Academic literature on the topic 'Meningeal stem cell'

Abstract BACKGROUND Meningiomas are the most common primary intracranial tumors in humans and dogs, but biologic drivers and cell types underlying meningeal tumorigenesis are incompletely understood. Here we integrate meningioma single-cell RNA sequencing with stem cell approaches to define a perivascular stem cell underlying vertebrate meningeal tumorigenesis. METHODS Single-cell RNA sequencing was performed on 57,114 cells from 8 human meningiomas, 54,607 cells from 3 dog meningiomas, and human meningioma xenografts in mice. Results were validated using immunofluorescence (IF), immunohistochemistry (IHC), and deconvolution of bulk RNA sequencing of 200 human meningiomas. Mechanistic and functional studies were performed using clonogenic and limiting dilution assays, xenografts, and genetically engineered mouse models. RESULTS Copy number variant identification from human meningioma single cells distinguished tumor cells with loss of chr22q from non-tumor cells with intact chr22q. A single cluster distinguished by expression of Notch3 and other cancer stem cell genes had an intermediate level of loss of chr22q, suggesting this cluster may represent meningioma stem cells. In support of this hypothesis, pseudotime trajectory analysis demonstrated transcriptomic progression starting from Notch3+ cells and encompassing all other meningioma cell types. Notch3+ meningioma cells had transcriptomic concordance to mural pericytes, and IF/IHC of prenatal and adult human meninges, as well as lineage tracing using a Notch3-CreERT2 allele in mice, confirmed Notch3+ cells were restricted to the perivascular stem cell niche in mammalian meningeal development and homeostasis. Integrating human and dog meningioma single cells revealed Notch3+ cells in tumor and non-tumor clusters in dog meningiomas. Notch3 IF/IHC and cell-type deconvolution of bulk RNA sequencing showed Notch3+ cells were enriched in high-grade human meningiomas. Notch3 overexpression in human meningioma cells increased clonogenic growth in vitro, and increased tumorigenesis and tumor growth in vivo, decreasing overall survival. CONCLUSIONS Notch3+ stem cells in the perivascular niche underlie vertebrate meningeal tumorigenesis.

Abstract There are no approved targeted therapies for meningiomas and the cell types underlying meningeal tumorigenesis are incompletely understood. To address these limitations, we performed single-cell RNA sequencing of 57,114 cells from 8 human meningiomas and 54,607 cells from 3 canine meningiomas. Pseudotime, gene ontology, and copy number variant analyses revealed a population of pericyte-like meningioma cells that were conserved across human and canine tumors and were enriched in expression of Notch3 and other cancer stem cell genes. Deconvolution of cell types from bulk RNA sequencing and DNA methylation profiling of 200 human meningiomas integrated with immunohistochemistry (IHC), immunofluorescence (IF), and RNAScope demonstrated Notch3+ pericytes and Notch3 expression were enriched in high grade or Immune-enriched meningiomas, which were distinguished from other meningioma DNA methylation groups by genes driving vasculature development. IHC and IF of human meninges integrated with lineage tracing approaches using Notch3-CreERT2 ROSAmT/mG alleles in mice demonstrated Notch3 expression was restricted to the perivascular stem cell niche during meningeal development and homeostasis. Mice harboring Notch3-CreERT2 Nf2fl/fl alleles developed meningeal hyperproliferation. Overexpression of constitutively activated Notch3 (Notch3AICD) in Immune-enriched human meningioma cells increased the expression of cancer stem cell genes, driving clonogenic growth in vitro, limiting dilution tumor-initiating capacity in vivo, and resistance to radiotherapy in vivo. A selective Notch3 neutralizing antibody (αNRR3) blocked meningioma cell proliferation and expression of Notch3 target genes, inhibiting meningioma xenograft growth and prolonging overall survival. Single-cell RNA sequencing of 187,366 cells from meningioma xenografts after αNRR3 or radiotherapy treatment ± Notch3AICD overexpression revealed distinct meningioma cell-intrinsic or cell-extrinsic mechanisms driving responses to radiotherapy or αNRR3, respectively. Combined treatment with αNRR3 and radiotherapy additively blocked meningioma xenograft growth and extended survival benefit. In sum, these data shed light on a novel cell type, molecular mechanism, and therapeutic vulnerability in the most common primary intracranial tumor.

Abstract The meningeal and perivascular spaces of the central nervous system (CNS) are inhabited by specialized macrophages. Under steady state conditions, we discovered two populations: immature macrophages (MHC-II−) and mature macrophages (MHC-II+). Microarray analyses revealed that IL-10 and TGFb were upstream regulators of the immature macrophage transcriptome, which included stem cell-specific genes. These data suggest that immature macrophages represent local progenitors maintained by anti-inflammatory cytokines within the meninges. Interestingly, in naïve mice MHC-II+ macrophages were enriched upon aging and upregulated inflammatory genes, suggesting age-based maturation. To better understand the dynamics of these macrophages, we triggered CNS inflammation by inoculating mice with lymphocytic choriomeningitis virus (LCMV). Both myeloid populations were infected by the virus, and intravital imaging studies revealed that they were targeted by infiltrating virus-specific CD8+ T cells, which promoted their depletion. Following viral clearance, myeloid repopulation of the meninges was derived largely from infiltrating monocytes that engrafted this CNS niche and adopted a transcriptomic signature of mature resident meningeal macrophages. In stark contrast, sterile depletion of meningeal macrophages without infection induced massive local proliferation of immature macrophages that transformed into mature macrophages and repopulated the meninges. This occurred in the absence of peripheral monocyte engraftment. Collectively, these data indicate that the CNS meninges are inhabited by two macrophage populations with a differential ability to repopulate the niche based on the inflammatory milieu.

Abstract Although the bone marrow contains most hematopoietic activity during adulthood, hematopoietic stem and progenitor cells can be recovered from various extramedullary sites. Cells with hematopoietic progenitor properties have even been reported in the adult brain under steady-state conditions, but their nature and localization remain insufficiently defined. Here, we describe a heterogeneous population of myeloid progenitors in the leptomeninges of adult C57BL/6 mice. This cell pool included common myeloid, granulocyte/macrophage, and megakaryocyte/erythrocyte progenitors. Accordingly, it gave rise to all major myelo-erythroid lineages in clonogenic culture assays. Brain-associated progenitors persisted after tissue perfusion and were partially inaccessible to intravenous antibodies, suggesting their localization behind continuous blood vessel endothelium such as the blood-arachnoid barrier. Flt3Cre lineage tracing and bone marrow transplantation showed that the precursors were derived from adult hematopoietic stem cells and were most likely continuously replaced via cell trafficking. Importantly, their occurrence was tied to the immunologic state of the central nervous system (CNS) and was diminished in the context of neuroinflammation and ischemic stroke. Our findings confirm the presence of myeloid progenitors at the meningeal border of the brain and lay the foundation to unravel their possible functions in CNS surveillance and local immune cell production.

Abstract Purpose To determine the characteristics of Non-Hodgkin’s lymphoma (NHL) patients with concomitant systemic and central nervous system localization at diagnosis, as well as the impact of consolidation with high dose chemotherapy followed by hematopoietic stem cell rescue on outcome Patients and Methods Newly diagnosed NHL patients with concomitant systemic and cerebral or meningeal involvement at diagnosis have been included in this study. Patients were retrieved from the database of the participating centers of the LYSA and LOC study groups Results Sixty-five patients (37 males; 28 females) were included. Median age was 60 years (23-85). Histological subtype was mainly diffuse large B-cell lymphoma (n=54; 83%). The IPI was >2 in 43 (66%) patients. LDH level was elevated in 27 (55%) patients. Median number of extranodal positive sites was 2 (1-5) and bone marrow involvement was documented in 30 (46%) patients. CNS involvement was documented in 51 patients. Paravertebral and epidural compressive mass with (n=5) or without (n=2) CSF involvement were present. Five patients had both CNS and peripheral nervous system involvement. Anthracycline-based chemotherapy with high dose metothrexate with or without cytarabine was the most chemotherapy used. Autologous stem cell transplantation was performed in 21 patients in response. BEAM (n=9) or thiotepa-based (n=9) conditioning regimen was the most intensive chemotherapy used before autologous SCT Post-chemotherapy ORR was 77%(CR69%;PR8%). 3-year overall survival (OS) and progression free survival (PFS) were 48±7% and 46±7% respectively. The consolidation strategy using high dose chemotherapy and autologous stem cell transplant positively impacted patient’s outcome. For the whole group as well as for patients ≤65 years, the 3-year OS and PFS were (77%vs29%;p=0.002) and (77%vs25%;p=0.001) and (75% vs 37%;p=0.002) and (75%vs32%;p=0.007) respectively. In multivariate analysis and for the whole group of patients, the absence high dose therapy had a negative impact on 3-year OS and PFS [p=0.003;HR=5.05[1.76-14.49]) and PFS by [p=0.002;HR=5.46(1.91-15.26)] respectively. This is remained true for younger patients 65 years or less who had poorer 3-year OS [p=0.036;HR=3.41 (1.08-10.75)]. Conclusion First line consolidative high dose therapy followed by autologous SCT in patients with systemic and neuro-meningeal NHL improve patient’s outcome Disclosures No relevant conflicts of interest to declare.

Il nostro gruppo ha dimostrato per la prima volta che una nuova nicchia di cellule precursori/staminali con potenziale di differenziamento neuronale risiede nelle meningi cerebrali di ratti in età postnatale. Grazie alla loro locazione superficiale, le meningi possono rappresentare una un nuovo ed accessibile tessuto ospitante cellule neuronali precursori/staminali nel Sistema Nervoso Centrale (SNC). Questo rappresenta un importante aspetto che può aprire nuove prospettive per la possibile estrazione e collezione di Cellule Staminali Neuronali (CSN) per la medicina rigenerativa e il trapianto autologo. Inoltre, ogni vaso nel SNC è circondato dallo Spazio Perivascolare (spazio di Virchow-Robin) formato da estroflessioni delle meningi e riempito da liquido cerebrospinale. Ciò suggerisce che le cellule precursori/staminali possono essere ampiamente distribuite anche nel parenchima del SNC. Per questo, noi ipotizziamo che le cellule neuronali precursori/staminali residenti nelle meningi possono contribuire alla omeostasi del SNC in situazioni normali e di malattia. La verifica di questa ipotesi può offrire nuove prospettive per la generazione di nuovi approcci farmacologici per il trattamento di malattie neurodegenerative. Basandosi sugli ottimi potenziali e sulla rilevanza delle nostre precedenti scoperte, durante il mio PhD, ho indirizzato i miei studi nelle seguenti principali domande: Come si distribuiscono le cellule meningee precursori/staminali nel cervello e nel midollo spinale di adulto? La nicchia meningea di cellule precursori/staminali è modificata da condizioni patologiche? Il piano sperimentale di questi due anni di PhD è stato focalizzato nello studio delle cellule meningee precursori/staminali e nella nicchia staminale meningea di organismi modello (ratti e topi). Al fine di analizzare la nicchia meningea a livello cellulare e molecolare, abbiamo usato la combinazione di diverse tecniche come la microscopia confocale ad immunofluorescenza, la real time PCR, il western blot e la coltura di cellule in vitro. Per descrivere le caratteristiche cellulari e molecolari della nicchia staminale meningea, abbiamo analizzato l’espressione e la distribuzione di markers per le cellule progenitrici/staminali (nestina, dcx, cxcr4), per la proliferazione (ki67), l’auto-rinnovamento (oct4, BrdU) e per la matrice extracellulare (laminina, fibronectina). Abbiamo trovato che cellule precursori/staminali con capacità di auto-rinnovamento sono presenti nelle meningi del cervello adulto. Inoltre, abbiamo dimostrato che la presenza di una popolazione di cellule immature nestina positive è una caratteristica conservata tra le speci, compresa quella umana. Il complesso equilibrio presente nel CNS adulto include anche la partecipazione di nicchie NSC funzionali. per studiare l'influenza del SNC in condizioni di malattia nella nicchia staminale meningea, abbiamo analizzato le meningi del cervello di topi affetti da una severa immunodeficienza (SCID) e le meningi del midollo spinale di ratti lesionati (SCI). La nicchia staminale meningea nei topi SCID era profondamente cambiata. Il numero di cellule precursori/staminali era statisticamente diminuita e ciò era associato ad un drammatico aumento delle componenti della matrice cellulare ed extracellulare (fibroblasti, fibronectina e collagene). Oltre a questo, le cellule precorsori/staminali delle meningi di topi SCID hanno dimostrato una velocità proliferativa diminuita in vitro. Questi risultati indicano che la mancanza del sistema immunitario adattativo porta ad una diminuzione delle proprietà staminali della nicchia staminale meningea. Nei ratti SCI abbiamo invece trovato che la nicchia di cellule precursori/staminali aumenta in spessore, e queste cellule aumentano la loro capacità proliferativa e il loro numero. Inoltre, la lesione induce un globale aumento della staminalità legata al profilo di espressione genica. Questa osservazione suggerisce che la SCI induce nelle meningi del midollo spinale un'amplificazione delle proprietà di staminalità della nicchia. In conclusione, i principali risultati di questo lavoro sono: 1) Una popolazione di cellule Precursori/staminali è presente nelle meningi adulte ed è conservata tra le specie. 2) La nicchia meningea, compresa la popolazione di cellule nestina positive del cervello di topo adulto risulta perturbata in modelli di immunodeficienza; 3) La nicchia meningea del midollo spinale di ratto adulto è attivata da un trauma di natura contusiva: le cellule precursori/staminali proliferano ed aumentano in numero. Tutti assieme questi risultati suggeriscono un nuovo ruolo delle meningi come una potenziale nicchia di cellule precorsori/staminali endogene che possono essere modificate in condizione di malattia. Sarà necessaria un ulteriore valutazioni dei meccanismi molecolari coinvolti in condizioni fisiopatologiche delle cellule precursori/staminali delle meningi. Altri risultati potranno aprire interessanti prospettive nella ricerca di nuovi trattamenti farmacologici e nella medicina rigenerativa applicata alle malattie del SNC.
Our group have demonstrated for the first time that a new niche for stem/precursor cells with neural differentiation potential resides in brain meninges (arachnoid and pia mater) of postnatal rats. Meningeal stem/progenitor cells express the neural stem progenitor marker nestin and can be extracted and expanded in vitro as neurospheres. Moreover, they can be induced to differentiate into neurons both in vitro and in vivo (Bifari et al., 2009). Thanks to their superficial location, meninges might represent a new easy accessible tissue hosting neural stem/progenitor cell in the Central Nervous system (CNS). This represents an important aspect that may open new perspective for the possible collection of Neural Stem Cells (NSCs) for regenerative medicine and autologous transplantation. Moreover, every parenchymal vessels inside the CNS are surrounded by a perivascular space (Virchow–Robin space) formed by the extroflexions of meninges filled with cerebrospinal fluid suggesting that meningeal stem/progenitor cells might be widely distributed also in CNS parenchyma. Thus, we hypothesized that meningeal stem/progenitor cells may contribute to CNS homeostasis in health and disease. Verifying this hypothesis could offer new insights for the generation of novel pharmacological approaches to treat neurodegenerative diseases. Based on the great potential and the relevance of our previous finding, during my PhD period, I addressed the following main questions: How is the distribution of the meningeal stem/progenitor cell niche in adult brain and spinal cord? Is the meningeal stem/progenitor cell niche modified by pathological conditions? The experimental plan of these two years of PhD has been focused on the study of the meningeal stem/progenitor cells and the meningeal stem cell niche in healthy and disease animal models (rat and mice). To analyze the meningeal niche at the cellular and molecular levels, we used the combinations of different technical approaches such as immunofluorescence confocal microcopy, real time PCR, western blot and in vitro cell culture. To describe the molecular and cellular features of the meningeal stem/progenitor cells and the organization of the meningeal stem cell niche in adult animals, we analyzed the expression and 4 distribution of markers of stem/progenitor cells (nestin/dcx/cxcr4), proliferation (ki67), self renewal (oct4, BrdU) and extracellular matrix components (laminin, fibronectin, condroitin sulphate, collagen 1a). We found that stem/progenitor cells with self-renewal and proliferative properties are present in adult brain and spinal cord meninges. Moreover, we have shown that the presence of immature nestin/positive cells population is a conserved feature across species including human. The complex dynamic equilibrium present in healthy adult CNS also involves the participation of functional NSC niches. In CNS, various pathogenic events acting by different mechanisms may cause neural cell loss and chronic inflammation. Several agents and mediators sustaining these mechanisms also act on niche homeostasis and it is therefore expected that these conditions may have a deep impact on NSC biology and NSC niche properties. To investigate the influence of CNS disease conditions on the meningeal stem cell niche, we have analyzed meninges of severe combined immunodeficient (SCID) mice and spinal cord injured (SCI) rats. Meningeal stem cell niche in SCID mice was deeply changed. The number of the stem/progenitor cells was statistically significantly decreased associated with a dramatically increase in the cellular and extracellular matrix components related to fibrosis (i.e. fibroblasts, fibronectin and collagene). Furthermore, stem/progenitor cells of meninges have shown a lower proliferation rate in vitro. These data indicate that the lack of the adaptive immune system decreases the stemness properties of the meningeal stem cell niche. In SCI mice model we found that meningeal stem/progenitor cell niche is activated. Following the contusion the meningeal niche increase in thickens, stem/progenitor cells largely increase their proliferation and number. Moreover, we found that SCI induced a global increase in the stemness related gene expression profile. This observation suggests that SCI induces in spinal cord meninges an amplification of the stemness properties of the niche. In conclusion the main results of this work are: I) A stem/precursor cell population, is present in adult meninges and is conserved across species; II) The meningeal niche, including the immature nestin positive cell population, of adult mice brain result perturbed in immunodeficient animal model; 5 III) Meningeal niche is activated by contusive spinal cord injury: meningeal stem/precursor cells proliferate and increase in number. All together our data suggest a novel role for meninges as a potential niche harboring endogenous stem/precursor cells that can be functionally modulated in disease conditions. Depending on specific disease-related stimuli, the meningeal stem cell niche can react both by increasing or decreasing its stem cell properties. This differential response to specific conditions, suggests a potential role and contribution of the meningeal stem/progenitor cells in the physiopathological events occurring in CNS diseases. Further evaluation of the molecular mechanisms involved in the meningeal stem/progenitor cells contribution to the physiopathology of different diseases, will open new prospective for the research on pharmacological treatments and regenerative medicine applied to CNS disease.

Le cellule staminali neurali sono caratterizzate dall'espressione di nestina e risiedono in regioni specifiche del cervello, in particolare l'ippocampo, la zona subventricolare (SVZ) e il bulbo olfattivo. Altre strutture cerebrali che contribuiscono al corretto sviluppo della corteccia, come le leptomeningi, non sono ancora conosciute contenere cellule staminali. Le leptomeningi, che comprendono l’aracnoide e la pia madre, rivestono tutto il sistema nervoso centrale e sono bagnate da liquido cerebrospinale prodotto dai plessi coroidei. Il nostro gruppo di ricerca ha identificato nelle leptomeningi la presenza di cellule staminali nestina positive. In questo lavoro di tesi si sono caratterizzate le popolazioni cellulari e le componenti della matrice extracellulare che costituiscono il tessuto delle leptomeningi durante lo sviluppo embrionale fino all’età adulta. Utilizzando l’immunofluorescenza in microscopia confocale si è descritta l’organizzazione cellulare delle leptomeningi di ratto agli stadi embrionali di 14 e 20 giorni (E14, E20), postnatale di 0 e 15 giorni (P0, P15) e in età adulta (2 mesi). La quantificazione del numero di cellule staminali nestina positive rileva che durante lo sviluppo si ha una diminuzione progressiva dal 30,6% al 13,1% rispetto al totale delle cellule presenti nella leptomeninge (p<0,05). Inoltre si sono caratterizzate anche le cellule nestina/Ki67-positive (marcatore di proliferazione) e quelle nestina/0ct4-positive (marcatore di selfrenewal). Mediante l’utilizzo della Laser Capture Microscopy è stato possibile confermare l’espressione di geni di staminalità nel tessuto leptomeningeo. In questo studio abbiamo inoltre esaminato i cambiamenti della nicchia dove risiedono le cellule staminali nelle leptomeningi durante lo sviluppo. Abbiamo analizzato la distribuzione di differenti popolazioni cellulari incluse le cellule endoteliali e i periciti. Abbiamo inoltre caratterizzato le componenti della matrice extracellulare come la laminina, e l’eparan solfato che contribuiscono a regolare e a mantenere nello sviluppo la nicchia di cellule staminali. In conclusione in nostri risultati mostrano che le cellule staminali nestina positive sono presenti nelle leptomeningi durante lo sviluppo fino nell’età adulta. Le leptomeningi, quindi sono un tessuto composto da diverse popolazioni cellulari e da componenti della matrice extracelllulare che cotrnibusicono a creare una nicchia favorevole ad ospitare cellule staminali /precursori durante tutto lo sviluppo fino all’età adulta.
Stem cells capable of generating neural differentiated cells are recognized by the expression of nestin and reside in specific regions of the brain, namely hippocampus, subventricular zone (SVZ), and olfactory bulb. For other brain structures, such as leptomeninges, which contribute to the correct cortex development and functions, there is no evidence so far that they may contain stem/precursor cells. Leptomeninges, which include arachnoid and pia mater, cover the entire CNS and are filled with cerebrospinal fluid produced by choroid plexi. They are present since the early embryonic stages of cortical development. In this work we characterize in more details this nestin positive cell population and its niche during development. Confocal immunomicroscopy on rat brain tissue slices was used to describe the cellular organization of the rat leptomeninges at E14, E20, P0, P15 and adulthood. The nestin-positive cell layer is identified since the E20 and is located outside the basal lamina (stained by anti-laminin, marker of basal lamina) in the leptomeningeal compartment. We quantify the number of nestin positive cells in meninges and we observe that they are decreasing from 30,6% to 13,1% during development among the total number. Cluster of proliferating Ki67 - nestin positive cells were found in the leptomeningeal tissue during all developmental stages but they decrease to a lower level in a adult stage. We also analyzed the expression pattern of the self renewal marker Oct4. Clusters of nestin positive cells coexpressing Oct4 were found in all the developmental stages analysed. We further analyzed the expression pattern of other neural stem cells related markers and extracellular matrix components in the leptomeningeal niche. We analyzed the distribution of vimentin, an intermediate filament and marker of neural stem cells. At all stages vimentin-positive cells were observed in leptomeninges. Sox2 was expressed by rare leptomeningeal cells at embryonic stage 14 and 20; we could not observe expression of this stem cell marker by postnatal and adult leptomeningeal cells. DCX (marker of neuroblasts) was abundant in embryonic stages; in postnatal rats we observe rare DCX positive cells expressed in meninges. Expression of GFAP, marker of glia cells was undetectable in leptomeninges at all developmental stages analyzed. Neurotrophin receptor p75 was expressed by cells that are lining pia mater basal lamina layer and mostly not localized with nestin cells.Tuj 1 (neuroblast marker) was present in all developmental stages in brain parenchyma lying underneath basal lamina. Meninges are crossed by vessels and we studied the expression of vascular cell markers. NG2, marker of microvascular pericytes, was expressed at all developmental stages by cells surrounding blood vessels. The endothelial marker CD31 was expressed by cells lining blood vessels. Neither of the two markers were co-expressed by nestin-positive cells. In the niche, nestin positive cells are in tight contact with several extracellular matrix components such as laminin, fibronectin and heparin sulphate which provide proper regulation and maintainence of stem cells. In conclusion we show that the leptomeninges is a putative new neural stem cell niche capable of housing and maintaining up to adulthood a population of stem/progenitor cells (LeSCs) expressing stemness related genes. During development, proliferating and quiescent LeSC are in direct contact with resident niche cells and extracellular matrix molecules, which may provide the proper regulation and maintenance of the stem cell.

La neurogenesi nel cervello dei mammiferi prosegue durante il corso di tutta la vita (Eriksson et al., 1998; Gage, 2000) in due nicchie germinative: la zona sottoventricolare e il giro dentato dell’ippocampo (Gage and Temple, 2013). Le cellule della glia radiale (Kriegstein and Alvarez-Buylla, 2009) sono le cellule staminali che, durante lo sviluppo embrionale e postnatale, danno origine a diversi tipi cellulari tra cui neuroblasti, neuroni, oligodendrociti, astrociti e cellule ependimali (Kriegstein and Alvarez-Buylla, 2009). Oltre a queste nicchie di cellule staminali ampiamente caratterizzate, è stata descritta l’esistenza di nicchie neurali ectopiche in seguito a condizioni traumatiche (Pluchino et al., 2010) e in condizioni fisiologiche in regioni specifiche, come la retina, il cervelletto e i bulbi olfattivi (Menezes et al., 1995; Ponti et al., 2008; Tropepe et al., 2000). Diversi gruppi hanno recentemente identificato una nuova funzione per le meningi, da semplice struttura protettiva del sistema nervoso centrale a nicchia contenente cellule staminali endogene. E’ stato infatti dimostrato che le meningi contengono cellule che presentano un potenziale differenziativo in senso neurale nel sistema nervoso centrale dell’adulto (Bifari et al., 2009, 2015; Decimo et al., 2011; Nakagomi et al., 2011, 2012; Petricevic et al., 2011). Infatti, i precursori identificati nelle meningi sono in grado di differenziare in neuroni con elevata efficienza, sia in vitro che dopo trapianto in vivo (Bifari et al., 2009; Decimo et al., 2011). Inoltre, è stata osservata un’attivazione di questa popolazione in seguito a lesioni ischemiche cerebrali, contribuendo ad una notevole espansione del pool di cellule staminali e progenitori presenti (Nakagomi et al., 2012). I precursori neurali delle meningi contribuiscono alla reazione parenchimale che si verifica dopo una lesione al midollo spinale, migrando verso l’area lesionata ed esprimendo i marcatori (come nestina e DCX) che sono espressi anche dai precursori neurali all’interno delle nicchie staminali classiche (Decimo et al., 2011). L’identificazione di questa nuova popolazione nelle meningi, che presenta caratteristiche tipiche delle cellule staminali neurali, suggerisce un potenziale ruolo per i precursori delle meningi nel mantenimento dell’omeostasi cerebrale. Tuttavia, il possibile contributo delle cellule delle meningi alla neurogenesi in condizioni fisiologiche non è ancora stato investigato. Durante il corso dei miei studi, ho studiato il contributo delle cellule delle meningi alla neurogenesi in vivo. Abbiamo sviluppato una tecnica che ci permettesse di marcare le cellule delle meningi, in modo da poterle visualizzare e seguire nel tempo, combinando iniezioni di coloranti vitali o marcatori genetici (lentivirus o plasmidi) con l’utilizzo di modelli transgenici. Abbiamo osservato che le cellule delle meningi migrano nel periodo postnatale, dalla loro sede esterna al parenchima cerebrale fino alle cortecce restrospleniale e visuo-motoria. In seguito, queste cellule differenziano in neuroni che sono funzionali dal punto di vista elettrofisiologico, integrati nel network neuronale esistente e responsivi a stimoli farmacologici. Inoltre, abbiamo dimostrato che queste cellule neurogeniche appartengono alla popolazione perivascolare delle meningi che esprime PDGFRß. Nonostante non sia stata ancora chiarita l’origine embrionale di queste cellule, i nostri dati preliminari suggeriscono una possibile provenienza dalla popolazione della cresta neurale. In conclusione, una riserva di progenitori residente nelle meningi, di origine embrionale, contribuisce alla neurogenesi corticale nel periodo postnatale. Queste cellule rappresentano una riserva di cellule staminali endogene e potrebbero quindi essere utilizzate in medicina rigenerativa nel trattamento dei disordini neurodegenerativi.
Neurogenesis continues throughout life in mammalian brain (Eriksson et al., 1998; Gage, 2000) in two germinal niches: the subventricular zone lining the lateral ventricle and subgranular zone in the dentate gyrus of the hippocampus (Gage and Temple, 2013). Radial glial cells (Kriegstein and Alvarez-Buylla, 2009) are the neural stem cells that, during embryonic and postnatal development, give rise to various cell types including neuroblasts, neurons, oligodendrocytes, astrocytes and ependymal cells (Kriegstein and Alvarez-Buylla, 2009). In adult mice, newly formed neuroblasts migrate through the rostral migratory stream to the olfactory bulb, where they continually replace local interneurons (Imayoshi et al., 2008). Apart from these well-established neural stem niches, the existence of ectopic neural stem cell niches has been reported following injury (Pluchino et al., 2010), as well as in selected physiological conditions in the retina, cerebellum and olfactory bulb (Menezes et al., 1995; Ponti et al., 2008; Tropepe et al., 2000). Interestingly, several independent groups have recently identified a novel role for meninges as a potential niche harbouring endogenous stem cells with neural differentiation potential in the adult central nervous system (Bifari et al., 2009, 2015; Decimo et al., 2011; Nakagomi et al., 2011, 2012; Petricevic et al., 2011). Surprisingly, meningeal neural precursors are able to differentiate both in vitro and, after transplantation in vivo, into neurons with extremely high efficiency (Bifari et al., 2009; Decimo et al., 2011). Moreover, these cells can be activated by central nervous system parenchymal injuries, undergoing an extensive expansion of stem cells and progenitors (Nakagomi et al., 2012). Meningeal neural precursors contribute to neural parenchymal reaction after spinal cord injury, migrating to the perilesioned area, while expressing the same markers (nestin and DCX) that are transiently expressed by neural precursors within classic neurogenic niches (Decimo et al., 2011). The finding of this new cell population in the meninges, with stem cell features, provides new insights into the complexity of the parenchymal reaction to a traumatic injury and suggests a potential role for meningeal progenitor cells in the maintainance of brain homeostasis. However, the possible contribution of meningeal neural precursors to neurogenesis in physiological conditions has not previously been investigated. During the course of my studies, I explored the hypothesis that meningeal cells may contribute to neurogenesis in vivo. We were able to specifically tag meningeal cells in P0 pups and track them during time, combining injection of cell tracers in the meningeal subarachnoid space and transgenic mouse lines. We found that neurogenic meningeal cells migrate from their location outside the brain parenchyma, along the meningeal substructures, to the retrosplenial and visual motor cortices during the neonatal period. Subsequently, meningeal-derived cells differentiate into cortical neurons that are electrophysiologically functional, integrated in the existing network and responsive to pharmacological stimuli. In addition we found that these meningeal neurogenic cells belongs to the perivascular PDGFRß+ lineage and are mainly additive to the well-characterized neurogenic parenchymal radial glia. Although the developmental origin of these cells still has to be elucidated, our preliminary data indicate a possible neural crest-derivation. Thus, a reservoir of embryonic derived progenitors residing in the meninges contributes to postnatal cortical neurogenesis. These cells may have a role as endogenous stem cell pool that can be exploited in regenerative medicine for neurodegenerative diseases.

Nonostante le lesioni traumatiche del midollo spinale siano abbastanza frequenti e comportino una grave sofferenza per l’individuo (sia livello fisico che psicologico) e pesanti costi di assistenza diretta ed indiretta, per questa patologia la terapia è ancora limitata ad interventi di minimizzazione del danno in fase acuta e di supporto e sostegno fisico nella fase cronica. Lo sviluppo della medicina rigenerativa ha ovviamente prodotto una grande aspettativa in questo settore; la scoperta della presenza di cellule neurali staminali nel sistema nervoso centrale dell’adulto e l’ampliarsi della conoscenza dei meccanismi che ne regolano il destino hanno infatti fatto intravedere la possibilità di applicazioni terapeutiche di queste cellule nel danno al midollo spinale. Di conseguenza, un buon numero di trials clinici basati sul trapianto di cellule staminali di diversa origine è stato attivato anche se, al momento, non è ancora emersa una soluzione definitiva. La maggior parte dei trapianti sperimentati è stata condotta sinora con cellule staminali non neurali in quanto queste sono di facile reperimento anche da individui adulti; per quanto infatti le cellule staminali neurali siano efficientemente prelevabili da blastocisti e da embrioni, nell'adulto le uniche fonti consistenti sono rappresentate solo da alcune nicchie localizzate in vicinanza dell'ippocampo e dei ventricoli, quindi in zone di difficile accesso per un prelievo autologo o in donatore vivente. Il nostro gruppo ha recentemente dimostrato che le leptomeningi ospitano una popolazione di cellule con proprietà staminali neurali presenti nel roditore anche in età adulta. Queste cellule possono essere coltivate ed espanse in vitro come neurosfere e possono essere indotte a differenziare in neuroni ed oligodendrociti. Se trapiantate in area ippocampale o ventricolare, queste cellule si integrano con il tessuto normale, entrando apparentemente a far parte dell'esistente rete neuronale. Inoltre, a seguito di danno traumatico del midollo spinale, le cellule delle leptomeningi si attivano e migrano all’interno del parenchima, dove partecipano alla reazione al trauma. Considerata la facile accessibilità chirurgica delle meningi e la loro presenza in età adulta, le cellule staminali delle leptomeningi (LeSCs) rappresentano un potenziale candidato per la terapia rigenerativa del danno al midollo spinale; altrettanto importante è l'osservazione che le LeSCs possono essere isolate anche da biopsie di meningi umane prelevate nel corso di interventi neurochirurgici (asportazione di tumori). Questo progetto di tesi indaga in profondità la possibile applicazione di LeSCs per terapie rigenerative di traumi del midollo spinale; considerato il fatto che i fenomeni di demielinizzazione post-traumatica giocano un ruolo fondamentale nella patogenesi del danno al midollo spinale e che studi condotti con cellule di diversa origine hanno dimostrato come un approccio di medicina rigenerativa basato sulla stimolazione dei processi di rimielinizzazione possa portare a risultati promettenti, nel mio lavoro ho innanzitutto sviluppato ed ottimizzato un metodo in grado di amplificare le LeSCs in vitro e di differenziarle efficacemente in oligodendrociti. É stato quindi messo a punto un protocollo innovativo di crescita e differenziamento delle LeSCs e, mediante l’analisi dell’espressione proteica e genica, è stato analizzato e dimostrato come queste cellule acquisiscano sia la tipica morfologia degli oligodendrociti, sia un’elevata espressione di diversi geni mielina-specifici. Il potenziale rigenerativo degli oligodendrociti derivati dalle LeSCs è stato quindi verificato in vivo in un modello animale di danno contusivo al midollo spinale. Nelle nostre condizioni sperimentali il trapianto degli oligodendrociti è associato ad un significativo aumento del recupero di alcune funzioni motorie, come determinato dalla valutazione con BBB score e analisi CatWalk. In conclusione, questo lavoro suggerisce per la prima volta che le cellule staminali delle leptomeningi possono rappresentare una risorsa nella terapia cellulare del danno del midollo spinale e apre la strada per futuri studi di medicina rigenerativa applicabili all’uomo.
Spinal cord injury (SCI) is a single event with devastating effects on the life of patients both in physiological and psychological terms and for which only supportive and damage-limiting interventions are available at the moment. In the last decades, regenerative therapies based on cell transplantation have generated increasing attention as a potential therapeutic approach for degenerative diseases such as spinal cord injury. In addition, the discovery of neural stem cells in the adult central nervous system and the expansion of the knowledge of the mechanisms regulating their fate have increased the expectations for therapeutic application of these cells to spinal cord injury. Indeed, a considerable number of potential cell-based regenerative therapies have reached the stage of clinical trial, but a clear solution has not emerged yet. We have recently shown that the leptomeninges host a cell population with neural stem/progenitor properties both in vitro and in vivo: isolated leptomeningeal cells can be propagated in vitro as neurospheres and induced to differentiate into neurons and oligodendrocytes. Moreover, they have been shown to become activated by injury to both the brain and the spinal cord and to migrate in the parenchyma, where they participate in the reaction to the injury. Considering the easily accessible anatomical location of the meninges, leptomeningeal stem/progenitor cells (LeSCs) represent a potential candidate for regenerative cell therapy for spinal cord injury. With this work, we provide a first evidence that leptomeningeal cells might indeed play a role in regenerative therapies applied to SCI. Considering the pathogenetic role of demyelination in SCI and that remyelination is a promising therapeutic approach, we first developed and optimized a method for efficient in vitro production of LeSCs and differentiation into mature oligodendrocytes; protein and gene expression analysis showed that by the end of the protocol cultured LeSCs acquired both the typical morphology of mature oligodendrocytes and the elevated expression of different myelin-specific genes. In addition, we performed a pilot study of the regenerative potential of LeSCs-derived oligodendrocyte precursors in an animal model of contusive spinal cord injury. In our conditions, cells transplantation was associated with a significant improvement of some of the motor functions, as determined by behavioural evaluation through BBB score and CatWalk gait analysis. This work indicates for the first time that leptomeningeal stem/progenitor cells could represent an asset in both transplantational and pharmacological therapy for spinal cord injury and paves the way to further studies of regenerative medicine in human SCI.