Transplantation of ex vivo proliferated cardiac stem cells (CSCs) is an emerging therapy for ischemic cardiomyopathy but outcomes are limited by modest engraftment and poor long-term survival. As such, we explored the effect of single cell microencapsulation to increase CSC engraftment and survival after myocardial injection. Transcript and protein profiling of human atrial appendage sourced CSCs revealed strong expression the pro-survival integrin dimers αVβ3 and α5β1-thus rationalizing the integration of fibronectin and fibrinogen into a supportive intracapsular matrix. Encapsulation maintained CSC viability under hypoxic stress conditions and, when compared to standard suspended CSC, media conditioned by encapsulated CSCs demonstrated superior production of pro-angiogenic/cardioprotective cytokines, angiogenesis and recruitment of circulating angiogenic cells. Intra-myocardial injection of encapsulated CSCs after experimental myocardial infarction favorably affected long-term retention of CSCs, cardiac structure and function. Single cell encapsulation prevents detachment induced cell death while boosting the mechanical retention of CSCs to enhance repair of damaged myocardium.

First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1a, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1a. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromalcell derived factor 1a over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR41 cardiac stem cells. Stromal-cell derived factor 1a enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1a enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1a content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1a transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. STEM CELLS 2016;34:1826-1835 SIGNIFICANCE STATEMENTFirst generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines (such as stromal-cell derived factor 1a) which opens the prospect of maximizing up-front paracrine-mediated repair. In this report, we demonstrate that paracrine engineering of human cardiac stem cells to over-express stromal-cell derived factor 1a enhances recruitment of endogenous stem cells, new myocyte/vessel formation and salvage of reversibly damaged myocardium to enhance cardiac repair after experimental myocardial infarction. Thus providing a simple novel means to enhance the efficacy of a cell products current already under clinical investigation.

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

Although patient-sourced cardiac explant-derived stem cells (EDCs) provide an exogenous source of new cardiomyocytes post-myocardial infarction, poor long-term engraftment indicates that the benefits seen in clinical trials are likely paracrine-mediated. Of the numerous cytokines produced by EDCs, interleukin-6 (IL-6) is the most abundant; however, its role in cardiac repair is uncertain. In this study, a custom short-hairpin oligonucleotide lentivirus was used to knockdown IL-6 in human EDCs, revealing an unexpected pro-healing role for the cytokine.Methods: EDCs were cultured from atrial appendages donated by patients undergoing clinically indicated cardiac surgery. The effects of lentiviral mediated knockdown of IL-6 was evaluated using in vitro and in vivo models of myocardial ischemia.Results: Silencing IL-6 in EDCs abrogated much of the benefits conferred by cell transplantation and revealed that IL-6 prompts cardiac fibroblasts and macrophages to reduce myocardial scarring while increasing the generation of new cardiomyocytes and recruitment of blood stem cells.Conclusions: This study suggests that IL-6 plays a pivotal role in EDC-mediated cardiac repair and may provide a means of increasing cell-mediated repair of ischemic myocardium.

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The value of preserving high quality bio specimens for fundamental research is significant as linking cellular and molecular changes to clinical and epidemiological data has fueled many recent advances in medicine. Unfortunately, storage of traditional biospecimens is limited to fixed samples or isolated genetic material. Here, we report the effect of cryopreservation of routine myocardial biopsies on explant derived cardiac stem cell (EDC) culture outcomes. We demonstrate that immediate cryopreservation or delayed cryopreservation after suspension within cardioplegia for 12 hours did not alter EDC yields, proliferative capacity, antigenic phenotype or paracrine signature. Cryopreservation had negligible effects on the ability of EDCs to adopt a cardiac lineage, stimulate new vessel growth, attract circulating angiogenic cells and repair injured myocardium. Finally, cryopreservation did not influence the ability of EDCs to undergo genetic reprogramming into inducible pluripotent stem cells. This study establishes a means of storing cardiac samples as a retrievable live cell source for cardiac repair or disease modeling.

Background: Although recent trials have demonstrated that cardiac-derived cell products repair damaged myocardium, the influence of age and medical co-morbidities is unknown. Therefore we investigated the effect of medical comorbidities on the functional activity of human explant-derived cardiac stem cells (EDCs). Methods/Results: Human EDCs were cultured from atrial appendages obtained during cardiac surgery. The Long Term Stratification model (LTS) was chosen as the best means of discriminating between patients with few or extensive comorbidities. Increasing LTS score demonstrated a moderate negative correlation with: 1) EDC conditioned media HUVEC tubule formation (R 2 =0.53; r=-4±1, p=0.003), 2) transwell recruitment of circulating angiogenic cells (R 2 =0.48; r=-15±5, p=0.016), 3) decreased echocardiographic ejection fraction (R 2 =0.64, r=-0.7±0.2, p=0.003) and increasing scar burden (R 2 =0.59, r=-0.5±0.2, p=0.04) 3 weeks after myocardial injection of EDCs into a SCID mouse model of myocardial infarction. These effects may be attributable to LTS score dependent decreases in SDF-1 (R 2 =0.33; r=-0.2±0.1, p=0.02) or exosome (R 2 =0.59; r=-2.4±0.8X10 7 , p=0.02) content within conditioned media. Interestingly, the most abundant cytokine in EDC conditioned media (IL-6) demonstrated a positive correlation with increasing LTS scores (R 2 =0.33, r=1.2±0.5, p=0.03). Lentiviral mediated knock down of IL-6 (IL-6KD) by EDCs suggests that IL-6 supports therapeutic regeneration as intra-myocardial injection of IL-6 KD EDCs from low (<3) and high (>5) LTS score patients reduced improvements in ejection fraction by 1.4±0.1 fold (p≤0.05) while increasing scar burden by 1.3±0.1 fold (p≤0.05). Immunohistochemistry demonstrated that IL-6KD reduced newly generated BrdU+ myocytes (p=0.03 vs. scramble treated EDCs) while promoting the persistence of maladaptive myofibroblasts (p=0.01 vs. scramble treated EDCs). Conclusions: The regenerative performance of the earliest cell population cultured from human tissue declines with accumulating medical co-morbidities. This effect is associated with diminished production of pro-cardiogenic cytokines and exosomes while IL-6 plays a novel pivotal role in EDC-mediated repair of damaged myocardium.

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