Gli autori desiderano ringraziare il Dott. Philip H. Schwartz del National Human Resource neurale delle cellule staminali presso l&#39;Ospedale dei Bambini, Orange County Istituto di Ricerca per la fornitura di culture hNSPC e il Dr. Gary Bassell alla Emory University per l&#39;istruzione iniziale nella tecnica di colorazione parafilm.

Giorno 1: Preparazione di cellule coprioggetto di vetro tedesco e Semina Nota: coprioggetto di vetro tedesco sono spesso preferibili per la coltura delle cellule staminali neurali primarie e neuroni. Usiamo il seguente protocollo di pulizia per migliorare l&#39;adesione cellulare e la diffusione. Coprioggetto posizione in un rack di piccole dimensioni che consentirà l&#39;accesso liquido entrambi i lati del vetrino. In primo luogo, coprioggetto incubare 1% Liquinox per 10 minuti, seguita da 3 lavaggi con acqua deionizzata. Assicurarsi che non rimangano bolle di sapone. Poi, scivola incubare in HCl 1M per 30 minuti, seguita da 3 lavaggi con acqua deionizzata. Coprioggetto secco a 65 ° C durante la notte. Trasferimento coprioggetto per un piatto di vetro e autoclave per la sterilizzazione. <ol><li> In una cappa colture di tessuti, luogo coprioggetto sterili in una piastra di dimensioni adeguate bene. </li><li> Coprioggetto cappotto con laminina per l&#39;adesione cellulare. Coprioggetto incubare a 10 mcg / ml poli D-lisina (PDL) in acqua sterile per 5 minuti a temperatura ambiente. Rimuovere coprioggetto PDL e incubare a 20 mg / ml in laminina Emem per 4 ore, o durante la notte, a 37 ° C di coltura tissutale incubatore. <ol><li> Nota: Fare riferimento all&#39;articolo Passaging neurali Cellule staminali umane ( <a href="/index/Details.stp?ID=263">https://www.jove.com/index/Details.stp?ID=263</a> ) e il conteggio umani articolo Neural Stem Cells ( <a href="/index/Details.stp?ID=262">https://www.jove.com / index / Details.stp? ID = 262</a> ) per imparare a risospendere e contare hNSPCs. </li></ol></li><li> Sciacquare laminina rivestita vetrini con PBS e cellule seme in ogni coprioggetto contenenti oltre a una certa densità (spesso usiamo una densità iniziale di placcatura 100,000-300,000 cellule / ml per le cellule che stiamo placcatura per immunostaining). </li><li> Piastra in 37 ° C di coltura tissutale incubatore e consentono alle cellule di aderire al coprioggetti (di solito un minimo di 4 ore è richiesto). </li></ol> Giorno 2: Fissaggio, Permeabilizing, blocco, e aggiunta di anticorpo primario alle celle Nota: utilizzare il fissativo seguente paraformaldeide 4% per preservare la morfologia cellulare. La ricetta prevede una transizione di pH per consentire al paraformaldeide di andare in soluzione. Noi preferiamo questo metodo piuttosto che l&#39;utilizzo del calore per sciogliere paraformaldeide perché temperature più elevate possono portare alla generazione di acido formico, che può aumentare la colorazione di fondo quando si usa fluorescenza. Alcuni componenti del fissativo aiutare a preservare la struttura del citoscheletro (MgCl 2 e EGTA), mentre altri aiutano a mantenere la morfologia complessiva (saccarosio). Fissativo paraformaldeide 4% per le celle <table style="border-collapse: collapse; border: medium none" border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Reagenti e procedura: </td><td> Importo per 100 ml di fissativo: </td></tr><tr><td> MilliQ acqua </td><td> 75 ml </td></tr><tr><td> Paraformaldeide (pesare nella cappa) </td><td> 4 g </td></tr><tr><td> 10N NaOH, mescolare e aggiungere goccia a goccia fino a quando la soluzione cancella </td><td> secondo necessità </td></tr><tr><td> 10X PBS </td><td> 10 ml </td></tr><tr><td> 1M MgCl 2 </td><td> 0,5 ml </td></tr><tr><td> 0,5 M EGTA </td><td> 2 ml </td></tr><tr><td> Saccarosio </td><td> 4 g </td></tr><tr><td> HCl 6N, titolare a pH 7,4 </td><td> secondo necessità </td></tr><tr><td> MilliQ acqua </td><td> portare a 100 ml </td></tr><tr><td colspan="2"> Conservare a 4 ° C per un massimo di 1 settimana. Calda a 37 ° C prima dell&#39;uso </td></tr></tbody></table><ol><li> In panchina, rimuovere il supporto e aggiungere fissativo preriscaldata. Incubare per 5-15 minuti (a seconda della antigene per essere rilevato, usiamo di solito 10 minuti). Eseguire questa e le seguenti operazioni a temperatura ambiente. </li><li> Scartare il fissativo in un apposito contenitore, come molte istituzioni paraformaldeide gestire come rifiuti pericolosi. Lavare le cellule 3 volte con PBS, a 5 minuti ogni volta. Quando si rimuove soluzioni da parte delle cellule, fare attenzione che di aspirazione non si asciuga le cellule. Inoltre, sempre la prossima soluzione a portata di mano per aggiungere alle cellule prima di rimuovere la soluzione in modo che ricopre le cellule non sono lasciati ad asciugare. </li><li> Se l&#39;antigene di interesse all&#39;interno della cellula, la membrana cellulare deve essere permeabile per consentire l&#39;ingresso degli anticorpi. Per permeabilize celle, utilizzare un nuovo 0,3% Triton X-100 soluzione in PBS. Pipetta lentamente perché Triton è viscoso, e assicurarsi che il detergente è completamente dissolto in PBS prima di aggiungere alle cellule. Permeabilize cellule per 5 minuti, seguito dal lavaggio delle cellule 3 volte con PBS, a 5 minuti ogni volta. </li><li> Le cellule devono essere trattati con un agente di blocco per impedire legame non specifico degli anticorpi. Noi usiamo sieroalbumina bovina (BSA) come agente di blocco. La soluzione bloccante è preparata sciogliendo 5% BSA in PBS (su un bilanciere o dei rotatori, perché ci vuole tempo per sciogliere), quindi filtrare il soldistribuzione pesi con un filtro a siringa da 0,45 micron. Bloccare le cellule nel 5% BSA / PBS soluzione per 1 ora a temperatura ambiente. </li></ol> Diluire l&#39;anticorpo primario (che si spera sia specifico per la proteina di interesse) per un pre-determinato la concentrazione di 1% BSA / PBS (preparate per diluizione dal 5% BSA / PBS). Luogo diluito anticorpo primario (30 microlitri per 25 coprioggetto mm, 20 l 18 per coprioggetto mm, 15 microlitri per 12 coprioggetto mm) su una lastra di vetro coperto con parafilm. Sollevare il vetrino con le cellule fisse, invertire tale che le cellule a faccia in giù, e adagiarla sulla soluzione di anticorpi in modo che le cellule sono in contatto con l&#39;anticorpo. Inserire una seconda striscia parafilm oltre il costrutto intero. Incubare a 4 ° C durante la notte. 3 ° giorno: lavaggio, Incubazione anticorpo secondario, Mordente nucleare e di montaggio <ol><li> Rimuovere con attenzione la striscia in alto parafilm e raccogliete il coprioggetti, negarli, e rimetterla nella piastra con PBS in ciascun pozzetto di lavaggio in modo che le cellule sono rivolti verso l&#39;alto. </li><li> Lavare le cellule 3 volte con PBS, a 5 minuti ogni volta. </li><li> Scegli un anticorpo secondario che rileverà l&#39;anticorpo primario, per esempio, se l&#39;anticorpo primario è stato fatto in un coniglio, l&#39;anticorpo secondario dovrebbe riconoscere IgG di coniglio. Fare attenzione a corrispondere anche gli anticorpi isotipo (IgG, IgM, ecc.) Se si usano più anticorpi primari, assicurarsi che siano di specie diverse o isotipi, e il piano da rilevare con marcatori diversi attribuiti agli anticorpi secondari. Per esempio, è possibile utilizzare un anti-coniglio accoppiato secondaria ad un fluoroforo rosso con un accoppiamento anti-topo secondaria ad un fluoroforo verde. Incubare cellule nella soluzione di anticorpo secondario diluito ad una concentrazione appropriata in BSA 1% per 2 ore al buio a temperatura ambiente, utilizzando la stessa tecnica impiegata per primaria (passaggi non mostrato nel video). Il volume di anticorpo secondario necessari per l&#39;incubazione può essere leggermente superiore a quello per l&#39;anticorpo primario se il secondario è memorizzato come una soluzione di glicerolo (40 microlitri per 25 coprioggetto mm, 30 microlitri per coprioggetto 18 mm, 25 microlitri per 12 coprioggetto mm) . <ul><li> Nota: Se si utilizza molecole fluorescenti per visualizzare l&#39;anticorpo secondario, il campione deve essere protetta dalla luce, una volta che l&#39;anticorpo secondario è stato aggiunto. Incubare al buio, o in un piatto foglio coperto. </li></ul></li><li> Rimuovere con attenzione i coprioggetti dai parafilm come descritto per l&#39;anticorpo primario. Lavare le cellule 2 volte con PBS, 5 minuti ogni volta, seguita da una incubazione di 1 minuto in 2 mg / ml Hoechst nucleare macchia diluito in PBS (se di contrasto nucleare è preferito). Se la macchia Hoechst è vecchio e il segnale è troppo debole, è possibile aumentare il tempo di incubazione e / o la concentrazione. Dopo l&#39;incubazione con Hoechst, lavare 1 volta con PBS per 5 minuti. </li><li> Il coprioggetto deve essere montato su un vetrino con mezzi di montaggio per la visualizzazione su un microscopio. Nei casi in cui viene utilizzato una molecola fluorescente per la visualizzazione dei anticorpo secondario, i mezzi di montaggio dovrebbe contenere agenti per minimizzare photobleaching. Noi usiamo Vectashield come mezzo di montaggio per fluorescenza. Inserire un calo di dimensioni adeguate Vectashield su un vetrino da microscopio (12 microlitri per 25 coprioggetto mm, 6 microlitri per 18 coprioggetto mm, 3 ml per 12 coprioggetto mm). </li><li> Cura ritirare il vetrino e lavare la schiena con acqua deionizzata per eliminare i sali (dal PBS). Tamponare l&#39;acqua in eccesso su un tovagliolo di carta o kimwipe, e giaceva sul goccia di Vectashield con le cellule verso il basso. Posizionare il coprioggetto in un angolo e permettono di scendere lentamente per evitare bolle d&#39;aria cattura. Etichettare il vetrino con la data e le informazioni sul campione. </li><li> Permettere che i vetrini con coprioggetto per asciugare (al buio) di aspirazione poi via Vectashield eccesso intorno al bordo coprioggetto. I bordi coprioggetto può ora essere sigillato con smalto, se lo si desidera (particolarmente utile se coprioggetto saranno visualizzati su un microscopio invertito). Di solito il nostro negozio di diapositive in un congelatore di -20 ° C. </li></ol>

<ol>
	<li>Flanagan, L. A., Chou, J., Falet, H., Neujahr, R., Hartwig, J. H., Stossel, T. P., Filamin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamin+A,+the+Arp2/3+complex,+and+the+morphology+and+function+of+cortical+actin+filaments+in+human+melanoma+cells.">Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells.</a> J. Cell Biol. 155, 511-518 (2001).</li><li>Flanagan, L. A., Rebaza, L. M., Derzic, S., Schwartz, P. H., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+neural+precursor+cells+by+laminin+and+integrins.">Regulation of human neural precursor cells by laminin and integrins.</a> J. Neurosci. Res. 83, 845-856 (2006).</li><li>Flanagan, L. A., Ziaeian, B., Palmer, T., Schwartz, P. H., Loring, J. F., Wesselschmidt, R. W., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+analysis+of+stem+cells.">Immunocytochemical analysis of stem cells.</a> In Human Stem Cell Manual: A Laboratory Guide. , (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+Human+Neural+Stem+Cells.">Passaging Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+Human+Neural+Stem+Cells.">Counting Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li></ol>

Questo protocollo descrive una procedura per immunocolorazione hNSPCs che riduce al minimo il volume di anticorpi necessari e dà colorazione affidabile cellulare. La procedura descritta è meglio per gli antigeni intracellulari, ma può essere modificato per macchiare le molecole della superficie cellulare o per migliorare la colorazione del citoscheletro.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>German glass (Deutsche Spiegelglas) coverslips</td>
<td>Tool</td>
<td>Carolina Biological Supply Company</td>
<td>WW-63-3029</td>
<td>12,15,18, 22, 25 mm coverslips available</td>
</tr>
<tr>
<td>Liquinox phosphate-free detergent</td>
<td>Reagent</td>
<td>Sigma</td>
<td>Z273279</td>
<td>very viscous</td>
</tr>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>Laminin, natural mouse</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>EMEM (1X)</td>
<td>Reagent</td>
<td>Mediatech</td>
<td>MT-10-010-CV</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>24 well plate</td>
<td>Tool</td>
<td>Fisher</td>
<td>08-772-1</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP151-500</td>
<td>very viscous</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6148</td>
<td>potential carcinogen, use caution when using it in powder and solution form</td>
</tr>
<tr>
<td>Bovine Serum Albumin, IgG-free</td>
<td>Reagent</td>
<td>Jackson Immunoresearch</td>
<td>001-000-161</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst 33342</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>H-1399</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>H-1000</td>
<td>viscous</td>
</tr>		</tbody>
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immunocytochemistry: human neural stem cells

Immunocitochimica è un metodo molto potente e molto semplice per la determinazione della presenza, localizzazione subcellulare e l&#39;abbondanza relativa di un antigene di interesse, più comunemente una proteina, in cellule in coltura. Questo protocollo presenta un facile da seguire serie di passi che permetterà ai ricercatori di conservare gli anticorpi primari e secondari mentre ottenere alta qualità, qualità riproducibile e dati quantitativi dalla loro colorazione. Ci sono due aspetti di questo protocollo che consente di preservare il volume di anticorpi necessari per la colorazione. Per uno, le cellule sono coltivate su piccole lamelle circolari che sono posti in pozzetti di una piastra di coltura dei tessuti. Dopo la fissazione, le cellule su vetrini possono essere rimossi dai pozzetti della piastra. Per la colorazione degli anticorpi, il vetrino con le cellule è invertita su una piccola goccia di soluzione di anticorpi su parafilm ed è coperto con un secondo pezzo di parafilm per evitare l&#39;essiccazione. Utilizzando questo metodo, solo ~ 25 ml di soluzione di anticorpi è necessario per ogni coprioggetto (o campione) per essere colorato. Questo protocollo descrive immunocolorazione di staminali neurali umane / precursore cellule (hNSPCs), ma può essere usato per molti altri tipi di cellule.

Watch this Scientific Journal Video about Immunocytochemistry: Human Neural Stem Cells at JoVE.com

Immunocytochemistry: Human Neural Stem Cells

Immunocitochimica è un potente metodo per determinare la presenza, localizzazione subcellulare e l&#39;abbondanza relativa di un antigene di interesse in cellule in coltura. Questo protocollo presenta un facile da seguire serie di passaggi che permettono di conservare gli anticorpi e ottenere il massimo della propria colorazione.

Immunocitochimica: cellule staminali neuronali

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

immunocytochemistry-human-neural-stem-cells

Research

JoVE Journal

Biology

27.3K Views.  University of California, Irvine (UCI). Immunocitochimica è un potente metodo per determinare la presenza, localizzazione subcellulare e l'abbondanza relativa di un antigene di interesse in cellule in coltura. Questo protocollo presenta un facile da seguire serie di passaggi che permettono di conservare gli anticorpi e ottenere il massimo della propria colorazione.

Video: Immunocytochemistry: Human Neural Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Propagazione di staminali embrionali umane (ES) Cellule

Ricerca

Biologia

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Trasfezione delle cellule staminali neuronali con il Nucleofector Amaxa

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

Conteggio cellule staminali neuronali

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

Passaging cellule staminali neuronali

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivazione di cellule staminali embrionali umane da Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Topo Epidermal cresta neurale delle cellule staminali (EPI-NCSC) Culture

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Immunoprecipitazione della cromatina di cellule staminali embrionali umane

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelamento e scongelamento cellule staminali embrionali umane

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Differenziazione delle cellule staminali embrionali in precursori degli oligodendrociti

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Trasfettando e Nucleofecting umani cellule staminali pluripotenti indotte

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...