Name: isolating nasal olfactory stem cells from rodents or humans
Uploaded: 2011-08-22T12:00:00
Duration: 9 min 19 s
Description: Watch this Scientific Journal Video about Isolating Nasal Olfactory Stem Cells from Rodents or Humans at JoVE.com

Questo lavoro è stato sostenuto finanziariamente dalla ANR (Agence Nationale de la Recherche), AFM (Association Française contre les Myopathies), FESR in PACA e Irme (Institut de Recherche sur la Moelle épinière et l&#39;Encéphale). Si ringrazia Marie Pierre Blanchard (Jean Roche Institute) per il suo aiuto efficiente durante la registrazione temporizzata.

1. Raccolta di mucosa olfattiva in topi <ol><li> Iniziate preparando tre piatti 35 millimetri Petri riempite con DMEM / HAM F12 terreno di coltura in una cappa di cultura pulito. </li><li> Qualsiasi metodo di eutanasia deve essere approvato preventivamente dalla cura degli animali dell&#39;istituto e sul comitato per l&#39;uso e svolte da personale qualificato. Dopo che il ratto è entrato anestesia profonda con sodio pentobarbital o altre forme di anestesia iniettabili come la chetamina / xylazina, decapitare e togliere la pelle. Anestetici inalatori dovrebbero essere evitati. L&#39;adeguatezza di anestesia sarà valutata pizzico piedi prima di decapitazione. Rimuovere la mascella inferiore con le forbici e con l&#39;aiuto di una pinza ossivora, eliminare i muscoli facciali su entrambi i lati. </li><li> A partire dalla parte posteriore degli incisivi, rimuovere con una pinza ossivora l&#39;osso che copre la cavità nasale, da un lato alla volta. I turbinati olfattivo entrare in vista come arancio / marrone organi che si trovano nella parte posteriore del naso. </li><li> Delicatamente scartare il turbinati con una pinza. Utilizzando un ago 26 gauge, isolare la mucosa olfattiva sdraiato sul setto tagliando il tessuto lungo tre direttrici: l&#39;arco della piastra perpendicolare, la lamina cribrosa e il soffitto della cavità nasale. </li><li> Raccogliere biopsie su entrambi i lati e trasferirli in un DMEM / HAM F12 piena di Petri. Questa procedura non dovrebbe richiedere più di 10 minuti dalla comparsa eutanasia. </li><li> Ora, al fine di rimuovere il muco, trasferire le biopsie due volte in media riempito piastre di Petri. </li></ol> 2. Raccolta di mucosa olfattiva negli esseri umani <ol><li> Questa procedura deve essere effettuata da un Otorinolaringoiatria (ORL), chirurgo, in conformità con le pertinenti locale comitato etico (s), e ogni ambulatoriale deve firmare un modulo di consenso informato. </li><li> Usando una 0 ° o 30 ° endoscopio rigido (4 mm di diametro), ispezionare sia cavità nasali e valutare la presenza putativo di polipi o lesioni infiammatorie. Scegli il miglior cavità nasale, tenendo conto della deviazione del setto. </li><li> Usando un applicatore di cotone, applicare un anestetico locale, come la lidocaina con adrenalina, per 10 minuti. </li><li> Con una pinza throughcut etmoide, raccogliere a due millimetri quadrati biopsia sia alla radice del mediale del turbinato medio o sul setto nella zona dorsomediale. </li><li> La biopsia olfattivo viene trasferito, utilizzando un ago sterile, in una sterile 2 ml di tubo riempito con 1 ml di DMEM / HAM F12. Punta il tubo a testa in giù per assicurarsi che la biopsia è immerso nel mezzo di coltura. </li><li> Inserire il tubo in un contenitore refrigerato e il trasporto al laboratorio di ricerca. In questa fase, la biopsia può essere utilizzato di per sé per gli studi comparativi molecolare focalizzata sulle malattie cerebrali specifici o trattati per la generazione di cellule staminali. </li></ol> 3. Isolamento di cellule staminali da olfattivi dell&#39;uomo e Rat Mucosa <ol><li> Lavare le biopsie in DMEM / HAM F12. Incubare le biopsie in piastre di Petri riempite con 1 ml di soluzione dispasi II (2,4 UI / ml), per 1 ora a 37 ° C. </li><li> Poi, sotto un microscopio da dissezione con una luce diffratta invertito, l&#39;epitelio olfattivo viene rimosso dal lamina propria sottostante con una spatola micro. </li><li> L&#39;epitelio olfattivo è più sottile e sembra trasparente su uno sfondo nero rispetto alla lamina propria, che è a strisce arancio / marrone. Su uno sfondo bianco, l&#39;epitelio appare grigia e la lamina propria, marrone. </li><li> Una volta purificate, trasferire la lamina propria in una capsula di Petri riempite con DMEM / HAM F12. </li><li> Se il tessuto è di un roditore, quindi tagliare la lamina propria in piccoli pezzi con due aghi di calibro 25. Poi, il trasferimento dei pezzi ad un tubo da 15 ml riempita con 1 ml di collagenasi IA. </li><li> Nel tubo, usando una pipetta sterile in plastica, dissociare il tessuto. Poi, incubare la provetta per 10 minuti a 37 ° C. </li><li> Per terminare la dissociazione, agitare delicatamente il tubo e aggiungere 9 ml di Ca-Mg libero e senza PBS e centrifugare a 200 g per 5 minuti. </li><li> Risospendere il pellet cellulare in DMEM / HAM F12 mezzo di coltura supplementato con 10% di siero fetale bovino, antibiotici e piastra su piatti di plastica cultura. </li><li> Ora, se il tessuto è umano, allora fetta della lamina propria in 3 a 4 pezzi con uno spessore da 200 a 500 micron. </li><li> Inserire ogni striscia il meglio di sé 2 piatto cultura cm di diametro e coprire con il tessuto sterile 1,3 scivola in vetro diametro cm copertura. </li><li> Quindi, aggiungere 500 microlitri di terreno di coltura (DMEM / HAM F12 supplementato con 10% di siero fetale bovino e antibiotici) per ogni piatto cultura. </li><li> Per entrambi i tipi di tessuto, rinnovare il terreno di coltura ogni 2 o 3 giorni. </li><li> Cinque a sette giorni dopo, le cellule staminali cominciano a invadere il piatto cultura e dopo due settimane dovrebbero essere confluenti. Quando confluenza viene raggiunto, passaggio e trasferimento alle cellule di fiaschi cultura. </li></ol> 4. Sfera Formazione e differe neuronalentiation delle cellule staminali olfattive <ol><li> Per generare sfere di cellule staminali, incubare i palloni per due ore a 37 ° C con poli-L-lisina. (TESTO: 5 g/cm2). </li><li> Piastra le cellule ad una densità di 16.000 cellule per centimetro quadrato in fiaschi trattati. </li><li> Ogni due giorni, nutrire le cellule con 0,2 ml per centimetro quadrato di media integrati (DMEM / HAM F12 integrato con l&#39;insulina, transferrina, selenio (ITS-X, 1%), EGF (50 ng / ml) e FGF2 (50 ng / ml)). </li><li> Da due a cinque giorni dopo, raccogliere le sfere delle cellule galleggianti e realizzato una nuova targa o dissociare prima innesto in modelli animali di terapia cellulare. </li><li> Per differenziare le cellule staminali in neuroni olfattivi-come le cellule, li coltivano per 21 giorni in media Neurobasal contenente B-27, penicillina, streptomicina, glutammina e glutammato. </li><li> Quindi apportare le modifiche di media ogni 3 giorni. Neurone-come le cellule dovrebbero apparire dopo due o tre settimane. </li></ol> 5. Rappresentante dei risultati: Nasale umano espianto-superando le cellule staminali (Figura 2A) si dividono rapidamente e confluenza può essere raggiunto in una o due settimane. Una caratteristica chiave di staminalità, espressione nestina, è stata valutata (Figura 2B). Quando coltivate su poli-L-lisina con un siero privo di mezzo di coltura integrato con EGF (50 ng / ml) e FGF2 (50 ng / ml), le cellule staminali olfattive dar luogo a sfere (figura 2C). Quando coltivate in siero contenente terreno di coltura di nuova sfere placcati dare origine a cellule che esprimono GFAP (~ 50%), tubulina cellule che esprimono (~ 10-15%) e O4 cellule che esprimono (~ 2-5%) 9 (Figure 2D-F). Tuttavia, il destino della sfera cellule derivate possono essere modificati. Per esempio, quando coltivate in un terreno di coltura Neurobasal integrato con B27 e il glutammato, la maggior parte delle cellule staminali olfattive nasale in neurone-come le cellule che esprimono β-tubulina III (Figura 2G) e MAP2 (Fig. 2H). <img src="/files/ftp_upload/2762/2762fig1.jpg" alt="Figura 1"/> Figura 1. Schema generale dell&#39;esperimento. Biopsie della mucosa olfattiva sono asportati dal ratto o umano cavità nasale. Espianti può essere utilizzato di per sé per gli studi comparativi molecolare al fine di individuare biomarcatori in malattie del cervello. Per isolare le cellule staminali olfattive, le interazioni tra la lamina propria e il neuro-epitelio sono interrotti con la II dispasi enzima e, dopo 45 minuti, l&#39;epitelio viene rimosso con un micro-spatola. Le cellule staminali olfattive roditori vengono ulteriormente selezionati dalla dissociazione della lamina propria con collagenasi IA. Per i tessuti umani, pezzi di lamina propria olfattive sono coltivate sotto vetro coprioggetto fino cellule staminali superando invadere l&#39;intero bene. Dopo la proliferazione, utilizzando un mezzo appropriato coltura, le cellule staminali olfattive possono generare sfere o differenziarsi in neuroni-come le cellule. Le cellule staminali olfattive possono essere usati per i) patologie cerebrali riparazione o trauma o ii) identificare marcatori molecolari di malattie del sistema nervoso centrale. Le illustrazioni sono realizzate con l&#39;ausilio di Arte Servier medica. <img src="/files/ftp_upload/2762/2762fig2.jpg" alt="Figura 2"/> Figura 2. Cultura e la differenziazione delle cellule staminali nasali olfattive. Cellule staminali umane a crescere senza la lamina propria a espianto (A) dividono rapidamente, quando coltivate in un siero contenente mezzo. Le cellule staminali esprimono la staminalità marcatore nestina (B). Quando placcato in poli-L-lisina rivestite di plastica e coltivati ​​in un siero privo di mezzo di coltura integrato con EGF e FGF2, le cellule staminali olfattive generare sfere (C). Sfera cellule derivate, quando placcato in siero contenente terreno di coltura, danno luogo a cellule che esprimono GFAP (~ 50%), tubulina cellule che esprimono (~ 10-15%) e O4 cellule che esprimono (~ 2-5%) 9 (DF). Quando coltivate in un terreno di coltura Neurobasal integrato con B27 e glutammato, essi si differenziano in neuroni-come le cellule che esprimono β-tubulina III (G) e MAP2 (H).

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Le tecniche qui presentate fanno il roditore e umano mucosa olfattiva un modello utile per la ricerca clinica sulle cause di malattie dello sviluppo neurologico e neurodegenerative, nonché uno strumento per riparare il cervello patologica o traumatizzati. Il protocollo è relativamente semplice e può essere facilmente effettuata da un esperto biologo cellulare. Il tasso di successo per le tecniche di biopsia e la cultura è alto. Passaggi critici <ul><li> Per la raccolta di roditore mucosa olfattiva, si consiglia di non superare un limite di tempo di 10 minuti tra l&#39;eutanasia e l&#39;escissione finale del tessuto olfattivo. </li><li> La dissociazione della lamina propria a olfattivo dei roditori si raggiunge solitamente dopo 10 min. incubazione in collagenasi IA. In caso contrario, si consiglia di raccogliere il giorno dopo il supernatante in cui i bit non dissociato del galleggiante lamina propria, si centrifugare a 200 g e meccanicamente dissociare la lamina mobile usando una pipetta Pasteur prima replating le cellule in un nuovo bene. Il primo pozzo che contiene le cellule già fissato è piena di terreno di coltura fresco. </li><li> Per la lamina propria umano, che è più compatto rispetto al tessuto roditore, si sconsiglia di uno dissociazione enzimatica. Il tessuto viene tagliato e ogni espianto è inserita tra il fondo del piatto di plastica e un copri bicchiere. Culture di successo sono espianti il ​​cui spessore va da 200 a 500 micron. </li></ul> Eventuali modifiche <ul><li> Il protocollo attuale può essere leggermente modificata in modo da generare neuroni olfattivi in ​​vitro. A tal fine, il neuro-epitelio non viene rimosso e la mucosa olfattiva è tutta a fette con un chopper McIlwain (200 micron di spessore). Ogni espianto è placcato in un piatto, parzialmente essiccato per un&#39;ora e poi reidratato con FCS contenenti terreno di coltura. Durante il primo messaggio placcatura giorni, le cellule epiteliali e mesenchimali crescere fuori l&#39;espianto. Poi, progenitori dei neuroni migrerà sulla parte superiore di questo strato di cellule e differenziarsi in neuroni. </li><li> Purificazione di cellule staminali olfattive possono essere raggiunti in citometria a flusso. Specifici marcatori di superficie possono essere recuperate dalla lista pubblicata sul giornale di caratterizzazione 11. </li><li> Per gli esperimenti di trapianto, è possibile utilizzare un ceppo di topi le cui cellule staminali olfattive sono GFP-positive. Questo ceppo ratto (Sprague Dawley, eGFP guidato dal promotore PGK) sono disponibili all&#39;indirizzo ITERT (Nantes, Francia). Per l&#39;inserimento del gene GFP in cellule staminali olfattive, usiamo un metodo basato sulla infezione lentivirus. </li><li> Questo documento si concentra su olfattivo ecto-le cellule staminali mesenchimali. Tuttavia, un altro tipo di cellula di interesse, le cellule olfattive ensheathing, può essere purificati dal tessuto stesso. Abbiamo descritto un metodo per la loro raccolta e la depurazione 1,17 e abbiamo usato cellule nasali ensheathing per uno studio di fase I / IIa clinica in pazienti paraplegici 18. </li><li> In qualità di membro della superfamiglia delle cellule staminali mesenchimali, OE-MSC sono in grado, in appropriate condizioni di coltura, di differenziarsi in adipociti, osteociti e miociti 11. </li></ul> Applicazioni future <ul><li> Abbiamo già usato biopsie olfattivo umano per studiare le anomalie cellulari e molecolari in pazienti affetti da autismo, disturbo bipolare, disautonomia familiare, morbo di Parkinson, morbo di Alzheimer e la schizofrenia 2-7. Teoricamente, tutte le malattie del cervello possono essere studiate usando biopsie nasali o periferico delle cellule staminali olfattive. </li><li> Roditore e cellule staminali olfattive nasali sono già stati innestati in modelli animali di amnesia, morbo di Parkinson, danni cocleari e traumi del midollo spinale 12-16. E &#39;concepibile di trapiantare queste cellule in modelli animali della malattia di Alzheimer, l&#39;ischemia cerebrale, sclerosi multipla. </li></ul>

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td colspan="3"> Raccolta della mucosa olfattiva nei ratti </td></tr><tr><td> DMEM / PROSCIUTTO F12 </td><td> Invitrogen </td><td> 31331-028 </td></tr><tr><td> Sodio pentobarbital </td><td></td><td></td></tr><tr><td> Pinza ossivora </td><td> FST </td><td></td></tr><tr><td> 26 gauge </td><td> Terumo </td><td> NN-2613R </td></tr><tr><td> Forcipe </td><td></td><td></td></tr><tr><td colspan="3"> Raccolta della mucosa olfattiva nell&#39;uomo </td></tr><tr><td> Endoscopio rigido </td><td> Karl Storz Medical o Richard Wolf </td><td></td></tr><tr><td> Lidocaina </td><td></td><td></td></tr><tr><td> Epinefrina </td><td></td><td></td></tr><tr><td> Throughcut etmoide forcipe </td><td> Karl Storz Medical o Richard Wolf </td><td></td></tr><tr><td colspan="3"> Isolamento di cellule staminali olfattive </td></tr><tr><td> Dispasi II </td><td> Roche </td><td> 10 295 825 001 </td></tr><tr><td> Microscopio da dissezione </td><td></td><td></td></tr><tr><td> Spatola Micro </td><td> FST </td><td></td></tr><tr><td> Collagenasi IA </td><td> Sigma-Aldrich </td><td> C9891 </td></tr><tr><td> Ca-free/Mg-free PBS </td><td> Invitrogen </td><td> 14190-250 </td></tr><tr><td> Siero di vitello fetale </td><td> Invitrogen </td><td> 10270098 </td></tr><tr><td> Vetro coprioggetto </td><td> Knittel Glaser </td><td> 001/35 </td></tr><tr><td colspan="3"> Sfera formazione e la differenziazione neuronale </td></tr><tr><td> Poli-L-lisina </td><td> Sigma-Aldrich </td><td> P-1274 </td></tr><tr><td> Insulina transferrina selenio (ITS) </td><td> Invitrogen </td><td> 51500056 </td></tr><tr><td> FEG </td><td> R &amp; D Systems </td><td> 236-EG </td></tr><tr><td> FGF2 </td><td> R &amp; D Systems </td><td> 233-FB </td></tr><tr><td> Medio Neurobasal </td><td> Invitrogen </td><td> 21103-049 </td></tr><tr><td> B-27 senza siero Supplemento </td><td> Invitrogen </td><td> 17504-044 </td></tr><tr><td> Penicillina / streptomicina </td><td> Invitrogen </td><td> 15140122 </td></tr><tr><td> Glutammina </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> Glutammato </td><td> Sigma-Aldrich </td><td></td></tr></tbody></table>

isolating nasal olfactory stem cells from rodents or humans

La mucosa olfattiva, che si trova nella cavità nasale, si occupa di rilevare gli odori. E &#39;anche l&#39;unico tessuto nervoso che è esposta all&#39;ambiente esterno e facilmente accessibile in ogni individuo vivente. Come risultato, questo tessuto è unico per chiunque al fine di individuare le anomalie molecolari nel cervello patologico o di isolare le cellule staminali adulte per la terapia cellulare. Anomalie molecolari nelle malattie del cervello sono spesso studiati utilizzando campioni di tessuto nervoso raccolti post-mortem. Tuttavia, questo materiale ha numerose limitazioni. Al contrario, la mucosa olfattiva è facilmente accessibile e può essere sottoposte a biopsia in modo sicuro senza alcuna perdita di olfatto 1. Di conseguenza, la mucosa olfattiva fornisce una &quot;finestra aperta&quot; nell&#39;essere umano adulto attraverso il quale si può studiare di sviluppo (es. autismo, schizofrenia) 2-4 o neurodegenerative (ad esempio, Parkinson, Alzheimer), le malattie 4,5. Mucosa olfattiva possono essere utilizzati sia per studi comparativi molecolari 4,6 o esperimenti in vitro sulla neurogenesi 3,7. L&#39;epitelio olfattivo è anche un tessuto nervoso che produce ogni giorno nuovi neuroni per sostituire quelli che sono danneggiati da inquinamento batterico delle infezioni virali. Questo neurogenesi permanente è sostenuta da progenitori, ma anche le cellule staminali che risiedono all&#39;interno di entrambi i compartimenti della mucosa, cioè il neuroepitelio e la sottostante lamina propria a 8-10. Abbiamo recentemente sviluppato un metodo per purificare le cellule staminali adulte si trova nella lamina propria e, dopo aver dimostrato che essi sono strettamente legate alle cellule staminali del midollo osseo mesenchimali (MSC-BM), abbiamo chiamato le olfattiva ecto-le cellule staminali mesenchimali (OE- MSC) 11. Interessante notare che, rispetto a BM-MSC, OE-MSC visualizzare un alto tasso di proliferazione, un clonogenicità elevato e una tendenza a differenziarsi in cellule neurali. Abbiamo approfittato di queste caratteristiche per svolgere studi dedicati a svelare nuovi geni candidati nella schizofrenia e 4 morbo di Parkinson. Noi e gli altri hanno anche dimostrato che OE-MSC sono promettenti candidati per la terapia cellulare, dopo un trauma del midollo spinale 12,13, un danno cocleare 14 o in un modello animale del morbo di Parkinson 15 o amnesia 16. In questo studio, presentiamo i metodi per la biopsia della mucosa olfattiva nei ratti e gli esseri umani. Dopo la raccolta, la lamina propria è enzimaticamente separato dal epitelio e le cellule staminali sono purificate utilizzando un enzimatici o un non-metodo enzimatico. Purificato le cellule staminali olfattive possono quindi essere coltivate in gran numero e sopraelevate in azoto liquido o indotto a formare sfere o differenziate in cellule nervose. Queste cellule staminali possono essere utilizzati anche per omiche comparativa (genomica, trascrittomica, epigenomic, proteomica) studi.

Watch this Scientific Journal Video about Isolating Nasal Olfactory Stem Cells from Rodents or Humans at JoVE.com

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

Descriviamo qui un metodo per biopsying mucosa olfattiva di ratto e umano cavità nasali. Queste biopsie può essere usato sia per identificare le anomalie molecolari nelle malattie cerebrali o isolare le cellule staminali adulte multipotenti che possono essere utilizzati per il trapianto di cellule in modelli animali di trauma cerebrale / malattia

Isolare cellule staminali olfattivo nasale da roditori o Umani

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

isolating-nasal-olfactory-stem-cells-from-rodents-or-humans

Research

JoVE Journal

Neuroscience

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

Le misurazioni elettrofisiologiche da un sistema olfattivo Moth

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

Ricerca

Neuroscienze

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolando LacZ cellule che esprimono da tessuti dell&#39;orecchio interno del mouse in citometria a flusso

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Ampliamento della embrionali e cellule staminali neurali adulte per<em&gt; In Utero</em&gt; Elettroporazione o Viral iniezione stereotassica

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolamento e la coltura delle cellule della cresta neurale da Embryonic Murine tubo neurale

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

Un metodo di espansione cGMP-applicabile per Aggregati di neurali umane staminali e cellule progenitrici derivate da cellule staminali pluripotenti o fetale tessuto cerebrale

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Derivazione libero-Feeder della cresta neurale progenitrici Cellule pluripotenti da cellule staminali umane

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Regia neuroni dopaminergici differenziazione da cellule staminali pluripotenti umane

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modellazione Astrocitoma Patogenesi<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Utilizzo corticale astrociti o cellule staminali neurali da condizionale, topi geneticamente ingegnerizzati

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

I neuroni olfattivi ottenuti attraverso nasale biopsia combinato con laser-Capture Microdissection: un approccio potenziale per studiare risposta al trattamento in Disturbi Mentali

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Differenziazione neuronale dalle cellule staminali embrionali del topo in vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...