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Summary Current leukaemia therapy focuses on increasing chemotherapy efficacy. Mesenchymal stromal cells (MSCs) have been proposed for carrying and delivery drugs to improve killing of cancer cells. We have shown that MSCs loaded with Paclitaxel (PTX) acquire a potent anti-tumour activity. We investigated the effect of human MSCs (hMSCs) and mouse SR4987 loaded with PTX (hMSCsPTX and SR4987PTX) on MOLT-4 and L1210, two leukaemia cell (LCs) lines of human and mouse origin, respectively. SR4987PTX and hMSCsPTX showed strong anti-LC activity. hMSCsPTX, co-injected with MOLT-4 cells or intra-tumour injected into established subcutaneous MOLT-4 nodules, strongly inhibited growth and angiogenesis. In BDF1-mice-bearing L1210, the intraperitoneal administration of SR4987PTX doubled mouse survival time. In vitro, both hMSCs and hMSCsPTX released chemotactic factors, bound and formed rosettes with LCs. In ultrastructural analysis of rosettes, hMSCsPTX showed no morphological alterations while the attached LCs were apoptotic and necrotic. hMSCs and hMSCsPTX released molecules that reduced LC adhesion to microvascular endothelium (hMECs) and down-modulated ICAM1 and VCAM1 on hMECs. Priming hMSCs with PTX is a simple procedure that does not require any genetic cell manipulation. Once the effectiveness of hMSCsPTX on established cancers in mice is proven, this procedure could be proposed for leukaemia therapy in humans.

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 ∼7 h of active time. Endothelial cell clusters are readily visible after 48 h, and expansion of BMVECs occurs over the course of ∼60 d.

OBJECTIVE: End-stage coagulation and the structure/function of fibrin are implicated in the pathogenesis of ischemic stroke. We explored whether genetic variants associated with end-stage coagulation in healthy volunteers account for the genetic predisposition to ischemic stroke and examined their influence on stroke subtype.METHODS: Common genetic variants identified through genome-wide association studies of coagulation factors and fibrin structure/function in healthy twins (n = 2,100, Stage 1) were examined in ischemic stroke (n = 4,200 cases) using 2 independent samples of European ancestry (Stage 2). A third clinical collection having stroke subtyping (total 8,900 cases, 55,000 controls) was used for replication (Stage 3).RESULTS: Stage 1 identified 524 single nucleotide polymorphisms (SNPs) from 23 linkage disequilibrium blocks having significant association (p < 5 × 10(-8)) with 1 or more coagulation/fibrin phenotypes. The most striking associations included SNP rs5985 with factor XIII activity (p = 2.6 × 10(-186)), rs10665 with FVII (p = 2.4 × 10(-47)), and rs505922 in the ABO gene with both von Willebrand factor (p = 4.7 × 10(-57)) and factor VIII (p = 1.2 × 10(-36)). In Stage 2, the 23 independent SNPs were examined in stroke cases/noncases using MOnica Risk, Genetics, Archiving and Monograph (MORGAM) and Wellcome Trust Case Control Consortium 2 collections. SNP rs505922 was nominally associated with ischemic stroke (odds ratio = 0.94, 95% confidence interval = 0.88-0.99, p = 0.023). Independent replication in Meta-Stroke confirmed the rs505922 association with stroke, beta (standard error, SE) = 0.066 (0.02), p = 0.001, a finding specific to large-vessel and cardioembolic stroke (p = 0.001 and p = < 0.001, respectively) but not seen with small-vessel stroke (p = 0.811). INTERPRETATION: ABO gene variants are associated with large-vessel and cardioembolic stroke but not small-vessel disease. This work sheds light on the different pathogenic mechanisms underpinning stroke subtype.

2039Background: In the two stage phase I-II study DENDR-I, 24 patients with first diagnosis of glioblastoma (GBM) were treated with DC immunotherapy associated to standard radio-chemotherapy. To go...

Stem cells isolated from the CNS of both embryonic and adult mice undergo extensive proliferation in the presence of epidermal growth factor (EGF). Removal of EGF determines the differentiation of these cells into neurons and glia. We have recently demonstrated that basic fibroblast growth factor (bFGF) regulates the proliferation of EGF-generated progenitors of the embryonic mouse striatum. We report here that bFGF induces proliferation of some EGF-generated precursors of the adult mouse striatum which, in turn, differentiate in vitro into cells possessing neuron-like morphology and neuronal antigenic properties. These results demonstrate that EGF and bFGF can act sequentially to regulate the de novo generation of neurons from the adult mouse CNS in vitro and suggest the existence of a lineage relationship between EGF- and bFGF-responsive progenitor cells of the adult murine brain.

A critical requirement of an efficient cancer immunotherapy is the generation of long-lasting, specific memory. In a clinical trial active at Istituto Besta, first diagnosis glioblastoma (GBM) patients, with post-surgery volume ≤10 cc, underwent leukapheresis before radiotherapy and chemotherapy with the alkylating agent temozolomide (TMZ). Three intradermal injections of mature dendritic cells (DCs) loaded by autologous tumor lysates were done before adjuvant chemotherapy. Subsequent 4 injections were performed in association with six cycles of adjuvant TMZ. Peripheral blood lymphocytes (PBLs) were analyzed by flow cytometry to characterize immune response before and after DC vaccines. The ratio of vaccination/baseline frequencies and counts (V/B ratio) of all of the immunological parameters for each patient was calculated, and the median of all of the observations used as the cut off value to separate patients. V/B ratio was correlated with the progression free survival (PFS) of each patient. Preliminary results from the interim analysis on 24 patients showed that an increased NK, but not CD8+ T cell response was significantly associated with prolonged survival (p In a group of responder patients (PFS>12), during the first three vaccines, CD8+ T cells underwent a rapid expansion and produced higher levels of IFN-γ compared to the baseline (p After the third vaccine and TMZ administration, primed CD8+ T cells failed to form an effector memory phenotype (CCR7negCD45RAnegCD62Llow/neg). Our results support a possible interference of adjuvant chemotherapy on effector memory formation. Further investigations are required to understand how memory T cells behave in response to repeated exposure to TMZ. Citation Format: Serena Pellegatta, Marica Eoli, Elena Anghileri, Sara Pessina, Carlo Antozzi, Simona Frigerio, Gabriele Cantini, Maria Grazia Bruzzone, Bianca Pollo, Eugenio A. Parati, Gaetano Finocchiaro. CD8+T cells fail to form an effector memory in glioblastoma patients treated with dendritic cell immunotherapy in combination with chemotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A031.

Background Percutaneous patent foramen ovale (PFO) closure is accepted as treatment for cryptogenic ischemic stroke/transient ischemic attack in young subjects. However, a thorough evaluation of residual right-to-left shunt (rRLS) after PFO closure is needed. Our aims were to analyze the characteristics related to PFO diagnosis and closure, focusing on rRLS and clinical recurrences until 24-month follow-up. Data were extrapolated from the 12-month Italian PFO Survey. Methods In all, 1035 patients were included. PFO diagnosis and right-to-left shunt (RLS) were assessed by contrast-enhanced transesophageal and/or transthoracic echocardiography and/or transcranial Doppler. Results PFO diagnosis with RLS data were available in 894 of 1035 (86.4%) patients. rRLS was investigated in 49.6% (6 months), 27.1% (12 months), and 3.5% (24 months), and observed in 19.5% (6 months) and 18.2% (12 months) of subjects. Large permanent rRLS was observed in less than 3% of RLS-positive patients after 1 year. Eleven of 14 and 3 of 14 neurological recurrences were observed in 10 of 444 (2.25%) and 2 of 243 (0.8%) patients within the 6- and 12-month follow-up, respectively. Among these, no large rRLS was reported. There were no neurological events at 2-year follow-up. Forty of 444 subjects had non-neurological complications, mostly cardiac arrhythmias within the sixth month. Conclusions PFO closure is a safe procedure. rRLS is not uncommon but large rRLS is rare. Clinical complications, mostly related to cardiac arrhythmias, are not unusual. Evaluation of the data of the whole survey is underway.

From the literature, the prevalence of fluorodeoxyglucose (FDG) uptake in large artery atherosclerotic plaques shows great heterogeneity. We retrospectively reviewed 100 consecutive patients who underwent FDG-positron emission tomography-computed tomography (PET/CT) imaging of their whole body, to evaluate FDG uptake in the arterial wall.We retrospectively evaluated 100 whole-body PET-CT scans. The PET images coregistered with CT were reviewed for abnormal 18F-FDG uptake. The mean standard uptake value (SUV) was measured in regions of interest (ROIs). The prevalence of PET+ plaques was determined based on the qualitative PET review, used as the gold standard in a receiver-operating characteristic (ROC) curve analysis to determine an optimal threshold for the quantitative PET analysis.The qualitative, visual assessment demonstrated FDG uptake in the arterial walls of 26 patients. A total of 85 slices exhibited FDG uptake within the arterial wall of 37 artery locations. 11, 17, and 2 patients exhibited FDG uptake within the wall of carotid arteries, of the aorta, and of the iliac arteries, respectively. Only 4 of the 26 patients had positive FDG uptake in more than one artery location. In terms of quantitative analysis, a threshold of 2.8 SUV was associated with a negative predictive value of 99.4% and a positive predictive value of 100% to predict qualitative PET+ plaques. A threshold of 1.8 SUV was associated with a negative predictive value of 100% and a positive predictive value of 99.4%. Area under the ROC curve was .839.The prevalence of PET uptake in arterial walls in a consecutive population of asymptomatic patients is low and usually confined to one type of artery, and its clinical relevance in terms of vulnerability to ischemic events remains to be determined.

Background and Purpose— Ischemic stroke (IS) shares many common risk factors with coronary artery disease (CAD). We hypothesized that genetic variants associated with myocardial infarction (MI) or CAD may be similarly involved in the etiology of IS. To test this hypothesis, we evaluated whether single-nucleotide polymorphisms (SNPs) at 11 different loci recently associated with MI or CAD through genome-wide association studies were associated with IS. Methods— Meta-analyses of the associations between the 11 MI-associated SNPs and IS were performed using 6865 cases and 11 395 control subjects recruited from 9 studies. SNPs were either genotyped directly or imputed; in a few cases a surrogate SNP in high linkage disequilibrium was chosen. Logistic regression was performed within each study to obtain study-specific βs and standard errors. Meta-analysis was conducted using an inverse variance weighted approach assuming a random effect model. Results— Despite having power to detect odds ratio of 1.09–1.14 for overall IS and 1.20–1.32 for major stroke subtypes, none of the SNPs were significantly associated with overall IS and/or stroke subtypes after adjusting for multiple comparisons. Conclusions— Our results suggest that the major common loci associated with MI risk do not have effects of similar magnitude on overall IS but do not preclude moderate associations restricted to specific IS subtypes. Disparate mechanisms may be critical in the development of acute ischemic coronary and cerebrovascular events.

Hepatocellular carcinoma (HCC) is a very angiogenic and malignant cancer. Conventional chemotherapy is poorly effective because of the abnormal structural organization of HCC-infiltrating vessels. In previous work, we demonstrated that HCC angiogenesis is driven by transforming growth factor beta-1(TGF-β1)/CD105 axis, stimulating liver-derived microvascular endothelial cells (Ld-MECs) migration. As TGF-β1 also affects mural cells (MCs) recruitment and maturation, we asked whether it may contribute to HCC-induced vascular abnormalities. HCC and adjacent non-neoplastic liver (nNL) biopsies obtained from 12 patients were analyzed by immunohistochemistry for angiogenic markers CD105, TGF-β1, CD44 and vascular endothelial growth factor-a (VEGFa) and for MC markers NG2, α-smooth muscle actin (αSMA) and neural cell adhesion molecule (NCAM). The same markers were also investigated by immunocytochemistry on cultured HCC-derived stromal cells (HCC-StCs) and nNL-derived StCs (nNL-StCs) isolated from the same liver biopsies. Angiogenic factors released by StCs were analyzed by ELISA and the interaction between StCs and Ld-MECs by adhesion assay. Compared with nNL, HCC biopsies showed increased angiogenic markers and αSMA that was localized in vessels. By contrast, NG2 and NCAM were substantially localized in tumor cells but absent in vessels and stroma. Cultured HCC-StCs showed less expression of NG2, αSMA and NCAM. They also demonstrated a lower capacity to release angiogenic factors and adhered on Ld-MECs. HCC-StCs and nNL-StCs treated with TGF-β1 or with of HepG2 (a human hepatoma cell line) derived conditioned medium (CM), down-modulated NCAM expression, whereas anti-NCAM antibodies significantly reduced the adhesion of StCs to Ld-MECs. By further blocking TGF-β1 with anti-TGF-β1 antibodies or with Ly-364947 (a specific inhibitor TGF-β1-receptor) adhesion to Ld-MECs and NCAM expression respectively was partially restored. TGF-β1 contributes to HCC-induced vascular alterations by affecting the interaction between HCC-StCs and Ld-MECs through a down-modulation of NCAM expression.

Complex microenvironmental stimuli influence neural cell properties. To study this, we developed a three-dimensional (3-D) neural culture system, composed of different populations including neurons, astrocytes, and neural stem cells (NSCs). In particular, these last-mentioned cells represent a source potentially exploitable to test drugs, to study neurodevelopment and cell-therapies for neuroregenerations. On seeding on matrigel in a medium supplemented with serum and mitogens, cells obtained from human fetal brain tissue formed 3-D self-organizing neural architectures. Immunocytochemical analysis demonstrated the presence of undifferentiated nestin+ and CD133+ cells, surrounded by β-tub-III+ and GFAP+ cells, suggesting the formation of niches containing potential human NSCs (hNSCs). The presence of hNSCs was confirmed by both neurosphere assay and RT-PCR, and their multipotentiality was demonstrated by both immunofluorescent staining and RT-PCR. Flow cytometry analysis revealed that neurosphere forming cells originating from at least two different subsets expressing, respectively, CD133 and CD146 markers were endowed with different proliferative and differentiation potential. Our data implicate that the complexity of environment within niches and aggregates of heterogeneous neural cell subsets may represent an innovative platform for neurobiological and neurodevelopmental investigations and a reservoir for a rapid expansion of hNSCs.

The goal of this study was to compare the rate of cerebral microembolization during carotid artery stenting (CAS) with proximal versus distal cerebral protection in patients with high-risk, lipid-rich plaque.Cerebral protection with filters partially reduces the cerebral embolization rate during CAS. Proximal protection has been introduced to further decrease embolization risk.Fifty-three consecutive patients with carotid artery stenosis and lipid-rich plaque were randomized to undergo CAS with proximal protection (MO.MA system, n = 26) or distal protection with a filter (FilterWire EZ, n = 27). Microembolic signals (MES) were assessed by using transcranial Doppler during: 1) lesion wiring; 2) pre-dilation; 3) stent crossing; 4) stent deployment; 5) stent dilation; and 6) device retrieval/deflation. Diffusion-weighted magnetic resonance imaging was conducted before CAS, after 48 h, and after 30 days.Patients in the MO.MA group had higher percentage diameter stenosis (89 ± 6% vs. 86 ± 5%, p = 0.027) and rate of ulcerated plaque (35% vs. 7.4%; p = 0.019). Compared with use of the FilterWire EZ, MO.MA significantly reduced mean MES counts (p0.0001) during lesion crossing (mean 18 [interquartile range (IQR): 11 to 30] vs. 2 [IQR: 0 to 4]), stent crossing (23 [IQR: 11 to 34] vs. 0 [IQR: 0 to 1]), stent deployment (30 [IQR: 9 to 35] vs. 0 [IQR: 0 to 1]), stent dilation (16 [IQR: 8 to 30] vs. 0 [IQR: 0 to 1]), and total MES (93 [IQR: 59 to 136] vs. 16 [IQR: 7 to 36]). The number of patients with MES was higher with the FilterWire EZ versus MO.MA in phases 3 to 5 (100% vs. 27%; p0.0001). By multivariate analysis, the type of brain protection was the only independent predictor of total MES number. No significant difference was found in the number of patients with new post-CAS embolic lesion in the MO.MA group (2 of 14, 14%) as compared with the FilterWire EZ group (9 of 21, 42.8%).In patients with high-risk, lipid-rich plaque undergoing CAS, MO.MA led to significantly lower microembolization as assessed by using MES counts.

BACKGROUND: Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation. METHODS AND PRINCIPAL FINDINGS: Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth. CONCLUSIONS: These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.

Physical stress induces changes in immune system parameters; these changes depend on effort schedule and are influenced by customary training. The mechanisms whereby they take place are not fully elucidated: sympathetic activation-mediated mobilization of cells of lymphoid organs, including the spleen, has been suggested. We studied exercise-induced changes (20' of cycloergometer test conducted up to 80% of maximal expected heart rate) in white blood cells (WBC), lymphocyte subsets, plasma catecholamine and cortisol levels in three groups of subjects: swimmers, untrained controls and splenectomized individuals. Physical exercise induced increase of WBC and significant changes in the percentage of most investigated lymphocyte subsets (NK, CD3+, CD4+, CD8+ and CD4/CD8 ratio), except for DR+ cells. No changes occurred in the percentage of CD14+ cells. Norepinephrine (NE) levels increased after effort, while epinephrine (E) and cortisol levels were unchanged. Splenectomized patients displayed similar effort-induced changes in investigated parameters as controls and swimmers. The results support the interactions between physical work and immune response and minimize the role of the spleen in this context.

Primary malignant central nervous system (CNS) tumors only represent about 2% of all cancers. However, they are very often associated with high morbidity and mortality. Despite current standard-of-care therapy, such as surgery, irradiation, and chemotherapy, neither cure nor any toxic therapy against malignant CNS tumors has been developed so far. Nanotechnology may alter this situation. It offers a new promise for cancer diagnosis and treatment. This emerging technology, by developing and manufacturing materials using atomic and molecular elements, can provide a platform for the combination of diagnostics, therapeutics and delivery to the tumor, with subsequent monitoring of the response. This review focuses on recent developments in cancer nanotechnology with particular attention to nanoparticle systems, important tools for the improvement of drug delivery in brain tumor. The latest advances in both the research sector and in recent patents for cancer imaging and therapy are discussed.

In the present study, we investigated whether cultured astrocytes derived from adult neural precursor cells (NPCs) obtained from the subventricular zone (SVZ) of wobbler mice display metabolic traits of the wobbler astrocytes in situ and in primary culture. We also utilized NPC-derived astrocytes as a tool to investigate the involvement of astrocytes in the molecular mechanism of MND focusing on the possible alteration of glutamate reuptake since excitotoxicity glutamate-mediated may be a contributory pathway. NPC-derived wobbler astrocytes are characterized by high immunoreactivity for GFAP, significant decrease of glutamate uptake and reduced immunoreactivity for glutamate transporters GLT1 and GLAST. Spinal cord motor neurons obtained from healthy mouse embryos, when co-cultured with wobbler NPC-derived astrocytes, show reduced viability and morphologic alterations. These suffering motor neurons are caspase-7 positive, and treatment with anti-apoptotic drug V5 increases cell survival. Physical contact with wobbler astrocytes is not essential because purified motor neurons display reduced survival also when treated with the medium conditioned by wobbler NPC-derived astrocytes. Toxic levels of glutamate were revealed by HPLC assay in the extracellular medium of wobbler NPC-derived astrocytes, whereas the level of intracellular glutamate is reduced if compared with controls. Moreover, glutamate receptor antagonists are able to enhance motor neuron survival. Therefore, our results demonstrate that astrocytes derived from wobbler neural precursor cells display impaired glutamate homeostasis that may play a crucial role in motor neuron degeneration. Finally, the cultured astrocytes derived from NPCs of adult mice may offer a useful alternative in vitro model to study the molecular mechanisms involved in neurodegeneration.

Caliber fluctuations of extra- and intracranial arteries, mostly related to vasospasm, are often recognized in various neurological conditions. We report a case of a 33-year-old woman affected by migraine with and without aura who exhibited a possible cervical internal carotid artery vasospasm, detected by ultrasound, before a typical migraine aura.

Many lines of research have suggested a relationship between migraine with aura (MA) and patent foramen ovale. Right-to-left shunt (RLS) of blood might explain both the occurrence of MA attacks, as well as the increased risk of ischaemic stroke in these patients. We evaluated the prevalence and the characteristics of RLS in a series of 120 MA patients, who were studied with contrast-enhanced Transcranial Doppler examination. We found RLS in 61 of them. A latent RLS was found in 28%, a permanent RLS in 72%, a shower-curtain pattern was detected in 52% of the studied patients.

Over the last decade, improvements in cancer therapies have prolonged the lives of cancer patients. Despite dramatic advances in imaging technology, surgical techniques, and adjuvant radio- and chemotherapy, the overall prognosis of this disease remains dismal. In light of this, there is an urgent need for the development of more effective therapies that can target residual disseminated tumor burden. Given the heterogeneity of tumors in general, no one strategy is likely to provide a satisfactory treatment regimen. Until the middle of the 20th century, medical treatments were limited to options like drugs, surgery, antibiotics, and radiation, but in the last years stem cells, due to their pathotropism, have become particularly attractive candidates not only to replace damaged tissue in degenerative pathologies, but also to deliver therapeutic molecules in patients with disseminated metastatic cancer. Worldwide there have been over 2000 patent applications involving human and non-human stem cells, of which one quarter refer to embryonic stem cells. Over one third of all stem cell applications and one quarter of all embryonic stem cell applications have been granted. The aim of this review is primarily to focus on the recent development of stem cell patents in cancer treatments.

To report experience on the use of self-closing nitinol U-Clips for different types of intracranial arterial microanastomosis.We treated 7 patients (3 females and 4 males, age ranging from 25 to 68 yo) admitted from November 2005 to January 2008 to the Neurological Institute C. Besta of Milan. One patient had cerebral hypoperfusion and the others a complex intracranial aneurysm. In each patient a bypass procedure was completed by using self-closing Nitinol U-Clips for intracranial arterial microanastomoses.The total time of temporary occlusion was 15.71 +/- 4.386 min. Bypass patency was confirmed intraoperatively by near-infrared indocyanine green videoangiography and microdoppler in each patient. No spasm of the graft was encountered and immediate post-operative bypass patency was confirmed in 6/7 patients. The graft thrombosed in 1 patient with antiphospholipid syndrome. 1 patient died from a massive Subarachnoid Hemorrhage due to rupture of an aneurysm while waiting for an endovascular procedure. In the 5 patients at the last follow-up, long-term patency of the bypass was confirmed and no neurological deficits occurred related to the procedure.This is the first report of the use of U-Clips for intracranial microanastomosis. Our data indicated that it is a safe technique, reduces the time taken to perform an anastomosis and the risk of an ischemic complication. Further studies of the longer-term patency of bypass as performed with U-Clips are required.

Response: We wish to thank Dr Tsivgoulis and colleagues for their kind appreciation of our article, and we are pleased to answer their questions. During TCD testing, blood pressure and heart rate were not systematically investigated. However, no patients reported subjective complaints that could be attributable to orthostatic intolerance, and heart rate, as recorded on the spectral display, did not change appreciably from the recumbent …

Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human tumors. Transforming growth factor-β1 (TGF-β1) and its coreceptor CD105 have been shown to contribute to HCC malignant progression. TGF-β1 and CD105 have also been implicated in angiogenesis, but their role in the vascularization of HCC has not been investigated. To fill this gap, we studied the effect of TGF-β1 and CD105 on HCC-derived endothelium. By using immunomagnetic beads, we isolated and cultured endothelial cells (ECs) from HCC (HCC-EC) and adjacent nonneoplastic tissue (nNL-ECs) obtained from 24 liver biopsies. HCC and nNL biopsies were also analyzed by immunohistochemistry for the expression of CD105, TGF-β1, Ve-cadherin (Ve-cad), CD44, β-catenin, and E-cadherin. Compared with nNL-ECs, HCC-ECs had higher expression of CD105, enhanced spontaneous motility, and greater capacity to migrate in response to TGF-β1 (5 ng/mL), particularly in the presence of a fibronectin matrix. The chemotactic effect of TGF-β1 was blocked by anti-CD105 antibodies and correlated with the grade of HCC malignancy. Histologic examination of HCC biopsies showed that HCCs with the worse malignant features had the highest expression of TGF-β1, CD105, and angiogenic markers (Ve-cad and CD44). Because CD105 was highly expressed in microvessels at the tumor periphery and TGF-β1 staining was only found in neoplastic hepatocytes, we conclude that HCC-derived TGF-β1 may act as a chemoattractant for CD105-expressing ECs and as a promoter of tumor angiogenesis. Thus, drugs that selectively target the TGF-β1/CD105 axis may interfere with HCC-related angiogenesis and HCC progression. [Cancer Res 2008;68(20):8626–34]

Background and Purpose— Right to left shunt is involved in conditions in which postural changes may be pathogenically relevant. The aim of this work was to assess the frequency of posturally dependent right to left shunt. Methods— In 109 consecutive right to left shunt-positive subjects (male/female=40/69, age 43±12 years), we assessed with contrast-enhanced transcranial Doppler the bubble load during normal breathing and after the Valsalva maneuver in both standing and recumbent position randomizing the order of testing. Results— During normal breathing, the average bubble count was 11±20 in the recumbent and 26±60 in the standing position. After the Valsalva maneuver, it was 40±38 and 42±37, respectively. The increase of bubble load in standing position occurred in 42% of patients and was independent of the order of testing. Conclusions— The amount of permanent right to left shunt is posture-dependent in 40% of patients. Testing in the sitting position may thus be warranted in doubtful or inconclusive results obtained with the subject in the horizontal position.

Background Treatment with neural stem cells represents a potential strategy to improve functional recovery of post-ischemic cerebral injury. The potential benefit of such treatment in acute phases of human ischemic stroke depends on the therapeutic viability of a systemic vascular delivery route. In spite of the large number of reports on the beneficial effects of intracerebral stem cells injection in experimental stroke, very few studies demonstrated the effectiveness of the systemic intravenous delivery approach. Metodology/Principal Findings We utilized a novel in vitro model of transient focal ischemia to analyze the brain distribution of neurosphere-derived cells (NCs) in the early 3 hours that follow transient occlusion of the medial cerebral artery (MCA). NCs obtained from newborn C57/BL6 mice are immature cells with self-renewal properties that could differentiate into neurons, astrocytes and oligodendrocytes. MCA occlusion for 30 minutes in the in vitro isolated guinea pig brain preparation was followed by arterial perfusion with 1×106 NCs charged with a green fluorescent dye, either immediately or 60 minutes after reperfusion onset. Changes in extracellular pH and K+ concentration during and after MCAO were measured through ion-sensitive electrodes. Conclusion/Significance It is demonstrated that NCs injected through the vascular system do not accumulate in the ischemic core and preferentially distribute in non-ischemic areas, identified by combined electrophysiological and morphological techniques. Direct measurements of extracellular brain ions during and after MCA occlusion suggest that anoxia-induced tissue changes, such as extracellular acidosis, may prevent NCs from entering the ischemic area in our in vitro model of transitory focal ischemia and reperfusion suggesting a role played by the surrounding microenviroment in driving NCs outside the ischemic core. These findings strongly suggest that the potential beneficial effect of NCs in experimental focal brain ischemia is not strictly dependent on their homing into the ischemic region, but rather through a bystander mechanism possibly mediated by the release of neuroprotective factors in the peri-infarct region.

(1990). Role of Physical Training on Immune Function: Preliminary Data. International Journal of Neuroscience: Vol. 51, No. 3-4, pp. 249-252.

Cell transplantation to repair or regenerate injured myocardium is a new frontier in the treatment of cardiovascular disease. Most studies on stem cell transplantation therapy in both experimental heart infarct and in phase-I human clinical trials have focused on the use of undifferentiated stem cells. Based on our previous observations demonstrating the presence of multipotent progenitor cells in human adult skeletal muscle, in this study we investigated the capacity of these progenitors to differentiate into cardiomyocytes. Here we show an efficient protocol for the cardiomyogenic differentiation of human adult skeletal muscle stem cells in vitro. We found that treatment with Retinoic Acid directed cardiomyogenic differentiation of skeletal muscle stem cells in vitro. After Retinoic Acid treatment, cells expressed cardiomyocyte markers and acquired spontaneous contraction. Functional assays exhibited cardiac-like response to increased extracellular calcium. When cocultured with mouse cardiomyocytes, Retinoic Acid-treated skeletal muscle stem cells expressed connexin43 and when transplanted into ischemic heart were detectable even 5 weeks after injection. Based on these results, we can conclude that human adult skeletal muscle stem cells, if opportunely treated, can transdifferentiate into cells of cardiac lineage and once injected into infarcted heart can integrate, survive in cardiac tissue and improve the cardiac function.

Purpose: Self-closing nitinol U-clips (Medtronic, Inc., Minneapolis, USA) have been used to create vascular microanastomoses by vascular surgeons. This device eliminates the need for suture management, knot tying, and surgical assistance. Therefore, a high-quality interrupted microvascular anastomosis can be obtained in a shorter time. Method: At the Istituto Nazionale Neurologico Carlo Besta of Milano in 2006, nitinol self-closing U-clips were used to create an intracranial interrupted microvascular anastomosis in three patients harboring large or giant unruptured aneurysms of the anterior circulation. In two patients an end-to-side microanastomosis between the fish-mouthed radial artery and the middle cerebral artery (M2 segment) was performed during a high-flow external carotid artery-internal carotid artery bypass procedure. The third patient required a pericallosal artery-pericallosal artery side-to-side bypass. Result: Intraoperative near-infrared indocyanine green videoangiography evidenced intraoperatively the bypass patency in all cases. The postoperative course was always uneventful. In all of the cases the aneurysm could be safely trapped or coiled after the bypass procedure. Postoperative angiography documented the successful exclusion of the aneurysm and the presence of revascularization of the distal branches of the bypassed arteries in all of the cases. Conclusion: This small series shows that U-clips can be easily employed for the microsuture of intracranial vessels. We estimate that the use of this or similar devices can contribute in the future to the reduction of temporary occlusion time and related intraoperative ischemic complications in cerebral bypass procedures. Further studies investigating the long-term patency rate of U-Clip bypass are required.

Dendritic cell (DC) immunotherapy is gaining momentum in clinical cancer immunotherapy. We set-up a phase I-II study in patients with first diagnosis of glioblastoma (GBM) in which DC immunotherapy was associated to standard radiochemotherapy. The primary goal was to evaluate progression free survival (PFS) 12 months after surgery (PFS-12) as defined by RANO criteria. Safety, feasibility and evidence of immune response were also considered. Twenty-four patients were assessed. After gross-total resection (

Mesenchymal stromal cells (MSCs) are considered an important therapeutic tool in cancer therapy. Because MSCs may home tumor microenvironment they have the possibility to directly delivery molecules to cancer cells. We have demonstrated that MSCs, upon in vitro priming with anti-cancer drug, become drug-releasing mesenchymal cells (Dr-MCs) able to strongly inhibit cancer cells growth. In order to expand our studies we here investigated whether intravenous (i.v) injection of MSCs loaded with Paclitaxel (PTX) were able to reduce B16 melanoma lung metastasis formation. To this end, as Dr-MCs, the murine MSC line SR4987 was used. SR4987 cells were loaded with PTX (SR4987PTX) by incubating for 24h with 2000ng/ml PTX. The anti-metastatic activity of SR4987PTX was evaluated in mice injected i.v. with 2.5 × 105 B16 melanoma cells. Tracking of the SR4987 in the lung was studied by immunohistochemistry using anti-Sca-1 antibodies. Adhesion and migration experiments were performed to elucidated the mechanism of SR4987PTX homing. In summary, we found that three i.v. injections of SR4987PTX on day 5, 10 and 15 after tumor injection almost completely (>90% inhibition) abolished B16 lung metastasis. This effect was significantly superior (p Citation Format: Giulio Alessandri, Carlo Leonetti, Simona Artuso, Augusto Orlandi, Daniela Passeri, Anna Benetti, Angiola Berenzi, Enrico Dessy, Luisa Pascucci, Piero Ceccarelli, Arianna Bonomi, Valentina Coccè, Nazario Portolani, Valentina Ceserani, Eugenio Parati, Augusto Pessina. Drug-releasing mesenchymal cells strongly suppress B16 lung metastasis in a syngeneic murine model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4237. doi:10.1158/1538-7445.AM2015-4237

Background and Purpose— Despite moderate heritability, the phenotypic heterogeneity of ischemic stroke has hampered gene discovery, motivating analyses of diagnostic subtypes with reduced sample sizes. We assessed evidence for a shared genetic basis among the 3 major subtypes: large artery atherosclerosis (LAA), cardioembolism, and small vessel disease (SVD), to inform potential cross-subtype analyses. Methods— Analyses used genome-wide summary data for 12 389 ischemic stroke cases (including 2167 LAA, 2405 cardioembolism, and 1854 SVD) and 62 004 controls from the Metastroke consortium. For 4561 cases and 7094 controls, individual-level genotype data were also available. Genetic correlations between subtypes were estimated using linear mixed models and polygenic profile scores. Meta-analysis of a combined LAA–SVD phenotype (4021 cases and 51 976 controls) was performed to identify shared risk alleles. Results— High genetic correlation was identified between LAA and SVD using linear mixed models ( r g =0.96, SE=0.47, P =9×10 −4 ) and profile scores ( r g =0.72; 95% confidence interval, 0.52–0.93). Between LAA and cardioembolism and SVD and cardioembolism, correlation was moderate using linear mixed models but not significantly different from zero for profile scoring. Joint meta-analysis of LAA and SVD identified strong association ( P =1×10 −7 ) for single nucleotide polymorphisms near the opioid receptor μ1 ( OPRM1 ) gene. Conclusions— Our results suggest that LAA and SVD, which have been hitherto treated as genetically distinct, may share a substantial genetic component. Combined analyses of LAA and SVD may increase power to identify small-effect alleles influencing shared pathophysiological processes.

We investigated in vitro the properties of selected populations of cancer stem-like cells defined as tumorospheres that were obtained from human glioblastoma. We also assessed their potential and capability of differentiating into mature cells of the central nervous system. In vivo, their tumorigenicity was confirmed after transplantation into the brain of non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice. The angiogenic potential of tumorospheres and glioblastoma-derived cells grown as adherent cells was revealed by evaluating the release of angiogenic factors such as vascular endothelial growth factor and CXCL12 by ELISA, as well as by rat aortic ring assay. The proliferative response of tumorospheres in the presence of CXCL12 was observed for the first time. Multidrug resistance-associated proteins 1 and 3 as well as other molecules conferring multidrug resistance were higher when compared with primary adherent cells derived from the same tumor. Finally, we obtained cells from the tumor developing after grafting that clearly expressed the putative neural stem cell marker CD133 as shown by FACS analysis and also nestin and CXCR4. The cells' positivity for glial fibrillary acidic protein was very low. Moreover these cells preserved their angiogenic potential. We conclude that human glioblastoma could contain tumor cell subsets with angiogenic and chemoresistance properties and that this chemoresistance potential is highly preserved by immature cells whereas the angiogenic potential is, to a higher extent, a property of mature cells. A better understanding of the features of these cell subsets may favor the development of more specifically targeted therapies.

The objective was to evaluate pravastatin modulation on peripheral blood mononuclear cell (PBMC) migration across endothelial monolayers. Eleven hypercholesterolaemic patients were treated with pravastatin 20 mg/day. At baseline (T0), after 40 days (T40) and after 6 months (T 6 months) of treatment total serum cholesterol, low-density lipoprotein (LDL), high-density lipoprotein, triglycerides, C-reactive protein, as well as tumour necrosis factor-alpha (TNF-alpha) and metalloproteinases-9 plasma levels were evaluated. At the same time points the effect of pravastatin on migration of PBMCs through a monolayer of murine brain endothelial cells was studied both in basal conditions and after endothelial stimulation with recombinant mouse TNF-alpha 10 ng/ml for 24 h. Seven volunteers were used as healthy controls. Significant decreases in total cholesterol, LDL and triglycerides as well as inhibition of transmigration were observed. PBMCs transmigration in patients prior to pravastatin therapy was higher than in healthy controls. These results suggest that pravastatin could be of benefit in a spectrum of diseases characterised by extravasation of PBMCs into the central nervous system.

Human lymphatic endothelial cells (LECs) have isolated prevalently from human derma and tumors. As specialized lymphatic organs within the oropharynx, palatine tonsils are easily obtained and rich in lymphatic venules. Using a two-step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1 (UEA-1)-coated beads, followed by purification with monoclonal antibody D2-40, we successfully purified LECs from human palatine tonsils. The LECs were expanded on flasks coated with collagen type 1 and fibronectin for up to 8-10 passages and then analyzed for phenotypic and functional properties. Cultured cells retained the phenotypic pattern of the lymphatic endothelium of palatine tonsils and expressed functional VEGFR-3 molecules. In fact, stimulation with VEGFR-3 ligand, the vascular endothelium grow factor C, induced a marked increase in cell proliferation. Similarly to blood endothelial cells (BECs), LECs were able to form tube-like structure when seeded in Cultrex basement membrane extract. Comparative studies performed on LECs derived from palatine tonsils and iliac lymphatic vessels (ILVs), obtained with the same procedures, showed substantial discrepancies in the expression of various lymphatic markers. This points to the existence of micro- and macrovessel-derived LECs with different phenotypes, possibly involving different biological activities and functions. Palatine tonsil- and ILV-derived LECs may, therefore, represent new models for investigating function and biochemical properties of these lymphatic endothelia.

OBJECTIVES: In vitro, neural stem cells (NSCs) proliferate as undifferentiated spheroids and differentiate into neurons, astrocytes and oligodendrocytes. These features make NSCs suitable for spinal cord (SC) reconstruction. However, in vivo experiments have demonstrated that in the injured SC transplanted NSCs either remain undifferentiated or differentiate into the astrocytic phenotype. The microenvironment of the injured SC is believed to play a crucial role in driving the differentiation of the engrafted NSCs. Here, we tested the hypothesis that inflammatory cytokines (ICs) may be involved in the restricted differentiation of NSCs after grafting onto the injured SC. METHODS: As the first step, we used immunohistochemistry to analyse the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and interferon (IFN)-gamma in the normal SC of mice and following traumatic injury. Then, we investigated whether a combination of TNF-alpha, IL-1beta and IFN-gamma may affect the phenotype of murine NSCs in vitro. RESULTS: We found that TNF-alpha, IL-1beta and IFN-gamma, which are absent in the normal SC, are all expressed in the injured SC and the expression of these cytokines follows a timely tuned fashion with IFN-gamma being detectable as long as 4 weeks after injury. In culture, exposure of proliferating NSCs to a combination of TNF-alpha, IL-1beta and IFN-gamma was per se sufficient to induce the astrocytic differentiation of these cells even in the absence of serum. CONCLUSIONS: In the traumatically injured SC, differentiation of engrafted NSCs is restricted towards the astrocytic lineage because of the inflammatory environment. ICs are likely to play a major role in differentiation of NSCs in the in vivo conditions.

The use of neural progenitor cells (NPCs) is limited by the incomplete knowledge of the extracellular signals regulating their proliferation and survival. We report that cultured mouse NPCs express functional mGlu3 and mGlu5 metabotropic glutamate receptors. Pharmacological blockade of both receptors reduced NPC proliferation and survival, whereas activation of mGlu5 receptors substantially enhanced cell proliferation. Adult mice lacking mGlu5 receptors or treated with mGlu5 or mGlu3 receptor antagonists showed a dramatic reduction in the number of dividing neuroprogenitors present in the subventricular zone and in the dentate gyrus of the hippocampus. These data disclose a novel function of mGlu receptors and offer new potential strategies for the optimization of cell replacement therapy in neurodegenerative disorders.

The mechanisms that control neural stem and progenitor cell survival are unknown. In several pathological conditions, death receptor (DR) ligands and inflammatory cytokines exert a deleterious effect on neurons, whereas primitive neural cells migrate and survive in the site of lesion. Here, we show that even in the presence of inflammatory cytokines, DRs are unable to generate death signals in primitive neural cells. Neural stem and progenitor cells did not express caspase 8, the presence of which is required for initiating the caspase cascade. However, exogenous or cytokine-mediated expression of caspase 8 was not sufficient to restore their DR sensitivity. Searching for molecules potentially able to block DR death-inducing signaling complex (DISC), we found that primitive neural cells expressed high levels of the death effector domain-containing protein PED (also known as PEA-15). PED localized in the DISC and prevented caspase 8 recruitment and activation. Moreover, lentiviral-mediated delivery of PED antisense DNA resulted in dramatic down-regulation of the endogenous gene expression and sensitization of primitive neural cells to apoptosis mediated by inflammatory cytokines and DRs. Thus, absence of caspase 8 and high expression of PED constitute two levels of protection from apoptosis induced by DRs and inflammatory cytokines in neural stem and progenitor cells.

Purpose: Deep venous thrombosis/pulmonary embolism (DVT/PE) is a frequent complication in the course of cancer, particularly in brain tumors. We investigated genetic and plasma factors possibly associated with risk of DVT/PE in patients with high-grade glioma. Experimental Design: In a case-control study, we studied polymorphisms of the genes coding for factor II (G20210A), factor V (G1691A), methylenetetrahydrofolate-reductase (C677T), tissue-type plasminogen activator (tPA; insertion/deletion), plasminogen activator inhibitor-1 (PAI-1; 4G/5G), and vascular endothelial growth factor (VEGF; C936T). We also measured plasma levels of d-dimer, lipoprotein (lp) (a), homocysteine, VEGF, tPA, and PAI-1, comparing healthy control patients with patients with glioma or with patients with neurological nonneoplastic disease (multiple sclerosis). Results: Genotype frequencies of polymorphisms analyzed were similar in patients with glioma and in healthy matched population. d-dimer, lp (a), homocysteine, VEGF, tPA, and PAI-1 plasma levels were significantly higher in patients with glioma than in healthy controls, whereas patients having neurological nonneoplastic disease had plasma values of these molecules not significantly different from healthy controls. VEGF, tPA, and PAI-1 were also found at high-plasma levels in patients carrying genotypes that, in healthy controls, were associated with “low-producing” phenotypes. Conclusions: Genetic risk factors alone did not explain the high incidence of DVT/PE observed in patients with glioma. Higher plasma levels of molecules influencing the coagulation pathways indicate that the tumor itself might confer an increased risk of DVT/PE; thus, d-dimer, homocysteine, lp (a), VEGF, tPA, and PAI-1 look like good candidates to be evaluated as DVT/PE prognostic factors.

High-grade gliomas are aggressive tumors of the central nervous system characterized by endothelial cell proliferation and a high degree of vascularity. Conventional antitumoral treatments (i.e., surgery, radiotherapy, and chemotherapy) do not achieve satisfactory results (median survival in glioblastoma 12-18 months). It has been suggested that immunotherapy with xenogenic endothelial cells could slow tumor growth rate in a number of tumors in a murine model, but the study did not include gliomas. In experiments performed in our laboratory, vaccination with proliferating bovine aortic endothelium increased survival time in Fischer rats inoculated intracerebrally with 9L. Immunotherapy was also able to reduce the growth of subcutaneously injected 9L gliosarcoma cells in Fischer rats and to decrease microvessel density within the tumors, in the absence of major organ toxicity. Immunoglobulins (Ig) in the sera from vaccinated rats stained bovine aortic endothelium as well as human umbilical vein endothelium in active proliferation. Moreover, immune sera from immunized rats stained microvessels of human malignant glioma specimens and vessels of intracerebrally implanted tumors. Two proteins of MW of 11 and 19 kDa were identified by Western blot as targets of Ig elicited by vaccination. A possible future development is to select peptides/proteins suitable for vaccination in humans, avoiding the biohazards connected with xenogenic whole-cell vaccination.

Levels of homovanillic acid (MVA) were measured in lumbar cerebrospinal fluid from 24 patients affected by amyotrophic lateral sclerosis (ALS) and compared with those found in 11 patients with Parkinson's disease (PD) and 10 patients with lumbar disc herniations who served as controls. Mean HVA levels were significantly decreased in ALS and PD patients. These findings are consistent with impairment of central dopaminergic systems in ALS as well as suggesting degeneration of neuroanatomical structures other than motor neurons.

2087 Background: DENDR1 is a phase I-II phase study aimed at evaluating dendritic cell (DC) immunotherapy in patients with first diagnosis of glioblastoma multiforme (GBM). Here we provide results ...

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.

Introduction: Silk fibroin (SF) scaffolds have been shown to be a suitable substrate for tissue engineering and to improve tissue regeneration when cellularized with mesenchymal stromal cells (MSCs). We here demonstrate, for the first time, that electrospun nanofibrous SF patches cellularized with human adipose-derived MSCs (Ad-MSCs-SF), or decellularized (D-Ad-MSCs-SF), are effective in the treatment of skin wounds, improving skin regeneration in db/db diabetic mice. Methods: The conformational and structural analyses of SF and D-Ad-MSCs-SF patches were performed by scanning electron microscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. Wounds were performed by a 5 mm punch biopsy tool on the mouse’s back. Ad-MSCs-SF and D-Ad-MSCs-SF patches were transplanted and the efficacy of treatments was assessed by measuring the wound closure area, by histological examination and by gene expression profile. We further investigated the in vitro angiogenic properties of Ad-MSCs-SF and D-Ad-MSCs-SF patches by affecting migration of human umbilical vein endothelial cells (HUVECs), keratinocytes (KCs) and dermal fibroblasts (DFs), through the aortic ring assay and, finally, by evaluating the release of angiogenic factors. Results: We found that Ad-MSCs adhere and grow on SF, maintaining their phenotypic mesenchymal profile and differentiation capacity. Conformational and structural analyses on SF and D-Ad-MSCs-SF samples, showed that sterilization, decellularization, freezing and storing did not affect the SF structure. When grafted in wounds of diabetic mice, both Ad-MSCs-SF and D-Ad-MSCs-SF significantly improved tissue regeneration, reducing the wound area respectively by 40% and 35%, within three days, completing the process in around 10 days compared to 15–17 days of controls. RT 2 gene profile analysis of the wounds treated with Ad-MSCs-SF and D-Ad-MSCs-SF showed an increment of genes involved in angiogenesis and matrix remodeling. Finally, Ad-MSCs-SF and D-Ad-MSCs-SF co-cultured with HUVECs, DFs and KCs, preferentially enhanced the HUVECs’ migration and the release of angiogenic factors stimulating microvessel outgrowth in the aortic ring assay. Conclusions: Our results highlight for the first time that D-Ad-MSCs-SF patches are almost as effective as Ad-MSCs-SF patches in the treatment of diabetic wounds, acting through a complex mechanism that involves stimulation of angiogenesis. Our data suggest a potential use of D-Ad-MSCs-SF patches in chronic diabetic ulcers in humans.

Vasculogenesis, the de novo formation of new blood vessels from undifferentiated precursor cells or angioblasts, has been studied with experimental in vivo and ex vivo animal models, but its mechanism is poorly understood, particularly in humans. We used the aortic ring assay to investigate the angioforming capacity of aortic explants from 11- to 12-week-old human embryos. After being embedded in collagen gels, the aorta rings produced branching capillary-like structures formed by mesenchymal spindle cells that lined a capillary-like lumen and expressed markers of endothelial differentiation (CD31, CD34, von Willebrand factor [vWF], and fms-like tyrosine kinase-1 [Flk-1]/vascular endothelial growth factor receptor 2 [VEGFR2]). The cell linings of these structures showed ultrastructural evidence of endothelial differentiation. The neovascular proliferation occurred primarily in the outer aspects of aortic rings, thus suggesting that the new vessels mainly arose from immature endothelial precursor cells localized in the outer layer of the aortic stroma, ie, a process of vasculogenesis rather than angiogenesis. The undifferentiated mesenchymal cells (CD34+/CD31-), isolated and cultured on collagen-fibronectin, differentiated into endothelial cells expressing CD31 and vWF. Furthermore, the CD34+/CD31+ cells were capable of forming a network of capillary-like structures when cultured on Matrigel. This is the first reported study showing the ex vivo formation of human microvessels by vasculogenesis. Our findings indicate that the human embryonic aorta is a rich source of CD34+/CD31- endothelial progenitor cells (angioblasts), and this information may prove valuable in studies of vascular regeneration and tissue bioengineering.

In this work, we show that the embryonic human brain contains multipotent central nervous system (CNS) stem cells, which may provide a continuous, standardized source of human neurons that could virtually eliminate the use of primary human fetal brain tissue for intracerebral transplantation. Multipotential stem cells can be isolated from the developing human CNS in a reproducible fashion and can be exponentially expanded for longer than 2 years. This allows for the establishment of continuous, nontransformed neural cell lines, which can be frozen and banked. By clonal analysis, reverse transcription polymerase chain reaction, and electrophysiological assay, we found that over such long-term culturing these cells retain both multipotentiality and an unchanged capacity for the generation of neuronal cells, and that they can be induced to differentiate into catechlaminergic neurons. Finally, when transplanted into the brain of adult rodents immunosuppressed by cyclosporin A, human CNS stem cells migrate away from the site of injection and differentiate into neurons and astrocytes. No tumor formation was ever observed. Aside from depending on scarce human neural fetal tissue, the use of human embryonic CNS stem cells for clinical neural transplantation should provide a reliable solution to some of the major problems that pertain to this field, and should allow determination of the safety characteristics of the donor cells in terms of tumorigenicity, viability, sterility, and antigenic compatibility far in advance of the scheduled day of surgery.

The subventricular zone (SVZ) of the adult mammalian forebrain contains kinetically distinct precursor populations that contribute new neurons to the olfactory bulb. Because among forebrain precursors there are stem-like cells that can be cultured in the presence of mitogens such as epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), we asked whether distinct subsets of stem-like cells coexist within the SVZ or whether the proliferation of a single type of SVZ stem-like cell is controlled by several GFs. We show that the latter is the case. Thus cells isolated from the SVZ coexpress the EGF and FGF receptors; by quantitative analysis, the number of stem-like cells isolated from the SVZ by either FGF2 or EGF is the same, whereas no additive effect occurs when these factors are used together. Furthermore, short-term administration of high-dose [3H]thymidinein vivodepletes both the EGF- and FGF2-responsive stem-like cell populations equally, showing they possess closely similar proliferation kinetics and likely belong to the constitutively proliferating SVZ compartment. By subcloning and population analysis, we demonstrate that responsiveness to more than one GF endows SVZ cells with an essential stem cell feature, the ability to vary self-renewal, that was until now undocumented in CNS stem-like cells. The multipotent stem cell-like population that expands slowly in the presence of FGF2 in culture switches to a faster growth mode when exposed to EGF alone and expands even faster when exposed to both GFs together. Analogous responses are observed when the GFs are used in the reverse order, and furthermore, these growth rate modifications are fully reversible.

Stem cells that can give rise to neurons, astroglia, and oligodendroglia have been found in the developing and adult central nervous system (CNS) of rodents. Yet, their existence within the human brain has not been documented, and the isolation and characterization of multipotent embryonic human neural stem cells have proven difficult to accomplish. We show that the developing human CNS embodies multipotent precursors that differ from their murine counterpart in that they require simultaneous, synergistic stimulation by both epidermal and fibroblast growth factor-2 to exhibit critical stem cell characteristics. Clonal analysis demonstrates that human C NS stem cells are multipotent and differentiate spontaneously into neurons, astrocytes, and oligodendrocytes when growth factors are removed. Subcloning and population analysis show their extensive self-renewal capacity and functional stability, their ability to maintain a steady growth profile, their multipotency, and a constant potential for neuronal differentiation for more than 2 years. The neurons generated by human stem cells over this period of time are electrophysiologically active. These cells are also cryopreservable. Finally, we demonstrate that the neuronal and glial progeny of long-term cultured human CNS stem cells can effectively survive transplantation into the lesioned striatum of adult rats. Tumor formation is not observed, even in immunodeficient hosts. Hence, as a consequence of their inherent biology, human CNS stem cells can establish stable, transplantable cell lines by epigenetic stimulation. These lines represent a renewable source of neurons and glia and may significantly facilitate research on human neurogenesis and the development of clinical neural transplantation.