Human adipose-derived mesenchymal stem cells as a new model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) or Kennedy's disease is an X-linked CAG/polyglutamine expansion motoneuron disease, in which an elongated polyglutamine tract (polyQ) in the N-terminal androgen receptor (ARpolyQ) confers toxicity to this protein. Typical markers of SBMA disease are ARpolyQ intranuclear inclusions. These are generated after the ARpolyQ binds to its endogenous ligands, which promotes AR release from chaperones, activation and nuclear translocation, but also cell toxicity. The SBMA mouse models developed so far, and used in preclinical studies, all contain an expanded CAG repeat significantly longer than that of SBMA patients. Here, we propose the use of SBMA patients adipose-derived mesenchymal stem cells (MSCs) as a new human in vitro model to study ARpolyQ toxicity. These cells have the advantage to express only ARpolyQ, and not the wild type AR allele. Therefore, we isolated and characterized adipose-derived MSCs from three SBMA patients (ADSC from Kennedy's patients, ADSCK) and three control volunteers (ADSCs). We found that both ADSCs and ADSCKs express mesenchymal antigens, even if only ADSCs can differentiate into the three typical cell lineages (adipocytes, chondrocytes and osteocytes), whereas ADSCKs, from SBMA patients, showed a lower growth potential and differentiated only into adipocyte. Moreover, analysing AR expression on our mesenchymal cultures we found lower levels in all ADSCKs than ADSCs, possibly related to negative pressures exerted by toxic ARpolyQ in ADSCKs. In addition, with proteasome inhibition the ARpolyQ levels increased specifically in ADSCKs, inducing the formation of HSP70 and ubiquitin positive nuclear ARpolyQ inclusions. Considering all of this evidence, SBMA patients adipose-derived MSCs cultures should be considered an innovative in vitro human model to understand the molecular mechanisms of ARpolyQ toxicity and to test novel therapeutic approaches in SBMA

Modulation of Neuroinflammation in the Central Nervous System: Role of Chemokines and Sphingolipids

Neuroinflammation is a process involved in the pathogenesis of different disorders, both autoimmune, such as neuropsychiatric systemic lupus erythematosus, and degenerative, such as Alzheimer\u2019s and Parkinson\u2019s disease. In the central nervous system, the local milieu is tightly regulated by different mediators, among which are chemoattractant cytokines, also known as chemokines. These small molecules are able to modulate trafficking of immune cells in the course of nervous system development or in response to tissue damage, and different patterns of chemokine molecule and receptor expression have been described in several neuroinflammatory disorders. In recent years, a number of studies have highlighted a pivotal role of sphingolipids in regulating neuroinflammation. Sphingolipids have different functions, among which are the control of leukocyte egress from lymphonodes into inflamed tissues, the expression of various mediators of inflammation and a direct effect on the cells of the central nervous system as regulators of neuroinflammation. In the future, a better knowledge of these two groups of mediators could provide insight into the pathogenesis of neuroinflammatory disorders and could help develop novel diagnostic tools and therapeutic strategies

Correlation of Preoperative Von Willebrand Factor with Magnetic Resonance Imaging Perfusion and Permeability Parameters as Predictors of Prognosis in Glioblastoma

Background: Angiogenesis has been shown to be strictly related to tumor malignancy. Glioblastoma (GBM) is highly vascularized and von Willebrand Factor (VWF) plays a potent proangiogenic role. Dynamic contrast-enhanced and dynamic susceptibility contrast magnetic resonance imaging (MRI) represent a widely accepted method to assess GBM microvasculature. Our aim was to investigate the correlation between plasma VWF:Ag, permeability, and perfusion MRI parameters and examine their potential in predicting GBM patient prognosis. Methods: We retrospectively analyzed preoperative dynamic contrast-enhanced, dynamic susceptibility contrast MRI, and VWF:Ag level of 26 patients with GBM. We assessed the maximum values of relative cerebral blood flow and volume, volume transfer constant K trans , plasma volume (V p ) and reflux rate constant between fractional volume of the extravascular space and blood plasma (K ep ). Nonparametric Mann-Whitney test and Kaplan-Meier survival analyses were conducted and a P value < 0.05 was considered statistically significant. Results: The median VWF:Ag value was 248 IU/dL and the median follow-up duration was about 13 months. We divided patients according to low-VWF:Ag and high-VWF:Ag and we found significant differences in the median follow-up duration (19 months vs. 10 months; P = 0.04) and in K trans (0.31/minute vs. 0.53/minute; P = 0.02), and K ep (1.79/minute vs. 3.89/minute; P = 0.005) values. The cumulative 1-year survival was significantly shorter in patients with high-VWF:Ag and high-K ep compared with patients with low-VWF:Ag and low-K ep (37.5% vs. 68%; P = 0.05). Conclusions: These findings, in a small group of patients, suggest a role for VWF:Ag, similar to K trans , and K ep as a prognostic indicator of postoperative survival of patients with GBM

A sphingosine-1-phosphate autocrine loop promotes proliferation and stemness of glioblastoma stem cells

Sphingosine-1-phosphate (S1P) is an onco-promoter lipid that, after interaction with specific membrane receptors, can influence different cell properties strictly related to cancer. Increasing evidence indicates that S1P acts as a key regulator of growth, invasion, and therapy-resistance in human glioblastoma (GBM), the most common and fatal intracranial cancer in adults. Recent studies support that GBM contain a subpopulation of cells, named glioblastoma stem cells (GSCs), that plays a crucial role in tumor initiation, maintenance, and malignant progression. So far, little is known on the origin and role of S1P in GBM. In this study, we investigated the possible role of S1P in GSC proliferation and stemness. To this purpose, among different GSC lines prepared in our laboratory from GBM specimens, we selected two lines, representative of slow- and fast-proliferating cells, and named S-GSCs and F-GSCs, respectively. These GSCs demonstrated heterogeneity not only in their proliferative potential, but also in the expression of stemness markers. Indeed, the fast proliferative status of the F-GSC population was paralleled by a significant higher expression of stemness parameters than that of S-GSCs. Metabolic studies revealed that both GSC lines constitutively exhibit the property to rapidly export S1P into the extracellular microenvironment. Intriguingly, the proliferative properties of GSCs were related to an efficient secretion of newly produced S1P. Indeed, in the fast-proliferating cells, the extracellular S1P level was found up to 10-fold higher than that of slow-proliferating ones, suggesting that the high extent of S1P released by F-GSCs reflects, and most probably participates to their proliferative and stemness features. In addition, the presence of EGF and bFGF potentiated the constitutive capacity of GSCs to secrete newly synthesized S1P, indicating that cooperation between S1P and these growth factors is of relevance in the maintenance and proliferation of GSCs. For the first time, we then report that S1P is able to act as a survival, proliferative, and pro-stemness factor for GSCs, promoting both cell cycle progression and stemness phenotypic profile. These effects were counteracted by FTY720 (a precursor of S1P receptor inhibitor), implying S1P specific receptors in its GSC-stimulating effects. In conclusion, this work implicates S1P to be an autocrine/paracrine mediator acting as a mitogenic and stemness-favoring factor, through direct effects in GSCs, and possibly through the induction of their niche. This suggests that the inhibition of S1P release from GSCs and/or of S1P receptors could be a valuable strategy to curtail GBM progression

Adipose mesenchymal stem cells and &#8220;regenerative adipose tissue graft&#8221; (lipogems&#8482;) for musculoskeletal regeneration

Regenerative medicine is a high-potential sector of strategic developments in medicine and health industry. The perspective to cure diseases up to now relied on medical treatments of long duration and limited effectiveness, and the possibility to avoid organ transplantation renders regenerative medicine attractive. In recent years, basic and translation research held great hope for this new field with significant progress in the modulation of stem cell commitment in vitro and providing protocols for targeted clinical applications. In line with this approach, mesenchymal stem cells (MSCs) have been introduced as potential therapeutic tools to correct the breakdown of musculoskeletal disorders. MSCs are able to secrete a large number of trophic factors capable of repairing the recipient tissue through angiogenic, anti-apoptotic and anti-fibrotic mechanisms. In this context, adipose tissue is emerging as a clinically relevant and easy to harvest source of multipotent progenitors to develop regenerative therapies. The present review focuses on the clinical application of MSCs, and in particular of adipose-derived stem cells, in the musculoskeletal disorders and on the current scientific challenges. In this perspective, we discuss future developments of an innovative system (Lipogems) for musculoskeletal regeneration, yielding a non-expanded and ready-touse microfractured fat tissue product harbouring MSCs and pericytes within a preserved stromal vascular niche. The Lipogems system may also pave the way for future off-the-shelf and large-scale approaches for reconstructive procedures and regenerative medicine

Autocrine sphingosine-1-phosphate fuels growth and stemness in glioblastoma stem cells

Sphingosine-1-phosphate (S1P) is an onco-promoter lipid that, after interaction with specific membrane receptors, acts as a key regulator of growth, invasion, and therapy-resistance of different tumors, including human glioblastomas (GBMs). These are the most common and lethal primary brain cancer in adults, exhibiting a dismal prognosis, despite diverse therapeutic approaches. Accumulating reports suggest that human GBMs contain glioblastoma stem cells (GSCs), a small subpopulation of cells determinant in tumor growth, and malignant progression. Little is known about the role of S1P in GSCs. Using GSCs derived from human GBM specimens with different proliferative index and stemness marker expression, we investigated the possible role of S1P in the proliferative and stemness properties of GSCs. Metabolic studies demonstrated that GSCs can rapidly export newly synthesized S1P, this process being enhanced in fast proliferating cells. Released S1P levels reached nM concentrations in response to increased extracellular sphingosine. Moreover, EGF and bFGF, recognized autocrine factors in GSC, potentiated the constitutive capacity of GSCs to secrete S1P, suggesting that cooperation between S1P and these growth factors is of relevance in GSC maintenance and proliferation. Of relevance, we then found that S1P is able to act as a proliferative and pro-stemness autocrine factor for GSCs, promoting both cell cycle progression and stemness phenotypic profile, in a receptor-dependent fashion. Overall, our results suggest that the GSC population is critically modulated by microenvironmental S1P, that acts as an autocrine signal to maintain a pro-stemness microenvironment and favoring GSC survival, proliferation and maintenance of stem properties. These findings could open novel opportunities for the development of effective treatments for GBMs

Significance and prognostic value of the coagulation profile in patients with glioblastoma: implications for personalized therapy

BACKGROUND: Coagulation is an important aspect of the vascular microenvironment in which brain tumors evolve. Tumor patients often show aberrant coagulation and fibrinolysis activation. In particular, Glioblastoma (GBM), the most aggressive primary brain tumor, is associated with a state of hypercoagulability, and venous thromboembolism (VTE) is a common complication of this cancer and its treatment. Our study aims to investigate the clinical and prognostic significance of routine laboratory tests to assess the coagulative state of patients with brain tumors, in order to identify potential new prognostic factors and targets for personalized therapy. METHODS: Blood samples were collected from GBM (n=58) and meningioma (MNG, n=22) patients, before any treatment. The parameters analysed were: prothrombin time (PT), activated partial thromboplastin time (aPTT), D-Dimer (DD), fibrinogen (FB), von Willebrand factor (VWF), leukocyte count and haemoglobin levels. RESULTS: Plasma levels of PT and aPTT were significantly reduced in GBMs compared to MNGs (p <0.05), whereas DD, VWF:Ag levels, and leukocyte count were significantly higher in GBMs than MNGs (p <0.01). Furthermore, we observed that GBM patients with reduced PT and aPTT and high levels of DD e VWF, defined as hypercoagulable patients, showed reduced overall survival (p<0.05) compared to non-hypercoagulable patients. CONCLUSION: Our data support the assumption that GBM patients show a plasma hypercoagulable profile and that coagulation profile is related to adverse outcome in patients with GBM. If confirmed, hypercoagulability could play an important role as a prognostic factor of the disease and in the decision of an antithrombotic prophylaxis

Isolation and expansion of human and mouse brain microvascular endothelial cells

Brain microvascular endothelial cells (BMVECs) have an important role in the constitution of the blood-brain barrier (BBB). The BBB is involved in the disease processes of a number of neurological disorders in which its permeability increases. Isolation of BMVECs could elucidate the mechanism involved in these processes. This protocol describes how to isolate and expand human and mouse BMVECs. The procedure covers brain-tissue dissociation, digestion and cell selection. Cells are selected on the basis of time-responsive differential adhesiveness to a collagen type I-precoated surface. The protocol also describes immunophenotypic characterization, cord formation and functional assays to confirm that these cells in endothelial proliferation medium (EndoPM) have an endothelial origin. The entire technique requires 3c7 h of active time. Endothelial cell clusters are readily visible after 48 h, and expansion of BMVECs occurs over the course of 3c60 d

Human and mouse brain-derived endothelial cells require high levels of growth factors medium for their isolation, in vitro maintenance and survival

BACKGROUND: Brain microvascular endothelial cells (BMVECs) constitute the primary limitation for passage of ions and molecules from the blood into the brain through the blood brain barrier. Numerous multi-step procedures for isolating and culturing BMVECs have been described. However, each one demonstrates major limitations in purity of culture and/or low proliferation rate. Our goal was to study the efficiency of our pending patent medium, Endothelial Proliferation Medium (EndoPM), on the isolation and purification of human and murine BMVECs. METHODS: BMVECs, cultured in EndoPM were compared to those cultured in a commercial medium EBM. Cultures were characterized by flow cytometric analysis, lineage differentiation, the ability to form tube-like structure, immunofluorescence, molecular analyses and also in an in vivo model assay. Moreover permeability was assayed by monitoring the passage of Dextran-FITC through a tight monolayer of BMVECs grown to confluence in Boyden chambers. One way Anova two-tailed test was utilized for all statistical analyses. RESULTS: The properties of ECs in human and murine BMVECs is confirmed by the expression of endothelial markers (CD31, CD105, CD146, Tie-2 and vWF), of representative proangiogenic genes (ICAM1, VCAM1 and integrin ITGAV), of considerable tube-forming ability, with low-density lipoprotein uptake, eNOS and GLUT-1 expression. Furthermore cells are able to express markers of the junctional architecture as VE-cadherin, beta-catenin and Claudin-5 and greatly reduce dextran permeability as barrier functional test. Moreover BMVECs spontaneously organize in vascular-like structures and maintain the expression of endothelial markers in an in vivo xenograft model assay. The significant effect of EndoPM is confirmed by the study of proliferation index, survival index and the behaviour of BMVECs and fibroblasts in co-culture conditions. CONCLUSION: Herein we describe a simple and reproducible method for the isolation and expansion of human and mouse BMVECs, based on a newly formulated medium (EndoPM) with optimized concentration of growth factors (EGF, FGF-2 and Bovine Brain Extract-BBE). This procedure should facilitate the isolation and expansion of human and mouse BMVECs with extended lifetime, good viability and purity. This approach may provide an effective strategy to aid phenotypical and functional studies of brain vessels under physiological and pathological conditions

Mechanical loading of intervertebral disc modulates microglia proliferation, activation, and chemotaxis

Objective: The aim of the study is to assess the effects of the neuroinflammatory microenvironment of a mechanically-induced degenerating intervertebral disc (IVD) on neuroinflammatory like cells such as microglia, in order to comprehend the role of microglial cells in degenerative disc disease. Methods: Bovine caudal IVDs were kept in culture in an ex vivo bioreactor under high frequency loading and limited nutrition or in free swelling conditions as control samples. Conditioned media (CM) were collected, analysed for cytokine and neurotrophin content and applied to microglial cells for neuroinflammatory activation assessment. Results: Degenerative conditioned medium (D-CM) induced a higher production of interleukin (IL)-8, nerve growth factor (NGF), interferon (IFN)-\u3b3 IL-17 from IVD cells than unloaded control conditioned medium (U-CM). Upon 48 h of co-incubation with microglia, D-CM stimulated microglia proliferation, activation, with increased expression of ionized calcium binding adaptor molecule 1 (IBA1) and CD68, and chemotaxis. Moreover, an increment of nitrite production was observed. Interestingly, D-CM caused an upregulation of IL-1\u3b2 IL-6, tumour necrosis factor \u3b1 (TNF\u3b1), inducible NO synthase (iNOS), IBA1, and vascular endothelial growth factor (VEGF) genes in microglia. Similar results were obtained when microglia were treated with the combination of the measured cytokines. Conclusions: Our findings show that in IVD degenerative microenvironment, IL-8, NGF, IFN-\u3b3 IL-17 drive activation of microglia in the spinal cord and increase upregulation of neuroinflammatory markers. This, in turn, enhances the inflammatory milieu within IVD tissues and in the peridiscal space, aggravating the cascade of degenerative events. This study provides evidence for an important role of microglia in maintaining IVD neuroinflammatory microenvironment and probably inducing low back pain