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Lipidomics is a lipid-targeted metabolomics approach that aims to the comprehensive analysis of lipids in biological systems in order to highlight the specific functions of lipid species in health and disease. Lipids play pivotal roles as they are major structural components of the cellular membranes and energy storage molecules but also, as most recently shown, they act as functional and regulatory components of intra- and intercellular signaling. Herein, emphasis is given to the recently highlighted roles of specific bioactive lipids species, as polyunsaturated fatty acids (PUFA)-derived mediators (generally known as eicosanoids), endocannabinoids (eCBs), and lysophospholipids (LPLs), and their involvement in the mesenchymal stem cells (MSCs)-related inflammatory scenario. Indeed, MSCs are a heterogenous population of multipotent cells that have attracted much attention for their potential in regulating inflammation, immunomodulatory capabilities, and reparative roles. The lipidomics of the inflammatory disease osteoarthritis (OA) and the influence of MSCs-derived lipids have also been addressed.

Undifferentiated pleomorphic sarcoma of bone (UPSb) is a rare primary bone sarcoma that lacks a specific line of differentiation. There is very little information about the genetic alterations leading to tumourigenesis or malignant transformation. Distinguishing between UPSb and other malignant bone sarcomas, including dedifferentiated chondrosarcoma and osteosarcoma, can be challenging due to overlapping features. To explore the genomic and transcriptomic landscape of UPSb tumours, whole-exome sequencing (WES) and RNA sequencing (RNA-Seq) were performed on UPSb tumours. All tumours lacked hotspot mutations in IDH1/2 132 or 172 codons, thereby excluding the diagnosis of dedifferentiated chondrosarcoma. Recurrent somatic mutations in TP53 were identified in four of 14 samples (29%). Moreover, recurrent mutations in histone chromatin remodelling genes, including H3F3A, ATRX and DOT1L, were identified in five of 14 samples (36%), highlighting the potential role of deregulated chromatin remodelling pathways in UPSb tumourigenesis. The majority of recurrent mutations in chromatin remodelling genes identified here are reported in COSMIC, including the H3F3A G34 and K36 hotspot residues. Copy number alteration analysis identified gains and losses in genes that have been previously altered in UPSb or UPS of soft tissue. Eight somatic gene fusions were identified by RNA-Seq, two of which, CLTC-VMP1 and FARP1-STK24, were reported previously in multiple cancers. Five gene fusions were genomically characterised. Hierarchical clustering analysis, using RNA-Seq data, distinctly clustered UPSb tumours from osteosarcoma and other sarcomas, thus molecularly distinguishing UPSb from other sarcomas. RNA-Seq expression profiling analysis and quantitative reverse transcription-polymerase chain reaction showed an elevated expression in FGF23, which can be a potential molecular biomarker for UPSb. To our knowledge, this study represents the first comprehensive WES and RNA-Seq analysis of UPSb tumours revealing novel protein-coding recurrent gene mutations, gene fusions and identifying a potential UPSb molecular biomarker, thereby broadening the understanding of the pathogenic mechanisms and highlighting the possibility of developing novel targeted therapeutics. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

In recent years, undifferentiated small round cell sarcomas (USRCSs) have been divided into a variety of new, rare, sarcoma subtypes, including the group of Ewing-like sarcomas, which have the morphological appearance of Ewing sarcomas, but carry CIC-DUX4, BCOR-CCNB3 and other gene fusions different from the classic EWSR1-ETS gene fusion. Using high-throughput RNA-sequencing (RNA-seq) analyses, we identified a novel recurrent gene fusion, CRTC1-SS18, in two cases of USRCS that lacked any known translocation. RNA-seq results were confirmed by reverse transcription polymerase chain reaction, long-range polymerase chain reaction, and fluorescence in situ hybridization. In vitro, we showed that the cells expressing the gene fusion were morphologically distinct and had enhanced oncogenic potential as compared with control cells. Expression profile comparisons with tumours of other sarcoma subtypes demonstrated that both cases clustered close to EWSR1-CREB1-positive tumours. Moreover, these analyses indicated enhanced NTRK1 expression in CRTC1-SS18-positive tumours. We conclude that the novel gene fusion identified in this study adds a new subtype to the USRCSs with unique gene signatures, and may be of therapeutic relevance. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

Low-grade chronic inflammation is a salient feature of obesity and many associated disorders. This condition frequently occurs in central obesity and is connected to alterations of the visceral adipose tissue (AT) microenvironment. Understanding how obesity is related to inflammation may allow the development of therapeutics aimed at improving metabolic parameters in obese patients. To achieve this aim, we compared the features of two subpopulations of adipose-derived stem cells (ASC) isolated from both subcutaneous and visceral AT of obese patients with the features of two subpopulations of ASC from the same isolation sites of non-obese individuals. In particular, the behavior of ASC of obese versus non-obese subjects during hypoxia, which occurs in obese AT and is an inducer of the inflammatory response, was evaluated. Obesity deeply influenced ASC from visceral AT (obV-ASC); these cells appeared to exhibit clearly distinguishable morphology and ultrastructure as well as reduced proliferation, clonogenicity and expression of stemness, differentiation and inflammation-related genes. These cells also exhibited a deregulated response to hypoxia, which induced strong tissue-specific NF-kB activation and an NF-kB-mediated increase in inflammatory and fibrogenic responses. Moreover, obV-ASC, which showed a less stem-like phenotype, recovered stemness features after hypoxia. Our findings demonstrated the peculiar behavior of obV-ASC, their influence on the obese visceral AT microenvironment and the therapeutic potential of NF-kB inhibitors. These novel findings suggest that the deregulated hyper-responsiveness to hypoxic stimulus of ASC from visceral AT of obese subjects may contribute via paracrine mechanisms to low-grade chronic inflammation, which has been implicated in obesity-related morbidity.

Tissue engineering is an emerging interdisciplinary field, born with the purpose to provide an alternative solution for the regeneration of lesioned or lost tissues, combining cells, biocompatible scaffolds and bioactive factors. The cells for this approach should be non-immunoreactive and non-tumorigenic. Moreover, they should be available in large amount and possess, or be able to acquire, a specific protein expression pattern similar to that of the damaged tissue and/or act as a pool of trophic factors for resident cells. All these reasons, make mesenchymal stem cells (MSCs) good candidates for applications in regenerative medicine. Although bone marrow is still the most common source of MSCs, these cells could be harvested from all vascularised tissues, and, interestingly, from tissues that are normally discarded, such as fat, placenta or umbilical cord. One of the most convenient source of MSCs, is unequivocally, the adipose tissue due to the easily accessible anatomical location and the abundance of subcutaneous adipose tissue. Adipose-derived stem cells (ASCs) are similar to MSCs isolated from bone marrow in morphology, immunophenotype, and differentiation ability, and own interesting features such as immunoregolatory and anti-inflammatory properties. In the recent years, many strategies for the cure of musculoskeletal tissues critical lesions, mainly in orthopaedic, oral and maxillo-facial surgery, have been under investigations. In this contest, the regeneration of structures including different tissues, such as the periodontium and the osteochondral unit, are particularly challenging. Periodontal regeneration is especially demanding, as it requires regeneration of three quite diverse and unique tissues such as the alveolar bone, the periodontal ligament and the cementum, that have to interface with each other to restore their complex structure. Since the promising results obtained with ASCs in preclinical studies of periodontal diseases arouse the curiosity of maxillofacial and dental surgeons, we decided to identify a novel source of ASCs, i.e, the buccal fat pad, convenient for these specialists. For this purpose, we studied human adipose derived-stem cells from buccal fat pad (BFP-ASCs), comparing them with cells from the subcutaneous adipose tissue (SC-ASCs) of the same donor (n=2). In parallel, considering the need for preclinical studies in which the effect of allogenic cells should be tested, and swine as an accepted animal model in tissue engineering applications, we also characterized porcine cells (n=6). With preclinical and clinical application prospective, we also investigated ASC interactions with oral tissues, natural and synthetic scaffolds and Amelogenin, an oral bioactive molecule. First of all, we showed that it is feasible to isolate ASCs even starting from very limited amounts of tissue (0,5 ml) and that the cellular yield is influenced by species, but not by the site of harvesting (1.1x105�1.4x104 human BFP-ASCs/ml and 1.15x105�7.1x103 human SC-ASCs/ml; 3.0x104�9.3x103 porcine BFP-ASCs/ml and 5.5x104�3.3x104 porcine SC-ASCs/ml). Despite the lower yield, the pASCs great proliferation rate allows to obtain high number of cells (potentially, 108 - 109) after few (3, 4) passages in culture. After the isolation, a great amount of cells deriving from all the tissues, adhered to cell culture plates showing the MSC fibroblast like morphology, with only mild shape differences constituted by the higher elongation and dimension of human SC-ASCs. Moreover, all the cells are easily expandable and showed good clonogenic ability at early passages. Cells of the same species, from both the harvesting site, displayed the same surface markers profile, that, in particular for human ASCs, was the typical one of hMSC (CD90+, CD105+, CD73+, CD14-, CD31-, and CD34-). Human and porcine BFP-ASCs, as SC-ASCs, are multipotent; indeed, when induced towards osteogenic and adipogenic lineages, they up-regulated significantly ALP activity, collagen and calcified extracellular matrix deposition and lipid vacuoles productions, respectively, already after 14 days of differentiation in vitro. Next, since cell/scaffold interaction is fundamental for the outcome of a tissue engineering approach, in sight of a preclinical study, we combined porcine BFP and SC-ASCs to both clinical grade (titanium) and innovative [silicon carbide?plasma-enhanced chemical vapor deposition (SIC-PECVD)] biomaterials, and studied cell adhesion and their differentiation ability. All the cells nicely grew on both scaffolds and, when osteoinduced, significantly increased the amount of calcified ECM compared to control cells; interestingly, titanium is osteoinductive even per se on pASCs (+284% and +91 for BFP- and SC-ASCs). Considering the importance of cell interaction with tissue of the lesion site, and with materials commonly used during surgical practices, we studied human BFP- and SC-ASC adherence to several supports. SEM analysis confirmed that both cell type nicely stick on alveolar bone, periodontal ligament, collagen membrane and polyglycolic acid filaments. Finally, we found that amelogenin, the most abundant enamel matrix protein seems to be an early osteoinductive factor for BFP-hASCs, whereas this effect is not manifested for SC-hASCs. For future cellular therapy, and since the use of FBS pose the risk of xenogenic contaminations leading to immunological complications during transplant, we tested cells growth in the presence of autologous supplements. Interestingly, both hASCs adapted rapidly to human serum, increasing their proliferation rates compared to standard culture condition, while porcine autologous or heterologous sera, did not improve pASC growth. In conclusion, we identified a cell population derived from a tissue easily available to dentists and maxillofacial surgeons, whose multipotent features and interaction with clinical grade scaffolds make proper candidate for future uses in tissue engineering approaches of periodontal diseases. In parallel, part of my PhD project was focused on the study of a critical osteochondral defect regeneration performed in a large animal preclinical model. The main obstacles for clinicians in treating this defect arises from the disparity concerning anatomy, composition and, most importantly, rate of healing of the articular cartilage (AC) and the subchondral bone. The key points of our study are the use of an innovative hydrogel of oligo(polyethylene glycol)fumarate (OPF) to fill the osteochondral defect, and of either porcine, or human ASCs, to create bioconstructs to be implanted in non-immuno-compromised minipigs. In particular, four critical osteochondral defects (diameter 9mm, depth 8mm) were created in the peripheral part of the trochlea of seven animals (defect n=28), and then treated with the different pre-made constructs. Untreated defects and defects filled by just scaffold were included as controls. No side-effects have been observed during the six-moths follow-up. At the end of this period, animals were sacrificed and knees explanted. Gross appearance analyses showed quite satisfactory filling of all the lesions, with the exception of one animal, whose joint appeared infected and not healed. MRI analyses revealed that in all the scaffold treated groups an overall improvement of the tissue quality at the osteochondral lesion site, was induced. More accurate evaluations (histological and immunohistochemistry analyses) revealed that some important tissue features were significantly improved by the association of OPF and ASCs. Indeed, regarding the subchondral bone, in all the OPF+ASCs groups, a mature bone appeared, with higher deposition of collagen type I compared to untreated or unseeded OPF groups. Moreover, the use of ASCs associated to scaffolds induced an improvement in newly formed cartilage features such as collagen type II deposition, and histological scores associated to these samples indicated a significant increase in matrix staining, tissue morphology and formation of tidemark, together with a reduction in vascularisation (a positive aspect in cartilage) compared to unseeded scaffolds. However, the histology indicated that in all the samples cartilage regeneration was still immature, most likely due to the limited time of follow up and/or the insufficient stimuli for cartilage complete regeneration. Despite this, biomechanical tests revealed that the neo-cartilage found in the cell-loaded scaffold groups possessed poroelastic behaviour, as well as indentation modulus and creep curves comparable to native cartilage. This important result suggest that the ASC presence at the lesion site, is able to enhance newly formed cartilage functionality. In conclusion, this in vivo study provide the evidence that both porcine and human adipose-derived stem cells associated to OPF hydrogel improve osteochondral defect regeneration, even though, at the moment, we are not able to define if the implanted ASCs are responsible per se of the new tissue formation or if they help spontaneous regeneration process by paracrine actions.

Neuropathic pain (NP) is a highly invalidating disease resulting as consequence of a lesion or disease affecting the somatosensory system. All the pharmacological treatments today in use give a long lasting pain relief only in a limited percentage of patients before pain reappears making NP an incurable disease. New approaches are therefore needed and research is testing stem cell usage. Several papers have been written on experimental neuropathic pain treatment using stem cells of different origin and species to treat experimental NP. The original idea was based on the capacity of stem cell to offer a totipotent cellular source for replacing injured neural cells and for delivering trophic factors to lesion site; soon the researchers agreed that the capacity of stem cells to contrast NP was not dependent upon their regenerative effect but was mostly linked to a bidirectional interaction between the stem cell and damaged microenvironment resident cells. In this paper we review the preclinical studies produced in the last years assessing the effects induced by several stem cells in different models of neuropathic pain. The overall positive results obtained on pain remission by using stem cells that are safe, of easy isolation, and which may allow an autologous transplant in patients may be encouraging for moving from bench to bedside, although there are several issues that still need to be solved.