Gianandrea Pasquinelli

A new nonenzymatic method and device to obtain a fat tissue derivative highly enriched in pericyte-like elements by mild mechanical forces from human lipoaspirates

Adipose tissue contains multipotent elements with phenotypic and gene expression profiles similar to human mesenchymal stem cells (hMSCs) and pericytes. The chance of clinical translation of the multilineage potential of these cells is delayed by the poor/negligible cell survival within cryopreserved lipoaspirates, the difficulty of ex vivo expansion, and the complexity of current Good Manufacturing Practice (cGMP) requirements for expanded cells. Hence, availability of a minimally manipulated, autologous, hMSC/pericyte-enriched fat product would have remarkable biomedical and clinical relevance. Here, we present an innovative system, named Lipogems, providing a nonexpanded, ready-to-use fat product. The system uses mild mechanical forces in a completely closed system, avoiding enzymes, additives, and other manipulations. Differently from unprocessed lipoaspirate, the nonexpanded Lipogems product encompasses a remarkably preserved vascular stroma with slit-like capillaries wedged between adipocytes and stromal stalks containing vascular channels with evident lumina. Immunohistochemistry revealed that Lipogems stromal vascular tissue included abundant cells with pericyte/hMSC identity. Flow cytometry analysis of nonexpanded, collagenase-treated Lipogems product showed that it was comprised with a significantly higher percentage of mature pericytes and hMSCs, and lower amount of hematopoietic elements, than enzymatically digested lipoaspirates. Differently from the lipoaspirate, the distinctive traits of freshly isolated Lipogems product were not altered by cryopreservation. Noteworthy, the features of fresh product were retained in the Lipogems product obtained from human cadavers, paving the way to an off-the-shelf strategy for reconstructive procedures and regenerative medicine. When placed in tissue culture medium, the Lipogems product yielded a highly homogeneous adipose tissue-derived hMSC population, exhibiting features of hMSCs isolated from other sources, including the classical commitment to osteogenic, chondrogenic, and adipogenic lineages. Moreover, the transcription of vasculogenic genes in Lipogems-derived adipose tissue hMSCs was enhanced at a significantly greater extent by a mixture of natural provasculogenic molecules, when compared to hMSCs isolated from enzymatically digested lipoaspirates.

Sodium butyrate inhibits platelet‐derived growth factor‐induced proliferation and migration in pulmonary artery smooth muscle cells through Akt inhibition

Sodium butyrate (BU) is a molecule that acts as a histone deacetylase inhibitor. As compared with its well‐known antineoplastic/antiproliferative effects, little is known about BU action on vascular cell dynamics. An imbalance of proliferation and migration in pulmonary arterial smooth muscle cells (PASMCs) is essential in the onset and progression of pulmonary arterial hypertension (PAH), a disease that is characterized by vascular lung derangement and that frequently has an unfavorable outcome. Here, we show that, in PASMCs of PAH rats, BU counteracted platelet‐derived growth factor (PDGF)‐induced Ki67 expression, and arrested the cell cycle, mainly at G0/G1. BU decreased proliferating cell nuclear antigen, c‐Myc and cyclin D1 transcription and protein expression, while increasing p21 expression. BU reduced the transcription of PDGF receptor‐β, and that of Ednra and Ednrb, two major receptors in PAH progression. Wound healing, migration and pulmonary artery ring assays indicated that BU inhibited PDGF‐induced PASMC migration. BU strongly inhibited PDGF‐induced Akt phosphorylation, an effect reversed by the phosphatase inhibitor calyculin A. BU‐treated cells showed a remarkable increase in acetylated Akt, indicating an inverse relationship between the levels of acetylated Akt and phospho‐Akt. These findings may provide novel perspectives on the use of histone deacetylase inhibitors in PAH.

Restored perfusion and reduced inflammation in the infarcted heart after grafting stem cells with a hyaluronan-based scaffold

The aim of this study is to investigate the blood perfusion and the inflammatory response of the myocardial infarct area after transplanting a hyaluronan‐based scaffold (HYAFF®11) with bone marrow mesenchymal stem cells (MSCs). Nine‐week‐old female pigs were subjected to a permanent left anterior descending coronary artery ligation for 4 weeks. According to the kind of the graft, the swine subjected to myocardial infarction were divided into the HYAFF®11, MSCs, HYAFF®11/MSCs and untreated groups. The animals were killed 8 weeks after coronary ligation. Scar perfusion, evaluated by Contrast Enhanced Ultrasound echography, was doubled in the HYAFF®11/MSCs group and was comparable with the perfusion of the healthy, non‐infarcted hearts. The inflammation score of the MSCs and HYAFF®11/MSCs groups was near null, revealing the role of the grafted MSCs in attenuating the cell infiltration, but not the foreign reaction strictly localized around the fibres of the scaffold. Apart from the inflammatory response, the native tissue positively interacted with the HYAFF®11/MSCs construct modifying the extracellular matrix with a reduced presence of collagene and increased amount of proteoglycans. The border‐zone cardiomyocytes also reacted favourably to the graft as a lower degree of cellular damage was found. This study demonstrates that the transplantation in the myocardial infarct area of autologous MSCs supported by a hyaluronan‐based scaffold restores blood perfusion and almost completely abolishes the inflammatory process following an infarction. These beneficial effects are superior to those obtained after grafting only the scaffold or MSCs, suggesting that a synergic action was achieved using the cell‐integrated polymer construct.

Human cadaver multipotent stromal/stem cells isolated from arteries stored in liquid nitrogen for 5 years

IntroductionRegenerative medicine challenges researchers to find noncontroversial, safe and abundant stem cell sources. In this context, harvesting from asystolic donors could represent an innovative and unlimited reservoir of different stem cells. In this study, cadaveric vascular tissues were established as an alternative source of human cadaver mesenchymal stromal/stem cells (hC-MSCs). We reported the successful cell isolation from postmortem arterial segments stored in a tissue-banking facility for at least 5 years.MethodsAfter thawing, hC-MSCs were isolated with a high efficiency (12 × 106) and characterized with flow cytometry, immunofluorescence, molecular and ultrastructural approaches.ResultsIn early passages, hC-MSCs were clonogenic, highly proliferative and expressed mesenchymal (CD44, CD73, CD90, CD105, HLA-G), stemness (Stro-1, Oct-4, Notch-1), pericyte (CD146, PDGFR-β, NG2) and neuronal (Nestin) markers; hematopoietic and vascular markers were negative. These cells had colony and spheroid-forming abilities, multipotency for their potential to differentiate in multiple mesengenic lineages and immunosuppressive activity to counteract proliferation of phytohemagglutinin-stimulated blood mononuclear cells.ConclusionsThe efficient procurement of stem cells from cadaveric sources, as postmortem vascular tissues, demonstrates that such cells can survive to prolonged ischemic insult, anoxia, freezing and dehydration injuries, thus paving the way for a scientific revolution where cadaver stromal/stem cells could effectively treat patients demanding cell therapies.

An innovative stand-alone bioreactor for the highly reproducible transfer of cyclic mechanical stretch to stem cells cultured in a 3D scaffold

Much evidence in the literature demonstrates the effect of cyclic mechanical stretch in maintaining, or addressing, a muscle phenotype. Such results were obtained using several technical approaches, useful for the experimental collection of proofs of principle but probably unsuitable for application in clinical regenerative medicine. Here we aimed to design a reliable innovative bioreactor, acting as a stand‐alone cell culture incubator, easy to operate and effective in addressing mesenchymal stem cells (MSCs) seeded onto a 3D bioreabsorbable scaffold, towards a muscle phenotype via the transfer of a controlled and highly‐reproducible cyclic deformation. Electron microscopy, immunohistochemistry and biochemical analysis of the obtained pseudotissue constructs showed that cells ‘trained’ over 1 week: (a) displayed multilayer organization and invaded the 3D mesh of the scaffold; and (b) expressed typical markers of muscle cells. This effect was due only to physical stimulation of the cells, without the need of any other chemical or genetic manipulation. This device is thus proposed as a prototypal instrument to obtain pseudotissue constructs to test in cardiovascular regenerative medicine, using good manufacturing procedures. Copyright

Dissecting histone deacetylase role in pulmonary arterial smooth muscle cell proliferation and migration.

Pulmonary Arterial Hypertension (PAH) is a rare and devasting condition characterized by elevated pulmonary vascular resistance and pulmonary artery pressure leading to right-heart failure and premature death. Pathologic alterations in proliferation, migration and survival of all cell types composing the vascular tissue play a key role in the occlusion of the vascular lumen. In the current study, we initially investigated the action of selective class I and class II HDAC inhibitors on the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) after exposure to Platelet Derived Growth Factor (PDGF). Class I HDAC inhibitors were able to counteract the hyperproliferative response to PDGF, reducing both proliferation and migration in PASMCs, while class II were ineffective. Selective silencing with siRNAs targeted against different HDACs revealed a major role of class I, and within this class, of HDAC1 in mediating PDGF-induced Akt Phosphorylation and Cyclin D1 (CycD1) expression. These results from these combinatorial approaches were further confirmed by the ability of a specific HDAC1 inhibitor to antagonize the PDGF action. The finding that HDAC1 is a major conductor of PDGF-induced patterning in PAH-PASMCs prompts the development of novel selective inhibitors of this member of class I HDACs as a potential tool to control lung vascular homeostasis in PAH.

Effect of different disinfection protocols on microbial and biofilm contamination of dental unit waterlines in community dental practices.

Output water from dental unit waterlines (DUWLs) may be a potential source of infection for both dental healthcare staff and patients. This study compared the efficacy of different disinfection methods with regard to the water quality and the presence of biofilm in DUWLs. Five dental units operating in a public dental health care setting were selected. The control dental unit had no disinfection system; two were disinfected intermittently with peracetic acid/hydrogen peroxide 0.26% and two underwent continuous disinfection with hydrogen peroxide/silver ions (0.02%) and stabilized chlorine dioxide (0.22%), respectively. After three months of applying the disinfection protocols, continuous disinfection systems were more effective than intermittent systems in reducing the microbial contamination of the water, allowing compliance with the CDC guidelines and the European Council regulatory thresholds for drinking water. P. aeruginosa, Legionella spp, sulphite-reducing Clostridium spores, S. aureus and β-haemolytic streptococci were also absent from units treated with continuous disinfection. The biofilm covering the DUWLs was more extensive, thicker and more friable in the intermittent disinfection dental units than in those with continuous disinfection. Overall, the findings showed that the products used for continuous disinfection of dental unit waterlines showed statistically better results than the intermittent treatment products under the study conditions.

Exploring the human mesenchymal stem cell tubule communication network through electron microscopy.

Abstract Cells use several mechanisms to transfer information to other cells. In this study, we describe micro/nanotubular connections and exosome-like tubule fragments in multipotent mesenchymal stem cells (MSCs) from human arteries. Scanning and transmission electron microscopy allowed characterization of sinusoidal microtubular projections (700 nm average size, 200 µm average length, with bulging mitochondria and actin microfilaments); short, uniform, variously shaped nanotubular projections (100 nm, bidirectional communication); and tubule fragments (50 nm). This is the first study demonstrating that MSCs from human arteries constitutively interact through an articulate and dynamic tubule network allowing long-range cell to cell communication.

Hepatocyte Growth Factor Effects on Mesenchymal Stem Cells Derived from Human Arteries: A Novel Strategy to Accelerate Vascular Ulcer Wound Healing

Vascular ulcers are a serious complication of peripheral vascular disease, especially in diabetics. Several approaches to treat the wounds are proposed but they show poor outcomes and require long healing times. Hepatocyte Growth Factor/Scatter Factor (HGF/SF) is a pleiotropic cytokine exerting many biological activities through the c-Met receptor. This study was aimed at verifying whether HGF/SF influences proliferation, migration, and angiogenesis on mesenchymal stem cells isolated from human arteries (hVW-MSCs). hVW-MSCs were exposed to NIBSC HGF/SF (2.5, 5, 10, and 70 ng/mL) from 6 hrs to 7 days. HGF and c-MET mRNA and protein expression, cell proliferation (Alamar Blue and Ki–67 assay), migration (scratch and transwell assays), and angiogenesis (Matrigel) were investigated. hVW-MSCs displayed stemness features and expressed HGF and c-MET. HGF/SF did not increase hVW-MSC proliferation, whereas it enhanced the cell migration, the formation of capillary-like structures, and the expression of angiogenic markers (vWF, CD31, and KDR). The HGF/SF effects on hVW-MSC migration and angiogenic potential are of great interest to accelerate wound healing process. Local delivery of HGF/SF could therefore improve the healing of unresponsive vascular ulcers.