium in patients after myocardial infarction (MI). However, whether the cells of the newly formed myocardium originate from the proliferation of adult cardiomyocytes or from the differentiation of endogenous stem cells remains unknown. Using genetic fate mapping to mark resident myocytes in combination with long-term BrdU pulsing, we investigated the origins of postnatal cardiomyogenesis in the normal, infarcted and cell-treated adult mammalian heart. In the normal mouse heart, cardiomyocyte turnover occurs predominantly through proliferation of resident cardiomyocytes at a rate of ∼1.3-4%/year. After MI, new cardiomyocytes arise from both progenitors as well as pre-existing cardiomyocytes. Transplantation of CDCs upregulates host cardiomyocyte cycling and recruitment of endogenous progenitors, while boosting heart function and increasing viable myocardium. The observed phenomena cannot be explained by cardiomyocyte polyploidization, bi/multinucleation, cell fusion or DNA repair. Thus, CDCs induce myocardial regeneration by differentially upregulating two mechanisms of endogenous cell proliferation., (Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

Aims: Cardiosphere-derived cells (CDCs) are in clinical development as a regenerative cell product which can be expanded ex vivo from patient cardiac biopsies. Cardiosphere-derived cells are clonogenic, exhibit multilineage differentiation, and exert functional benefits in preclinical models of heart failure. The origin of CDCs remains unclear: are these cells endogenous to the heart, or do they arise from cells that populate the heart via blood-borne seeding?, Methods and Results: Right ventricular endomyocardial biopsies were obtained from cardiac transplant recipients (n = 10, age 57 ± 15 years), and CDCs expanded from each biopsy. Donor-recipient mismatches were used to probe the origin of CDCs in three complementary ways. First, DNA analysis of short-tandem nucleotide repeats (STRs) was performed on genomic DNA from donor and recipient, then compared with the STR pattern of CDCs. Second, in two cases where the donor was male and the recipient female, CDCs were examined for the presence of X and Y chromosomes by fluorescence in situ hybridization. Finally, in two cases, quantitative PCR (qPCR) was performed for individual-specific polymorphisms of a major histocompatability locus to quantify the contribution of recipient cells to CDCs. In no case was recipient DNA detectable in the CDCs by STR analysis. In the two cases in which a female patient had received a male heart, all CDCs examined had an X and Y chromosome, similarly indicating exclusively donor origin. Likewise, qPCR on CDCs did not detect any recipient DNA., Conclusion: Cardiosphere-derived cells are of endogenous cardiac origin, with no detectable contribution from extra-cardiac seeding.

In order for bloodborne stem cells to be effective in tissue regeneration, cells must cross vessel walls and enter the parenchyma. Although such transmigration does occur, the mechanism remains elusive. Leukocytes invade tissue by diapedesis; stem cells are commonly assumed to do likewise, but evidence is lacking. Cardiac-derived regenerative cells and multicellular cardiospheres (CSPs) were infused into the coronary vessels of rat hearts. Serial histology revealed a novel mechanism of cell transmigration, "active vascular expulsion," which underlies the extravasation of infused cells and cell aggregates. In this mechanism, the vascular barrier undergoes extensive remodeling, while the cells themselves are relatively passive. The mechanism was confirmed in vivo by serial intravital microscopy of CSP extravasation in a dorsal skin flap model. Integrins and matrix metalloproteinases play critical roles in active vascular expulsion. In vitro models revealed that active vascular expulsion is generalizable to other stem cell types and to breast cancer cells. Recognition of active vascular expulsion as a mechanism for transvascular cell migration opens new opportunities to enhance the efficacy of vascularly delivered cell therapy., (Copyright © 2012 AlphaMed Press.)

Cardiac regenerative therapies seek to grow new myocardium after "irreversible" injury such as myocardial infarction. Various cell types and delivery techniques have been used in experimental models of human disease and clinical trials. When selecting a candidate stem cell type for clinical use, multiple factors need to be taken into consideration. The ability to regenerate myocardium without potentiating arrhythmogenesis is a critical property. Skeletal myoblasts engraft, differentiate, and are arrhythmogenic; in contrast, bone marrow-derived cells do not engraft long-term and have not been associated with excess arrhythmias. Neither cell type, however, achieves true myocardial regeneration. Recognition of the existence of cardiac stem cells and of the ability of mature myocytes to reenter the cell cycle and proliferate has motivated the development of new approaches to cardiac regenerative medicine. Cardiosphere-derived cells decrease scar mass and regenerate viable myocardium both in animal models and in the CADUCEUS (Cardiosphere-Derived Cells For Heart Regeneration After Myocardial Infarction) clinical trial. Although cardiosphere-derived cells fulfill the criteria for stem cells, their stemness appears not to mediate the therapeutic benefit; instead, indirect mechanisms lead to proliferation of the host myocardium. Being of endogenous origin, the newly grown heart muscle is electrically and mechanically well integrated with preexisting myocardial tissue. We hypothesize that cardiac arrhythmias are less likely to complicate cell therapy when the mechanisms of benefit involve secondary proliferation of endogenous myocardium. Conversely, arrhythmias will more likely bedevil therapeutic approaches (such as transplantation of skeletal myoblasts or pluripotent stem cells) that lead to exogenous grafts within the heart, with the degree of coupling and the extent of inhomogeneity being critical determinants of the net effect., (Copyright © 2012 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Stem cell-based methods for myocardial regeneration suffer from considerable cell attrition. Artificial matrices reproducing mechanical and structural properties of the native tissue may facilitate survival, retention and functional integration of adult stem or progenitor cells, by conditioning the cells prior to, and during, transplantation. Here we combined autologous cardiosphere-derived cells (CDCs) with nanotopographically defined hydrogels mimicking the native myocardial matrix, to form in vitro cardiac stem cell niches, and control cell function and fate. These platforms were used to produce cardiac patches that could be transplanted at the site of infarct. In culture, highly anisotropic, but not more randomized nanotopographic, control augmented cell adhesion, migration, and proliferation. It also dramatically enhanced early, and, in the presence of mature cardiomyocytes, late cardiomyogenesis. Nanotopography sensing and transcriptional response was mediated via p190RhoGAP. In a rat infarction model, engraftment of nanofabricated scaffolds with CDCs enhanced retention and growth of transplanted cells, and their integration with the host tissue. The infarcted ventricle wall increased in thickness, with higher cell viability and better collagen organization. These results suggest that nanostructured polymeric materials that closely mimic the extracellular matrix structure on which cardiac cells reside in vivo can be both very effective tools in investigating the mechanisms of cardiac differentiation and the basis for cardiac tissue engineering, thus facilitating stem cell-based therapy in the heart.

Trastuzumab (TZM), a monoclonal antibody against the ERBB2 protein, increases survival in ERBB2-positive breast cancer patients. Its clinical use, however, is limited by cardiotoxicity. We sought to evaluate whether TZM cardiotoxicity involves inhibition of human adult cardiac-derived stem cells, in addition to previously reported direct adverse effects on cardiomyocytes. To test this idea, we exposed human cardiosphere-derived cells (hCDCs), a natural mixture of cardiac stem cells and supporting cells that has been shown to exert potent regenerative effects, to TZM and tested the effects in vitro and in vivo. We found that ERBB2 mRNA and protein are expressed in hCDCs at levels comparable to those in human myocardium. Although clinically relevant concentrations of TZM had no effect on proliferation, apoptosis, or size of the c-kit-positive hCDC subpopulation, in vitro assays demonstrated diminished potential for cardiogenic differentiation and impaired ability to form microvascular networks in TZM-treated cells. The functional benefit of hCDCs injected into the border zone of acutely infarcted mouse hearts was abrogated by TZM: infarcted animals treated with TZM + hCDCs had a lower ejection fraction, thinner infarct scar, and reduced capillary density in the infarct border zone compared with animals that received hCDCs alone (n = 12 per group). Collectively, these results indicate that TZM inhibits the cardiomyogenic and angiogenic capacities of hCDCs in vitro and abrogates the morphological and functional benefits of hCDC transplantation in vivo. Thus, TZM impairs the function of human resident cardiac stem cells, potentially contributing to TZM cardiotoxicity.

The efficiency of stem cell transplantation is limited by low cell retention. Intracoronary (IC) delivery is convenient and widely used but exhibits particularly low cell retention rates. We sought to improve IC cell retention by magnetic targeting. Rat cardiosphere-derived cells labeled with iron microspheres were injected into the left ventricular cavity of syngeneic rats during brief aortic clamping. Placement of a 1.3 Tesla magnet ~1 cm above the heart during and after cell injection enhanced cell retention at 24 h by 5.2-6.4-fold when 1, 3, or 5 × 10(5) cells were infused, without elevation of serum troponin I (sTnI) levels. Higher cell doses (1 or 2 × 10(6) cells) did raise sTnI levels, due to microvascular obstruction; in this range, magnetic enhancement did not improve cell retention. To assess efficacy, 5 × 10(5) iron-labeled, GFP-expressing cells were infused into rat hearts after 45 min ischemia/20 min reperfusion of the left anterior coronary artery, with and without a superimposed magnet. By quantitative PCR and optical imaging, magnetic targeting increased cardiac retention of transplanted cells at 24 h, and decreased migration into the lungs. The enhanced cell engraftment persisted for at least 3 weeks, at which time left ventricular remodeling was attenuated, and therapeutic benefit (ejection fraction) was higher, in the magnetic targeting group. Histology revealed more GFP(+) cardiomyocytes, Ki67(+) cardiomyocytes and GFP(-)/ckit(+) cells, and fewer TUNEL(+) cells, in hearts from the magnetic targeting group. In a rat model of ischemia/reperfusion injury, magnetically enhanced intracoronary cell delivery is safe and improves cell therapy outcomes.

Cardiac stem cells (CSCs) are promising candidates for use in myocardial regenerative therapy. We test the hypothesis that growing cardiac-derived cells as three-dimensional cardiospheres may recapitulate a stem cell niche-like microenvironment, favoring cell survival and enhancing functional benefit after transplantation into the injured heart. CSCs and supporting cells from human endomyocardial biopsies were grown as cardiospheres and compared with cells cultured under traditional monolayer condition or dissociated from cardiospheres. Cardiospheres self-assembled into stem cell niche-like structures in vitro in suspension culture, while exhibiting greater proportions of c-kit(+) cells and upregulated expression of SOX2 and Nanog. Pathway-focused polymerase chain reaction (PCR) array, quantitative real-time PCR, and immunostaining revealed enhanced expression of stem cell-relevant factors and adhesion/extracellular-matrix molecules (ECM) in cardiospheres including IGF-1, histone deacetylase 2 (HDAC2), Tert, integrin-α(2), laminin-β(1), and matrix metalloproteinases (MMPs). Implantation of cardiospheres in severe combined immunodeficiency (SCID) mouse hearts with acute infarction disproportionately improved cell engraftment and myocardial function, relative to monolayer-cultured cells. Dissociation of cardiospheres into single cells decreased the expression of ECM and adhesion molecules and undermined resistance to oxidative stress, negating the improved cell engraftment and functional benefit in vivo. Growth of cardiac-derived cells as cardiospheres mimics stem cell niche properties with enhanced "stemness" and expression of ECM and adhesion molecules. These changes underlie an increase in cell survival and more potent augmentation of global function following implantation into the infarcted heart., (Copyright © 2010 AlphaMed Press.)

The adult heart contains reservoirs of progenitor cells that express embryonic and stem cell-related antigens. While these antigenically-purified cells are promising candidates for autologous cell therapy, clinical application is hampered by their limited abundance and tedious isolation methods. Methods that involve an intermediate cardiosphere-forming step have proven successful and are being tested clinically, but it is unclear whether the cardiosphere step is necessary. Accordingly, we investigated the molecular profile and functional benefit of cells that spontaneously emigrate from cardiac tissue in primary culture. Adult Wistar-Kyoto rat hearts were minced, digested and cultured as separate anatomical regions. Loosely-adherent cells that surround the plated tissue were harvested weekly for a total of five harvests. Genetic lineage tracing demonstrated that a small proportion of the direct outgrowth from cardiac samples originates from myocardial cells. This outgrowth contains sub-populations of cells expressing embryonic (SSEA-1) and stem cell-related antigens (c-Kit, abcg2) that varied with time in culture but not with the cardiac chamber of origin. This direct outgrowth, and its expanded progeny, underwent marked in vitro angiogenic/cardiogenic differentiation and cytokine secretion (IGF-1, VGEF). In vivo effects included long-term functional benefits as gauged by MRI following cell injection in a rat model of myocardial infarction. Outgrowth cells afforded equivalent functional benefits to cardiosphere-derived cells, which require more processing steps to manufacture. These results provide the basis for a simplified and efficient process to generate autologous cardiac progenitor cells (and mesenchymal supporting cells) to augment clinically-relevant approaches for myocardial repair.

Stem cell cytogenetic abnormalities constitute a roadblock to regenerative therapies. We investigated the possibility that reactive oxygen species (ROSs) influence genomic stability in cardiac and embryonic stem cells. Karyotypic abnormalities in primary human cardiac stem cells were suppressed by culture in physiological (5%) oxygen, but addition of antioxidants to the medium unexpectedly increased aneuploidy. Intracellular ROS levels were moderately decreased in physiological oxygen, but dramatically decreased by the addition of high-dose antioxidants. Quantification of DNA damage in cardiac stem cells and in human embryonic stem cells revealed a biphasic dose-dependence: antioxidants suppressed DNA damage at low concentrations, but potentiated such damage at higher concentrations. High-dose antioxidants decreased cellular levels of ATM (ataxia-telangiectasia mutated) and other DNA repair enzymes, providing a potential mechanistic basis for the observed effects. These results indicate that physiological levels of intracellular ROS are required to activate the DNA repair pathway for maintaining genomic stability in stem cells. The concept of an "oxidative optimum" for genomic stability has broad implications for stem cell biology and carcinogenesis.

In the heart, the proteomes secreted by both cardiac stem cells (CSCs) and cardiac myocytes could act synergistically, but the identification and functionality of the proteins comprising the individual secretomes have not yet been described. In this study, we have identified proteins present in the media obtained from cultured rat CSCs and from cultured neonatal rat ventricular myocytes (NRVMs) and compared them with proteins identified in the media alone. Briefly, 83 unique proteins were identified after analysis by RPLC and MS. In total 49 and 23% were NRVM-specific or CSC-specific proteins, respectively, and 63% of total 83 proteins were integral plasma membrane and/or known secreted proteins. Fifteen proteins met our criteria for paracrine/autocrine factors: (i) robust protein identification, (ii) cell specific and (iii) known to be secreted. Most of these proteins have not been previously linked to stem cells. NRVM-specific proteins atrial natriuretic factor (ANP) and connective tissue growth factor, and CSC-specific protein interleukin-1 receptor-like 1 (ST2) were found to affect rat CSC proliferation. These findings suggest that relative concentration of each protein may be crucial for cellular intertalk and for the final outcome of cardiac cell therapy.

Background: At least four laboratories have shown that endogenous cardiac progenitor cells (CPCs) can be grown directly from adult heart tissue in primary culture, as cardiospheres or their progeny (cardiosphere-derived cells, CDCs). Indeed, CDCs are already being tested in a clinical trial for cardiac regeneration. Nevertheless, the validity of the cardiosphere strategy to generate CPCs has been called into question by reports based on variant methods. In those reports, cardiospheres are argued to be cardiomyogenic only because of retained cardiomyocytes, and stem cell activity has been proposed to reflect hematological contamination. We use a variety of approaches (including genetic lineage tracing) to show that neither artifact is applicable to cardiospheres and CDCs grown using established methods, and we further document the stem cell characteristics (namely, clonogenicity and multilineage potential) of CDCs., Methodology/principal Findings: CPCs were expanded from human endomyocardial biopsies (n = 160), adult bi-transgenic MerCreMer-Z/EG mice (n = 6), adult C57BL/6 mice (n = 18), adult GFP(+) C57BL/6 transgenic mice (n = 3), Yucatan mini pigs (n = 67), adult SCID beige mice (n = 8), and adult Wistar-Kyoto rats (n = 80). Cellular yield was enhanced by collagenase digestion and process standardization; yield was reduced in altered media and in specific animal strains. Heparinization/retrograde organ perfusion did not alter the ability to generate outgrowth from myocardial sample. The initial outgrowth from myocardial samples was enriched for sub-populations of CPCs (c-Kit(+)), endothelial cells (CD31(+), CD34(+)), and mesenchymal cells (CD90(+)). Lineage tracing using MerCreMer-Z/EG transgenic mice revealed that the presence of cardiomyocytes in the cellular outgrowth is not required for the generation of CPCs. Rat CDCs are shown to be clonogenic, and cloned CDCs exhibit spontaneous multineage potential., Conclusions/significance: This study demonstrates that direct culture and expansion of CPCs from myocardial tissue is simple, straightforward, and reproducible when appropriate techniques are used.

New approaches for cardiac repair have been enabled by the discovery that the heart contains its own reservoir of stem cells. These cells are positive for various stem/progenitor cell markers, are self-renewing, and exhibit multilineage differentiation potential. Recently we developed a method for ex vivo expansion of cardiac-derived stem cells from human myocardial biopsies with a view to subsequent autologous transplantation for myocardial regeneration. Here we review the state of the cardiac stem cell field and our own work on cardiosphere-derived stem cells from human hearts. The first of this two-part review outlines emerging preclinical data on the application of cardiac stem cells. Part 2 continues with a discussion of other stem cell sources with clinical potential, a summary of the critical issues surrounding stem cell therapy (with an emphasis on the crucial issue of how cell transplantation may influence arrhythmias), our perception of clinical stem cell trials to date, and the issues facing the clinical application of cardiac stem cells.

Cardiosphere-derived resident cardiac stem cells (CDCs) are readily isolated from adult hearts and confer functional benefit in animal models of heart failure. To study cardiogenic differentiation in CDCs, we developed a method to genetically label and selectively enrich for cells that have acquired a cardiac phenotype. Lentiviral vectors achieved significantly higher transduction efficiencies in CDCs than any of the nine adeno-associated viral (AAV) serotypes tested. To define the most suitable vector system for reporting cardiogenic differentiation, we compared the cell specificity of five commonly-used cardiac-specific promoters in the context of lentiviral vectors. The promoter of the cardiac sodium-calcium exchanger (NCX1) conveyed the highest degree of cardiac specificity, as assessed by transducing seven cell types with each vector and measuring fluorescence intensity by flow cytometry. NCX1-GFP-positive CDC subpopulations, demonstrating prolonged expression of a variety of cardiac markers, could be isolated and expanded in vitro. Finally, we used chemical biology to validate that lentiviral vectors bearing the cardiac NCX1-promoter can serve as a highly accurate biosensor of cardiogenic small molecules in stem cells. The ability to accurately report cardiac fate and selectively enrich for cardiomyocytes and their precursors has important implications for drug discovery and the development of cell-based therapies.

In the past few years it has been established that the heart contains a reservoir of stem and progenitor cells. These cells are positive for various stem/progenitor cell markers (Kit, Sca-1, Isl-1, and Side Population (SP) properties). The relationship between the various cardiac stem cells (CSC) and progenitor cells described awaits clarification. Furthermore, they may open a new therapeutic strategies of cardiac repair based on the regeneration potential of cardiac stem cells. Currently, cellular cardiomyoplasty is actively explored as means of regenerating damaged myocardium using several different cell types. CSCs seem a logical cell source to exploit for cardiac regeneration therapy. Their presence into the heart, the frequent co-expression of early cardiac progenitor transcription factors, and the capability for ex vivo and in vivo differentiation toward the cardiac lineages offer promise of enhanced cardiogenicity compared to other cell sources. CSCs, when isolated from various animal models by selection based on c-Kit, Sca-1, and/or MDR1, have shown cardiac regeneration potential in vivo following injection in the infracted myocardium. Recently, we have successfully isolated CSCs from small biopsies of human myocardium and expanded them ex vivo by many folds without losing differentiation potential into cardiomyocytes and vascular cells, bringing autologous transplantation of CSCs closer to clinical evaluation. These cells are spontaneously shed from human surgical specimens and murine heart samples in primary culture. This heterogeneous population of cells forms multi-cellular clusters, dubbed cardiospheres (CSs), in suspension culture. CSs are composed of clonally-derived cells, consist of proliferating c-Kit positive cells primarily in their core and differentiating cells expressing cardiac and endothelial cell markers on their periphery. Although the intracardiac origin of adult myocytes has been unequivocally documented, the potential of an extracardiac source of cells, able to repopulate the lost CSCs in pathological conditions (infarct) cannot be excluded and will be discussed in this review. The delivery of human CSs or of CSs-derived cells into the injured heart of the SCID mouse resulted in engraftment, migration, myocardial regeneration and improvement of left ventricular function. Our method for ex vivo expansion of resident CSCs for subsequent autologous transplantation back into the heart, may give these cell populations, the resident and the transplanted one, the combined ability to mediate myocardial regeneration to an appreciable degree, and may change the way in which cardiovascular disease will be approached in the future.

Cardiac differentiation of embryonic stem cells (ESCs) can give rise to de novo chamber cardiomyocytes and nodal pacemaker cells. Compared with our understanding of direct differentiation toward atrial and ventricular myocytes, the mechanisms for nodal pacemaker cell commitment are not well understood. Taking a cue from the prominence of canonical Wnt signaling during cardiac pacemaker tissue development in chick embryos, we asked if modulations of Wnt signaling influence cardiac progenitors to bifurcate to either chamber cardiomyocytes or pacemaker cells. Omitting an exogenous Wnt inhibitor, which is routinely added to maximize cardiac myocyte yield during differentiation of mouse and human ESCs, led to increased yield of spontaneously beating cardiomyocytes with action potential properties similar to those of native sinoatrial node pacemaker cells. The pacemaker phenotype was accompanied by enhanced expression of genes and gene products that mark nodal pacemaker cells such as Hcn4, Tbx18, Tbx3, and Shox2. Addition of exogenous Wnt3a ligand, which activates canonical Wnt/β-catenin signaling, increased the yield of pacemaker-like myocytes while reducing cTNT-positive pan-cardiac differentiation. Conversely, addition of inhibitors of Wnt/β-catenin signaling led to increased chamber myocyte lineage development at the expense of pacemaker cell specification. The positive impact of canonical Wnt signaling on nodal pacemaker cell differentiation was evidenced in direct differentiation of two human ESC lines and human induced pluripotent stem cells. Our data identify the Wnt/β-catenin pathway as a critical determinant of cardiac myocyte subtype commitment during ESC differentiation: endogenous Wnt signaling favors the pacemaker lineage, whereas its suppression promotes the chamber cardiomyocyte lineage.

Sudden death is the most common mode of exodus in patients with heart failure and preserved ejection fraction (HFpEF). Cardiosphere-derived cells (CDCs) reduce inflammation and fibrosis in a rat model of HFpEF, improving diastolic function and prolonging survival. We tested the hypothesis that CDCs decrease ventricular arrhythmias (VAs) and thereby possibly contribute to prolonged survival. Dahl salt-sensitive rats were fed a high-salt diet to induce HFpEF. Allogeneic rat CDCs (or phosphate-buffered saline as placebo) were injected in rats with echo-verified HFpEF. CDC-injected HFpEF rats were less prone to VA induction by programmed electrical stimulation. Action potential duration (APD) was shortened, and APD homogeneity was increased by CDC injection. Transient outward potassium current density was upregulated in cardiomyocytes from CDC rats relative to placebo, as were the underlying transcript (Kcnd3) and protein (Kv4.3) levels. Fibrosis was attenuated in CDC-treated hearts, and survival was increased. Sudden death risk also trended down, albeit nonsignificantly. CDC therapy decreased VA in HFpEF rats by shortening APD, improving APD homogeneity, and decreasing fibrosis. Unlike other stem/progenitor cells, which often exacerbate arrhythmias, CDCs reverse electrical remodeling and suppress arrhythmogenesis in HFpEF.

Pulmonary arterial hypertension (PAH) is a progressive condition characterized by occlusive pulmonary arteriopathy, in which survival remains poor despite pharmacologic advances. The aim of this study was to evaluate the ability of cardiosphere-derived cells (CDCs), cardiac progenitor cells with potent anti-inflammatory and immunomodulatory properties, to attenuate hemodynamic and morphometric remodeling of the right ventricle (RV) and pulmonary arterioles in rats with established monocrotaline (MCT)-induced PAH. Animals were divided into 3 groups: 1) Control (CTL), 2) PAH in which CDCs were centrally infused (CDC) and 3) PAH in which saline was given (Sham). Significant increments in RV systolic pressure (RVSP) and RV hypertrophy were noted in Sham animals compared to CTL. In CDC rats at day 35, RSVP fell (- 38%; p< 0.001) and RV hypertrophy decreased (-26%; p< 0.01). TAPSE and cardiac output were preserved in all 3 groups at day 35. Pulmonary arteriolar wall thickness was greater in Sham rats compared to CTL, and reduced in CDC animals for vessels 20–50 μm (P<0.01; back to CTL levels) and 50–80μm (P<0.01) in diameter. The macrophage population was increased in Sham animals compared to CTL (P< 0.001), but markedly reduced in CDC rats. In conclusion, infusion of CDCs markedly attenuated several key pathophysiologic features of PAH. As adjunctive therapy to PAH-specific agents, CDCs have the potential to impact on the pathobiology of adverse pulmonary arteriolar remodeling, by acting on multiple mechanisms simultaneously. [ABSTRACT FROM AUTHOR]

Cardiosphere-derived cells ( CDCs) reduce myocardial infarct size via secreted extracellular vesicles ( CDC-EVs), including exosomes, which alter macrophage polarization. We questioned whether short non-coding RNA species of unknown function within CDC-EVs contribute to cardioprotection. The most abundant RNA species in CDC-EVs is a Y RNA fragment (EV- YF1); its relative abundance in CDC-EVs correlates with CDC potency in vivo. Fluorescently labeled EV- YF1 is actively transferred from CDCs to target macrophages via CDC-EVs. Direct transfection of macrophages with EV- YF1 induced transcription and secretion of IL-10. When cocultured with rat cardiomyocytes, EV- YF1-primed macrophages were potently cytoprotective toward oxidatively stressed cardiomyocytes through induction of IL-10. In vivo, intracoronary injection of EV- YF1 following ischemia/reperfusion reduced infarct size. A fragment of Y RNA, highly enriched in CDC-EVs, alters Il10 gene expression and enhances IL-10 protein secretion. The demonstration that EV- YF1 confers cardioprotection highlights the potential importance of diverse exosomal contents of unknown function, above and beyond the usual suspects (e.g., micro RNAs and proteins). [ABSTRACT FROM AUTHOR]

Exosomes are nanosized membrane particles that are secreted by cells that transmit information from cell to cell. The information within exosomes prominently includes their protein and RNA payloads. Exosomal microRNAs in particular can potently and fundamentally alter the transcriptome of recipient cells. Here we summarize what is known about exosome biogenesis, content, and transmission, with a focus on cardiovascular physiology and pathophysiology. We also highlight some of the questions currently under active investigation regarding these extracellular membrane vesicles and their potential in diagnostic and therapeutic applications. [ABSTRACT FROM AUTHOR]

Background: Epicardial injection of heart-derived cell products is safe and effective post-myocardial infarction (MI), but clinically-translatable transendocardial injection has never been evaluated. We sought to assess the feasibility, safety and efficacy of percutaneous transendocardial injection of heart-derived cells in porcine chronic ischemic cardiomyopathy. Methods and Results: We studied a total of 89 minipigs; 63 completed the specified protocols. After NOGA-guided transendocardial injection, we quantified engraftment of escalating doses of allogeneic cardiospheres or cardiosphere-derived cells in minipigs (n = 22) post-MI. Next, a dose-ranging, blinded, randomized, placebo-controlled (“dose optimization”) study of transendocardial injection of the better-engrafting product was performed in infarcted minipigs (n = 16). Finally, the superior product and dose (150 million cardiospheres) were tested in a blinded, randomized, placebo-controlled (“pivotal”) study (n = 22). Contrast-enhanced cardiac MRI revealed that all cardiosphere doses preserved systolic function and attenuated remodeling. The maximum feasible dose (150 million cells) was most effective in reducing scar size, increasing viable myocardium and improving ejection fraction. In the pivotal study, eight weeks post-injection, histopathology demonstrated no excess inflammation, and no myocyte hypertrophy, in treated minipigs versus controls. No alloreactive donor-specific antibodies developed over time. MRI showed reduced scar size, increased viable mass, and attenuation of cardiac dilatation with no effect on ejection fraction in the treated group compared to placebo. Conclusions: Dose-optimized injection of allogeneic cardiospheres is safe, decreases scar size, increases viable myocardium, and attenuates cardiac dilatation in porcine chronic ischemic cardiomyopathy. The decreases in scar size, mirrored by increases in viable myocardium, are consistent with therapeutic regeneration. [ABSTRACT FROM AUTHOR]

Objective: We sought to understand the cellular and tissue-level changes underlying the attenuation of adverse remodeling by cardiosphere transplantation in acute myocardial infarction (MI). Background: Cardiospheres (CSps) are heart-derived multicellular clusters rich in stemness and capable of multilineage differentiation. Post-MI CSp transplantation improves left ventricular (LV) function and attenuates remodeling in both small and large animal studies. However, the mechanisms of benefit have not yet been fully elucidated. Methods: Four groups were studied: 1) “Sham” (Wistar Kyoto rats with thoracotomy and ligature without infarction); 2) “MI” (proximal LAD ligation with peri-infarct injection of vehicle); 3) “MI+CSp” (MI with cardiospheres injected in the peri-infarct area); 4) “Small MI” (mid-LAD ligation only). Results: In vivo 1 week after CSp transplantation, LV functional improvement was associated with an increase in cardiomyocyte proliferation. By 3 weeks, microvessel formation was enhanced, while cardiomyocyte hypertrophy and regional fibrosis were attenuated. Collagen deposition was reduced, collagen degradation was enhanced, and MMPs were upregulated. The beneficial effects of CSp transplantation were not observed in the Small MI group, indicating that the effects are not solely due to CSp-induced cardioprotection. In vitro, CSp-conditioned media reduced collagen production in coculture with fibroblasts and triggered neoangiogenesis in an ex vivo aortic ring assay. Conclusion: Cardiospheres enhance cardiomyocyte proliferation and angiogenesis, and attenuate hypertrophy and fibrosis, in the ischemic myocardium. These synergistic effects underlie the attenuation of adverse remodeling by cardiospheres. [ABSTRACT FROM AUTHOR]

Despite mounting pre-clinical and clinical evidence of the beneficial effects of cell-based therapy, optimal cell dosing and delivery approaches have not been identified. Cardiospheres are self-assembling three-dimensional (3 D) microtissues formed by cardiac stem cells and supporting cell types. The ability of cardiospheres to augment cardiac function has been demonstrated in animal models of ischemic cardiomyopathy. In this study, we studied the dose dependence of the benefits of human cardiospheres, delivered via intramyocardial injection, upon cardiac function and ventricular remodelling in SCID mice with acute myocardial infarction. Four doses of cardiospheres were used: 1 × 104, 5 × 104, 1 × 105 and 5 × 105 (expressed as number of plated cardiosphere-forming cells). Acute (24 hr) cell retention rates in all groups were similar. Functional assessment and quantitative heart morphometry indicated benefit from higher cell doses (≥5 × 104) in terms of ejection fraction, infarct size and capillary density. Histological analysis indicated that the dose-dependent benefit was primarily because of indirect effects of transplanted cells. The results provide scalable data on cardiosphere dosing for intramyocardial injection. [ABSTRACT FROM AUTHOR]

Introduction Stem cell therapy has emerged as a promising strategy for the treatment of ischemic cardiomyopathy. Sources of data Multiple candidate cell types have been used in preclinical animal models and in clinical trials to repair or regenerate the injured heart either directly (through formation of new transplanted tissue) or indirectly (through paracrine effects activating endogenous regeneration). Areas of agreement (i) Clinical trials examining the safety and efficacy of bone marrow derived cells in patients with heart disease are promising, but results leave much room for improvement. (ii) The safety profile has been quite favorable. (iii) Efficacy has been inconsistent and, overall, modest. (iv) Tissue retention of cells after delivery into the heart is disappointingly low. (v) The beneficial effects of adult stem cell therapy are predominantly mediated by indirect paracrine mechanisms. Areas of controversy The cardiogenic potential of bone marrow-derived cells, the mechanism whereby small numbers of poorly-retained cells translate to measurable clinical benefit, and the overall impact on clinical outcomes are hotly debated. Growing points/areas timely for developing research This overview of the field leaves us with cautious optimism, while motivating a search for more effective delivery methods, better strategies to boost cell engraftment, more apt patient populations, safe and effective ‘off the shelf’ cell products and more potent cell types. [ABSTRACT FROM AUTHOR]

Aims The ex vivo expansion of cardiac stem cells from minimally invasive human heart biopsies yields tens of millions of cells within 3–4 weeks, but chromosomal abnormalities were frequently detected in preliminary production runs. Here we attempt to avoid aneuploidy and improve cell quality by expanding human cardiac stem cells in physiological low-oxygen (5% O2) conditions, rather than in traditional culture in a general CO2 incubator (20% O2). Methods and results Human heart biopsies (n = 16) were divided and processed in parallel to expand cardiac stem cells under 5% or 20% O2. Compared with 20% O2, 5% O2 culture doubled the cell production and markedly diminished the frequency of aneuploidy. Cells expanded in 5% O2 showed lower intracellular levels of reactive oxygen species, less cell senescence, and higher resistance to oxidative stress than those grown in 20% O2, although the expression of stem cell antigens and adhesion molecules was comparable between groups, as was the paracrine secretion of growth factors into conditioned media. In vivo, the implantation of 5% O2 cells into infarcted hearts of mice resulted in greater cell engraftment and better functional recovery than with conventionally cultured cells. Conclusion The expansion of human adult cardiac stem cells in low oxygen increased cell yield, and the resulting cells were superior by various key in vitro and in vivo metrics of cell quality. Physiological oxygen tensions in culture facilitate the ex vivo expansion of healthy, biologically potent stem cells. [ABSTRACT FROM AUTHOR]

Stem cell cytogenetic abnormalities constitute a roadblock to regenerative therapies. We investigated the possibility that reactive oxygen species (ROSs) influence genomic stability in cardiac and embryonic stem cells. Karyotypic abnormalities in primary human cardiac stem cells were suppressed by culture in physiological (5%) oxygen, but addition of antioxidants to the medium unexpectedly increased aneuploidy. Intracellular ROS levels were moderately decreased in physiological oxygen, but dramatically decreased by the addition of high-dose antioxidants. Quantification of DNA damage in cardiac stem cells and in human embryonic stem cells revealed a biphasic dose-dependence: antioxidants suppressed DNA damage at low concentrations, but potentiated such damage at higher concentrations. High-dose antioxidants decreased cellular levels of ATM (ataxia-telangiectasia mutated) and other DNA repair enzymes, providing a potential mechanistic basis for the observed effects. These results indicate that physiological levels of intracellular ROS are required to activate the DNA repair pathway for maintaining genomic stability in stem cells. The concept of an “oxidative optimum” for genomic stability has broad implications for stem cell biology and carcinogenesis. STEM CELLS 2010;28:1178–1185 [ABSTRACT FROM AUTHOR]

The article presents a study on the use of cardiosphere-derived cells (CDCs) labeled with superparamagnetic microspheres (SPMs) for retention of transplanted cells in the heart. It is reported that cells were attracted towards the magnet and accumulated around the ischemic zone with magnetic targeting. It concludes that cell retention, engraftment and functional benefit are enhanced with magnetic targeting.

The article reviews the study which examines the myocardial regeneration of stem and progenitor cell transplantation into the heart. Accordingly, engraftment and low cell retention would limit the successful application of cell therapy. However, engraftment can be improved by using accurate methods of tracking cells and focusing to quantify cell survival.

Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) is one of the most sensitive, economical and widely used methods for evaluating gene expression. However, the utility of this method continues to be undermined by a number of challenges including normalization using appropriate reference genes. The need to develop tailored and effective strategies is further underscored by the burgeoning field of extracellular vesicle (EV) biology. EVs contain unique signatures of small RNAs including microRNAs (miRs). In this study we develop and validate a comprehensive strategy for identifying highly stable reference genes in a therapeutically relevant cell type, cardiosphere-derived cells. Data were analysed using the four major approaches for reference gene evaluation: NormFinder, GeNorm, BestKeeper and the Delta Ct method. The weighted geometric mean of all of these methods was obtained for the final ranking. Analysis of RNA sequencing identified miR-101-3p, miR-23a-3p and a previously identified EV reference gene, miR-26a-5p. Analysis of a chip-based method (NanoString) identified miR-23a, miR-217 and miR-379 as stable candidates. RT-qPCR validation revealed that the mean of miR-23a-3p, miR-101-3p and miR-26a-5p was the most stable normalization strategy. Here, we demonstrate that a comprehensive approach of a diverse data set of conditions using multiple algorithms reliably identifies stable reference genes which will increase the utility of gene expression evaluation of therapeutically relevant EVs. [ABSTRACT FROM PUBLISHER]

Background A single dose of allogeneic cardiosphere-derived cells (CDCs) improves cardiac function and reduces scarring, and increases viable myocardium in the infarcted rat and pig heart without eliciting a detrimental immune response. Clinical trials using single doses of allogeneic human CDCs are underway. It is unknown whether repeat dosing confers additional benefit or if it elicits an immune response. Methods Wistar–Kyoto rats underwent coronary artery ligation and intramyocardial injection of CDCs, with a second thoracotomy and repeat CDC injection 3 weeks later. Treatment permutations included 2 doses of allogeneic Brown–Norway CDCs ( n = 24), syngeneic Wistar–Kyoto CDCs ( n = 24), xenogeneic human CDCs ( n = 24) or saline ( n = 8). Cardiac function was assessed by transthoracic echocardiography, infarct size and inflammatory infiltration by histology, and cellular and humoral immune responses by lymphocyte proliferation and alloantibody assays. Results Repeat dosing of allogeneic and syngeneic CDCs improved ejection fraction by 5.2% (95% CI 2.1 to 8.3) and 6.8% (95% CI 3.8 to 9.8) after the first dose, and by 3.4% (95% CI 0.1% to 6.8%) and 6.4% (95% CI 4.2% to 8.6%) after the second dose. Infarct size was equally reduced with repeat dosing of syngeneic and allogeneic CDCs relative to xenogeneic and control treatments ( p < 0.0001). Significant rejection-like infiltrates were present only in the xenogeneic group; likewise, lymphocyte proliferation and antibody assays were positive in the xenogeneic and negative in syngeneic and allogeneic groups. Conclusions Repeat dosing of allogeneic CDCs in immunocompetent rats is safe and effective, consistent with the known immunomodulatory and anti-inflammatory properties of CDCs. These findings motivate clinical testing of repeatedly dosed CDCs for chronic heart disease. [ABSTRACT FROM AUTHOR]

Objectives: This study sought to report full 1-year results, detailed magnetic resonance imaging analysis, and determinants of efficacy in the prospective, randomized, controlled CADUCEUS (CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction) trial. Background: Cardiosphere-derived cells (CDCs) exerted regenerative effects at 6 months in the CADUCEUS trial. Complete results at the final 1-year endpoint are unknown. Methods: Autologous CDCs (12.5 to 25 × 106) grown from endomyocardial biopsy specimens were infused via the intracoronary route in 17 patients with left ventricular dysfunction 1.5 to 3 months after myocardial infarction (MI) (plus 1 infused off-protocol 14 months post-MI). Eight patients were followed as routine-care control patients. Results: In 13.4 months of follow-up, safety endpoints were equivalent between groups. At 1 year, magnetic resonance imaging revealed that CDC-treated patients had smaller scar size compared with control patients. Scar mass decreased and viable mass increased in CDC-treated patients but not in control patients. The single patient infused 14 months post-MI responded similarly. CDC therapy led to improved regional function of infarcted segments compared with control patients. Scar shrinkage correlated with an increase in viability and with improvement in regional function. Scar reduction correlated with baseline scar size but not with a history of temporally remote MI or time from MI to infusion. The changes in left ventricular ejection fraction in CDC-treated subjects were consistent with the natural relationship between scar size and ejection fraction post-MI. Conclusions: Intracoronary administration of autologous CDCs did not raise significant safety concerns. Preliminary indications of bioactivity include decreased scar size, increased viable myocardium, and improved regional function of infarcted myocardium at 1 year post-treatment. These results, which are consistent with therapeutic regeneration, merit further investigation in future trials. (CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction [CADUCEUS]; NCT00893360) [ABSTRACT FROM AUTHOR]

Background--Cardiosphere-derived cells (CDCs) are an attractive cell type for tissue regeneration, and autologous CDCs are being tested clinically. However, autologous therapy necessitates patient-specific tissue harvesting and cell processing, with delays to therapy and possible variations in cell potency. The use of allogeneic CDCs, if safe and effective, would obviate such limitations. We compared syngeneic and allogeneic CDC transplantation in rats from immunologically-mismatched inbred strains. Methods and Results--In vitro, CDCs expressed major histocompatibility complex class I but not class II antigens or B7 costimulatory molecules. In mixed-lymphocyte cocultures, allogeneic CDCs elicited negligible lymphocyte proliferation and inflammatory cytokine secretion. In vivo, syngeneic and allogeneic CDCs survived at similar levels in the infarcted rat heart 1 week after delivery, but few syngeneic (and even fewer allogeneic) CDCs remained at 3 weeks. Allogeneic CDCs induced a transient, mild, local immune reaction in the heart, without histologically evident rejection or systemic immunogenicity. Improvements in cardiac structure and function, sustained for 6 months, were comparable with syngeneic and allogeneic CDCs. Allogeneic CDCs stimulated endogenous regenerative mechanisms (cardiomyocyte cycling, recruitment of c-kit+ cells, angiogenesis) and increased myocardial vascular endothelial growth factor, insulin-like growth factor-l, and hepatocyte growth factor equally with syngeneic CDCs. Conclusions--Allogeneic CDC transplantation without immunosuppression is safe, promotes cardiac regeneration, and improves heart function in a rat myocardial infarction model, mainly through stimulation of endogenous repair mechanisms. The indirect mechanism of action rationalizes the persistence of benefit despite the evanescence of transplanted cell survival. This work motivates the testing of aliogeneic human CDCs as a potential off-the-shelf product for cellular cardiomyoplasty. [ABSTRACT FROM AUTHOR]

Objectives: The purpose of this study was to test the safety and efficacy of direct injection of cardiosphere-derived cells (CDCs) and their 3-dimensional precursors, cardiospheres, for cellular cardiomyoplasty in a mini-pig model of heart failure after myocardial infarction. Background: Intracoronary administration of CDCs has been demonstrated to reduce infarct size and improve hemodynamic indexes in the mini-pig model, but intramyocardial injection of CDCs or cardiospheres has not been assessed in large animals. Methods: Autologous cardiospheres or CDCs grown from endomyocardial biopsies were injected through thoracotomy 4 weeks after anteroseptal myocardial infarction. Engraftment optimization with luciferase-labeled CDCs guided the choice of cell dose (0.5 million cells/site) and target tissue (20 peri-infarct sites). Pigs were randomly allocated to placebo (n = 11), cardiospheres (n = 8), or CDCs (n = 10). Functional data were acquired before injection and again 8 weeks later, after which organs were harvested for histopathology. Results: Beyond the immediate perioperative period, all animals survived to protocol completion. Ejection fraction was equivalent at baseline, but at 8 weeks was higher than placebo in both of the cell-treated groups (placebo vs. CDC, p = 0.01; placebo vs. cardiospheres, p = 0.01). Echocardiographic and hemodynamic indexes of efficacy improved disproportionately with cardiospheres; likewise, adverse remodeling was more attenuated with cardiospheres than with CDCs. Provocative electrophysiologic testing showed no differences among groups, and no tumors were found. Conclusions: Dosage-optimized direct injection of cardiospheres or CDCs is safe and effective in preserving ventricular function in porcine ischemic cardiomyopathy. Although CDCs and cardiospheres have equivalent effects on left ventricular ejection fraction, cardiospheres are superior in improving hemodynamics and regional function, and in attenuating ventricular remodeling. [ABSTRACT FROM AUTHOR]

Objectives: We examined the sodium-iodide symporter (NIS), which promotes in vivo cellular uptake of technetium 99m (99mTc) or iodine 124 (124I), as a reporter gene for cell tracking by single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. Background: Stem cells offer the promise of cardiac repair. Stem cell labeling is a prerequisite to tracking cell fate in vivo. Methods: The human NIS complementary deoxyribonucleic acid was transduced into rat cardiac-derived stem cells (rCDCs) using lentiviral vectors. Rats were injected intramyocardially with up to 4 million NIS+-rCDCs immediately after left anterior descending coronary artery ligation. Dual isotope SPECT (or PET) imaging was performed, using 99mTc (or 124I) for cell detection and thallium 201 (or ammonia 13) for myocardial delineation. In a subset of animals, high resolution ex vivo SPECT scans of explanted hearts were obtained to confirm that in vivo signals were derived from the cell injection site. Results: NIS expression in rCDCs did not affect cell viability and proliferation. NIS activity was verified in isolated transduced cells by measuring 99mTc uptake. NIS+ rCDCs were visualized in vivo as regions of 99mTc or 124I uptake within a perfusion deficit in the SPECT and PET images, respectively. Cells could be visualized by SPECT up to 6 days post-injection. Ex vivo SPECT confirmed that in vivo 99mTc signals were localized to the cell injection sites. Conclusions: Ectopic NIS expression allows noninvasive in vivo stem cell tracking in the myocardium, using either SPECT or PET. The general approach shows significant promise in tracking the fate of transplanted cells participating in cardiac regeneration, given its ability to observe living cells using clinically applicable imaging modalities. [Copyright &y& Elsevier]

Cardiosphere-derived resident cardiac stem cells (CDCs) are readily isolated from adult hearts and confer functional benefit in animal models of heart failure. To study cardiogenic differentiation in CDCs, we developed a method to genetically label and selectively enrich for cells that have acquired a cardiac phenotype. Lentiviral vectors achieved significantly higher transduction efficiencies in CDCs than any of the nine adeno-associated viral (AAV) serotypes tested. To define the most suitable vector system for reporting cardiogenic differentiation, we compared the cell specificity of five commonly-used cardiac-specific promoters in the context of lentiviral vectors. The promoter of the cardiac sodium-calcium exchanger (NCX1) conveyed the highest degree of cardiac specificity, as assessed by transducing seven cell types with each vector and measuring fluorescence intensity by flow cytometry. NCX1-GFP-positive CDC subpopulations, demonstrating prolonged expression of a variety of cardiac markers, could be isolated and expanded in vitro. Finally, we used chemical biology to validate that lentiviral vectors bearing the cardiac NCX1-promoter can serve as a highly accurate biosensor of cardiogenic small molecules in stem cells. The ability to accurately report cardiac fate and selectively enrich for cardiomyocytes and their precursors has important implications for drug discovery and the development of cell-based therapies.Molecular Therapy (2008); 16 5 957–964 doi:10.1038/mt.2008.30 [ABSTRACT FROM AUTHOR]

Introduction: Central IV infusion of cardiosphere-derived cells (CDCs) in rats with pulmonary arterial hypertension (PAH) attenuated pulmonary arteriolar wall thickness and inflammation while improving RV hemodynamics and morphometry in a rescue paradigm (PLoS ONE 2017; Aug 24;12(8): e0183557). This led to an IND and Phase 1A and B clinical trial of CDCs in patients with PAH (ALPHA study: AL logeneic cdcs for Pulmonary arterial Hypertension therapy). Objective: The primary objective is to determine the safety profile of CAP-1002 (allogeneic CDCs manufactured by Capricor Inc.) administered by infusion in the RV outflow tract of patients with idiopathic PAH, heritable PAH, and PAH associated with connective tissue disease or HIV. The secondary objective is to further assess safety and exploratory efficacy endpoints over 1 year, with 4-months post infusion the designated key exploratory efficacy endpoint. Methods: Phase 1A is a safety and dosing study in which 3 patients received 50 million CDCs followed by 100 million CDCs in a further 3 patients. Phase 1B is a double-blind placebo controlled trial using the maximal dose of CDCs from Phase 1A (anticipated enrollment of 20 subjects). Primary safety endpoints include measures of gas exchange, hemodynamics, arrhythmia and unexpected morbidity and mortality. Serial long-term secondary safety endpoints include time to clinical worsening, BNP and other biomarkers, transthoracic echo (TTE), 6-minute walk test (6MWT), immunologic surveillance and CAMPHOR quality of life (QOL) tool. Exploratory efficacy endpoints include serial TTE, cardiac MRI, BNP, right heart catheterization parameters, pulmonary function, 6MWT, cytokine arrays and QOL measures. Results: We have safely infused the first 6 patients with either 50 or 100 million CDCs as part of Phase 1A of ALPHA. Enrollment for Phase 1B is ongoing. Complete 4-month safety and efficacy for Phase 1A will be available in November. Conclusion: ALPHA represents the largest cell therapy trial for patients with PAH. We hypothesize that allogeneic CDCs will be well tolerated for both short and long-term safety endpoints and that positive exploratory efficacy endpoints will emerge. [ABSTRACT FROM AUTHOR]