Centro Of Biotecnologie Per La Ricerca Medica Applicata Brma

Novara, Italy

Centro Of Biotecnologie Per La Ricerca Medica Applicata Brma

Novara, Italy
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Oltolina F.,University of Piemonte Orientale | Zamperone A.,University of Piemonte Orientale | Zamperone A.,Yeshiva University | Colangelo D.,University of Piemonte Orientale | And 16 more authors.
PLoS ONE | Year: 2015

A major obstacle to an effective myocardium stem cell therapy has always been the delivery and survival of implanted stem cells in the heart. Better engraftment can be achieved if cells are administered as cell aggregates, which maintain their extra-cellular matrix (ECM). We have generated spheroid aggregates in less than 24 h by seeding human cardiac progenitor cells (hCPCs) onto methylcellulose hydrogel-coated microwells. Cells within spheroids maintained the expression of stemness/mesenchymal and ECM markers, growth factors and their cognate receptors, cardiac commitment factors, and metalloproteases, as detected by immunofluorescence, q-RT-PCR and immunoarray, and expressed a higher, but regulated, telomerase activity. Compared to cells in monolayers, 3D spheroids secreted also bFGF and showed MMP2 activity. When spheroids were seeded on culture plates, the cells quickly migrated, displaying an increased wound healing ability with or without pharmacological modulation, and reached confluence at a higher rate than cells from conventional monolayers. When spheroids were injected in the heart wall of healthy mice, some cells migrated from the spheroids, engrafted, and remained detectable for at least 1 week after transplantation, while, when the same amount of cells was injected as suspension, no cells were detectable three days after injection. Cells from spheroids displayed the same engraftment capability when they were injected in cardiotoxin-injured myocardium. Our study shows that spherical in vivo ready-To-implant scaffold-less aggregates of hCPCs able to engraft also in the hostile environment of an injured myocardium can be produced with an economic, easy and fast protocol. Copyright: © 2015 Oltolina et al.

Pietronave S.,University of Piemonte Orientale | Zamperone A.,University of Piemonte Orientale | Oltolina F.,University of Piemonte Orientale | Colangelo D.,University of Piemonte Orientale | And 10 more authors.
Stem Cells and Development | Year: 2014

Electrical stimulation (ES) of cells has been shown to induce a variety of responses, such as cytoskeleton rearrangements, migration, proliferation, and differentiation. In this study, we have investigated whether monophasic and biphasic pulsed ES could exert any effect on the proliferation and differentiation of human cardiac progenitor cells (hCPCs) isolated from human heart fragments. Cells were cultured under continuous exposure to monophasic or biphasic ES with fixed cycles for 1 or 3 days. Results indicate that neither stimulation protocol affected cell viability, while the cell shape became more elongated and reoriented more perpendicular to the electric field direction. Moreover, the biphasic ES clearly induced the upregulation of early cardiac transcription factors, MEF2D, GATA-4, and Nkx2.5, as well as the de novo expression of the late cardiac sarcomeric proteins, troponin T, cardiac alpha actinin, and SERCA 2a. Both treatments increased the expression of connexin 43 and its relocation to the cell membrane, but biphasic ES was faster and more effective. Finally, when hCPCs were exposed to both monophasic and biphasic ES, they expressed de novo the mRNA of the voltage-dependent calcium channel Cav 3.1(α1G) subunit, which is peculiar of the developing heart. Taken together, these results show that ES alone is able to set the conditions for early differentiation of adult hCPCs toward a cardiac phenotype. © Copyright 2014, Mary Ann Liebert, Inc.

De Luca G.,Centro Of Biotecnologie Per La Ricerca Medica Applicata Brma | Verdoia M.,Centro Of Biotecnologie Per La Ricerca Medica Applicata Brma | Cassetti E.,Centro Of Biotecnologie Per La Ricerca Medica Applicata Brma | Schaffer A.,Centro Of Biotecnologie Per La Ricerca Medica Applicata Brma | And 7 more authors.
Blood Coagulation and Fibrinolysis | Year: 2013

Platelets play a central role in the pathogenesis of coronary artery disease (CAD). Mean platelet volume (MPV) is an indicator of platelet activation, and has been demonstrated to be correlated with platelet reactivity. Diabetic patients have been shown to have larger MPV, that may contribute to higher platelet reactivity and atherothrombotic complications observed in these patients. Therefore, the aim of the current study was to investigate whether MPV is associated with platelet reactivity and the extent of CAD among diabetic patients. We performed a cohort study including 1016 consecutive diabetic patients undergoing coronary angiography at the University Hospital 'Maggiore della Carita', Novara, Italy. CAD is defined as stenosis above 50% in at least one coronary vessel at coronary angiography. Platelet reactivity was evaluated in 50 diabetic patients without history of CAD and who were free (in the past month) from medications which may affect platelet aggregation. Platelet aggregation was evaluated by light transmission aggregometry after stimulation with 1μg/ml collagen type I. We additionally evaluated platelet surface expression of P-selectin after stimulation with U46619 (a stable synthetic analogue of the prostaglandin PGH2) and plasma concentration of thromboxane B2 (TxB2). Patients were grouped according to tertile values of MPV (<10.6 fl, group 1; 10.6-11.3 fl, group 2; >11.4 fl, group 3). MPV was associated with age (P=0.011), baseline fasting glucose (P=0.044), glycosylated haemoglobin (P=0.005), creatinine (P=0.052) and haemoglobin (P=0.003), but inversely related to platelet count (P0.001) and triglycerides (P=0.031). Larger MPV was associated with therapy with statins (P=0.012) and diuretics (P=0.021). CAD was observed in 826 patients (81.3%). MPV was not associated with the prevalence of CAD [odds ratio (OR), 0.85 (0.7-1.03), P=0.11]. The results were confirmed in terms of severe CAD [OR, 1.03 (0.88-1.21), P=0.7]. The absence of any significant relationship between MPV and CAD was confirmed after correction for baseline confounding factors [OR, 0.9 (0.75-1.08), P=0.19]. Finally, MPV was not related to platelet reactivity. This is the first study showing that in diabetic patients MPV is not related to platelet reactivity and the prevalence and extent of CAD. Therefore, MPV may not be considered a risk factor for CAD among diabetic patients. © 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Zamperone A.,University of Piemonte Orientale | Pietronave S.,University of Piemonte Orientale | Merlin S.,University of Piemonte Orientale | Colangelo D.,University of Piemonte Orientale | And 9 more authors.
Stem Cells and Development | Year: 2013

The emerging field of tissue engineering and regenerative medicine is a multidisciplinary science that is based on the combination of a reliable source of stem cells, biomaterial scaffolds, and cytokine growth factors. Adult mesenchymal stem cells are considered important cells for applications in this field, and adipose tissue has revealed to be an excellent source of them. Indeed, adipose-derived stem cells (ASCs) can be easily isolated from the stromal vascular fraction (SVF) of adipose tissue. During the isolation and propagation of murine ASCs, we observed the appearance of a spontaneously immortalized cell clone, named m17.ASC. This clone has been propagated for more than 180 passages and stably expresses a variety of stemness markers, such as Sca-1, c-kit/CD117, CD44, CD106, islet-1, nestin, and nucleostemin. Furthermore, these cells can be induced to differentiate toward osteogenic, chondrogenic, adipogenic, and cardiogenic phenotypes. m17.ASC clone displays a normal karyotype and stable telomeres; it neither proliferates when plated in soft agar nor gives rise to tumors when injected subcutaneously in NOD/SCID-γ null mice. The analysis of gene expression highlighted transcriptional traits of SVF cells. m17.ASCs were genetically modified by lentiviral vectors carrying green fluorescent protein (GFP) as a marker transgene and efficiently engrafted in the liver, when injected in the spleen of NOD/SCID-γ null monocrotaline-treated mice. These results suggest that this non-tumorigenic spontaneously immortalized ASC line may represent a useful tool (cell model) for studying the differentiation mechanisms involved in tissue repair as well as a model for pharmacological/toxicological studies. © Mary Ann Liebert, Inc.

Pavesi A.,Polytechnic of Milan | Soncini M.,Polytechnic of Milan | Zamperone A.,University of Piemonte Orientale | Pietronave S.,University of Piemonte Orientale | And 6 more authors.
Biotechnology and Bioengineering | Year: 2014

In tissue engineering, several factors play key roles in providing adequate stimuli for cells differentiation, in particular biochemical and physical stimuli, which try to mimic the physiological microenvironments. Since electrical stimuli are important in the developing heart, we have developed an easy-to-use, cost-effective cell culture platform, able to provide controlled electrical stimulation aimed at investigating the influence of the electric field in the stem cell differentiation process. This bioreactor consists of an electrical stimulator and 12 independent, petri-like culture chambers and a 3-D computational model was used to characterize the distribution and the intensity of the electric field generated in the cell culture volume. We explored the effects of monophasic and biphasic square wave pulse stimulation on a mouse adipose-derived stem cell line (m17.ASC) comparing cell viability, proliferation, protein, and gene expression. Both monophasic (8V, 2ms, 1Hz) and biphasic (+4V, 1ms and -4V, 1ms; 1Hz) stimulation were compatible with cell survival and proliferation. Biphasic stimulation induced the expression of Connexin 43, which was found to localize also at the cell membrane, which is its recognized functional mediating intercellular electrical coupling. Electrically stimulated cells showed an induced transcriptional profile more closely related to that of neonatal cadiomyocytes, particularly for biphasic stimulation. The developed platform thus allowed to set-up precise conditions to drive adult stem cells toward a myocardial phenotype solely by physical stimuli, in the absence of exogenously added expensive bioactive molecules, and can thus represent a valuable tool for translational applications for heart tissue engineering and regeneration. © 2014 Wiley Periodicals, Inc.

Zamperone A.,University of Piemonte Orientale | Zamperone A.,Yeshiva University | Pietronave S.,University of Piemonte Orientale | Colangelo D.,University of Piemonte Orientale | And 7 more authors.
Food and Function | Year: 2014

Cocoa contains phenolic compounds with known antioxidant and antiradical properties beneficial in different pathologies, including cardiovascular diseases. Herein, we have evaluated the protective effects of clovamide, a minor cocoa component, against oxidative stress induced in the rat cardiomyoblast cell line, also comparing it to its bio-isosteric form, rosmarinic acid, and to the main monomeric flavan-3-ol from low-molecular-weight polyphenol in cocoa, i.e. epicatechin. At nano-micro-molar concentrations, the three compounds inhibited the production of reactive oxygen species and apoptosis, evaluated under different aspects, namely, annexin V positivity, DNA fragmentation, caspase release and activation. These molecules can, thus, be considered for their bioactive beneficial activity in the context of cardiovascular pathologies and, particularly, in the protection towards oxidative stress that follows ischemic injury. Clovamide may, thus, be the primary compound for the development of innovative nutraceutical strategies towards cardiovascular diseases. © 2014 the Partner Organisations.

Pietronave S.,University of Piemonte Orientale | Prat M.,Centro Of Biotecnologie Per La Ricerca Medica Applicata Brma
Canadian Journal of Physiology and Pharmacology | Year: 2012

Direct reprogramming of somatic cells into pluripotent cells is an emerging technology for creating patient-specific cells, and potentially opens new scenarios in medical and pharmacological fields. From the discovery of Shinya Yamanaka, who first obtained pluripotent cells from fibroblasts by retrovirus-derived ectopic expression of defined embryonic transcription factors, new methods have been developed to generate safe induced pluripotent stem (iPS) cells without genomic manipulations. This review will focus on the recent advances in iPS technology and their application in pharmacology and medicine.

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