Littérature scientifique sur le sujet « Leptomeningeal Stem Cells »

Leptomeningeal dissemination (LMD) is the defining pattern of metastasis for medulloblastoma. Although LMD is responsible for virtually 100% of medulloblastoma deaths, it remains the least well-understood part of medulloblastoma pathogenesis. The fact that medulloblastomas rarely metastasize outside the CNS but rather spread almost exclusively to the spinal and intracranial leptomeninges has fostered the long-held belief that medulloblastoma cells spread directly through the CSF, not the bloodstream. In this paper the authors discuss selected molecules for which experimental evidence explains how the effects of each molecule on cell physiology contribute mechanistically to LMD. A model of medulloblastoma LMD is described, analogous to the invasion–metastasis cascade of hematogenous metastasis of carcinomas. The LMD cascade is based on the molecular themes that 1) transcription factors launch cell programs that mediate cell motility and invasiveness and maintain tumor cells in a stem-like state; 2) disseminating medulloblastoma cells escape multiple death threats by subverting apoptosis; and 3) inflammatory chemokine signaling promotes LMD by creating an oncogenic microenvironment. The authors also review recent experimental evidence that challenges the belief that CSF spread is the sole mechanism of LMD and reveal an alternative scheme in which medulloblastoma cells can enter the bloodstream and subsequently home to the leptomeninges.

Abstract INTRODUCTION Leptomeningeal carcinomatosis remains one of the most lethal forms of central nervous system metastasis, with a median survival of only 4 months. Effective new therapies are urgently needed to treat this highly aggressive cancer. In this study, we used models of both prophylactic and established leptomeningeal disease to investigate the efficacy of engineered tumor-homing neural stem cells (NSCs) therapy for breast cancer leptomeningeal carcinomatosis. METHODS Personalized NSC carriers were created using Sox2 overexpression to transdifferentiate human fibroblasts into induced NSCs (iNSCs) that home to cancer cells and carry therapeutic agents to induce tumor kill. Leptomeningeal models were created by engineering MDA-MB231-Br human breast cancer cells with fluorescent and bioluminescent reporters, then using intracisternal injection to inoculate Nude mice with the tumor cells. iNSC therapy was evaluated by infusing iNSCs releasing the pro-apoptotic agent TRAIL into the lateral ventricle of mice either 1 week prior to or 3 days after tumor inoculation for prophylactic or established tumor treatment respectively. Tumor progression in the brain and spinal cord was monitored by serial bioluminescence imaging (BLI). RESULTS Serial BLI showed that intracerebroventricular (ICV) iNSC-TRAIL therapy reduced the volume of metastatic tumor burden 99.49% in the brain and 99.80% in the spine within 2 weeks post-infusion and extended survival from 24 to 42 days. Additionally, prophylactic iNSC-TRAIL therapy delivered ICV markedly delayed tumor development, with tumors in the brain remaining >1000-fold smaller than control through 1-month post-treatment, below the limit of detection in the spinal cord through 1 month, and eliminating mortality through 50 days post-therapy. CONCLUSION These data suggest that iNSC therapy could be a promising treatment option for breast cancer patients with leptomeningeal carcinomatosis.

Abstract INTRODUCTION Non-small cell lung cancer (NSCLC) is the most common cancer to spread to the brain, and spread to the leptomeninges is particularly devastating, with a median survival of only months. While radiation may offer symptomatic relief, new adjuvant therapies are needed for more durable tumor kill. Spheroidal, human induced neural stem cells (hiNeuroS) transdifferentiated from fibroblasts are inherently tumoritropic. When engineered to secrete the cytotoxic protein TRAIL, they provide the potential for a personalized, targeted approach to NSCLC leptomeningeal metastases. METHODS hiNeuroS-TRAIL in vivo efficacy was determined by tracking the progression and survival of mice with NSCLC leptomeningeal tumors treated with intracerebroventricular hiNeuroS, radiation, or both. To determine the impact of radiation on the tumor tropism of hiNeuroS, we performed 2-dimensional motion assays on hiNeuroS with and without the presence of NSCLC pre- and post-radiation. Migrational capacity in vivo was determined by infusing hiNeuroS into the lateral ventricles of mice with established NSCLC tumors and monitoring hiNeuroS accumulation using post-mortem fluorescent analysis. RESULTS/CONCLUSION Mice treated with the combination of hiNeuroS-TRAIL and 2 Gy showed a significantly reduced mean tumor signal (2.7%) compared to controls (100%) or 2 Gy-only (54.9%). Mice treated with 2 Gy alone showed no significant survival difference compared to controls. Both combination and hiNeuroS-TRAIL-only-treated mice showed a significant improvement in median survival compared to controls (36.6% and 46.3% improvement, respectively). hiNeuroS showed enhanced directionality and displacement in the presence of NSCLC in 2-dimensional motion assays, indicating directional migration, and they maintained this ability following exposure to radiation. Co-localization of hiNeuroS with NSCLC was also observed in vivo. These results suggest the potential of hiNeuroS-TRAIL as a powerful adjuvant to radiation in the treatment of leptomeningeal NSCLC.

Abstract Allogeneic stem cell-based therapy is a promising tool for the treatment of a range of human degenerative and inflammatory diseases. Many reports highlighted the immune modulatory properties of some stem cell (SC) types, such as mesenchymal stromal cells (MSCs), but a comparative study with SCs of different origin, to assess whether immune regulation is a general SC property, is still lacking. To this aim, we applied highly standardized methods employed for MSC characterization to compare the immunological properties of bone marrow-MSCs, olfactory ecto-mesenchymal stem cells, leptomeningeal stem cells, and three different c-Kit-positive SC types, i.e. amniotic fluid SCs, cardiac SCs, and lung SCs. We found that all the analyzed human SCs share a common pattern of immunological features, in terms of expression of activation markers, modulatory activity towards immune effector cells, immunogenicity and molecular inhibitory pathways, with some SC type-related peculiarities. In addition, we found that the inhibitory behaviour is not a constitutive property of SCs, but is acquired as a consequence of immune effector cell activation, as previously described for MSCs. Thus, immune regulation is a general property of stem cells and the characterization of this phenomenon may be useful for a proper therapeutical use of SCs. Disclosures: No relevant conflicts of interest to declare.

Abstract A detailed autopsy and autoradiographic study was performed after the death of a patient undergoing intrathecal, antibody-guided irradiation for carcinomatous meningitis. The results demonstrated tumor cells infiltrating the surface meninges and a severe astrocytic reaction associated with oedema in the periventricular and brain stem subpial white matter. This was not seen in cortical or other gray matter structures. Autoradiographic examination correlated well, demonstrating isotope within the oedematous areas of the white matter in addition to the expected concentration in the leptomeningeal layers. These findings are discussed in the context of antibody binding to tumor tissue and the possible benefits conferred in the treatment of infiltrating tumor cells.

Engineered tumor-homing neural stem cells (NSCs) have shown promise in treating cancer. Recently, we transdifferentiated skin fibroblasts into human-induced NSCs (hiNSC) as personalized NSC drug carriers. Here, using a SOX2 and spheroidal culture-based reprogramming strategy, we generated a new hiNSC variant, hiNeuroS, that was genetically distinct from fibroblasts and first-generation hiNSCs and had significantly enhanced tumor-homing and antitumor properties. In vitro, hiNeuroSs demonstrated superior migration to human triple-negative breast cancer (TNBC) cells and in vivo rapidly homed to TNBC tumor foci following intracerebroventricular (ICV) infusion. In TNBC parenchymal metastasis models, ICV infusion of hiNeuroSs secreting the proapoptotic agent TRAIL (hiNeuroS-TRAIL) significantly reduced tumor burden and extended median survival. In models of TNBC leptomeningeal carcinomatosis, ICV dosing of hiNeuroS-TRAIL therapy significantly delayed the onset of tumor formation and extended survival when administered as a prophylactic treatment, as well as reduced tumor volume while prolonging survival when delivered as established tumor therapy.

Abstract INTRODUCTION Breast cancer brain metastasis, including leptomeningeal carcinomatosis (LC), remains one of the most lethal CNS diseases. New therapies are urgently needed to treat this highly aggressive disease. Here we used models of both breast cancer brain parenchymal metastasis and leptomeningeal metastasis to investigate the efficacy of engineered tumor-homing neural stem cells (NSCs) therapy. METHODS Personalized NSCs were created using Sox2 overexpression to transdifferentiate human fibroblasts into induced NSCs (iNSCs), followed by genetic engineering to enable iNSCs to secrete cytotoxic TRAIL (iNSC-TRAIL). For the parenchymal metastasis study, iNSC-TRAIL therapy was infused intracerebroventricularly (ICV) into Nude mice bearing established intracranial MDA-MB-231-Br human breast cancer cells expressing fluorescent and bioluminescent reporters. For LC studies, we established the disease model by inoculating Nude mice with MDA-MB-231-Br tumor cells via intracisternal infusion. iNSC-TRAIL therapy was evaluated by infusing therapy ICV either 1 week prior to or 3 days after tumor inoculation to mirror prophylactic or established tumor treatment, respectively. Tumor progression in the brain and spine was monitored by serial bioluminescence imaging (BLI), and survival was analyzed. RESULTS Serial BLI showed ICV-infused iNSC-TRAIL reduced parenchymal tumor volumes by 72% 3 weeks post-ICV infusion, and extended median survival from 37 to 52 days. Testing iNSC-TRAIL therapy against established LC tumors, serial BLI showed ICV iNSC-TRAIL therapy reduced established tumors 196-fold in the brain and 500-fold in the spine within 2 weeks post-infusion, while extending median survival from 25 to 47 days. In the prophylactic LC model, iNSC-TRAIL therapy markedly delayed tumor development with tumors in the brain remaining > 1000-fold smaller than control, and tumors in the spine below the limit of detection through 1 month post-treatment. The therapy also eliminated mortality through 50 days post-therapy. CONCLUSION These data suggest iNSC therapy could be a promising treatment option for breast cancer brain metastasis patients.

Staminali neuronali adulte (NSC), sono state trovate nelle primcipali aree neurogeniche del cervello, per esempio ippocampo, regione subventricolare (SVZ), bulbi olfattivi, e in alcune regioni non neurogeniche come ad esempio il midollo spinale. Altre regioni del cervello possono ospitare nicchie di NSC e, in particolare, considerando il ruolo delle meningi nel corretto sviluppo della corteccia cerebrale, è nostro interesse esplorare la regione delle leptomeningi che si estende dall’aracnoide fino al primo strato della corteccia cerebrale. Lo scopo di questo progetto è caratterizzare le leptomeningi come potenziale nicchia di cellule staminali neuronali. La regione delle leptomeningi è stata caratterizzata mediante immunoistochimica, in ratti di diversa età, dall’embrione E20, a ratti in età postnatale P0, P15 e nell’adulto. Cellule positive per il marcatore di cellule staminali neuronali nestina, sono state individuate in leptomeninge. Queste cellule sono distribuite fuori dalla membrane basale (positive per il marker Laminina), come una popolazione distinta dagli astrociti (cellule GFAP positive) e dai precursori oligodendrocitari (cellule NG2 positive ), che risiedono nel tessuto circostante. Le cellule nestine positive sono state prelevate dale leptomeningi di ratti P0, P15 e adulti ed espanse in vitro. Le cellule così prelevate sono state espanse in aderenza come una popolazione omogena di cellule nestina positive. Se sottoposto a stimuli differentiativi neuranali, le cellule nestine positive sono in grado di differenziare principalmente in neuroni (positive per MAP2), ma anche in astrociti ed oligodendrociti (positive per O4). Come primo approcio di analisi funzionale delle cellule differenziate in vitro, è stata valutata la loro capacità di rispondere a stimuli depoarizzanti mediante calico imaging, dopo incubazione delle cellule con Fura2. I neuroni ottenuti dal differenziamento in vitro delle cellule nestine positive sono in grado di rispondere all’applicazione dell’agente depolarizzante KCl, suggerendo l’espressione di canali del calico voltaggio dipendenti, come i neuroni funzionali. Il potenziale differenziativo in vivo di queste cellule è stato valutato mediante infusione stereotassica in ippocampo di ratti adulti, di cellule nestine positive estratte dalle leptomeningi di ratti transgenici EGFP. L’ippocampo dei ratti iniettati sono stati analizzati mediante immunofluorescenza a due mesi dall’iniezione delle cellule EGFP. Circa metà delle cellule EGFP identificate in ippocampo esprimevano markers neuronali (DCX, MAP2, NeuN, Neurofilament-160, GAD67). Vista la persistenza di queste cellule nestina positive nelle meningi di ratto durante lo sviluppo fino all’età adulta, dato il loro potenziale proliferativo in vitro ed il loro potenziale differenziativo neuronale sia in vitro che in vivo, queste cellule sono state proposte come nuova entità con il nome di Leptomeningeal stem/progenitor cells (LeSC). Dall’anatomia delle meningi si evince che ricoprono l’intero sistema nervosa centrale, il che comprende anche il midollo spinale. Per questo motivo sono state analizzate anche le leptomeningi che ricoprono il midollo spinale. Come osservato in precedenza per il cervello, cellule positive per il marcatore delle cellule staminali neuronali nestina, sono state individuate in leptomeninge. Queste cellule sono distribuite fuori dalla membrane basale (positive per il marker Laminina), come una popolazione distinta dagli astrociti (cellule GFAP positive) e dai precursori oligodendrocitari (cellule NG2 positive ), che risiedono nel tessuto circostante. Un nuovo studio in collaborazione con la professoressa M. Schwartz group (Weizmann Institute, Rehovot, Israel) è in corso sul potenziale ruolo del sistema immunitario nel regolare le leptomeningi ed in particolare le LeSC (come suggerito da precedenti pubblicazioni del gruppo della prof. Schwartz). Risultati preliminary sul confronto ex vivo della proliferazione delle LeSC in topi SCID e wt, mostrano una significativa diminuzione dl numero di LeSC nestinepositive in topi SCID. Nonostante questa diminuzione di cellule nestine positive, il numero totale di cellule che risiedono in leptomeninge è comparabile in entrambi I topi SCID e wt. E’ in corso una più estensiva caratterizzazione delle leptomeningi dei topi SCID e wt per capire la natura delle cellule nestine negative che risiedono nelle leptomeningi dei topi SCID. L’importanza delle LeSC risiede nella posizione facilmente raggiungibile rispetto alle già note nicchie di staminali neuronali, ed inoltre nell’elevato potenziale differenziativo neuronale. Queste peculiarità apriranno nuovi studi nell’ambito della medicina rigenerativa
Adult neural stem cells (NSC), have been found in the main neurogenic regions of brain, i.e. hippocampus, sub ventricular zone (SVZ), olfactory bulb, and in some non-neurogenic regions, i.e. spinal cord. Other brain sites could host NSC niches and, in particular, considering the role of meninges in correct cortex development we were interested in exploring the region residing between arachnoide and the first layers of the cerebral cortex, called Leptomeninges. Aim of this project is characterized the leptomeningeal compartment as potential niche for neural stem cells with ex vivo and in vitro approaches. The leptomeningeal compartment has been characterized by immunohistochemistry at different rat ages, from embryo E20, postnatal day 0 (P0), P15 and adult. We found a(nestin) neuro-epithelial stem cells marker positive cells layer with decreasing thickness from embryo up to adult. Nestin positive cells were distributed outside the basal lamina (marked by laminin), and as a distinct population from astrocytes (stained with GFAP) and oligodendrocytes (stained with NG2). Nestin positive cells were dissected and expanded in vitro from P0, P15 and adult rats leptomeninges. We were able to culture them as homogeneus nestin positive cells population in adherent condition In neuronal differentiating conditions, nestin positive cells mainly differentiate into MAP2 positive cells but also GFAP and O4 (marker for mature oligodendrocyte) positive cells were detected in culture. As a first level of functional evaluation of differentiated cells, their ability to depolarize has been analyzed by calcium imaging assay after Fura-2 loading. In vitro differentiated neurones responded to fast applications of the depolarizing agent KCl suggesting the expression of voltage dependent calcium channels, similar to that of functional neurons. As following step, the in vivo neuronal differentiation potential was assessed by infusion of expanded EGFP LeSC in rat hippocampus. Engrafted LeSC were monitored by immunofluorescence up two months and during this period LeSC were able to survive after injection. About half of EGFP cells engrafted in hippocampus, expressed neuronal markers (DCX, MAP2, NeuN, Neurofilament-160, GAD67) and shown differentiated neuronal morphology. Because of the persistence of these cells up to adulthood, their proliferation capability in vitro, and their differentiation potential into neuronal cells in vitro and in vivo, we suggest to name them leptomeningeal stem/progenitor cells (LeSC) as a new population never described before. Since meninges cover whole brain, also Leptomeninges from rat spinal cord has been analyzed. Nestin positive cells were distributed as previously observed in the brain, outside the basal lamina, and as a distinct population from astrocytes and oligodendrocytes. Cells were dissected and kept in culture as neurosphere and resulted positive for nestin, MAP2, GFAP, O4, and Oct4. A new study In collaboration with professor M. Schwartz group (Weizmann Institute, Rehovot, Israel) is ongoing to understand the potential role of immune system in regulating leptomeninges and LeSC (as suggested by previous publications from Schwartz’s group). Preliminary results Comparison of LeSC proliferation and nestin expression by immunohistochemistry in SCID vs wt mice, revealed a significant decrease of nestin positive LeSC in SCID mice. However total cell number and proliferating cells in leptomeninges were not changed. Further characterizations are ongoing to understand the phenotype of proliferating nestin negative cells in meninges. The importance of Leptomeningeal stem cells reside in the easier reachable localization compared to the already known neural stem cell niches, and in their high neuronal differentiation potential. These characteristics will open novel studies in regenerative medicine.

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.

La terapia mediante l’uso di cellule staminali allogeniche è una potenziale alternativa per la cura di malattie infiammatorie e degenerative. Vari gruppi di ricerca hanno messo in luce le proprietà immunomodulatorie di varie cellule staminali, come per esempio le cellule mesenchimali stromali, ma al momento non è stato ancora fatto uno studio comparativo mostrante differenze qualitative e quantitative delle proprietà immunoregolatorie di staminali di diversa origine. Lo scopo del lavoro è stato quindi quello di confrontare le proprietà immunologiche di varie cellule staminali, tra cui cellule mesenchimali stromali di origine midollare, cellule staminali olfattorie, cellule staminali isolate da leptomeningi, e tre tipi di cellule staminali esprimenti il recettore c-Kit, ovvero cellule staminali del fluido amniotico, cellule staminali cardiache e cellule staminali polmonari. Tutte queste tipologie cellulari mostravano caratteristiche immunologiche comuni, come per esempio l’espressione dei marcatori attivatori ICAM-1, VCAM-1, HLA-ABC, e HLA-DR, e quando coltivate con linfociti T, NK e B purificati, erano in grado di regolarne la proliferazione. Il trattamento con citochine infiammatorie riduceva l’immunogenicità delle staminali analizzate nei confronti di linfociti NK rispetto alle cellule controllo, e induceva l’espressione dell’indolamina-2,3 diossigenasi (IDO) responsabile dell’immunosoppressione dei linfociti T. Infine, tutte le cellule staminali analizzate mostravano un effetto anti-apoptotico nei confronti delle cellule effettrici immuni non stimolate. Inoltre, in questo lavoro abbiamo mostrato che l’effetto immunosoppressivo non è una proprietà costitutiva delle cellule staminali, ma è una conseguenza dell’induzione mediata da citochine infiammatorie, effetto precedente mostrato per le cellule mesenchimali stromali. L’immunoregolazione è una proprietà condivisa da vari tipi di cellule staminali, e una caratterizzazione più approfondita di questo meccanismo potrebbe essere fondamentale per il loro uso terapeutico.
Allogeneic stem cell (SC)-based therapy is a promising tool for the treatment of a range of human degenerative and inflammatory diseases. Many reports highlighted the immune modulatory properties of some SC types, such as mesenchymal stromal cells (MSCs), but a comparative study with SCs of different origin, to assess whether immune regulation is a general SC property, is still lacking. To this aim, we applied highly standardized methods employed for MSC characterization to compare the immunological properties of bone marrow (BM)-MSCs, olfactory ectomesenchymal SCs (OE-MSCs), leptomeningeal SCs (LeSCs), and three different c-Kit-positive SC types, that is, amniotic fluid SCs (AFSCs), cardiac SCs (CSCs), and lung SCs (LSCs). We found that all the analyzed human SCs share a common pattern of immunological features, in terms of expression of activation markers ICAM-1, VCAM-1, HLA-ABC, and HLA-DR, modulatory activity toward purified T, B, and NK cells, lower immunogenicity of inflammatory-primed SCs as compared to resting SCs, and indoleamine-2,3-dioxygenase (IDO)-activation as molecular inhibitory pathways, with some SC type-related peculiarities. Moreover, the SC types analyzed exert an anti- apoptotic effect toward not-activated immune effector cells (IECs). In addition, we found that the inhibitory behavior is not a constitutive property of SCs, but is acquired as a consequence of IEC activation, as previously described for MSCs. Thus, immune regulation is a general property of SCs and the characterization of this phenomenon may be useful for a proper therapeutic use of SCs.

Cellule staminali con potenzialità differenziativa neurale sono cararatterizzate dall’espressione di nestina e risiedono in specifiche aree dell’encefalo, quali l’ippocampo, la zona sottoventricolare (SVZ) e il bulbo olfattivo. Questo lavoro si basa sul’ipotesi che altre strutture encefaliche possano contenere nicchie di cellule staminali neurali. Noi ci siamo focalizzati nell’esplorare la porzione comprendente i primi strati corticali e le leptomeningi, poichè in questa regione, interazioni spazio-temporali tra le cellule assicurano la corretta corticogenesi. Il nostro lavoro ha identificato la presenza di una popolazione di cellule nestina-positive nelle leptomeningi di ratto durante lo sviluppo fino all’età adulta. Queste cellule nestina-positive possono essere estratte ed espanse in vitro sia come neurosfere, mostrando elevata similitudine con le neurosfere ottenute da staminali neurali di SVZ, che come coltura omogenea di cellule con caratteristiche di staminalità. La popolazione di cellule staminali espansa in vitro può essere indotta a differenziare con elevata efficienza in cellule eccitabili con fenotipo e morfologia neuronale. Trapiantate in un encefalo di ratto adulto, queste cellule sopravvivono e differenziano in neuroni, mostrando quindi che il potenziale differenziativo neurale è mantenuto anche in vivo. In conclusione, questi dati evidenziano l’esistenza di una popolazione di cellule immature con potenziale differenziativo neuronale residenti nelle leptomeningi per tutta la durata della vita. Considerando che le leptomeningi ricoprono l’intero sistema nervoso centrale, questi risultati potrebbero avere ripercussioni importanti nell’ambito della medicina rigenerativa applicata alle patologie neurologiche e per studi concernenti lo sviluppo corticale.
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. Our work hypotesis is based on the assumption that other brain sites could host NSC niches. We were interested in exploring the region between leptomeninges and the first layers of the cerebral cortex. In this region, spatial-temporal interactions amongst environmental cells ensure the correct cortex development. In this work, we show that nestin-positive cells are present in rat leptomeninges during development up to adulthood. The newly identified nestin-positive cells can be extracted and expanded in vitro both as neurospheres, displaying high similarity with SVZ-derived neural stem cells, and as homogeneous cell population with stem cell features. In vitro expanded stem cell population can differentiate with high efficiency into excitable cells with neuronal phenotype and morphology. Once injected into adult brain, these cells survive and differentiate into neurons, thus showing that their neuronal differentiation potential is operational also in vivo. In conclusion, our data provide evidence that a specific population of immature cells endowed of neuronal differentiation potential is resident in the leptomeninges throughout the life. As leptomemniges cover the entire central nervous system, these findings could have relevant implications for studies on cortical development and for regenerative medicine applied to neurological disorders.

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.

Usual diagnostic methods of leptomeningeal metastases (LM) in CerebroSpinal fluid (CSF), lack both specificity and sensitivity. The Veridex CellSearch® technique quantifying circulating tumour cells (CTCs) in blood was adapted to detect Tumour Cells (CSFTCs) in CSF from cancer patients with LM. CSF samples from 60 patients with established or suspected breast cancer or lung cancer LM and/or melanoma were evaluated. 5 mL CSF samples were collected on CellSave® preservative and analyzed within 3 days after CSF sampling. Gold Standard cytological analysis on 1 to 10 mL CSF samples from patients with established LM allowed sometimes the detection but usually not the quantification of TCs. In established LM, EpCAM+/cytokeratin+ or CD146+/HMW-MAA+ nucleated (DAPI+) cells were observed and enumerated with precision from one to up to 10 000 cells/mL. Their morphology on digital images galleries could be discriminant between breast and lung cancer. This methodology, established on a limited volume of CSF compared to the Gold Standard and allowing delayed processing, is of great interest in the diagnosis and follow-up of cancer patients with LM. The reliability of the method also opens new fields of investigation for other biological fluids and to precise the stem cell potential of metastatic cells in CSF.