Eugenio Parati

Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells

Glioblastoma is a highly angiogenetic malignancy, the neoformed vessels of which are thought to arise by sprouting of pre-existing brain capillaries. The recent demonstration that a population of glioblastoma stem-like cells (GSCs) maintains glioblastomas indicates that the progeny of these cells may not be confined to the neural lineage. Normal neural stem cells are able to differentiate into functional endothelial cells. The connection between neural stem cells and the endothelial compartment seems to be critical in glioblastoma, where cancer stem cells closely interact with the vascular niche and promote angiogenesis through the release of vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (refs 5–9). Here we show that a variable number (range 20–90%, mean 60.7%) of endothelial cells in glioblastoma carry the same genomic alteration as tumour cells, indicating that a significant portion of the vascular endothelium has a neoplastic origin. The vascular endothelium contained a subset of tumorigenic cells that produced highly vascularized anaplastic tumours with areas of vasculogenic mimicry in immunocompromised mice. In vitro culture of GSCs in endothelial conditions generated progeny with phenotypic and functional features of endothelial cells. Likewise, orthotopic or subcutaneous injection of GSCs in immunocompromised mice produced tumour xenografts, the vessels of which were primarily composed of human endothelial cells. Selective targeting of endothelial cells generated by GSCs in mouse xenografts resulted in tumour reduction and degeneration, indicating the functional relevance of the GSC-derived endothelial vessels. These findings describe a new mechanism for tumour vasculogenesis and may explain the presence of cancer-derived endothelial-like cells in several malignancies.

Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation

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.

Genetic risk factors for ischaemic stroke and its subtypes (the METASTROKE collaboration): a meta-analysis of genome-wide association studies.

Summary Background Various genome-wide association studies (GWAS) have been done in ischaemic stroke, identifying a few loci associated with the disease, but sample sizes have been 3500 cases or less. We established the METASTROKE collaboration with the aim of validating associations from previous GWAS and identifying novel genetic associations through meta-analysis of GWAS datasets for ischaemic stroke and its subtypes. Methods We meta-analysed data from 15 ischaemic stroke cohorts with a total of 12 389 individuals with ischaemic stroke and 62 004 controls, all of European ancestry. For the associations reaching genome-wide significance in METASTROKE, we did a further analysis, conditioning on the lead single nucleotide polymorphism in every associated region. Replication of novel suggestive signals was done in 13 347 cases and 29 083 controls. Findings We verified previous associations for cardioembolic stroke near PITX2 (p=2·8×10−16) and ZFHX3 (p=2·28×10−8), and for large-vessel stroke at a 9p21 locus (p=3·32×10−5) and HDAC9 (p=2·03×10−12). Additionally, we verified that all associations were subtype specific. Conditional analysis in the three regions for which the associations reached genome-wide significance (PITX2, ZFHX3, and HDAC9) indicated that all the signal in each region could be attributed to one risk haplotype. We also identified 12 potentially novel loci at p<5×10−6. However, we were unable to replicate any of these novel associations in the replication cohort. Interpretation Our results show that, although genetic variants can be detected in patients with ischaemic stroke when compared with controls, all associations we were able to confirm are specific to a stroke subtype. This finding has two implications. First, to maximise success of genetic studies in ischaemic stroke, detailed stroke subtyping is required. Second, different genetic pathophysiological mechanisms seem to be associated with different stroke subtypes. Funding Wellcome Trust, UK Medical Research Council (MRC), Australian National and Medical Health Research Council, National Institutes of Health (NIH) including National Heart, Lung and Blood Institute (NHLBI), the National Institute on Aging (NIA), the National Human Genome Research Institute (NHGRI), and the National Institute of Neurological Disorders and Stroke (NINDS).

Genome-wide association study identifies a variant in HDAC9 associated with large vessel ischemic stroke.

Genetic factors have been implicated in stroke risk, but few replicated associations have been reported. We conducted a genome-wide association study (GWAS) for ischemic stroke and its subtypes in 3,548 affected individuals and 5,972 controls, all of European ancestry. Replication of potential signals was performed in 5,859 affected individuals and 6,281 controls. We replicated previous associations for cardioembolic stroke near PITX2 and ZFHX3 and for large vessel stroke at a 9p21 locus. We identified a new association for large vessel stroke within HDAC9 (encoding histone deacetylase 9) on chromosome 7p21.1 (including further replication in an additional 735 affected individuals and 28,583 controls) (rs11984041; combined P = 1.87 × 10−11; odds ratio (OR) = 1.42, 95% confidence interval (CI) = 1.28–1.57). All four loci exhibited evidence for heterogeneity of effect across the stroke subtypes, with some and possibly all affecting risk for only one subtype. This suggests distinct genetic architectures for different stroke subtypes.

Isolation and culture of human muscle-derived stem cells able to differentiate into myogenic and neurogenic cell lineages.

BACKGROUND Skeletal-muscle-derived stem cells seem to be a distinct population of immature progenitors of satellite cells, but their functional properties remain unclear, especially in human adult tissue. We investigated their differentiation in samples of skeletal muscle obtained from adults undergoing cardiovascular surgery. METHODS Samples were obtained from the brachioradialis muscle of 12 patients in whom the radial artery was the conduit for myocardial revascularisation. The stem cells were isolated by a procedure similar to that used for rat gastrocnemius and cultured in medium optimised for growth of neural stem cells. Cytometry was used for phenotypic characterisation and immunocytochemistry and RT-PCR to assess differentiation. Immunohistochemistry was used to examine engraftment of skeletal-muscle-derived stem cells into injured rat spinal cord. FINDINGS The skeletal-muscle stem cells consisted of two distinct types: one with the typical spindle morphology of satellite cells, the other of rounded cells. Some cultures could be maintained for longer than 6 months. The cells were mainly positive for desmin and to a lesser extent CD105, vimentin, and AC133/CD133, but negative for FLK-1/KDR, CD34, CD31, CD45, von Willebrand factor, Ve-cadherins, and BCL2. After in-vitro differentiation, the cells were able to organise skeletal-muscle fibres and stained positively for striated-muscle actin, smooth-muscle actin, and desmin. Moreover, they differentiated into astrocytes and neurons, as confirmed by positive staining for characteristic proteins. INTERPRETATION Adult human skeletal muscle includes a population of progenitor stem cells that can generate cells of the same lineage and cells with neurogenic properties. Muscle may therefore be a tissue source for the isolation of pluripotent stem cells for development of cell-based therapies for human myogenic and neurogenic diseases.

Microembolization during carotid artery stenting in patients with high-risk, lipid-rich plaque: A randomized trial of proximal versus distal cerebral protection

OBJECTIVES 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. BACKGROUND Cerebral protection with filters partially reduces the cerebral embolization rate during CAS. Proximal protection has been introduced to further decrease embolization risk. METHODS 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. RESULTS 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 (p < 0.0001) 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%; p < 0.0001). 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%). CONCLUSIONS In patients with high-risk, lipid-rich plaque undergoing CAS, MO.MA led to significantly lower microembolization as assessed by using MES counts.

Human skin-derived stem cells migrate throughout forebrain and differentiate into astrocytes after injection into adult mouse brain.

Recent evidence indicates that neural stem cell properties can be found among a mammalian skin‐derived multipotent population. A major barrier in the further characterization of the human skin‐derived neural progenitors is the inability to isolate this population based on expression of cell surface markers. Our work has been devoted to purified human skin‐derived stem cells that are capable of neural differentiation, based on the presence or absence of the AC133 cell surface marker. The enriched skin‐derived AC133+ cells express the CD34 and Thy‐1 antigens. These cells cultured in a growth medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) proliferate, forming spheres, and differentiate in vitro into neurons, astrocytes, and rarely into oligodendrocytes. Single cells from sphere cultures initiated from human purified AC133+ cells were replated as single cells and were able to generate new spheres, demonstrating the self‐renewing ability of these stem cell populations. Brain engraftment of cells obtained from human purified AC133+‐derived spheres generated different neural phenotypes: immature neurons and a most abundant population of well differentiated astrocytes. The AC133‐derived astrocytes assumed perivascular locations in the frontal cortex. No donor‐derived oligodendrocytes were found in the transplanted mouse brains. Several donor small, rounded cells that expressed endothelial markers were found close to the host vessel and near the subventricular zone. Thus, mammalian skin AC133‐derived cells behave as a multipotent population with the capacity to differentiate into neural lineages in vitro and, prevalently, endothelium and astrocytes in vivo, demonstrating the great plasticity of these cells and suggesting potential clinical application.

Basic fibroblast growth factor supports the proliferation of epidermal growth factor-generated neuronal precursor cells of the adult mouse CNS.

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.

Neurosphere-Derived Cells Exert a Neuroprotective Action by Changing the Ischemic Microenvironment

Background Neurosphere-derived cells (NC), containing neural stem cells, various progenitors and more differentiated cells, were obtained from newborn C57/BL6 mice and infused in a murine model of focal ischemia with reperfusion to investigate if: 1) they decreased ischemic injury and restored brain function; 2) they induced changes in the environment in which they are infused; 3) changes in brain environment consequent to transient ischemia were relevant for NC action. Methodology/Principal Findings NC were infused intracerebroventricularly 4 h or 7 d after 30 min middle cerebral artery occlusion. In ischemic mice receiving cells at 4 h, impairment of open field performance was significantly improved and neuronal loss significantly reduced 7–14 d after ischemia compared to controls and to ischemic mice receiving cells at 7 d. Infusion of murine foetal fibroblast in the same experimental conditions was not effective. Assessment of infused cell distribution revealed that they migrated from the ventricle to the parenchyma, progressively decreased in number but they were observable up to 14 d. In mice receiving NC at 7 d and in sham-operated mice, few cells could be observed only at 24 h, indicating that the survival of these cells in brain tissue relates to the ischemic environment. The mRNA expression of trophic factors such as Insulin Growth Factor-1, Vascular Endothelial Growth Factor-A, Transforming Growth Factor-β1, Brain Derived Neurotrophic Factor and Stromal Derived Factor−1α, as well as microglia/macrophage activation, increased 24 h after NC infusion in ischemic mice treated at 4 h compared to sham-operated and to mice receiving cells at 7 d. Conclusions/Significance NC reduce functional impairment and neuronal damage after ischemia/reperfusion injury. Several lines of evidence indicate that the reciprocal interaction between NC and the ischemic environment is crucial for NC protective actions. Based on these results we propose that a bystander control of the ischemic environment may be the mechanism used by NC to rapidly restore acutely injured brain function.

Endogenous activation of metabotropic glutamate receptors supports the proliferation and survival of neural progenitor cells.

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.