Michael E. Coleman

Effect of freshly isolated autologous tissue resident stromal cells on cardiac function and perfusion following acute myocardial infarction

BACKGROUND The aim of this study was to investigate the effect of intracoronary administration of freshly isolated, uncultured autologous tissue-derived stromal cells on cardiac function and perfusion after acute infarction in pigs. METHODS A transmural myocardial infarction in a porcine model was induced by occlusion of the mid LAD with an angioplasty balloon for 3 h. Upon reperfusion, freshly isolated, uncultured autologous stromal cells (1.5×10⁶ cells/kg) or control solution was injected into the infarct artery. Cardiac function and area at risk were determined by (99m)Tc-SPECT. RESULTS Eight weeks after infarction, cell treated pigs showed a 20% smaller myocardial perfusion defect compared to control animals (35±9% vs. 44±5% of LV, treated vs. control, respectively, p<0.05). The reduction of the perfusion defect was associated with a significantly higher myocardial salvage index in the cell group as well as a significant increase in ejection fraction compared to control (EF at 8 weeks 43±7% vs. 35±3%, treated vs. control, respectively, p<0.05). This functional improvement was reflected by an increased wall thickness of the infarct and border zone in the treated group (11.2±2.2 mm) compared to control (8.6±1.6 mm, p<0.05) as well as an increased capillary density in the border zone (treated vs. control; 41.6±17.9 vs. 32.9±12.6 capillaries per 0.1 mm², p<0.05). CONCLUSIONS This study demonstrates for the first time that recovery and intracoronary delivery of uncultured autologous tissue derived stromal cells at time of vessel reperfusion is feasible and improves ventricular function.

Human adipose tissue-derived stem cells exhibit proliferation potential and spontaneous rhythmic contraction after fusion with neonatal rat cardiomyocytes

Various types of stem cells have been shown to have beneficial effects on cardiac function. It is still debated whether fusion of injected stem cells with local resident cardiomyocytes is one of the mechanisms. To better understand the role of fusion in stem cell‐based myocardial regeneration, the present study was designed to investigate the fate of human adipose tissue‐derived stem cells (hASCs) fused with neonatal rat cardiomyocytes in vitro. hASCs labeled with the green fluorescent probe Vybrant DiO were cocultured with neonatal rat cardiomyocytes labeled with the red fluorescent probe Vybrant Dil and then treated with fusion‐inducing hemagglutinating virus of Japan (HVJ). Cells that incorporated both red and green fluorescent signals were considered to be hASCs that had fused with rat cardiomyocytes. Fusion efficiency was 19.86 ± 4.84% at 5 d after treatment with HVJ. Most fused cells displayed cardiomyocyte‐like morphology and exhibited spontaneous rhythmic contraction. Both immunofluorescence staining and lentiviral vector labeling showed that fused cells contained separate rat cardiomyocyte and hASC nuclei. Immunofluorescence staining assays demonstrated that human nuclei in fused cells still expressed the proliferation marker Ki67. In addition, hASCs fused with rat cardiomyocytes were positive for troponin I. Whole‐cell voltage‐clamp analysis demonstrated action potentials in beating fused cells. RT‐PCR analysis using rat‐ or human‐specific myosin heavy chain primers revealed that the myosin heavy‐chain expression in fused cells was derived from rat cardiomyocytes. Real‐time PCR identified expression of human troponin T in fused cells and the presence of rat cardiomyocytes induced a cardiomyogenic protein expression of troponin T in human ASCs. This study illustrates that hASCs exhibit both stem cell (proliferation) and cardiomyocyte properties (action potential and spontaneous rhythmic beating) after fusion with rat cardiomyocytes, supporting the theory that fusion, even if artificially induced in our study, could indeed be a mechanism for cardiomyocyte renewal in the heart.—Metzele, R., Alt, C., Bai, X., Yan, Y., Zhang, Z., Pan, Z., Coleman, M., Vykoukal, J., Song, Y.‐H., Alt, E. Human adipose tissue‐derived stem cells exhibit proliferation potential and spontaneous rhythmic contraction after fusion with neonatal rat cardiomyocytes. FASEB J. 25, 830–839 (2011).

Two sides of the same coin: stem cells in cancer and regenerative medicine

Multipotent stromal cells (MSCs) derived from bone marrow, adipose tissue, cord blood, and other origins have recently received much attention as potential therapeutic agents with beneficial immunomodulatory and regenerative properties. In their native tissue environment, however, such cells also appear to have essential functions in building and supporting tumor microenvironments, providing metastatic niches, and maintaining cancer hallmarks. Here, we consider the varied roles of these tissue‐resident stroma‐associated cells, synthesize recent and emerging discoveries, and discuss the role, potential, and clinical applications of MSCs in cancer and regenerative medicine.—Ilmer, M., Vykoukal, J., Recio Boiles, A., Coleman, M., Alt, E. Two sides of the same coin: stem cells in cancer and regenerative medicine. FASEB J. 28, 2748–2761 (2014). www.fasebj.org

Improved Method for Isolation of Neonatal Rat Cardiomyocytes with Increased Yield of C-Kit+ Cardiac Progenitor Cells.

Cell therapy represents a promising new paradigm for treatment of heart disease, a major cause of death in the industrialized world. The recent discovery of tissue resident c-Kit+ cardiac progenitor cells (CPCs) has fueled scientific efforts to exploit these cells therapeutically for regenerative interventions, and primary culture of cardiomyocytes is a common in-vitro model to investigate basic molecular mechanisms underlying cardiac degeneration and regeneration. Current protocols for cardiomyocyte isolation frequently result in low cell yield and insufficient depletion of fibroblasts, which then overgrow the cardiomyocytes in culture. In this protocol we describe an improved method for the isolation of neonatal rat cardiomyocytes that also enables enhanced yields of CPCs. Gentle techniques of enzymatic and mechanical tissue processing ensure high cell numbers and viability, while subsequent Percoll density gradient centrifugation minimizes fibroblasts. We compared the advantages of different enzymes and found that Collagenase 2 alone leads to very high yields of cardiomyocytes, whereas the application of Matrase™ enzyme blend increases the relative yield of c-Kit+ CPCs to up to 35%. Cardiomyocytes and CPCs isolated with this protocol may constitute an important cell source for investigating heart disease as well as cell based therapeutic approaches.

High yield recovery of equine mesenchymal stem cells from umbilical cord matrix/Wharton’s jelly using a semi- automated process

Umbilical cord is an abundant source of perinatal, plastic adherent mesenchymal stem cells (UC-MSCs). UC-MSCs exhibit robust stemness and strong immunosuppressive and regenerative effects in vivo. This protocol describes enzymatic and mechanical dissociation of umbilical cord matrix (Whartons jelly) that results in efficient isolation of large numbers of fresh nucleated umbilical cord regenerative cells (UC-RCs) that, when cultured on plastic, exhibit similar characteristics of UC-MSCs. This protocol potentially alleviates the need for culture expansion to obtain large numbers of cells required for clinical application. Dissociation is achieved with a blend of collagenase and neutral proteases with agitation at 37 °C in a semi-automatic system. Average expected yield is 1.65 × 10(6) cells/g tissue with 93 % viability. This protocol has been successfully used to isolate an uncultured nucleated regenerative cell population (also referred to as stromal vascular fraction or SVF) from surgically debrided skin and from human, equine, and canine adipose tissue. The procedure requires less than 30 min for tissue dissection and less than 100 min for cell extraction. Quickly obtaining a large number of UC-RCs that have pluripotent differentiation capacity without the complexity and risks of culture expansion could simplify and expand the use of UC-RCs in clinical as well as research applications.