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Cox S.N.,University of Bari | Serino G.,University of Bari | Sallustio F.,University of Bari | Blasi A.,Medestea Research and Production Laboratories | And 3 more authors.
Nephrology Dialysis Transplantation | Year: 2015

Background The main defect of immunoglobulin A nephropathy (IgAN) lies within the immune system and in peripheral blood mononuclear cells rather than in the kidney. Previously, we found an altered gene expression in monocytes compared with B and T cells isolated from IgAN patients; thus, our aim here has been to study this subset at a genome-wide and functional level. Methods A total of 39 IgAN patients and 37 healthy blood donors (HBDs) were included in this study, and microarray technology was used to evaluate global gene expression differences in monocytes isolated from IgAN patients and HBDs. Aberrantly expressed genes and pathways were then validated on an independent set of IgAN patients with RT-PCR western blot and flow cytometric analysis. Results Gene expression differences in monocytes from IgAN patients and HBDs primarily involved apoptosis signalling, mitochondrial dysfunction, tnfr2/1 and death receptor signalling. Both the extrinsic and intrinsic apoptotic pathways seem to be implicated; in particular, the protein levels of NDUFS3 and TNFRSF1A were upregulated thus confirming the altered mitochondrial and death receptor homeostasis. Furthermore, the basal intracellular protein levels of TNF in monocytes were lower in IgAN patients compared with HBDs. We validated at protein level an enhanced apoptotic phenotype and a different subset distribution in monocytes from IgAN patients. We found that the non-classical monocyte subset (CD14+CD16+) was significantly expanded in all IgAN patients tested even though the total monocyte count remained unchanged. Conclusions Our findings demonstrate, for the first time, an aberrant modulation of the mitochondrial respiratory system in monocytes isolated from IgAN patients. Furthermore, the aberrant expansion of the (CD14+CD16+) subset could explain the enhanced apoptotic phenotype seen in these cells thus revealing their potential role in the pathogenesis of IgAN. © 2015 The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.


De Siena R.,Medestea Research and Production Laboratories | Balducci L.,Medestea Research and Production Laboratories | Blasi A.,Medestea Research and Production Laboratories | Montanaro M.G.,Medestea Research and Production Laboratories | And 14 more authors.
Experimental Cell Research | Year: 2010

Cell-based therapy could be a valid option to treat myocardial infarct (MI). Adipose-derived stromal cells (ADStCs) have demonstrated tissue regenerative potential including cardiomyogenesis. Omentum is an extremely rich source of visceral fat and its accumulation seems to correlate with cardiovascular diseases. We investigated the capacity of human fat Omentum-derived StCs (FOStCs) to affect heart function upon acute infarct in pigs induced by permanent ligation of the anterior interventricular artery (IVA). We demonstrated for the first time that the local injection of 50×106 of FOStCs ameliorates the functional parameters of post-infarct heart. Most importantly, histology of FOStCs treated hearts demonstrated a substantial improvement of cardiomyogenesis. In culture, FOStCs produced an impressive number and amount of angiogenic factors and cytokines. Moreover, the conditioned medium of FOStCs (FOStCs-CM) stimulates in vitro cardiac endothelial cells (ECs) proliferation and vascular morphogenesis and inhibits monocytes, EC activation and cardiomyocyte apoptosis. Since FOStCs in vivo did not trans-differentiate into cardiomyocyte-like cells, we conclude that FOStCs efficacy was presumably mediated by a potent paracrine mechanism involving molecules that concomitantly improved angiogenesis, reduced inflammation and prevented cardiomyocytes death. Our results highlight for the first time the important role that human FOStCs may have in cardiac regeneration. © 2010 Elsevier Inc.


PubMed | Imperial College London, R.Ø.S.A., Medestea Research and Production Laboratories and University of Bari
Type: Comparative Study | Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association | Year: 2015

The main defect of immunoglobulin A nephropathy (IgAN) lies within the immune system and in peripheral blood mononuclear cells rather than in the kidney. Previously, we found an altered gene expression in monocytes compared with B and T cells isolated from IgAN patients; thus, our aim here has been to study this subset at a genome-wide and functional level.A total of 39 IgAN patients and 37 healthy blood donors (HBDs) were included in this study, and microarray technology was used to evaluate global gene expression differences in monocytes isolated from IgAN patients and HBDs. Aberrantly expressed genes and pathways were then validated on an independent set of IgAN patients with RT-PCR western blot and flow cytometric analysis.Gene expression differences in monocytes from IgAN patients and HBDs primarily involved apoptosis signalling, mitochondrial dysfunction, tnfr2/1 and death receptor signalling. Both the extrinsic and intrinsic apoptotic pathways seem to be implicated; in particular, the protein levels of NDUFS3 and TNFRSF1A were upregulated thus confirming the altered mitochondrial and death receptor homeostasis. Furthermore, the basal intracellular protein levels of TNF in monocytes were lower in IgAN patients compared with HBDs. We validated at protein level an enhanced apoptotic phenotype and a different subset distribution in monocytes from IgAN patients. We found that the non-classical monocyte subset (CD14(+)CD16(+)) was significantly expanded in all IgAN patients tested even though the total monocyte count remained unchanged.Our findings demonstrate, for the first time, an aberrant modulation of the mitochondrial respiratory system in monocytes isolated from IgAN patients. Furthermore, the aberrant expansion of the (CD14(+)CD16(+)) subset could explain the enhanced apoptotic phenotype seen in these cells thus revealing their potential role in the pathogenesis of IgAN.


Blasi A.,Medestea Research and Production Laboratories | Martino C.,Medestea Research and Production Laboratories | Balducci L.,Medestea Research and Production Laboratories | Saldarelli M.,Medestea Research and Production Laboratories | And 7 more authors.
Vascular Cell | Year: 2011

Background: Mesenchymal stem cells (MSCs) are multipotent stem cells able to differentiate into different cell lineages. However, MSCs represent a subpopulation of a more complex cell composition of stroma cells contained in mesenchymal tissue. Due to a lack of specific markers, it is difficult to distinguish MSCs from other more mature stromal cells such as fibroblasts, which, conversely, are abundant in mesenchymal tissue. In order to find more distinguishing features between MSCs and fibroblasts, we studied the phenotypic and functional features of human adipose-derived MSCs (AD-MSCs) side by side with normal human dermal fibroblasts (HNDFs) in vitro. Methods. AD-MSCs and HNDFs were cultured, expanded and phenotypically characterized by flow cytometry (FC). Immunofluorescence was used to investigate cell differentiation. ELISA assay was used to quantify angiogenic factors and chemokines release. Cultures of endothelial cells (ECs) and a monocyte cell line, U937, were used to test angiogenic and anti-inflammatory properties. Results: Cultured AD-MSCs and HNDFs display similar morphological appearance, growth rate, and phenotypic profile. They both expressed typical mesenchymal markers-CD90, CD29, CD44, CD105 and to a minor extent, the adhesion molecules CD54, CD56, CD106 and CD166. They were negative for the stem cell markers CD34, CD146, CD133, CD117. Only aldehyde dehydrogenase (ALDH) was expressed. Neither AD-MSCs nor HNDFs differed in their multi-lineage differentiation capacity; they both differentiated into osteoblast, adipocyte, and also into cardiomyocyte-like cells. In contrast, AD-MSCs, but not HNDFs, displayed strong angiogenic and anti-inflammatory activity. AD-MSCs released significant amounts of VEGF, HGF and Angiopoietins and their conditioned medium (CM) stimulated ECs proliferation and tube formations. In addition, CM-derived AD-MSCs (AD-MSCs-CM) inhibited adhesion molecules expression on U937 and release of RANTES and MCP-1. Finally, after priming with TNF, AD-MSCs enhanced their anti-inflammatory potential; while HNDFs acquired pro-inflammatory activity. Conclusions: AD-MSCs cannot be distinguished from HNDFs in vitro by evaluating their phenotypic profile or differentiation potential, but only through the analysis of their anti-inflammatory and angiogenic properties. These results underline the importance of evaluating the angiogenic and anti-inflammatory features of MSCs preparation. Their priming with inflammatory cytokines prior to transplantation may improve their efficacy in cell-based therapies for tissue regeneration. © 2011 Blasi et al; licensee BioMed Central Ltd.


Balducci L.,Medestea Research and Production Laboratories | Blasi A.,Medestea Research and Production Laboratories | Saldarelli M.,Medestea Research and Production Laboratories | Soleti A.,Medestea Research and Production Laboratories | And 10 more authors.
Stem Cell Research and Therapy | Year: 2014

Introduction. Human adipose-derived stromal cells (hASCs), due to their relative feasibility of isolation and ability to secrete large amounts of angiogenic factors, are being evaluated for regenerative medicine. However, their limited culture life span may represent an obstacle for both preclinical investigation and therapeutic use. To overcome this problem, hASCs immortalization was performed in order to obtain cells with in vitro prolonged life span but still maintain their mesenchymal marker expression and ability to secrete angiogenic factors. Methods. hASCs were transduced with the human telomerase reverse transcriptase (hTERT) gene alone or in combination with either SV-40 or HPV E6/E7 genes. Mesenchymal marker expression on immortalized hASCs lines was confirmed by flow cytometry (FC), differentiation potential was evaluated by immunocytochemistry and ELISA kits were used for evaluation of angiogenic factors. Green fluorescent protein (GFP) gene transduction was used to obtain fluorescent cells. Results: We found that hTERT alone failed to immortalize hASCs (hASCs-T), while hTERT/SV40 (hASCs-TS) or hTERT/HPV E6/E7 (hASCs-TE) co-transductions successfully immortalized cells. Both hASCs-TS and hASCs-TE were cultured for up to one year with a population doubling level (PDL) up to 100. Comparative studies between parental not transduced (hASCs-M) and immortalized cell lines showed that both hASCs-TS and hASCs-TE maintained a mesenchymal phenotypic profile, whereas differentiation properties were reduced particularly in hASCs-TS. Interestingly, hASCs-TS and hASCs-TE showed a capability to secrete significant amount of HGF and VEGF. Furthermore, hASCs-TS and hASCs-TE did not show tumorigenic properties in vitro although some chromosomal aberrations were detected. Finally, hASCs-TS and hASCs-TE lines were stably fluorescent upon transduction with the GFP gene. Conclusions: Here we demonstrated, for the first time, that hASCs, upon immortalization, maintain a strong capacity to secrete potent angiogenic molecules. By combining hASCs immortalization and their paracrine characteristics, we have developed a "hybridoma-like model" of hASCs that could have potential applications for discovering and producing molecules to use in regenerative medicine (process scale-up).In addition, due to the versatility of these fluorescent-immortalized cells, they could be employed in in vivo cell-tracking experiments, expanding their potential use in laboratory practice. © 2014Balducci et al.; licensee BioMed Central Ltd.


PubMed | Medestea Research and Production Laboratories and Fondazione IRCCS Neurological Institute Carlo Besta
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2016

Human adipose tissue has proven to be an abundant, accessible, and rich source of adult mesenchymal stromal cells, suitable for tissue engineering and regenerative medicine. However, a major complication in fully investigating these cells may derive from their limited life span.Although methods to isolate, expand, and immortalize these cells have been widely reported in the literature, exhaustive explanations on the problems that can be encountered during these processes and how these can be solved have never been described. It is of fundamental importance to follow a common protocol to achieve reliable and reproducible results. Here, we describe a protocol to isolate and expand human adipose stromal cells from specimens obtained from tissue biopsies and liposuction surgical interventions. Finally, we broadly describe the cell immortalization technique, and particular attention is paid to some of the apparently secondary aspects.


Desantis S.,University of Bari | Accogli G.,University of Bari | Zizza S.,University of Bari | Mastrodonato M.,University of Bari | And 5 more authors.
Annals of Anatomy | Year: 2015

Ovine bone marrow-derived mesenchymal stromal cells (oBM-MSCs) represent a good animal model for cell-based therapy and tissue engineering. Despite their use as a new therapeutic tool for several clinical applications, the morphological features of oBM-MSCs are yet unknown. Therefore, in this study the ultrastructural phenotype of these cells was analysed by transmission electron microscopy (TEM). The oBM-MSCs were isolated from the iliac crest and cultured until they reached near-confluence. After trypsinization, they were processed to investigate their ultrastructural features as well as specific surface marker proteins by flow cytometry and immunogold electron microscopy. Flow cytometry displayed that all oBM-MSCs lacked expression of CD31, CD34, CD45, HLA-DR whereas they expressed CD44, CD58, HLAI and a minor subset of the cell population (12%) exhibited CD90. TEM revealed the presence of two morphologically distinct cell types: cuboidal electron-lucent cells and spindle-shaped electron-dense cells, both expressing the CD90 antigen. Most of the electron-lucent cells showed glycogen aggregates, dilated cisternae of RER, moderately developed Golgi complex, and secretory activity. The electron-dense cell type was constituted by two different cell-populations: type A cells with numerous endosomes, dense bodies, rod-shaped mitochondria and filopodia; type B cells with elongated mitochondria, thin pseudopodia and cytoplasmic connectivity with electron-lucent cells. These morphological findings could provide a useful support to identify "in situ" the cellular components involved in the cell-therapy when cultured oBM-MSCs are injected. © 2015 Elsevier GmbH.


PubMed | Medestea Research and Production Laboratories
Type: Journal Article | Journal: Experimental cell research | Year: 2010

Cell-based therapy could be a valid option to treat myocardial infarct (MI). Adipose-derived stromal cells (ADStCs) have demonstrated tissue regenerative potential including cardiomyogenesis. Omentum is an extremely rich source of visceral fat and its accumulation seems to correlate with cardiovascular diseases. We investigated the capacity of human fat Omentum-derived StCs (FOStCs) to affect heart function upon acute infarct in pigs induced by permanent ligation of the anterior interventricular artery (IVA). We demonstrated for the first time that the local injection of 50x10(6) of FOStCs ameliorates the functional parameters of post-infarct heart. Most importantly, histology of FOStCs treated hearts demonstrated a substantial improvement of cardiomyogenesis. In culture, FOStCs produced an impressive number and amount of angiogenic factors and cytokines. Moreover, the conditioned medium of FOStCs (FOStCs-CM) stimulates in vitro cardiac endothelial cells (ECs) proliferation and vascular morphogenesis and inhibits monocytes, EC activation and cardiomyocyte apoptosis. Since FOStCs in vivo did not trans-differentiate into cardiomyocyte-like cells, we conclude that FOStCs efficacy was presumably mediated by a potent paracrine mechanism involving molecules that concomitantly improved angiogenesis, reduced inflammation and prevented cardiomyocytes death. Our results highlight for the first time the important role that human FOStCs may have in cardiac regeneration.

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