Priyadharshini Sivakumaran

Electrical Stimulation Promotes Cardiac Differentiation of Human Induced Pluripotent Stem Cells

Background. Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes. Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs) for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days. Results. In iPS(Foreskin)-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression of ACTC1, TNNT2, MYH7, and MYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner. Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used.

Trichostatin A Enhances Differentiation of Human Induced Pluripotent Stem Cells to Cardiogenic Cells for Cardiac Tissue Engineering

Human induced pluripotent stem (iPS) cells are a promising source of autologous cardiomyocytes to repair and regenerate myocardium for treatment of heart disease. In this study, we have identified a novel strategy to enhance cardiac differentiation of human iPS cells by treating embryoid bodies (EBs) with a histone deacetylase inhibitor, trichostatin A (TSA), together with activin A and bone morphogenetic protein 4 (BMP4). Over a narrow window of concentrations, TSA (1 ng/ml) directed the differentiation of human iPS cells into a cardiomyocyte lineage. TSA also exerted an additive effect with activin A (100 ng/ml) and BMP4 (20 ng/ml). The resulting cardiomyocytes expressed several cardiac‐specific transcription factors and contractile proteins at both gene and protein levels. Functionally, the contractile EBs displayed calcium cycling and were responsive to the chronotropic agents isoprenaline (0.1 μM) and carbachol (1 μM). Implanting microdissected beating areas of iPS cells into tissue engineering chambers in immunocompromised rats produced engineered constructs that supported their survival, and they maintained spontaneous contraction. Human cardiomyocytes were identified as compact patches of muscle tissue incorporated within a host fibrocellular stroma and were vascularized by host neovessels. In conclusion, human iPS cell‐derived cardiomyocytes can be used to engineer functional cardiac muscle tissue for studying the pathophysiology of cardiac disease, for drug discovery test beds, and potentially for generation of cardiac grafts to surgically replace damaged myocardium.

Cardiac Repair With a Novel Population of Mesenchymal Stem Cells Resident in the Human Heart

Cardiac resident stem cells (CRSCs) hold much promise to treat heart disease but this remains a controversial field. Here, we describe a novel population of CRSCs, which are positive for W8B2 antigen and were obtained from adult human atrial appendages. W8B2+ CRSCs exhibit a spindle‐shaped morphology, are clonogenic and capable of self‐renewal. W8B2+ CRSCs show high expression of mesenchymal but not hematopoietic nor endothelial markers. W8B2+ CRSCs expressed GATA4, HAND2, and TBX5, but not C‐KIT, SCA‐1, NKX2.5, PDGFRα, ISL1, or WT1. W8B2+ CRSCs can differentiate into cardiovascular lineages and secrete a range of cytokines implicated in angiogenesis, chemotaxis, inflammation, extracellular matrix remodeling, cell growth, and survival. In vitro, conditioned medium collected from W8B2+ CRSCs displayed prosurvival, proangiogenic, and promigratory effects on endothelial cells, superior to that of other adult stem cells tested, and additionally promoted survival and proliferation of neonatal rat cardiomyocytes. Intramyocardial transplantation of human W8B2+ CRSCs into immunocompromised rats 1 week after myocardial infarction markedly improved cardiac function (∼40% improvement in ejection fraction) and reduced fibrotic scar tissue 4 weeks after infarction. Hearts treated with W8B2+ CRSCs showed less adverse remodeling of the left ventricle, a greater number of proliferating cardiomyocytes (Ki67+cTnT+ cells) in the remote region, higher myocardial vascular density, and greater infiltration of CD163+ cells (a marker for M2 macrophages) into the border zone and scar regions. In summary, W8B2+ CRSCs are distinct from currently known CRSCs found in human hearts, and as such may be an ideal cell source to repair myocardial damage after infarction. Stem Cells 2015;33:3100–3113

In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation.

Pluripotent stem cells are a potential source of autologous cells for cell and tissue regenerative therapies. They have the ability to renew indefinitely while retaining the capacity to differentiate into all cell types in the body. With developments in cell therapy and tissue engineering these cells may provide an option for treating tissue loss in organs which do not repair themselves. Limitations to clinical translation of pluripotent stem cells include poor cell survival and low cell engraftment in vivo and the risk of teratoma formation when the cells do survive through implantation. In this study, implantation of human induced-pluripotent stem (hiPS) cells, suspended in Matrigel, into an in vivo vascularized tissue engineering chamber in nude rats resulted in substantial engraftment of the cells into the highly vascularized rat tissues formed within the chamber. Differentiation of cells in the chamber environment was shown by teratoma formation, with all three germ lineages evident within 4 weeks. The rate of teratoma formation was higher with partially differentiated hiPS cells (as embryoid bodies) compared to undifferentiated hiPS cells (100% versus 60%). In conclusion, the in vivo vascularized tissue engineering chamber supports the survival through implantation of human iPS cells and their differentiated progeny, as well as a novel platform for rapid teratoma assay screening for pluripotency.

Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro

Long-term culture of primary neonatal rat cardiomyocytes is limited by the loss of spontaneous contractile phenotype within weeks in culture. This may be due to loss of contractile cardiomyocytes from the culture or overgrowth of the non-cardiomyocyte population. Using the mitochondria specific fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM), we showed that neonatal rat cardiomyocytes enriched by fluorescence-activated cell sorting can be maintained as contractile cultures for long periods (24-wk culture vs. 2 wk for unsorted cardiomyocytes). Long-term culture of this purified cardiomyocyte (TMRM high) population retained the expression of cardiomyocyte markers, continued calcium cycling, and displayed cyclic electrical activity that could be regulated pharmacologically. These findings suggest that non-cardiomyocyte populations can negatively influence contractility of cardiomyocytes in culture and that by purifying cardiomyocytes, the cultures retain potential as an experimental model for longitudinal studies of cardiomyocyte biology in vitro.

Melatonin Protects Human Adipose-Derived Stem Cells from Oxidative Stress and Cell Death.

Background Adipose-derived stem cells (ASCs) have applications in regenerative medicine based on their therapeutic potential to repair and regenerate diseased and damaged tissue. They are commonly subject to oxidative stress during harvest and transplantation, which has detrimental effects on their subsequent viability. By functioning as an antioxidant against free radicals, melatonin may exert cytoprotective effects on ASCs. Methods We cultured human ASCs in the presence of varying dosages of hydrogen peroxide and/or melatonin for a period of 3 hours. Cell viability and apoptosis were determined with propidium iodide and Hoechst 33342 staining under fluorescence microscopy. Results Hydrogen peroxide (1–2.5 mM) treatment resulted in an incremental increase in cell death. 2 mM hydrogen peroxide was thereafter selected as the dose for co-treatment with melatonin. Melatonin alone had no adverse effects on ASCs. Co-treatment of ASCs with melatonin in the presence of hydrogen peroxide protected ASCs from cell death in a dose-dependent manner, and afforded maximal protection at 100 µM (n=4, one-way analysis of variance P<0.001). Melatonin co-treated ASCs displayed significantly fewer apoptotic cells, as demonstrated by condensed and fragmented nuclei under fluorescence microscopy. Conclusions Melatonin possesses cytoprotective properties against oxidative stress in human ASCs and might be a useful adjunct in fat grafting and cell-assisted lipotransfer.

Friedreich’s ataxia induced pluripotent stem cell-derived cardiomyocytes display electrophysiological abnormalities and calcium handling deficiency

We sought to identify the impacts of Friedreichs ataxia (FRDA) on cardiomyocytes. FRDA is an autosomal recessive degenerative condition with neuronal and non-neuronal manifestations, the latter including progressive cardiomyopathy of the left ventricle, the leading cause of death in FRDA. Little is known about the cellular pathogenesis of FRDA in cardiomyocytes. Induced pluripotent stem cells (iPSCs) were derived from three FRDA individuals with characterized GAA repeats. The cells were differentiated into cardiomyocytes to assess phenotypes. FRDA iPSC- cardiomyocytes retained low levels of FRATAXIN (FXN) mRNA and protein. Electrophysiology revealed an increased variation of FRDA- cardiomyocyte beating rates which was prevented by addition of nifedipine, suggestive of a calcium handling deficiency. Finally, calcium imaging was performed and we identified small amplitude, diastolic and systolic calcium transients confirming a deficiency in calcium handling. We defined a robust FRDA cardiac-specific electrophysiological profile in patient-derived iPSCs which could be used for high throughput compound screening. This cell-specific signature will contribute to the identification and screening of novel treatments for this life-threatening disease.

Enhancing Human Cardiomyocyte Differentiation from Induced Pluripotent Stem Cells with Trichostatin A.

Human induced pluripotent stem (iPS) cells are a promising source of autologous cardiomyocytes to repair and regenerate myocardium for treatment of heart disease. In this study, we describe a method for enhanced cardiomyocyte production from human iPS cells by treating embryoid bodies with a histone deacetylase inhibitor, trichostatin A (TSA), together with activin A and bone morphogenetic protein (BMP)-4. The resulting cardiomyocytes expressed cardiac-specific transcription factors and contractile proteins at both gene and protein levels. Functionally, the contractile embryoid bodies (EBs) displayed calcium cycling and were responsive to the chronotropic agents isoprenaline (0.1 μM) and carbachol (1 μM). The cardiomyocytes derived from human iPS cells may be used to engineer functional cardiac muscle tissue for studying pathophysiology of cardiac disease, for drug discovery test beds, and potentially for generation of cardiac grafts to surgically replace damaged myocardium.

Biologically active constituents of the secretome of human W8B2 + cardiac stem cells

The benefits of adult stem cells for repair of the heart have been attributed to the repertoire of salutary paracrine activities they appear to exert. We previously isolated human W8B2+ cardiac stem cells (CSCs) and found they powerfully influence cardiomyocytes and endothelial cells to collectively promote cardiac repair and regeneration. Here, the complexity of the W8B2+ CSC secretomes was characterised and examined in more detail. Using ion exchange chromatography to separate soluble proteins based on their net surface charge, the secreted factors responsible for the pro-survival activity of W8B2+ CSCs were found within the low and medium cation fractions. In addition to the soluble proteins, extracellular vesicles generated from W8B2+ CSCs not only exhibited pro-survival and pro-angiogenic activities, but also promoted proliferation of neonatal cardiomyocytes. These extracellular vesicles contain a cargo of proteins, mRNA and primary microRNA precursors that are enriched in exosomes and are capable of modulating collectively many of the cellular pathways involved in protein metabolism, cell growth, as well as cellular responses to stress and organisation of the extracellular matrix. Thus the W8B2+ CSC secretome contains a multitude of bioactive paracrine factors we have now characterised, that might well be harnessed for therapeutic application for cardiac repair and regeneration.

Mdivi-1 Protects Human W8B2+ Cardiac Stem Cells from Oxidative Stress and Simulated Ischemia-Reperfusion Injury

Cardiac stem cell (CSC) therapy is a promising approach to treat ischemic heart disease. However, the poor survival of transplanted stem cells in the ischemic myocardium has been a major impediment in achieving an effective cell-based therapy against myocardial infarction. Inhibiting mitochondrial fission has been shown to promote survival of several cell types. However, the role of mitochondrial morphology in survival of human CSC remains unknown. In this study, we investigated whether mitochondrial division inhibitor-1 (Mdivi-1), an inhibitor of mitochondrial fission protein dynamin-related protein-1 (Drp1), can improve survival of a novel population of human W8B2+ CSCs in hydrogen peroxide (H2O2)-induced oxidative stress and simulated ischemia-reperfusion injury models. Mdivi-1 significantly reduced H2O2-induced cell death in a dose-dependent manner. This cytoprotective effect was accompanied by an increased proportion of cells with tubular mitochondria, but independent of mitochondrial membrane potential recovery and reduction of mitochondrial superoxide production. In simulated ischemia-reperfusion injury model, Mdivi-1 given as a pretreatment or throughout ischemia-reperfusion injury significantly reduced cell death. However, the cytoprotective effect of Mdivi-1 was not observed when given at reperfusion. Moreover, the cytoprotective effect of Mdivi-1 in the simulated ischemia-reperfusion injury model was not accompanied by changes in mitochondrial morphology, mitochondrial membrane potential, or mitochondrial reactive oxygen species production. Mdivi-1 also did not affect mitochondrial bioenergetics of intact W8B2+ CSCs. Taken together, these experiments demonstrated that Mdivi-1 treatment of human W8B2+ CSCs enhances their survival and can be employed to improve therapeutic efficacy of CSCs for ischemic heart disease.