1. Scongelamento hES cellule Pre-set-up sperimentale <ol><li> Un giorno prima di scongelare le cellule hES, piatto uno strato alimentatore di fibroblasti di topo irradiati embrionali (MEF), CF1 sforzo, da un pozzetto di una gelatina rivestite 6 e piastra di coltura tissutale nel terreno di coltura MEF (D-MEM medio basale integrato con 10% di siero FBS e 1% di aminoacidi non essenziali). Incubare per una notte a 37 ° C e 5% di CO 2. </li><li> Prima di rimuovere le cellule hES da azoto liquido, essere sicuri di avere tutte le attrezzature necessarie e reagenti a posto e pronti da utilizzare per garantire un disgelo efficiente e di successo. </li></ol> Scongelamento delle Cellule hES <ol start="3"><li> Rimuovere una fiala criogenico di cellule hES dal serbatoio di stoccaggio di azoto liquido con pinze di metallo. </li><li> Stendere la fiala tra le mani guantate per 3-5 secondi per rimuovere il gelo. </li><li> Registrare le informazioni sull&#39;etichetta del flaconcino. </li><li> Utilizzando pinze di metallo, immergere il flacone in un bagno d&#39;acqua a 37 ° C. Ruotare delicatamente il flaconcino e osservare i progressi del disgelo spesso (ma velocemente!) Tenendo il flacone fino alla luce per vedere le dimensioni del cristallo di ghiaccio. Non immergere il tappo del flacone nel bagno d&#39;acqua in quanto ciò potrebbe contaminare le cellule. </li><li> Quando solo un cristallo di ghiaccio rimane piccolo, immergere la fiala intera in etanolo per sterilizzare. </li><li> In un armadio di sicurezza biologica sterile, trasferire il contenuto della fiala criogenico direttamente al fondo di un tubo da 15 ml. </li><li> Lentamente aggiungere 4 ml di terreno di coltura cellulare hES (D-MEM/F-12 medio basale completato con la sostituzione Knockout 20% siero, 1% di aminoacidi non essenziali, 1% di L-glutammina, 0,2% β-mercaptoetanolo e 4 ng / ml bFGF) al tubo. Scuotere delicatamente per mescolare continuamente le cellule come il nuovo mezzo si aggiunge al tubo. </li><li> Centrifugare le cellule per 5 minuti a 200 x g. </li></ol> Placcatura Celle hES <ol start="11"><li> Mentre le cellule hES sono nella centrifuga, preparare il piatto alimentatore MEF, mediante l&#39;etichettatura con l&#39;apposito info cella hES: linea cellulare, il numero di passaggio, e la data disgelo. </li><li> Quando vi è di circa 2 minuti dalla fine del periodo di rotazione, aspirare il terreno di coltura MEF e aggiungere 1,0 ml di PBS al pozzo. </li><li> Portare le cellule pellet hES torna alla cappa di sicurezza biologica, e con attenzione aspirare il surnatante. Fare attenzione a non aspirare il pellet, ma rimuovere il più surnatante possibile, in quanto questa soluzione contiene DMSO dal mezzo di congelamento. </li><li> Risospendere il pellet molto delicatamente con l&#39;aggiunta di 2,5 ml di terreno di coltura cellulare hES utilizzando una pipetta di vetro 5 ml e pipettare 3-4 volte. </li><li> Aspirare il PBS dall&#39;alimentatore MEF bene e aggiungere lentamente tutti i 2,5 ml di sospensione cellulare hES al preparato bene del 6 pozzetti. </li><li> Collocare la piastra nel 37 ° C incubatore e con attenzione scorrere la piastra in avanti a indietro, e un lato all&#39;altro per distribuire uniformemente le cellule in tutto il bene. Non agitare il piatto in un modello circolare per evitare di concentrare le colonie al centro. </li><li> Permettono alle cellule di fissare a 37 ° C e 5% CO 2 durante la notte. </li><li> Il giorno dopo il disgelo, rimuovere il supporto (con eventuali cellule galleggiante) e aggiungere 2,5 ml di fresca terreno di coltura cellulare hES al pozzo. </li><li> Opzionale: Il mezzo rimosso e le cellule possono essere trasferiti su un piatto preparato alimentatore separato MEF. Questa operazione genera spesso nuove colonie che può essere coltivato in parallelo con le cellule dal disgelo originale. </li><li> Incubare le piastre a 37 ° C di CO 2 e il 5% durante la notte. </li></ol> 2. Congelamento delle Cellule hES <ol><li> Mentre le cellule hES sono nella centrifuga, etichetta le fiale criogenica all&#39;interno della cappa di sicurezza biologica, inclusa la linea cellulare, il numero di passaggio, e la data di congelamento. La densità tipica per il congelamento delle cellule hES è un pozzo di cellule (da una a 6 pozzetti) per flaconcino criogenico. </li><li> Quando le cellule sono finiti hES centrifugazione, portare i tubi dietro alla cappa di sicurezza biologica. Aspirare il surnatante dal tubo, facendo attenzione a non disturbare il vagamente ricco di pellet. </li><li> Risospendere il pellet cellulare con la giusta quantità di terreno di coltura cellulare hES: 0,5 ml di cellule hES terreno di coltura per flaconcino criogenico. Siate molto gentile durante il pipettaggio, come le cellule hES recuperare meglio se congelati in pezzi colonia relativamente grandi. </li><li> Lentamente, mentre scuotendo leggermente il tubo, aggiungere la giusta quantità (0,5 ml per provetta Microbank ™) di media 2X congelamento delle cellule hES (60% FBS definito, il 20% delle cellule hES terreno di coltura, e il 20% DMSO) per le cellule. Una volta che il medium di congelamento è aggiunto, pipetta la soluzione estrema dolcezza 1-2 volte per mescolare. </li><li> Rapidamente, ma delicatamente, aggiungere 1 ml di sospensione cellulare per ogni flacone criogenici. </li><li> Trasferire le provette in un contenitore congelamento isopropanolo e posto inuno congelatore -80 ° C durante la notte. Le cellule si blocca a 1 ° C al minuto nel contenitore congelamento isopropanolo. </li><li> Il giorno seguente, trasferire rapidamente le fiale congelato in un serbatoio di stoccaggio di azoto liquido con pinze di metallo. </li></ol> Rappresentante Risultati Il primo giorno dopo cellule staminali embrionali umane sono scongelati, piccole colonie possono apparire trasparente e può essere difficile da vedere sotto il microscopio. Dato che le cellule ES appena scongelati lentamente tendono a proliferare, possono richiedere qualche giorno di apparire come stabilito colonie (Figura 1). Figura 1. Il recupero e la crescita cellulare. Cellule hES scongelato da azoto liquido sono stati imaged Giorni 1, 7 e 11 dopo lo scongelamento.

<ol>
	<li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Technique. , (2005).</li><li>Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1147 (1998).</li></ol>

Il video che accompagna illustra un metodo per lo scongelamento e congelamento delle cellule HES. Cellule congelate in azoto liquido deve essere scongelato bagno il più rapidamente possibile per ottenere il miglior recupero possibile. Ricordatevi di essere estremamente attenti quando pipettaggio cellule scongelamento: ridurre al minimo la manipolazione delle cellule e pipettare delicatamente. Un flaconcino di cellule hES può essere placcato su uno dei due e di un 6-pozzetti, o tutti i pozzetti di un 4-pozzetti e subito messo in incubatrice. Dal momento che la coltura in quattro pozzetti separati riduce la possibilità di perdere tutta la cultura alla contaminazione, e rende più facile individuare le colonie di cellule hES sulla superficie più piccola, il 4-pozzetti è consigliato quando scongelamento cellule hES per la prima volta. Il primo giorno dopo lo scongelamento cellule hES, le colonie sono piccole e possono essere trasparenti. Riempire la media delle cellule hES cultura ogni giorno, anche se le colonie non sono evidenti sotto il microscopio in quanto può richiedere qualche giorno in coltura per cellule hES di apparire come colonie stabilite. E &#39;importante utilizzare un fresco strato placcato alimentatore MEF quando lo scongelamento le cellule, come le colonie spesso non raggiungono una dimensione appropriata per passaging fino a 10 12 giorni dopo il disgelo. Quando lo scongelamento una fiala basso passaggio di cellule hES, è buona per espandere e congelare fiale aggiuntive per mantenere uno stock di cellule passaggio basso per la cultura e gli esperimenti futuri. E &#39;anche una buona idea di congelare regolarmente tutte le &quot;extra&quot; le cellule sane hES per mantenere scorte congelate. Il disgelo di maggior successo e le culture successive sono stati congelati in alta qualità, indifferenziato, attivamente dividendo colonie di cellule HES. cellule hES recuperare da un blocco in modo più efficiente se maneggiato con delicatezza come aggregati di cellule più grandi. La dimensione aggregata finale dovrebbe essere simile a quello (o leggermente più grande) le dimensioni degli aggregati placcato dopo un passaggio di routine.

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<tr>
<td>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965-092</td>
<td>For MEF mediumjavascript:void(0);</td>
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<tr>
<td>Fetal Bovine Serum (FBS), Certified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>Heat-inactivated 
For MEF medium</td>
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<tr>
<td>D-MEM/F-12</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-057</td>
<td>&nbsp;</td>
<tr>
<td>Knockout Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>Pre-screen to ensure support of hES cells</td>
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<td>bFGF</td>
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<td>Collagenase IV</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>Make a fresh solution at 1 mg/mL in D-MEM/F-12</td>
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<td>G1890</td>
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<td>DMSO</td>
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<td>Fetal Bovine Serum (FBS), Defined</td>
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<td>6-well plates</td>
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<td>4-well plates</td>
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<td>Nunc</td>
<td>176740</td>
<td>For thawing</td>
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<td>5 mL glass pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-27E</td>
<td>Individually wrapped</td>
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<tr>
<td>15 mL conical tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>Polypropylene</td>
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<tr>
<td>Cryogenic vials</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>5000-1020</td>
<td>1.5 mL capacity</td>
</tr>
<tr>
<td>Freezing container</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>5100-0001</td>
<td>&#x201C;Mr. Frosty&#x201D;</td>
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freezing and thawing human embryonic stem cells

Dal momento che James Thomson<em&gt; Et al</em&gt; Sviluppato una tecnica nel 1998 per isolare e far crescere hES in coltura, le cellule per un uso successivo congelamento e scongelamento ed espandere cellule da uno stock congelati sono diventati importanti procedure di routine effettuato in colture cellulari hES. Dato che le cellule hES sono molto sensibili alle sollecitazioni di gelo e disgelo, particolare cura deve intraprendere. Qui mostriamo la tecnica adeguata per il rapido scongelamento cellule hES provenienti dalle scorte di azoto liquido, placcatura loro sulle cellule embrionali di topo alimentatore, e lentamente li congelamento per la conservazione a lungo termine.

Watch this Scientific Journal Video about Freezing and Thawing Human Embryonic Stem Cells at JoVE.com

Freezing and Thawing Human Embryonic Stem Cells

Dal momento che James Thomson et al sviluppato una tecnica nel 1998 per isolare e far crescere hES in coltura, le cellule per un uso successivo congelamento e scongelamento ed espandere cellule da uno stock congelati sono diventati importanti procedure di routine effettuato in colture cellulari hES. Dato che le cellule hES sono molto sensibili alle sollecitazioni di gelo e disgelo, particolare cura deve intraprendere. Qui mostriamo la tecnica adeguata per il rapido scongelamento cellule hES provenienti dalle scorte di azoto liquido, placcatura loro sulle cellule embrionali di topo alimentatore, e lentamente li congelamento per la conservazione a lungo termine.

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 ...

freezing-and-thawing-human-embryonic-stem-cells

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45.7K Views.  Stemgent. Dal momento che James Thomson et al sviluppato una tecnica nel 1998 per isolare e far crescere hES in coltura, le cellule per un uso successivo congelamento e scongelamento ed espandere cellule da uno stock congelati sono diventati importanti procedure di routine effettuato in colture cellulari hES. Dato che le cellule hES sono molto sensibili alle sollecitazioni di gelo e disgelo, particolare cura deve intraprendere. Qui mostriamo la tecnica adeguata per il rapido sconge...

Video: Freezing and Thawing Human Embryonic Stem Cells

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

Congelamento cellule staminali embrionali umane

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

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Biologia

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

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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Sondaggio per mitocondriale complessa attività di cellule staminali embrionali umane

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Freezing, Thawing, and Packaging Cells for Transport

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks. 

Cultured mammalian cells are used extensively in cell biology studies. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.

Congelamento, lo scongelamento, e celle di imballaggio per il trasporto

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve 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 ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Etichettatura in vitro di cellule staminali embrionali umane per la Risonanza Magnetica

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Cultura e manutenzione di cellule staminali embrionali umane

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Preparazione Stendere cromosomiche di cellule staminali embrionali umane per cariotipo

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

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 ...

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Congelamento e scongelamento cellule staminali embrionali umane