Elisa Messina

Isolation and Expansion of Adult Cardiac Stem Cells From Human and Murine Heart

Cardiac myocytes have been traditionally regarded as terminally differentiated cells that adapt to increased work and compensate for disease exclusively through hypertrophy. However, in the past few years, compelling evidence has accumulated suggesting that the heart has regenerative potential. Recent studies have even surmised the existence of resident cardiac stem cells, endothelial cells generating cardiomyocytes by cell contact or extracardiac progenitors for cardiomyocytes, but these findings are still controversial. We describe the isolation of undifferentiated cells that grow as self-adherent clusters (that we have termed “cardiospheres”) from subcultures of postnatal atrial or ventricular human biopsy specimens and from murine hearts. These cells are clonogenic, express stem and endothelial progenitor cell antigens/markers, and appear to have the properties of adult cardiac stem cells. They are capable of long-term self-renewal and can differentiate in vitro and after ectopic (dorsal subcutaneous connective tissue) or orthotopic (myocardial infarction) transplantation in SCID beige mouse to yield the major specialized cell types of the heart: myocytes (ie, cells demonstrating contractile activity and/or showing cardiomyocyte markers) and vascular cells (ie, cells with endothelial or smooth muscle markers).

Regenerative Potential of Cardiosphere-Derived Cells Expanded From Percutaneous Endomyocardial Biopsy Specimens

Background— Ex vivo expansion of resident cardiac stem cells, followed by delivery to the heart, may favor regeneration and functional improvement. Methods and Results— Percutaneous endomyocardial biopsy specimens grown in primary culture developed multicellular clusters known as cardiospheres, which were plated to yield cardiosphere-derived cells (CDCs). CDCs from human biopsy specimens and from comparable porcine samples were examined in vitro for biophysical and cytochemical evidence of cardiogenic differentiation. In addition, human CDCs were injected into the border zone of acute myocardial infarcts in immunodeficient mice. Biopsy specimens from 69 of 70 patients yielded cardiosphere-forming cells. Cardiospheres and CDCs expressed antigenic characteristics of stem cells at each stage of processing, as well as proteins vital for cardiac contractile and electrical function. Human and porcine CDCs cocultured with neonatal rat ventricular myocytes exhibited biophysical signatures characteristic of myocytes, including calcium transients synchronous with those of neighboring myocytes. Human CDCs injected into the border zone of myocardial infarcts engrafted and migrated into the infarct zone. After 20 days, the percentage of viable myocardium within the infarct zone was greater in the CDC-treated group than in the fibroblast-treated control group; likewise, left ventricular ejection fraction was higher in the CDC-treated group. Conclusions— A method is presented for the isolation of adult human stem cells from endomyocardial biopsy specimens. CDCs are cardiogenic in vitro; they promote cardiac regeneration and improve heart function in a mouse infarct model, which provides motivation for further development for therapeutic applications in patients.

Relative Roles of Direct Regeneration Versus Paracrine Effects of Human Cardiosphere-Derived Cells Transplanted Into Infarcted Mice

Rationale: Multiple biological mechanisms contribute to the efficacy of cardiac cell therapy. Most prominent among these are direct heart muscle and blood vessel regeneration from transplanted cells, as opposed to paracrine enhancement of tissue preservation and/or recruitment of endogenous repair. Objective: Human cardiac progenitor cells, cultured as cardiospheres (CSps) or as CSp-derived cells (CDCs), have been shown to be capable of direct cardiac regeneration in vivo. Here we characterized paracrine effects in CDC transplantation and investigated their relative importance versus direct differentiation of surviving transplanted cells. Methods and Results: In vitro, many growth factors were found in media conditioned by human adult CSps and CDCs; CDC-conditioned media exerted antiapoptotic effects on neonatal rat ventricular myocytes, and proangiogenic effects on human umbilical vein endothelial cells. In vivo, human CDCs secreted vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor 1 when transplanted into the same SCID mouse model of acute myocardial infarction where they were previously shown to improve function and to produce tissue regeneration. Injection of CDCs in the peri-infarct zone increased the expression of Akt, decreased apoptotic rate and caspase 3 level, and increased capillary density, indicating overall higher tissue resilience. Based on the number of human-specific cells relative to overall increases in capillary density and myocardial viability, direct differentiation quantitatively accounted for 20% to 50% of the observed effects. Conclusions: Together with their spontaneous commitment to cardiac and angiogenic differentiation, transplanted CDCs serve as “role models,” recruiting endogenous regeneration and improving tissue resistance to ischemic stress. The contribution of the role model effect rivals or exceeds that of direct regeneration.

Cardiac tissue engineering using tissue printing technology and human cardiac progenitor cells.

Tissue engineering is emerging as a potential therapeutic approach to overcome limitations of cell therapy, like cell retention and survival, as well as to mechanically support the ventricular wall and thereby prevent dilation. Tissue printing technology (TP) offers the possibility to deliver, in a defined and organized manner, scaffolding materials and living cells. The aim of our study was to evaluate the combination of TP, human cardiac-derived cardiomyocyte progenitor cells (hCMPCs) and biomaterials to obtain a construct with cardiogenic potential for in vitro use or in vivo application. With this approach, we were able to generate an in vitro tissue with homogenous distribution of cells in the scaffold. Cell viability was determined after printing and showed that 92% and 89% of cells were viable at 1 and 7 days of culturing, respectively. Moreover, we demonstrated that printed hCMPCs retained their commitment for the cardiac lineage. In particular, we showed that 3D culture enhanced gene expression of the early cardiac transcription factors Nkx2.5, Gata-4 and Mef-2c as well as the sarcomeric protein TroponinT. Printed cells were also able to migrate from the alginate matrix and colonize a matrigel layer, thereby forming tubular-like structures. This indicated that printing can be used for defined cell delivery, while retaining functional properties.

Cardiac stem cells: isolation, expansion and experimental use for myocardial regeneration

Cellular cardiomyoplasty (myogenic cell grafting) is actively being explored as a novel method to regenerate damaged myocardium. The adult human heart contains small populations of indigenous committed cardiac stem cells or multipotent cardiac progenitor cells, identified by their cell-surface expression of c-kit (the receptor for stem cell factor), P-glycoprotein (a member of the multidrug resistance protein family), and Sca-1 (stem cell antigen 1, a mouse hematopoietic stem cell marker) or a Sca-1-like protein. Cardiac stem cells represent a logical source to exploit in cardiac regeneration therapy because, unlike other adult stem cells, they are likely to be intrinsically programmed to generate cardiac tissue in vitro and to increase cardiac tissue viability in vitro. Cardiac stem cell therapy could, therefore, change the fundamental approach to the treatment of heart disease.

Differentiation of human adult cardiac stem cells exposed to extremely low-frequency electromagnetic fields

AIMS Modulation of cardiac stem cell (CSC) differentiation with minimal manipulation is one of the main goals of clinical applicability of cell therapy for heart failure. CSCs, obtained from human myocardial bioptic specimens and grown as cardiospheres (CSps) and cardiosphere-derived cells (CDCs), can engraft and partially regenerate the infarcted myocardium, as previously described. In this paper we assessed the hypothesis that exposure of CSps and CDCs to extremely low-frequency electromagnetic fields (ELF-EMFs), tuned at Ca2+ ion cyclotron energy resonance (Ca2+-ICR), may drive their differentiation towards a cardiac-specific phenotype. METHODS AND RESULTS A significant increase in the expression of cardiac markers was observed after 5 days of exposure to Ca2+-ICR in both human CSps and CDCs, as evidenced at transcriptional, translational, and phenotypical levels. Ca2+ mobilization among intracellular storages was observed and confirmed by compartmentalized analysis of Ca2+ fluorescent probes. CONCLUSIONS These results suggest that ELF-EMFs tuned at Ca2+-ICR could be used to drive cardiac-specific differentiation in adult cardiac progenitor cells without any pharmacological or genetic manipulation of the cells that will be used for therapeutic purposes.

Ferritin as a reporter gene for in vivo tracking of stem cells by 1.5-T cardiac MRI in a rat model of myocardial infarction

The methods currently utilized to track stem cells by cardiac MRI are affected by important limitations, and new solutions are needed. We tested human ferritin heavy chain (hFTH) as a reporter gene for in vivo tracking of stem cells by cardiac MRI. Swine cardiac stem/progenitor cells were transduced with a lentiviral vector to overexpress hFTH and cultured to obtain cardiospheres (Cs). Myocardial infarction was induced in rats, and, after 45 min, the animals were subjected to intramyocardial injection of ∼200 hFTH-Cs or nontransduced Cs or saline solution in the border zone. By employing clinical standard 1.5-Tesla MRI scanner and a multiecho T2* gradient echo sequence, we localized iron-accumulating tissue only in hearts treated with hFTH-Cs. This signal was detectable at 1 wk after infarction, and its size did not change significantly after 4 wk (6.33 ± 3.05 vs. 4.41 ± 4.38 mm(2)). Cs transduction did not affect their cardioreparative potential, as indicated by the significantly better preserved left ventricular global and regional function and the 36% reduction in infarct size in both groups that received Cs compared with control infarcts. Prussian blue staining confirmed the presence of differentiated, iron-accumulating cells containing mitochondria of porcine origin. Cs-derived cells displayed CD31, α-smooth muscle, and α-sarcomeric actin antigens, indicating that the differentiation into endothelial, smooth muscle and cardiac muscle lineage was not affected by ferritin overexpression. In conclusion, hFTH can be used as a MRI reporter gene to track dividing/differentiating stem cells in the beating heart, while simultaneously monitoring cardiac morpho-functional changes.

Cardiosphere-derived cells improve function in the infarcted rat heart for at least 16 weeks--an MRI study.

Aims Endogenous cardiac progenitor cells, expanded from explants via cardiosphere formation, present a promising cell source to prevent heart failure following myocardial infarction. Here we used cine-magnetic resonance imaging (MRI) to track administered cardiosphere-derived cells (CDCs) and to measure changes in cardiac function over four months in the infarcted rat heart. Methods and Results CDCs, cultured from neonatal rat heart, comprised a heterogeneous population including cells expressing the mesenchymal markers CD90 and CD105, the stem cell marker c-kit and the pluripotency markers Sox2, Oct3/4 and Klf-4. CDCs (2×106) expressing green fluorescent protein (GFP+) were labelled with fluorescent micron-sized particles of iron oxide (MPIO). Labelled cells were administered to the infarcted rat hearts (n = 7) by intramyocardial injection immediately following reperfusion, then by systemic infusion (4×106) 2 days later. A control group (n = 7) was administered cell medium. MR hypointensities caused by the MPIOs were detected at all times and GFP+ cells containing MPIO particles were identified in tissue slices at 16 weeks. At two days after infarction, cardiac function was similar between groups. By 6 weeks, ejection fractions in control hearts had significantly decreased (47±2%), but this was not evident in CDC-treated hearts (56±3%). The significantly higher ejection fractions in the CDC-treated group were maintained for a further 10 weeks. In addition, CDC-treated rat hearts had significantly increased capillary density in the peri-infarct region and lower infarct sizes. MPIO-labelled cells also expressed cardiac troponin I, von Willebrand factor and smooth muscle actin, suggesting their differentiation along the cardiomyocyte lineage and the formation of new blood vessels. Conclusions CDCs were retained in the infarcted rat heart for 16 weeks and improved cardiac function.

Human cardiosphere-seeded gelatin and collagen scaffolds as cardiogenic engineered bioconstructs

Cardiac tissue engineering (CTE) aims at regenerating damaged myocardium by combining cells to a biocompatible and/or bioactive matrix. Collagen and gelatin are among the most suitable materials used today for CTE approaches. In this study we compared the structural and biological features of collagen (C-RGD) or gelatin (G-FOAM)-based bioconstructs, seeded with human adult cardiac progenitor cells in the form of cardiospheres (CSps). The different morphology between C-RGD (fibrous ball-of-thread-like) and G-FOAM (trabecular sponge-like) was evidenced by SEM analysis and X-ray micro-tomography, and was reflected by their different mechanical characteristics. Seeded cells were viable and proliferating after 1 week in culture, and a reduced expression of cell-stress markers versus standard CSp culture was detected by realtime PCR. Cell engraftment inside the scaffolds was assessed by SEM microscopy and histology, evidencing more relevant cell migration and production of extracellular matrix in C-RGD versus G-FOAM. Immunofluorescence and realtime PCR analysis showed down-regulation of vascular and stemness markers, while early-to-late cardiac markers were consistently and significantly upregulated in G-FOAM and C-RGD compared to standard CSps culture, suggesting selective commitment towards cardiomyocytes. Overall our results suggest that CSp-bioconstructs have suitable mechanical properties and improved survival and cardiogenic properties, representing promising tools for CTE.

Metformin increases APP expression and processing via oxidative stress, mitochondrial dysfunction and NF-κB activation: Use of insulin to attenuate metformin's effect.

Clinical and experimental biomedical studies have shown Type 2 diabetes mellitus (T2DM) to be a risk factor for the development of Alzheimers disease (AD). This study demonstrates the effect of metformin, a therapeutic biguanide administered for T2DM therapy, on β-amyloid precursor protein (APP) metabolism in in vitro, ex vivo and in vivo models. Furthermore, the protective role of insulin against metformin is also demonstrated. In LAN5 neuroblastoma cells, metformin increases APP and presenilin levels, proteins involved in AD. Overexpression of APP and presenilin 1 (Pres 1) increases APP cleavage and intracellular accumulation of β-amyloid peptide (Aβ), which, in turn, promotes aggregation of Aβ. In the experimental conditions utilized the drug causes oxidative stress, mitochondrial damage, decrease of Hexokinase-II levels and cytochrome C release, all of which lead to cell death. Several changes in oxidative stress-related genes following metformin treatment were detected by PCR arrays specific for the oxidative stress pathway. These effects of metformin were found to be antagonized by the addition of insulin, which reduced Aβ levels, oxidative stress, mitochondrial dysfunction and cell death. Similarly, antioxidant molecules, such as ferulic acid and curcumin, are able to revert metformins effect. Comparable results were obtained using peripheral blood mononuclear cells. Finally, the involvement of NF-κB transcription factor in regulating APP and Pres 1 expression was investigated. Upon metformin treatment, NF-κB is activated and translocates from the cytoplasm to the nucleus, where it induces increased APP and Pres 1 transcription. The use of Bay11-7085 inhibitor suppressed the effect of metformin on APP and Pres 1 expression.