Academic literature on the topic 'Cellule staminali neurali'

3D cell cultures are becoming more and more important in the field of regenerative medicine due to their ability to mimic the cellular physiological microenvironment. Among the different types of 3D scaffolds, we focus on the Nichoid, a miniaturized scaffold with a structure inspired by the natural staminal niche. The Nichoid can activate cellular responses simply by subjecting the cells to mechanical stimuli. This kind of influence results in different cellular morphology and organization, but the molecular bases of these changes remain largely unknown. Through RNA-Seq approach on murine neural precursors stem cells expanded inside the Nichoid, we investigated the deregulated genes and pathways showing that the Nichoid causes alteration in genes strongly connected to mechanobiological functions. Moreover, we fully dissected this mechanism highlighting how the changes start at a membrane level, with subsequent alterations in the cytoskeleton, signaling pathways, and metabolism, all leading to a final alteration in gene expression. The results shown here demonstrate that the Nichoid influences the biological and genetic response of stem cells thorough specific alterations of cellular signaling. The characterization of these pathways elucidates the role of mechanical manipulation on stem cells, with possible implications in regenerative medicine applications.

In questa seconda parte, l’attenzione viene focalizzata sulle “cellule staminali non embrionali”, cioè le cellule staminali somatiche (di origine fetale o adulta) e le cellule staminali del sangue del cordone ombelicale. Queste cellule, spesso definite “cellule staminali adulte”, sono state identificate prima delle cellule staminali embrionali. Infatti, l’espressione stessa di cellula “staminale” deriva dall’identificazione delle cellule staminali emopoietiche nel midollo osseo (1961). Più tardi le ricerche hanno evidenziato la presenza di tali cellule immature, multipotenti, che si auto-rinnovano e si auto-differenziano pressoché in tutti i tessuti ed organi del feto e dell’adulto. Appena scoperte, queste cellule staminali “adulte” hanno trovato subito un impiego terapeutico con i primi trapianti di midollo osseo per il trattamento di patologie, maligne e non, del sangue e del sistema linfoide. Oggi le cellule staminali emopoietiche sono usate anche nel trattamento di malattie auto-immuni, come la sclerosi multipla o il lupus erythematosus e nella medicina rigenerativa. Una seconda fonte importante di cellule staminali “adulte” è rappresentata dalle cellule staminali mesenchimali, situate principalmente nel midollo osseo, progenitrici di vari ceppi cellulari: osso, cartilagine, muscolo, tessuto adiposo e astrociti. Queste cellule sembrano avere un ruolo-chiave nella rigenerazione dei tessuti. Sono stati isolati diversi tipi di cellule mesenchimali multipotenti, con proprietà paragonabili a quelle delle cellule staminali embrionali. Il più noto è quello delle MAPCs di Catherine Verfaillie. Queste cellule sono usate clinicamente per vari scopi, tra cui la rigenerazione del miocardio infartuato, l’angiogenesi terapeutica in pazienti con ischemia periferica acuta (specialmente la malattia di Buerger) e il bioengineering (rivestimento cellulare di legamenti o di valvole cardiache sostitutive). In questo ambito si sono registrati risultati incoraggianti nell’animale per il trattamento delle malattie neurodegenerative, dell’ictus, del trauma cerebrale e dei danni del midollo spinale. Sono stati isolati molti altri tipi di cellule staminali “adulte” le cui proprietà riparatrici sono state verificate con successo nell’animale: cellule staminali neuronali (per il morbo di Parkinson, la sclerosi multipla, il morbo di Huntington, l’ictus, il trauma cerebrale, le lesioni del midollo spinale), cellule staminali muscolari (per l’incontinenza urinaria, il danno miocardico), cellule staminali endoteliali (per l’ischemia acuta periferica), cellule staminali cardiache, cellule staminali della retina (per la degenerazione maculare), cellule staminali del limbus della cornea (per il danno corneale). Allo stato attuale, i risultati clinici più promettenti si sono ottenuti con le cellule staminali del sangue del cordone ombelicale (UCB), che hanno portato allo sviluppo di un’area di mercato caratterizzata dalla creazione di banche private di UCB. Generalmente le cellule UCB provocano, al massimo, una reazione immune piuttosto blanda quando vengono trapiantate in soggetti con donatori non compatibili. Si usano con successo laddove sia necessaria una riparazione o rigenerazione nell’organismo del ricevente. I migliori risultati con cellule staminali UCB, fino ad ora, sono stati ottenuti nel trattamento di bambini con morbo di Krabbe. Benefici si sono ottenuti anche dal trapianto locale di cellule UCB in pazienti con danni al midollo spinale. ---------- In this second part of the article, the attention is focused on “non embryonic stem cells”, that is somatic stem cells (from fetus or adult organisms), and umbilical cord blood stem cells. These stem cells, sometimes referred to as “adult stem cells”, were known and recognized as such before the embryonic ones. In fact the mere expression “stem” cells to designate this particular type of immature cell, from which derive all the others, more differentiated cells, came from the identification of the hematopoietic stem cells, in bone marrow (1961). Later investigations have shown that there are such cells, immature, multipotent, self-renewing, and self-differentiating ones in almost all tissues and organs of fetus or adult organism. As soon as they were discovered, these “adult”, autologous stem cells were immediately put in the service of patients, with the first transplantations of bone marrow performed either for the treatment of malignancies, or for the treatment of hematologic disorders. Today, autologous hematopoietic stem cells are also used for the treatment of auto-immune diseases, such as multiple sclerosis or lupus erythematosus and for regenerative medicine. A second, important source of “adult” stem cells are the mesenchymal stem cells, found mainly in bone marrow, but also in blood, progenitors of multiple cell lineages, including bone, cartilage, muscle, adipose tissue and astrocytes, and which seem to hold the key to tissue regeneration. Different types of multipotent mesenchymal stem cells, with properties comparable to those of embryonic stem cells, have been isolated, the best known being the multipotent adult progenitor cells (MAPCs). These cells are used clinically mainly for the healing of the heart after myocardial infarction, with positive statistically significant results, for therapeutic angiogenesis in patients suffering of peripheric ischemic disease (especially Buerger’s disease), and for bioengineering (cellular coating of artificial ligaments or of prosthetic heart valves). They have given promising results in animals for the treatment of neurodegenerative diseases, ictus, brain trauma and spinal cord injuries. Many other types of “adult” stem cells have been isolated and their healing properties assessed with success in animals, such as neural stem cells (for Parkinson’s disease, multiple sclerosis, Huntington’s disease, ictus, brain trauma, spinal cord injury), muscle stem cells (for urinary incontinence, myocardial infarction), endothelial stem cells (for critical limb ischemia), cardiac stem cells, retinal stem cells (for macular degeneration), limbal stem cells (for damaged cornea). At the moment, the more promising results in patients have been obtained with umbilical cord blood stem cells (UCB), prompting the birth of a commercial trade based on private banks. Umbilical cord blood stem cells offer indeed the advantage of their immaturity: as such, they rarely trigger more than a mild immune reaction when transplanted in unrelated recipient organisms. They are used with profit wherever a healing or regenerative process is necessary in a given patient. Up to now, best results with the UCB cells have been obtained in the treatment of children with Krabbe’s disease. Some patients with injured spinal cords have also experienced benefits from UCB cells grafts.

Otto anni dopo l'inizio della ricerca sulle cellule staminali umane, sembra essere arrivato il momento di considerare oggettivamente quale possa essere il futuro di tale ricerca, e quali siano i problemi etici collegati. In questo articolo sono considerate le cellule staminali embrionali (ES) a livello tecnico e clinico. L'interesse particolare di tali cellule risiede nella loro capacità di continua proliferazione indifferenziata e di stabile sviluppo potenziale in un’ampia tipologia di cellule, anche dopo una coltura prolungata. Numerosi lavori mostrano, in particolare, che le cellule ES possono essere differenziate in neuroni e glia ed integrarsi nel tessuto neurale in animali riceventi. La differenziazione verso neuroni dopaminergici è stata ottenuta per le cellule staminali embrionali umane (hES) con promesse per il trattamento clinico della malattia di Parkinson. Le cellule ES hanno anche dimostrato la capacità di facilitare il recupero del danno del midollo spinale, nel topo. L'innesto di cellule ES in ratti con infarto miocardico provoca un miglioramento a lungo termine della funzione del cuore ed aumenta la percentuale di sopravvivenza. Tuttavia, ci sono molti ostacoli che devono essere superati prima di pensare ad un uso clinico di tali cellule. Il problema forse più complesso è di poter dirigere in modo efficiente e riproducibile la differenziazione delle cellule ES attraverso percorsi specifici. In secondo luogo, il rischio di difetti o instabilità epigenetiche nelle cellule ES è reale, tenendo conto della loro origine da embrioni ottenuti da fecondazione in vitro e del processo di coltura di tali cellule, una volta individuate. Terzo, le cellule ES allo stato indifferenziato sono cancerogeniche, il che, per un uso clinico, rende necessaria la loro differenziazione e l’attenta eliminazione di cellule ES rimaste indifferenziate. Infine, l'uso clinico delle cellule ES richiede la soluzione del problema immunologico della compatibilità HLA con il ricevente. A tale scopo sono state proposte varie soluzioni, per prima il trasferimento nucleare, detto anche “clonazione terapeutica”. Allo stato attuale essa non è applicabile ai primati ed alla specie umana. Inoltre sarebbe necessaria una quantità enorme ed irrealistica di ovociti umani. Ci si orienta oggi, anche per motivi etici, verso soluzioni "alternative" come il trasferimento nucleare modificato, nel quale si producono embrioni deficitari incapaci di svilupparsi correttamente, la partenogenesi, la raccolta di blastomeri in occasione della diagnosi preimpiantatoria, o la riprogrammazione delle cellule staminali somatiche. Ad oggi, lo studio delle cellule staminali embrionali rappresenta una promettente chiave per futuri progressi in ambito biologico (biologia dello sviluppo, biologia cellulare e biologia molecolare), nella misura in cui permette di capire meglio i processi ed i meccanismi della differenziazione e della rigenerazione dei tessuti. ---------- Eight years after the onset of the investigation on embryonic stem cells (ESCs), it seems that time has come to consider objectively what the future of such research can be, and what are the ethical issues that are involved. In this first part ESCs are considered at the technical and clinical level. The particular interest of such cells resides in their ability for endless undifferentiated proliferation and for potential development in a large array of various types of cells, even after prolonged culture. A large amount of studies show in particular that ESCs can differentiate in neurons and glia and integrate in the neural tissue of recipient animals. The promotion of such differentiation toward dopaminergic neurons has been obtained for human embryonic stem cells (hESCS), which is promising for possible future clinical application to the treatment of Parkinson's disease. The ESCs have also demonstrated their ability to facilitate the recovery of damaged spinal cord in mice. The graft of ESCs in the hearts of rats with myocardial infarction leads to an improvement of heart function and increases survival. Nevertheless, there are many obstacles that must be overcome before thinking to a clinical use of such cells. The problem perhaps more complex is to be able to direct in an efficient and reproducible way the differentiation of the ESCs in culture. Second, the risk of epigenetic defects or instability with ESCs is real, keeping in mind their origin from embryos created by in vitro fertilization, and the fact that they are kept proliferating in culture for a long period of time, once individualized. Third, ESCs in the undifferentiated state generate cancers when injected in tissues, and that makes necessary, for a clinical use, to start their differentiation in vitro and then to eliminate carefully from the end product these ESCs that are still undifferentiated. Finally, the clinical use of ESCs supposes resolved the immunological problem of their HLA compatibility with the patient who will receive them. Various solutions have been proposed for resolving this last problem, with, in first line, nuclear transfer, the so called "therapeutic cloning." Up to now this nuclear transfer has not been successful in primates and humans. Moreover, it would require the availability of unrealistically large amounts of human ovocytes. Today, also for ethical reasons, the tendency is to look after "alternative solutions" such as "altered nuclear transfer", in which are created disabled embryos, unable to develop correctly, parthenogenesis, the harvest of human blastomeres in the course of preimplantation diagnosis or the reprogramming of human somatic stem cells to an "embryonic state". At present time, the study of ESCs represents a promising key to progresses in the knowledge of cellular and molecular aspects of development, healing and tissue regeneration. These progresses may in turn lead to clinical applications, especially in the field of degenerative diseases and for the recovery of damaged tissues and organs.

The development of neural differentiation protocol aimed at obtaining functional neurons in vitro represents the future in medicine. The text will explain the protocol developed by Cattaneolab in order to obtain in vitro striatal neurons from human embryonic and pluripotent stem cells. The work has been published in 2013 on Development (Delli Carri A., Onorati M., et al.; 2013). Striatal neurons at ganglia level die in Huntington’s Disease, a neurodegenerative disorder of the central nervous system, characterized by a gradual development of involuntary muscle movements, progressive disorientation and confusion, personality disintegration, impairment of memory control.

In 2012 the Nobel Prize for Medicine was awarded to Sir John Gurdon and Shinya Yamanaka for their joined discovery that mature, specialized cells can be reprogrammed to become immature pluripotent cells capable of developing into all different tissues of the body. This seminal breakthrough changed our vision of developmental biology and made possible to generate in vitro from somatic cells of any healthy or affected individual Induced Pluripotent Stem Cells (iPSCs) and differentiate them into virtually all types of cells. Scientists around the world were provided with the tool of modelling human diseases, even those involving inaccessible tissues such as the nervous system, to study the pathological process. iPSCs also revealed an enormous potential for pharmaceutical and clinical applications as on these cells a myriad of small molecules or candidate drugs can be tested some of which hopefully will become new effective medicines for intractable diseases. We have generated the first iPSC-derived neuronal model for the Rubinstein-Taybi syndrome (RSTS), a mendelian neurodevelopmental disorder, characterized by facial dysmorphisms, growth and speech delay, skeletal dysplasia and intellectual disability often associated to behavior disorder. RSTS is caused by mutation in the homologous CREBBP and EP300 genes encoding proteins acting as chromatin regulators through their intertwined activity of transcriptional co-activators and acetyltransferases (KATs) on histone and non histone proteins. I summarize herein the workflow used to generate iPSC-derived neurons (iNeurons) from six RSTS patients with a variable cognitive impairment in parallel to iNeurons from four healthy controls. Immunohistochemical characterization of samples at the stages of iPSCs, neural rosettes, neural progenitors and post-mitotic cortical neurons did not reveal gross alterations in the expression of stage-specific differentiation markers across patients’ or between patients’ and controls’ neuronal cultures. Conversely, altered morphology of patients’ differentiating neurons, showing reduced branch length and increased branch number and hypoexcitability of differentiated neurons emerged as relevant “disease” biomarkers. Both the anomalous neuronal morphology and the impaired electrophysiological performance varied across iNeurons from different RSTS patients. possibly reflecting cognitive and behavioural impairment of the donor patients. To validate the identified morpho-functional markers we performed further studies on iNeurons from the RSTS patient with the most severe intellectual disability by using trichostatin A (TSA), an epidrug that by inhibiting histonedeacetylases (HDAcs), the KATs opposing enzymes, has been successfully applied in different cbp+/cbp- mouse models with improvement of some neurological abnormalities. Short acute and chronic TSA treatment of two independent neural progenitor lines from the selected RSTS patient determined in both replicas a consistent reversal of a few morphological abnormalities and a significant rescue of the defective electrophysiological performance, highlighting the potential postnatal intervention of epidrugs to ameliorate the cognitive impairment of RSTS patients.

Le cellule staminali neurali sono presenti, nel cervello dei mammiferi, in quelle aree dove la neurogenesi è mantenuta durante tutta la vita dell’organismo. Una caratteristica importante riguarda la loro abilità di proliferare e migrare nella materia cerebrale attratte dai siti di danno indotto, per esempio, da numerose malattie degenerative e dai tumori cerebrali. Il punto focale del mio dottorato è stato quello di studiare il ruolo di Reelin, proteina della matrice extracellulare fortemente implicata nello sviluppo cerebrale, nelle cellule staminali neurali murine. Mettendo a confronto le cellule “selvatiche” con quelle estratte da topi reeler, caratterizzate dalla mutazione negativa spontanea nel gene, ho osservato che l’assenza di Reelin rallenta la proliferazione delle cellule staminali neurali, così come la formazione delle neurosfere, aggregati sferici in sospensione tipici della crescita in vitro di queste cellule. Inoltre ho dimostrato un ruolo della proteina nel potenziale di differenziazione, favorendo la neuronogenesi in vitro senza modificarne la gliogenesi. Infine ho riscontrato l’incapacità delle cellule reeler di migrare nello stato aggregato chiamato “a catena”, la modalità di migrazione caratteristica delle cellule staminali neurali in vivo. Tutti questi effetti sono parzialmente recuperati dalla somministrazione della proteina esogena alle cellule reeler o con la diretta ingegnerizzazione per ripristinarne l’espressione endogena. Le conclusioni ricavate dal mio studio assegnano a Reelin un ruolo chiave nella biologia delle cellule staminali neurali adulte, intervenendo nella proliferazione, nel differenziamento e nella migrazione, le tre principali caratteristiche che guidano il processo di rigenerazione del tessuto danneggiato.
In the adult mammalian brain, multipotential neural stem cells persist throughout life in those areas where neurogenesis is maintained. A distinctive trait of these cells is their ability to self-renew and to migrate through brain matter to sites of injury, such as those of occurrence of gliomas and neurodegenerative deseases. The aim of my doctorate study was the role of Reelin, an extracellular matrix protein deeply involved in brain development, in newborn mouse neural stem cells (NSCs). By comparing wild type and Reelin knock out reeler stem cells, I show that the absence of Reelin negatively affects proliferation and neurosphere-forming ability, as well as in vitro neuronal differentiation potential. Notably, reeler NSCs are not able to migrate in chains, a migration mode typical of neural precursors homing to injury sites in adult CNS. All these effects are partially rescued by ectopic Reelin supplementation. Overall, my results indicate that Reelin affects all three major features of post-natal NSCs, and that it is required for the proper homing of NSCs to injury sites in adult brain.

In the mouse nervous system, Sox2 is expressed in stem cells and early precursors, and in few mature neurons (Zappone et al., 2000; Ferri et al., 2004). Adult Sox2-deficient mice, in which Sox2 expression is decreased by about 70%, exhibit neural stem/precursor cell proliferative defects in the hippocampus and periventricular zone (Ferri et al., 2004). I performed in vitro differentiation studies on neurosphere derived neural cells. Neural stem cells from Sox2-deficient mice produce reduced numbers of neural progenitors. Moreover, I found the SOX2 is important for the neural progenitors cell cycle progression. I demonstrated that SOX2 promoted the proliferation of neural stem cells through facilitating the G1/S transition with the levels of cyclin D1 and cyclin E expression in neural progenitors samples. Mash1 is an important regulator of neurogenesis in the ventral telencephalon, where it is required both to specify neuronal precursors and to control the timing of their production. Mash1 is required for the generation of neocortical neurons with characteristics of GABAergic interneurons. Moreover, in vivo Sox2 deficiency causes a reduction of GABAergic neurons (Cavallaro M. et al., 2008). These observations point to a possible role for SOX2 togheter with Mash1 in the genesis of neural progenitors of GABAergic neurons. The early neural progenitors, derive from Sox2-deficient mice produce reduced numbers of MASH1 positive cells. In vivo Chromatin immunoprecipitation assays reveal interactions of SOX2 and Mash1 in early neural progenitor cells. My data also suggest that the specific recruitment of these protein to the Mash1 in the ventral telencephalon defines the spatiotemporal activity of MASH1 in the developing nervous system.

Background: Zika virus (ZIKV), West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that generally cause mild or asymptomatic disease in humans. However, ZIKV infection has been associated with fetal microcephaly and Guillain-Barrè syndrome in adults; WNV infection may evolve to severe neuroinvasive disease in the elderly and immunocompromised subjects; DENV may rarely cause neurological complications in infected individuals. In addition, another emerging mosquito-borne flavivirus, Usutu virus (USUV), which may cause fatal neuroinvasive disease in different bird species, has been recently shown to infect humans but its pathogenicity is unknown. Aim of the study: In the context of the recent outbreak of ZIKV in the Americas and the increasing evidences of an association between ZIKV infection and the occurrence of fetal microcephaly, aim of this study was to investigate the effect of ZIKV infection in human neural cells in comparison with other flavivirus infections. To this aim, ZIKV infectivity, replication kinetics, cytopathic effect (CPE), and induction of innate antiviral responses were investigated in human induced pluripotent stem cells (hiPSCs), hiPSCs-derived neural stem cells (NSCs) and neurons and compared with other flaviviruses, i.e., WNV, DENV and USUV. Methods: NSCs and neurons were differentiated from hiPSCs. hiPSCs, NSCs, and neurons were infected with isolates of ZIKV Asian lineage (KU853013), WNV lineage 2 (KF179640), DENV serotype 2, and USUV Europe lineage 1 (AY453411). Time course experiments were performed to evaluate viral load by qRT-PCR and TCID50, expression of host genes involved in antiviral innate immunity by qRT-PCR, expression of cell differentiation markers by IF and qRT-PCR, cell viability and cell death by flow cytometry. The impact of ZIKV on embryogenesis and neurogenesis was evaluated by infection of hiPSCs and NSCs during neural differentiation and embryo body formation. Results: ZIKV infected and replicated efficiently in NSCs, neurons and hiPSCs. Infection led to typical CPE and cell death by apoptosis. ZIKV infection of hiPSCs, NSCs, and neurons induced the expression of innate immune response genes, especially the cellular pattern recognition receptor (PRR) IFH1 gene (MDA5), IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) and 2 (IFIT2). Infected embryoid bodies were massively destroyed by ZIKV infection and infected hiPSCs and NSCs died before ending the neural differentiation process. ZIKV replication efficiency in NSCs was significantly higher than that of DENV-2 and USUV, but lower than that of WNV. In particular, WNV replicated more efficiently, induced more cell death and higher levels of antiviral gene expression than ZIKV in NSCs, neurons and hiPSCs. The induction of innate immune response genes in NSCs after infection with ZIKV and DENV-2 infection was milder than after infection with WNV and USUV, in agreement with the adaptation of these viruses to the human host and their ability to shut down the antiviral response. Conclusion: ZIKV infects and replicates efficiently in NSCs and induces cell death abrogating neural development, although less efficiently than WNV. Because of the similarities between flaviviruses in their interactions with host neural cells, it is conceivable that infection of other human cells, such as those involved in the extablishment of the blood-placenta barrier, are crucial for ZIKV-induced damage of the fetal brain.
Presupposti dello studio: Zika virus (ZIKV), West Nile virus (WNV), dengue virus (DENV) e Usutu virus (USUV) sono trasmessi da zanzare ed appartengono al genere Flavivirus della famiglia Flaviviridae. L’infezione da ZIKV è associata a microcefalia fetale e sindrome di Guillan-Barrè; WNV può causare una grave sindrome neuroinvasiva nell’anziano e nei soggetti immunocompromessi; l’infezione da DENV raramente si associa a complicazioni neurologiche; USUV può causare una sindrome neuroinvasiva fatale in diverse specie di uccelli, è stato dimostrato che può infettare pure l’uomo, ma la sua patogenicità resta ancora da chiarire. Scopo: Alla luce della recente epidemia di ZIKV in America e di una probabile associazione tra l’infezione da ZIKV e lo sviluppo di microcefalia fetale, lo scopo di questo studio è stato confrontare l’infezione da ZIKV sulle cellule neurali umane con l’infezione da WNV, DENV e USUV. A tal fine, la cinetica di replicazione, l’effetto citopatico e l’immunità innata indotta dall’infezione virale sono state analizzate in cellule staminali pluripotenti indotte (hiPSCs), cellule staminali neurali derivate da iPSCs e neuroni. Materiali e metodi: Le NSCs ed i neuroni sono stati differenziati da hiPSCs. I diversi tipi cellulari sono stati infettati con l’isolato di ZIKV lignaggio asiatico (KU853013), WNV lignaggio 2 (KF179640), DENV sierotipo 2 e USUV lignaggio 1 europeo (AY453411). La carica virale è stata valutata a diversi tempi dall’infezione mediante qRT-PCR e TCID50, il livello di espressione dei geni coinvolti nell’immunità innata è stato analizzato mediante qRT-PCR e l’espressione dei markers di differenziamento cellulare mediante IF e qRT-PCR, la sopravvivenza cellulare e l’apoptosi mediante il saggio MTT e analisi dell’attivazione di caspasi-3. L’impatto dell’infezione da ZIKV sull’embriogenesi e la neurogenesi è stato valutato infettando le hiPSCs e le NSCs durante il differenziamento neurale e durante la formazione dei corpi embrioidi. Risultati: ZIKV era in grado di infettare e replicare efficientemente nelle NSCs, nei neuroni e nelle hiPSCs, causando un tipico effetto citopatico e morte cellulare per apoptosi. L’infezione ha indotto un significativo aumento dell’espressione dei geni dell’immunità innata, in particolare dei geni MDA5 (the cellular pattern recognition receptor (PRR) IFH1 gene), IFIT1 (IFN-induced protein with tetratricopeptide repeats 1) e IFIT2. I corpi embrioidi sono stati distrutti dal virus e le hiPSCs e le NSCs infettate sono morte prima di completare il differenziamento neurale. L’efficienza di replicazione di ZIKV nelle NSCs era maggiore rispetto a quella di DENV-2 e USUV, ma minore rispetto al WNV. Infatti, WNV replicava in modo più efficiente, induceva una maggiore morte cellulare e stimolava una più elevata risposta antivirale rispetto a ZIKV nei diversi tipi cellulari. Conclusione: ZIKV infetta e replica nelle NSCs, inducendo morte cellulare e impedendo lo sviluppo neurale, ma in modo meno efficiente rispetto al WNV. E’ probabile quindi che l’infezione di altri tipi cellulari sia determinante per il danno al sistema nervoso fetale indotto in modo specifico da ZIKV.

2008/2009
The NS culture system is an innovative yet not fully characterized method of culturing neural stem cells (NSCs). Previous reports have described the possibility of isolation of a virtually pure NSC culture from embryonic, fetal or adult NSCs (Conti et al., 2005; Pollard et al., 2006; Sun et al., 2008). These cells, grown in adhesion, are called NS cells and show radial glial characteristics. The present thesis aims to characterize the in vitro and in vivo behaviour of human and mouse NS cells. In the first study, we describe the isolation of a new strain of human striatal NS cells. They show unique features in vitro, and can be efficiently differentiated into neurons. After transplantation in newborn rats, they survive, migrate and differentiate in the host brain. In the second part of the thesis, we show that mouse ES-derived NS cells fuse with cortical pyramidal neurons after transplantation in mice or rats. This process is mediated by microglia, which first fuse with the NS cells and then with the neurons. This NS-microglia-neuron fusion has not been previously described, although it occurs both in vivo and in vitro. In summary, we report here previously undescribed features of the NS cells; our results are relevant to better understanding of NSCs in general and their behaviour after transplantation in the brain.
XXII Ciclo
1981

L’obiettivo del presente lavoro è stata l’identificazione di un ruolo per il recettore degli estrogeni (ER) nel neurosviluppo e nella neurodegenerazione, con particolare attenzione al coinvolgimento delle cellule gliali. E’ noto che gli estrogeni influenzano lo sviluppo, la maturazione ed il differenziamento dei neuroni nel sistema nervoso centrale, e che i suoi recettori mostrano un picco di espressione durante le fasi precoci di neurosviluppo. La zona subventricolare del cervello di topo adulto è una ricca fonte di progenitori neurali. Questi possono essere mantenuti in coltura, in un mezzo chimicamente definito contenente il fattore di crescita epidermico (EGF), sottoforma di neurosfere, che possono differenziarsi in neuroni e glia quando piastrate su laminina in assenza di EGF. Il presente studio ha mostrato che gli ER sono espressi nelle neurosfere sia in sospensione che in adesione, ed in particolare ER mostra un picco di espressione durante le fasi più precoci del differenziamento della neurosfera (6-24 ore). Il trattamento con 17-estradiolo 10nM (17-E2) non influenza significativamente la proliferazione nelle neurosfere in sospensione, ma modifica il differenziamento già dopo 6 ore di piastratura su laminina, con un significativo aumento della percentuale di neuroblasti PSA-NCAM-positivi e, successivamente, a 3 giorni, con un aumento nel numero di neuroni MAP2-positivi. Il trattamento con 17-E2 induce inoltre un aumento nella percentuale di cellule GFAP-positive ed un aumento dei livelli proteici di GFAP, con un effetto marcato a 24 ore di piastratura. In uno studio parallelo, è stata valutata la capacità della glia di mediare gli effetti neuroprotettivi degli estrogeni. Il 17-E2 esercita infatti effetti protettivi anche verso la tossicità da beta-amiloide (AP). Al fine di valutare il coinvolgimento degli astrociti in tale fenomeno, il terreno di coltura condizionato da astroglia pre-trattata con 17-E2 per 4 ore, è stato trasferito su neuroni corticali puri trattati per 24 ore con AP25-35 25M. I risultati ottenuti hanno mostrato una aumentata vitalità dei neuroni corticali, effetto che non appare modificato dal trattamento con l’antagonista dei recettori per gli estrogeni ICI 182,780 addizionato direttamente ai neuroni. Il TGF-1 è stato identificato quale fattore solubile responsabile della neuroprotezione indotta dal 17-E2. I livelli di TGF-1 intracellulare e rilasciato aumentano infatti in seguito al trattamento con 17-E2, ed il contenuto intracellulare di TGF-1 nelle cellule positive si riduce, suggerendo che il 17-E2 stimoli prevalentemente il rilascio di tale citochina. Infine, l’incubazione con anticorpo neutralizzante anti-TGF-1 incide significativamente sulla riduzione della morte neuronale indotta dal terreno condizionato da astrociti trattati con 17-E2. Nell’insieme, i risultati ottenuti puntano verso un ruolo chiave dei recettori degli estrogeni nel neurosviluppo e nella neuroprotezione, ed identificano la glia come target primario per l’azione degli estrogeni.
The aim of the present study was the identification of a role for estrogen receptor (ER) in neurodevelopment and neurodegeneration, focusing on the involvement of glial cells. Estrogen is in fact known to affect development, maturation and differentiation of neurons in the central nervous system and its receptors exhibit a peak of expression during early phases of neurodevelopment. The subventricular zone of the adult mouse brain is a source of progenitor cells which can be grown as neurospheres in a chemically defined medium supplemented with epidermal growth factor (EGF), and are able to differentiate into neurons and glia when plated on laminin in the absence of EGF. The present study has indicated that ERs are expressed by both floating and adherent neurospheres, with ER showing a peak of expression during the earlier phases of neurosphere differentiation (6-24 hrs). Treatment with 10 nM 17-Estradiol (17-E2) did not significantly affect proliferation in floating neurospheres, but modified progenitor differentiation as early as 6 hours after plating on laminin, with a marked increase in the percentage of PSA-NCAM-positive neuroblasts, and later on at 3 days post-plating with an increase in MAP2-positive neurons. Treatment with 17-E2 also increased the number of GFAP-positive cells and the levels of GFAP protein with a major effect at 24 hours. In a parallel study, the ability of glia to mediate the neuroprotective effect of estrogen has been evaluated. 17-E2 is known to exert neuroprotective activity also against ß-amyloid (ßAP). To evaluate the involvement of astroglia in this effect, the conditioned medium from astrocytes preexposed to 17-E2 for 4 h was transferred to pure rat cortical neurons challenged with 25M AP25-35 for 24 h. The results obtained have shown an increased viability of cortical neurons. This effect is not modified by treatment with the estrogen receptor antagonist ICI 182,780 added directly to neurons. TGF-1 has been identified as the soluble factor responsible for 17-E2-induced neuroprotection. Accordingly, the intracellular and released levels of TGF-1 are increased by 17-E2 treatment, and the intracellular content of TGF-1 in immunopositive cells is reduced, suggesting that 17-E2 stimulates mainly the release of the cytokine. Finally, incubation with a neutralizing anti-TGF-1 antibody significantly modifies the decrease in neuronal death induced by 17-E2 -treated astrocyte-conditioned medium. Taken together these results point to a key role for estrogen receptor both in neurodevelopment and neurodegeneration and identify glia as a major target for estrogen action.

Medium-sized spiny neurons (MSNs) are the only neostriatum-projection neurons, and their degeneration underlies some of clinical features of Huntington's disease. We used human developmental biology and exposure to key neurodevelopmental molecules to drive human pluripotent stem (hPS) cells into MSNs. In a feeder-free adherent culture, ventral-telencephalic specification is induced by BMP/TGF-β inhibition and subsequent SHH/DKK-1 treatment. The emerging FOXG1+/GSX2+ telencephalic progenitors are then terminally differentiated, resulting in the systematic line-independent generation of FOXP1+/FOXP2+/CTIP2+/calbindin+/DARPP-32+ MSNs. Similarly to mature MSNs, these neurons carry dopamine- and A2a-receptors, elicit typical firing pattern, and show inhibitory postsynaptic currents, as well as dopamine neuromodulation and synaptic integration ability in vivo. When transplanted into the striatum of quinolinic acid-lesioned rats, hPS-derived neurons survive and differentiate into DARPP-32+-neurons, leading to a restoration of apomorphine-induced rotation behaviour. In summary, hPS cells can be efficiently driven to acquire a functional striatal fate using an ontogeny-recapitulating stepwise method. Moreover, we have established stable HD-iPS cell lines that recapitulating, in vitro, features of the disease can be used for investigating disease mechanisms that underlie HD, representing a platform for in vitro human developmental neurobiology studies and drug screening approaches.

Inhibition of microglia-mediated neuroinflammation is an important terapeuthic target in order to avoid cognitive and motor impairment in brain ischemia . Reportedly, neural stem cell (NSC) brain grafts have neuroprotective effecs 1. It has been proposed that these positive effects are not caused only by NSC proliferation and generation of new neurons, but also by a modulation of the brain lesion environment 2. Our primary aim was to ascertain whether NSC were capable of modifying microglial activation in vitro. We used ATP as inflammatory stimuli, since it is massively released from damaged neurons and is responsible of activation of microglia during ischemia3. We demonstrated that N9 murine microglia cells incubated with conditioned media (CM) from NSC culture have a blunted response to ATP. In fact, ATP stimulation of N9 cells preincubated with CM at different passages induced a reduced release of intracellular calcium compared to controls (Fig.1). Moreover, CM preincubation significantly inhibited the expression of inflammatory cytokines like TNF-alfa, COX-2, and IL-10 that are up-regulated after ATP stimulation (Fig.2) Reportedly, high-dose ATP (>1mM) exposure is detrimental both for neurons and microglial cells4. We tested CM action of survival of N9 microglia treated with 3mM ATP for 24 hours. CM preincubation for 24 hours was capable of significantly reducing N9 mortality induced by ATP treatment (Fig.3). In conclusion NSC release soluble factors that have an antinfiammatory action blunting N9 response to ATP stimulation.

In the last decade, multipotent mesenchymal stromal cells (MSCs) demonstrated a significant therapeutic efficacy, particularly in cell therapy approaches aiming at tissue regeneration. MSCs exert their action via trophic support, induction of angiogenesis, immunomodulation and reduction of necrosis at affected tissues. Importantly, these regenerative and protective properties are largely associated to MSC secretome. Unfortunately, cell-based approaches not always meet the criteria for a smooth translation to the clinic. For instance, the use of stem cells in pathologies with a very short therapeutic window, such as few hours, is not compatible with the requested minimal criteria for MSC release, before administration to the patient. Notwithstanding, in the regenerative medicine field, the MSC mechanism of action paradigm was recently extended to include the action of extracellular vesicles (EVs), which are cytoplasm-containing cellular bodies secreted by a wide range of cell types. Intriguingly, many studies reported that EVs generated by MSCs are able to recapitulate the majority of the regenerative properties of parental MSCs. Starting from these premises, the objectives of the present doctoral research project were: to address EV-mediated cell-to-cell communication as novel MSC mechanism of action; to address reprogrammed MSC-EV generation; to define, for the first time in the literature, stem cell EV molecular content (e.g.: miRNome), comparing reprogrammed to non-reprogrammed MSC-EVs; to challenge stem cell-EV therapeutic potential in a model of acute tissue damage, as a proof-of-concept for feasibility and effectiveness of a stem cell-based albeit cell-free regenerative strategy. Intriguingly, EVs may be produced in a ready-to-use formulation, so that clinicians could use them as soon as a therapeutic need arises, also in the case of an urgent one. In this way, EV-shuttled MSC regenerative properties could exert beneficial effects also on pathologies currently lacking any cell therapy option. To develop this innovative therapeutic strategy, MSCs were isolated from different tissues and their biological properties were evaluated in order to choose the MSC source most suitable for the implementation of the project. Thus, both MSC transcriptome and immunophenotype were addressed. MSCs from adult sources (e.g.: bone marrow) showed senescence-related features in vitro, correlated to donor’s age in vivo. On the other hand, MSCs from perinatal tissues (e.g.: cord blood) showed a phenotype more similar to that of pericytes, which are the in vivo progenitors of MSCs. Therefore, cord blood was chosen as MSC source, also in the prospective of clinical translation, since public banking of cord blood units for clinical use already exists worldwide. Next, thanks to an extended analysis of the stromal populations present in cord blood, a MSC subpopulation showing higher proliferation properties and significantly longer telomeres was isolated. In addition, the standard cord blood MSC isolation protocol was improved, leading to an efficiency of 80%. Eventually, MSC secretome-associated anti-inflammatory and anti-apoptotic properties were observed in vitro and in vivo. In order to investigate if EVs contributed to MSC paracrine properties, MSC-EV secretion and regenerative properties were assessed. The MSC-EV therapeutic effectiveness was challenged in an in vitro model of acute tissue damage. Intriguingly, MSC-EVs could rescue damage-induced cell mortality, showing the same protective effect of parental MSCs. In spite of the use of a high proliferative cord blood MSC subpopulation, primary cultures still show a limited lifespan. In order to increase their replicative potential and to better exploit their EV production, induced cellular reprogramming was tested on MSCs as an alternative to traditional immortalization techniques. In this way, MSC-derived cell lines endowed with unlimited lifespan were generated, and permanent modification of their genome was avoided. The next step was to confirm the generation of EVs from reprogrammed MSCs, since reprogramming drastically changes cell identity. Furthermore, the EV miRNome load of reprogrammed and non-reprogrammed MSCs was addressed. Importantly, the majority of miRNAs were common between the two samples, indicating that reprogramming did not change the EV miRNA content. This result could have relevant consequences on the functional features of reprogrammed MSC-EVs, since EV-mediated miRNA transfer from donor to target cells was proposed as one of MSC mechanisms of action. In the last part of this doctoral research, stem cell (non-reprogrammed and reprogrammed MSCs)-EV therapeutic effectiveness was addressed and compared to that of parental MSCs. In order to do that, an organotypic ex vivo mouse model of brain ischemia was used. This model recapitulated the modulation of some ischemic damage-related parameters, including increased secretion of inflammatory cytokines, high tissue necrosis and the impairment of neuronal and astrocytic cell populations. Therefore, this model mimicked early phase events of brain ischemia, whose thrombolytic clinical treatment must be administered within 3-6 hours of first signs of ischemia. Notably, stem cell-EVs were tested for the first time in this pathological context to verify their potential role in tissue regeneration. Strikingly, stem cell-EV administration to affected tissues showed significant neuroprotective properties, which were comparable to those of parental MSCs. Importantly, the ischemic damage-related parameters previously described were rescued. In particular, inflammatory-associated parameters underwent the most statistically significant decrease, showing levels similar to or better than those of the uninjured brain tissue. This is of uttermost importance, considering that chronic inflammation is detrimental to tissue regeneration. To conclude, the results of the present PhD thesis confirmed the feasibility of stem cell EV-based therapies in regenerative medicine approaches. In the future, this innovative EV therapy may be applied to pathological contexts currently without a cell therapy option. In the framework of advanced therapy medicinal products, the new drug would be the EVs, rather than the parental stem cells. Finally, EVs could play the role of ready-to-use anti-inflammatory molecule carriers, in order to guarantee a rapid therapeutic action for the regeneration of injured tissues.
Negli ultimi anni, le cellule stromali mesenchimali multipotenti umane (CSM) hanno mostrato una grande efficacia terapeutica, soprattutto in approcci di terapia cellulare aventi come obiettivo la rigenerazione tissutale. L’azione delle CSM avviene attraverso supporto trofico, induzione di angiogenesi, modulazione della risposta immunitaria e diminuzione della necrosi a livello dei tessuti colpiti. Inoltre, recente letteratura ha dimostrato che queste capacità rigenerative e protettive sono in larga parte associate al secretoma delle CSM. Purtroppo, gli approcci di terapia cellulare non sono sempre traslabili alla clinica. Ad esempio, l’utilizzo di cellule staminali in patologie caratterizzate da una finestra terapeutica molto stretta, dell’ordine di poche ore, non è compatibile con la necessità di scongelare e valutare i minimi standard di qualità delle CSM prima della somministrazione al paziente. Nonostante ciò, il paradigma del meccanismo d’azione delle CSM nel campo della medicina rigenerativa si è ulteriormente arricchito. Infatti, molti recenti studi hanno dimostrato che le vescicole extracellulari, ossia porzioni di citoplasma delimitate da membrana cellulare secrete dalle CSM, sono in grado di riprodurre la maggior parte delle proprietà rigenerative delle CSM stesse. Date queste premesse, gli obiettivi del progetto di ricerca del presente Dottorato sono stati i seguenti: indagare la comunicazione intercellulare tramite vescicole extracellulari quale innovativo meccanismo d’azione delle CSM; studiare la produzione di vescicole extracellulari da parte di CSM riprogrammate, e, per la prima volta in letteratura, definirne il contenuto molecolare (es.: miRNoma), a confronto con le CSM d’origine; testare il potenziale terapeutico di vescicole extracellulari da cellule staminali in un modello di danno tissutale acuto, come proof-of-concept della funzionalità di una strategia terapeutica cell-free. Infatti, le vescicole extracellulari potrebbero essere prodotte in formulazioni pronte all’uso, a immediata disposizione per ogni richiesta clinica, anche urgente. In questo modo le proprietà rigenerative delle CSM potrebbero essere veicolate dalle vescicole extracellulari anche in contesti patologici attualmente senza alcuna opzione di terapia cellulare. Per lo sviluppo di questa innovativa strategia terapeutica, CSM isolate da vari tessuti sono state caratterizzate e confrontate in base al loro trascrittoma e al loro immunofenotipo, allo scopo di valutarne le proprietà biologiche e quindi scegliere le CSM più adatte all’implementazione del progetto di Dottorato. Le CSM da tessuti adulti (e.g.: midollo osseo) hanno mostrato in vitro caratteristiche di senescenza correlate all’età del donatore in vivo. Al contrario, le CSM da tessuti perinatali (e.g.: sangue di cordone ombelicale) hanno mostrato un fenotipo più simile a quello dei periciti, ossia i progenitori delle CSM in vivo. Quindi, tenuto conto anche della traslabilità clinica, il sangue di cordone ombelicale è stato scelto come fonte di CSM, visto che la raccolta e la crioconservazione di unità di sangue placentare a fini terapeutici è già una realtà clinica. In seguito, un’analisi estesa delle popolazioni stromali presenti nel sangue di cordone ombelicale ha portato alla definizione di una sottopopolazione di CSM dotata di maggiori capacità proliferative e con una lunghezza del telomero significativamente più alta. Inoltre il protocollo standard di isolamento delle CSM da sangue di cordone ombelicale è stato migliorato, arrivando ad un’efficienza di circa 80%. Infine, le proprietà anti-infiammatorie e anti-apoptotiche del secretoma delle CSM sono state studiate sia in vitro che in vivo. Al fine di verificare se le vescicole extracellulari contribuissero alle proprietà paracrine delle CSM, se ne è caratterizzata la secrezione e se ne sono indagate le proprietà rigenerative. Una chiara efficacia terapeutica da parte delle vescicole extracellulari di CSM è stata dimostrata in un modello in vitro di danno tissutale acuto, in cui le vescicole extracellulari hanno eguagliato i risultati ottenuti con le CSM stesse. Nonostante l’utilizzo di CSM dalle elevate proprietà proliferative, la loro lifespan in coltura, in quanto cellule primarie, rimane limitata. Allo scopo di aumentarne il potenziale replicativo e di sfruttarne al meglio così la produzione di vescicole extracellulari, le CSM sono state sottoposte alla riprogrammazione cellulare indotta. In questo modo sono state generate linee cellulari derivate da CSM dal potenziale di crescita illimitato, evitando però di modificarne il genoma come nelle tradizionali tecniche di immortalizzazione. Siccome la riprogrammazione implica una modificazione radicale dell’identità della cellula d’origine, il passo successivo è stato quello di confermare la capacità di questa nuova popolazione di generare vescicole extracellulari, poi opportunamente caratterizzate. In particolare, il miRNoma delle vescicole extracellulari da cellule riprogrammate è stato oggetto di studio e di confronto con quello delle vescicole extracellulari delle CSM d’origine. Si è così potuto dimostrare che la maggior parte dei miRNA era presente nelle vescicole extracellulari sia prima che dopo la riprogrammazione. Ciò indica che il processo di riprogrammazione non ne ha alterato in modo sostanziale il contenuto. Questo potrebbe avere importanti ricadute sugli aspetti funzionali delle vescicole extracellulari da CSM riprogrammate. Infatti è stato ipotizzato che il trasferimento di miRNA specifici da cellule donatrici a cellule target mediato dalle vescicole extracellulari sia uno dei meccanismi d’azione delle CSM. Nell’ultima parte di questo progetto di Dottorato, l’utilità terapeutica delle vescicole extracellulari da cellule staminali (CSM e CSM riprogrammate) è stata confrontata con quella delle CSM d’origine. A tale scopo è stato utilizzato un modello ex vivo di ischemia cerebrale, in cui è stato osservato il movimento di alcuni parametri di danno ischemico acuto, tra cui un picco di produzione di citochine infiammatorie, una forte necrosi tissutale e una riduzione delle popolazioni cellulari neuronali e astrocitiche. Questo particolare modello mima infatti la fase acuta di questa condizione patologica, il cui trattamento a base di agenti trombolitici deve avvenire entro 3-6 ore dall’insorgenza dei primi sintomi. Quindi le vescicole extracellulari prodotte dalle CSM riprogrammate sono state testate per la prima volta in questo contesto patologico per verificare se potessero esercitare una funzione rigenerativa. La loro somministrazione al tessuto colpito dal danno ischemico ha generato uno spiccato effetto neuroprotettivo, pari a quello delle CSM d’origine, che ha riportato a valori simili a quelli del tessuto cerebrale non danneggiato i parametri di danno sopra descritti. Il risultato più interessante e statisticamente significativo è stato soprattutto a carico di quei parametri legati ai processi infiammatori, i quali sfavoriscono il recupero del danno tissutale. In conclusione, i risultati presentati in questa tesi di Dottorato confermano la possibilità di utilizzo di vescicole extracellulari secrete da cellule staminali in strategie di medicina rigenerativa. Questa innovativa extracellular vesicle therapy potrebbe in futuro essere applicata in contesti patologici per i quali ad oggi non è praticabile una terapia cellulare. A questo punto, nel quadro dei prodotti medicinali per le terapie avanzate, il “farmaco” non sarebbe più la cellula staminale, ma le rispettive vescicole extracellulari. Queste acquisirebbero così il ruolo di carrier di molecole antinfiammatorie, pronte all’uso e capaci di garantire un’azione terapeutica tempestiva per la rigenerazione di tessuti danneggiati.

Generating neural stem cells and neurons from reprogrammed human astrocytes is a potential strategy for repair in neurological diseases. It has been recently showed that astrocytes from murine cerebral cortex can be differentiated into neurons by the forced expression of a single transcription factor (Heinrich et al., 2010). While these studies have evaluated astrocyte conversion in the murine context, a similar possibility has yet to be demonstrated in human cells. An essential element for developing such applications with therapeutic value is a thorough comprehension of the mechanisms that regulate reprogramming of adult cells into induced pluripotent stem cells (iPSCs) (Hanna et al., 2010) or directly into another committed lineage, such as fibroblasts converted into neurons and also specific neuronal subpopulations like dopaminergic neurons (Pang et al., 2011, Caiazzo et al., 2011). Here we demonstrate the possibility to obtain progenitors and mature cells of the neural fate directly from human cortical astrocytes with a dedifferentiation into neural stem/progenitor phenotype. Even if for the purpose of autologous cell transplantation in neurological disorders, fibroblasts from patients resemble a much more suitable source of neurons than astrocytes from patients, nevertheless, shading light in the mechanisms that make possible to reprogram astrocytes into NSCs is useful for the final goal of using these cells as endogenous cell source for in situ neural repair in the CNS without any invasive cell graft. Human astrocytes can be reprogrammed into iPSCs, with similar efficiencies to other cells, using the viral expression of four reprogramming factors (Oct4, Sox2, Klf4, and cMyc) (Riuz et al., 2008). Remarkably, overexpression of a single factor like OCT4 in adult cells can induce full reprogramming, as when it is expressed in NSCs (human and mouse) (Kim et al., 2009 a, b) or promote the formation of another phenotype, such as the generation of blood cells with its expression in human fibroblasts (Szabo et al., 2010). These data suggest that the effect of these stem reprogramming factors changes in relationship to the lineage and the differentiation stage of the cells expressing them. In the current work, using the individual expression of OCT4, SOX2, or NANOG, we demonstrated and characterized the direct neural fate conversion of human astrocytes into multipotent neural progenitors, in vitro and in vivo. These cells were generated in a manner that is independent of iPSC production. Individual ectopic expression of the reprogramming factors OCT4 or SOX2 or NANOG into astrocytes, together with specific cytokine/culture conditions, activated the neural stem gene program and induced the generation of cells expressing neural stem/precursors markers. This change of lineage commitment was obtained also in pure CD44+ mature astrocytes and did not require passing through a pluripotent state. These unique astrocytes-derived neural stem cells gave rise to neurons, astrocytes and oligodendrocytes, and showed in vivo engraftment properties. ASCL1 expression further promotes the acquisition of a neuronal phenotype in vitro and in vivo. ASCL1 expression further promotes the acquisition of a neuronal phenotype in vitro and in vivo (Kim et al., 2009). To develop a broader understanding of astrocytes reprogramming we performed a methylation analysis demonstrating that epigenetic modifications underlie this process. These observations indicated that the sites of epigenetic and gene expression changes during reprogramming of astrocytes to NSCs are tightly linked to genes that are functionally important for pluripotency. These data demonstrate restoration of multipotency from human astrocytes, and point out a possible application of cellular reprogramming to endogenous CNS cells for repair of neurological disorders.

Spinal muscular atrophy (SMA), characterized by selective loss of lower motor neurons, is an incurable genetic neurodegenerative disease.and represents one of the most common genetic causes of infant mortality. Patients with SMA exhibit muscle weakness and hypotonia. Stem cell transplantation is a potential therapeutic strategy for SMA and other motor neuronal diseases. In this study, we analized the therapeutic capacity of different stem cells sources in order to improve SMA phenotype in a SMA murine model. First of all we isolated spinal cord neural stem cells (NSCs) from mice expressing green fluorescent protein (GFP) only in motor neurons and assessed their therapeutic effects on the phenotype of SMA mice. Intrathecally grafted NSCs migrated into the parenchyma and generated a small proportion of motor neurons. Treated SMA mice exhibited improved neuromuscular function, increased life span, and improved motor unit pathology NSC transplantation positively affected the SMA disease phenotype, indicating that transplantation of NSCs may be a possible treatment for SMA. However primary NSC as stem cell source have limited translational value. Thus we used alternative stem cells sources, NSC derived from wild-type embryonic stem cells (wt-ESCs) and from a drug-selectable embryonic stem cell line (OSG-ESC. This cells have promise as an unlimited source of NSCs for transplantation. We found that ESC-derived NSCs can differentiated into motor neuron in vitro, and, when intrathecally transplanted into SMA mice survived, migrated, ameliorated behavioral and life-span and may confer neuroprotection in SMA mice. NSCs obtained using a drug-selectable ESC line (positively for neuroepithelial cells and negatively for undifferentiated cells) yielded the greatest improvements. As with cells originating from primary tissue, the ESC-derived NSCs integrated appropriately into the parenchyma, expressing neuron- and motor neuron-specific markers. Our results suggest translational potential for the use of pluripotent cells in NSC-mediated therapies and highlight potential safety improvements and benefits of drug-selection for neuroepithelial cells.