Lidia De Filippis

The EphA2 Receptor Drives Self-Renewal and Tumorigenicity in Stem-Like Tumor-Propagating Cells from Human Glioblastomas

In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs. Cytofluorimetric sorting into EphA2(High) and EphA2(Low) populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications.

A novel, immortal, and multipotent human neural stem cell line generating functional neurons and oligodendrocytes.

The discovery and study of neural stem cells have revolutionized our understanding of the neurogenetic process, and their inherent ability to adopt expansive growth behavior in vitro is of paramount importance for the development of novel therapeutics based on neural cell replacement. Recent advances in high‐throughput assays for drug development and gene discovery dictate the need for rapid, reproducible, long‐term expansion of human neural stem cells (hNSCs). In this view, the complement of wild‐type cell lines currently available is insufficient. Here we report the establishment of a stable human neural stem cell line (immortalized human NSCs [IhNSCs]) by v‐myc‐mediated immortalization of previously derived wild‐type hNSCs. These cells demonstrate three‐ to fourfold faster proliferation than wild‐type cells in response to growth factors but retain rather similar properties, including multipotentiality. By molecular biology, biochemistry, immunocytochemistry, fluorescence microscopy, and electrophysiology, we show that upon growth factor removal, IhNSCs completely downregulate v‐myc expression, cease proliferation, and differentiate terminally into three major neural lineages: astrocytes, oligodendrocytes, and neurons. The latter are functional, mature cells displaying clear‐cut morphological and physiological features of terminally differentiated neurons, encompassing mostly the GABAergic, glutamatergic, and cholinergic phenotypes. Finally, IhNSCs produce bona fide oligodendrocytes in fractions up to 20% of total cell number. This is in contrast to the negligible propensity of hNSCs to generate oligodendroglia reported so far. Thus, we describe an immortalized hNSC line endowed with the properties of normal hNSCs and suitable for developing the novel, reliable assays and reproducible high‐throughput gene and drug screening that are essential in both diagnostics and cell therapy studies.

The proliferative capacity of the subventricular zone is maintained in the parkinsonian brain

There are many indications that neurogenesis is impaired in Parkinsons disease, which might be due to a lack of dopamine in the subventricular zone. An impairment in neurogenesis may have negative consequences for the development of new therapeutic approaches in Parkinsons disease, as neural stem cells are a potential source for endogenous repair. In this study, we examined the subventricular zone of 10 patients with Parkinsons disease and 10 age- and sex-matched controls for proliferation and neural stem cell numbers. We also included five cases with incidental Lewy body disease, which showed Parkinsons disease pathology but no clinical symptoms and thus did not receive dopaminergic treatment. We quantified the neural stem cell number and proliferative capacity in the subventricular zone of these three donor groups. We found subventricular neural stem cells in each donor, with a high variation in number. We did not observe significant differences in neural stem cell number or in proliferation between the groups. Additionally, we were able to culture neural stem cells from post-mortem brain of several patients with Parkinsons disease, confirming the presence of viable neural stem cells in these brains. We have also examined the subventricular zone of a chronic, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsons disease mouse model, and again found no effect of dopaminergic denervation on precursor proliferation. Lastly, we investigated the proliferation capacity of two different human neural stem cell lines in response to dopamine. Both cell lines did not respond with a change in proliferation to treatment with dopamine agonists and an antagonist. In summary, the adult neural stem cell pool in the subventricular zone was not clearly affected in the human parkinsonian brain or a Parkinsons disease mouse model. Furthermore, we did not find evidence that dopamine has a direct effect on human neural stem cell proliferation in vitro. Thus, we conclude that the number of adult neural stem cells is probably not diminished in the parkinsonian brain and that dopamine depletion most likely has no effect on human neural stem cells.

Long-term survival of human neural stem cells in the ischemic rat brain upon transient immunosuppression.

Understanding the physiology of human neural stem cells (hNSCs) in the context of cell therapy for neurodegenerative disorders is of paramount importance, yet large-scale studies are hampered by the slow-expansion rate of these cells. To overcome this issue, we previously established immortal, non-transformed, telencephalic-diencephalic hNSCs (IhNSCs) from the fetal brain. Here, we investigated the fate of these IhNSCs immediate progeny (i.e. neural progenitors; IhNSC-Ps) upon unilateral implantation into the corpus callosum or the hippocampal fissure of adult rat brain, 3 days after global ischemic injury. One month after grafting, approximately one fifth of the IhNSC-Ps had survived and migrated through the corpus callosum, into the cortex or throughout the dentate gyrus of the hippocampus. By the fourth month, they had reached the ipsilateral subventricular zone, CA1-3 hippocampal layers and the controlateral hemisphere. Notably, these results could be accomplished using transient immunosuppression, i.e administering cyclosporine for 15 days following the ischemic event. Furthermore, a concomitant reduction of reactive microglia (Iba1+ cells) and of glial, GFAP+ cells was also observed in the ipsilateral hemisphere as compared to the controlateral one. IhNSC-Ps were not tumorigenic and, upon in vivo engraftment, underwent differentiation into GFAP+ astrocytes, and β-tubulinIII+ or MAP2+ neurons, which displayed GABAergic and GLUTAmatergic markers. Electron microscopy analysis pointed to the formation of mature synaptic contacts between host and donor-derived neurons, showing the full maturation of the IhNSC-P-derived neurons and their likely functional integration into the host tissue. Thus, IhNSC-Ps possess long-term survival and engraftment capacity upon transplantation into the globally injured ischemic brain, into which they can integrate and mature into neurons, even under mild, transient immunosuppressive conditions. Most notably, transplanted IhNSC-P can significantly dampen the inflammatory response in the lesioned host brain. This work further supports hNSCs as a reliable and safe source of cells for transplantation therapy in neurodegenerative disorders.

GFAPdelta in radial glia and subventricular zone progenitors in the developing human cortex.

A subpopulation of glial fibrillary acidic protein (GFAP)-expressing cells located along the length of the lateral ventricles in the subventricular zone (SVZ) have been identified as the multipotent neural stem cells of the adult mammalian brain. We have previously found that, in the adult human brain, a splice variant of GFAP, termed GFAPδ, was expressed specifically in these cells. To investigate whether GFAPδ is also present in the precursors of SVZ astrocytes during development and whether GFAPδ could play a role in the developmental process, we analyzed GFAPδ expression in the normal developing human cortex and in the cortex of foetuses with the migration disorder lissencephaly type II. We demonstrated for the first time that GFAPδ is specifically expressed in radial glia and SVZ neural progenitors during human brain development. Expression of GFAPδ in radial glia starts at around 13 weeks of pregnancy and disappears before birth. GFAPδ is continuously expressed in the SVZ progenitors at later gestational ages and in the postnatal brain. Co-localization with Ki67 proved that these GFAPδ-expressing cells are able to proliferate. Furthermore, we showed that the expression pattern of GFAPδ was disturbed in lissencephaly type II. Overall, these results suggest that the adult SVZ is indeed a remnant of the foetal SVZ, which develops from radial glia. Furthermore, we provide evidence that GFAPδ can distinguish resting astrocytes from proliferating SVZ progenitors.

Immortalization of human neural stem cells with the c-myc mutant T58A

Human neural stem cells (hNSC) represent an essential source of renewable brain cells for both experimental studies and cell replacement therapies. Their relatively slow rate of proliferation and physiological senescence in culture make their use cumbersome under some experimental and pre-clinical settings. The immortalization of hNSC with the v-myc gene (v-IhNSC) has been shown to generate stem cells endowed with enhanced proliferative capacity, which greatly facilitates the study of hNSCs, both in vitro and in vivo. Despite the excellent safety properties displayed by v-IhNSCs – which do not transform in vitro and are not tumorigenic in vivo – the v-myc gene contains several mutations and recombination elements, whose role(s) and effects remains to be elucidated, yielding unresolved safety concerns. To address this issue, we used a c-myc T58A retroviral vector to establish an immortal cell line (T-IhNSC) from the same hNSCs used to generate the original v-IhNSCs and compared their characteristics with the latter, with hNSC and with hNSC immortalized using c-myc wt (c-IhNSC). T-IhNSCs displayed an enhanced self-renewal ability, with their proliferative capacity and clonogenic potential being remarkably comparable to those of v-IhNSC and higher than wild type hNSCs and c-IhNSCs. Upon growth factors removal, T-IhNSC promptly gave rise to well-differentiated neurons, astrocytes and most importantly, to a heretofore undocumented high percentage of human oligodendrocytes (up to 23%). Persistent growth-factor dependence, steady functional properties, lack of ability to generate colonies in soft-agar colony-forming assay and to establish tumors upon orthotopic transplantation, point to the fact that immortalization by c-myc T58A does not bring about tumorigenicity in hNSCs. Hence, this work describes a novel and continuous cell line of immortalized human multipotent neural stem cells, in which the immortalizing agent is represented by a single gene which, in turn, carries a single and well characterized mutation. From a different perspective, these data report on a safe approach to increase human neural stem cells propagation in culture, without altering their basic properties. These T-IhNSC line provides a versatile model for the elucidation of the mechanisms involved in human neural stem cells expansion and for development of high throughput assays for both basic and translational research on human neural cell development. The improved proclivity of T-IhNSC to generate human oligodendrocytes propose T-IhNSC as a feasible candidate for the design of experimental and, perhaps, therapeutic approaches in demyelinating diseases.

Concise Review: Self-Renewal in the Central Nervous System: Neural Stem Cells from Embryo to Adult

The recent discovery of neural stem cells (NSCs) in the adult mammalian brain has fostered a plethora of translational and preclinical studies to investigate future therapeutic approaches for the cure of neurodegenerative diseases. These studies are finally at the clinical stage, and some of them are already under way. The definition of a bona fide stem cell has long been the object of much debate focused on the establishment of standard and univocal criteria to distinguish between stem and progenitor cells. It is commonly accepted that NSCs have to fulfill two basic requirements, the capacity for long‐term self‐renewal and the potential for differentiation, which account for their physiological role, namely central nervous system tissue homeostasis. Strategies such as immortalization or reprogramming of somatic cells to the embryonic‐like stage of pluripotency indicate the relevance of extensive self‐renewal ability of NSCs either in vitro or in vivo. Moreover, the discovery of stem‐like tumor cells in brain tumors, such as gliomas, accompanied by the isolation of these cells through the same paradigm used for related healthy cells, has provided further evidence of the key role that self‐renewal plays in the development and progression of neurodegenerative diseases and cancer. In this review we provide an overview of the current understanding of the self‐renewal capacity of nontransformed human NSCs, with or without immortalization or reprogramming, and of stem‐like tumor cells, referring to both research and therapeutic studies.

Isolation of neural stem cells from neural tissues using the neurosphere technique

This unit describes protocols for the derivation, characterization, and expansion of neural stem cell (NSC) lines from the adult mouse subventricular zone (mNSCs), embryonic mouse brain and from the human fetal brain (hNSCs). NSCs can be isolated by enzymatic digestion of specific regions (NSCs niches) of the central nervous system (CNS) and grown in suspension. By using this methodology, NSCs form spherical clusters called neurospheres, which are mechanically dissociated to a single-cell suspension and replated in the selective culture medium. Removal of growth factors and plating cells on an adherent substrate allows cells to differentiate into neurons, astrocytes, and oligodendrocytes, the main cell type of the CNS. Correct culturing of NSCs, according to this methodology, will allow cells to expand over 100 passages without alteration of cell karyotype, growth ability, and differentiation potential.

Histone acetylation in astrocytes suppresses GFAP and stimulates a reorganization of the intermediate filament network.

ABSTRACT Glial fibrillary acidic protein (GFAP) is the main intermediate filament in astrocytes and is regulated by epigenetic mechanisms during development. We demonstrate that histone acetylation also controls GFAP expression in mature astrocytes. Inhibition of histone deacetylases (HDACs) with trichostatin A or sodium butyrate reduced GFAP expression in primary human astrocytes and astrocytoma cells. Because splicing occurs co-transcriptionally, we investigated whether histone acetylation changes the ratio between the canonical isoform GFAP&agr; and the alternative GFAP&dgr; splice variant. We observed that decreased transcription of GFAP enhanced alternative isoform expression, as HDAC inhibition increased the GFAP&dgr;∶GFAP&agr; ratio. Expression of GFAP&dgr; was dependent on the presence and binding of splicing factors of the SR protein family. Inhibition of HDAC activity also resulted in aggregation of the GFAP network, reminiscent of our previous findings of a GFAP&dgr;-induced network collapse. Taken together, our data demonstrate that HDAC inhibition results in changes in transcription, splicing and organization of GFAP. These data imply that a tight regulation of histone acetylation in astrocytes is essential, because dysregulation of gene expression causes the aggregation of GFAP, a hallmark of human diseases like Alexanders disease.

Chronic exposure of astrocytes to interferon-α reveals molecular changes related to Aicardi-Goutieres syndrome

Aicardi-Goutières syndrome is a genetically determined infantile encephalopathy, manifesting as progressive microcephaly, psychomotor retardation, and in ∼25% of patients, death in early childhood. Aicardi-Goutières syndrome is caused by mutations in any of the genes encoding TREX1, RNASEH2-A, -B, -C and SAMHD1, with protein dysfunction hypothesized to result in the accumulation of nucleic acids within the cell, thus triggering an autoinflammatory response with increased interferon-α production. Astrocytes have been identified as a major source of interferon-α production in the brains of patients with Aicardi-Goutières syndrome. Here, we study the effect of interferon-α treatment on astrocytes derived from immortalized human neural stem cells. Chronic interferon-α treatment promoted astrocyte activation and a reduction in cell proliferation. Moreover, chronic exposure resulted in an alteration of genes and proteins involved in the stability of white matter (ATF4, eIF2Bα, cathepsin D, cystatin F), an increase of antigen-presenting genes (human leukocyte antigen class I) and downregulation of pro-angiogenic factors and other cytokines (vascular endothelial growth factor and IL-1). Interestingly, withdrawal of interferon-α for 7 days barely reversed these cellular alterations, demonstrating that the interferon-α mediated effects persist over time. We confirmed our in vitro findings using brain samples from patients with Aicardi-Goutières syndrome. Our results support the idea of interferon-α as a key factor in the pathogenesis of Aicardi-Goutières syndrome relating to the observed leukodystrophy and microangiopathy. Because of the sustained interferon-α effect, even after withdrawal, therapeutic targets for Aicardi-Goutières syndrome, and other interferon-α-mediated encephalopathies, may include downstream interferon-α signalling cascade effectors rather than interferon-α alone.