ATTREZZATURE <ul><li> Tessuto cultura incubatore, 37 ° C, 5% di CO 2 </li><li> Centrifuga </li><li> Set di micropipettors (100, 100μL) </li><li> Acqua del bagno 37 ° C </li><li> Bagnomaria a 90 ° C a essere fonte di vapore d&#39;acqua </li><li> Microscopio a contrasto di fase </li></ul> PRIMO PASSO Trattando le cellule con Colcemid In questa procedura abbiamo usato la linea hESC H9 colta su fibroblasti embrionali di topo (MEF) inattivato con Mitomicina C (Sigma) e mantenuti in DMEM/F12 (Invitrogen) integrata con la sostituzione ad eliminazione diretta il 20% di siero (KSR, Gibco) e 8ng/mL di fattore di crescita dei fibroblasti (FGF-2, R &amp; S). Al fine di ottenere un gran numero di metafasi si diffonde, si consiglia di utilizzare le culture con elevato indice mitotico in cui ci sono molte cellule in divisione. <ol><li> Prendere il pallone cultura dal termostato e, nel flusso laminare, aggiungere Colcemid ad una concentrazione finale di 0,1 mg / ml. </li><li> Ritorna il pallone l&#39;incubatore e attendere 3 ore in modo che il inibitori può causare arresto mitotico in metafase, quando i cromosomi sono ben condensati e possono essere facilmente visualizzati sotto il microscopio. </li></ol> Fissaggio delle cellule <ol start="3"><li> Estrarre il supporto e aggiungere tripsina / EDTA 0,05% sufficiente a coprire la superficie delle cellule. Consentono alle cellule di incubare per 5 minuti a temperatura ambiente e poi inattivare l&#39;enzima sia con l&#39;aggiunta di terreno di coltura contenente siero o con la sola aggiunta di siero alle cellule. NOTA: è importante fare in modo che una sospensione singola cella è realizzata per ottenere diffonde metafase bene. </li><li> Trasferire le cellule in una provetta da centrifuga da 15 ml e centrifugare a 122 g per 5 minuti. </li><li> Eliminare il supernatante, lasciando circa 200 mL e risospendere il pellet delicatamente toccando il tubo, come mostrato nel video. Assicurarsi che le cellule sono ben dissociato, quindi non è possibile visualizzare qualsiasi grumi nella soluzione. </li><li> Successivamente sarà necessario aggiungere 5 ml di soluzione ipotonica (KCl 75mm) pre-riscaldato a 37 ° C, lentamente, dalla parete del tubo come nella seguente descrizione. In primo luogo aggiungere 3 ml, invertire il tubo in posizione orizzontale solo per mescolare le cellule con la soluzione ipotonica quindi aggiungere i rimanenti 2 ml e tenere in caldo a 37 ° C per 15 minuti. NOTA: A questo punto la soluzione ipotonica causerà un aumento del volume cellulare e aiuta a districare i cromosomi. Il tempo di incubazione è importante acquisire diffonde cromosoma bene. Se i tempi sono troppo lunghi, la membrana cellulare può esplodere troppo presto e cromosomi vengono persi. Se troppo breve, può essere difficile ottenere diffonde cromosoma perché la membrana cellulare non può interrompere. </li><li> Aggiungere 3 gocce della soluzione di fissativo (metanolo / acido acetico glaciale 3:1), invertire il tubo delicatamente per mescolare la soluzione fissativa con le cellule e centrifugare a 122 g per 5 minuti, senza intervallo. NOTA: La soluzione fissativo dovrebbe essere preparato. NOTA: è importante usare centrifugazione senza interruzione per evitare attriti cellule, impedendo la fuoriuscita del materiale. </li><li> Eliminare il supernatante, lasciando circa 200 mL e risospendere il pellet delicatamente toccando il fondo della provetta. NOTA: Anche in questo caso, assicurarsi che le cellule sono ben dissociato, quindi non è possibile visualizzare qualsiasi grumi nella soluzione. </li><li> Aggiungere i primi 3 ml di soluzione fissativo, lentamente la parete del tubo, mentre si ruota il tubo. Invertire il tubo in posizione orizzontale solo per mescolare le celle e quindi aggiungere altre 2 mL della soluzione fissativo dalla parete del tubo di prendere le cellule verso il basso. Magazzino a 4 ° C durante la notte. NOTA: Le cellule fisso può essere conservato in soluzione fissante per mesi a 4 ° C. Oltre a proteggere le cellule nel loro stato gonfio, la soluzione fissante rimuove i lipidi e le proteine ​​denatura. Questi eventi rendono la membrana cellulare fragile e, di conseguenza, rendere il cromosoma diffonde facilmente. </li></ol> PULIZIA DEL vetrini Prima di iniziare rendendo il vostro diffonde cromosoma assicurarsi che le diapositive siano adeguatamente puliti o si rischia di perdere alcuni nuclei e anche metafasi. Questo passo è così importante se si vuole provare tutte le tecniche di ibridazione 9. <ol><li> Posizionare i vetrini in un bicchiere contenente HCl 6M per almeno 3 ore a temperatura ambiente. </li><li> Trascorso questo tempo, lavare i vetrini in acqua corrente per 10 minuti. </li><li> Sciacquare i vetrini in acqua distillata e lasciare che si asciugano a temperatura ambiente. </li><li> Ora le diapositive possono essere memorizzati in etanolo al 96% e asciugato con un panno immediatamente prima dell&#39;uso. </li></ol> NOTA: Per metodi di ibridazione in situ, le diapositive non può essere conservato per più di 2 giorni in etanolo al 96% SECONDO STEP Lavaggio delle cellule <ol start="10"><li> Il giorno dopo, centrifugare le cellule a 122 g per 5 minuti, senza intervallo. </li><li> Discard il surnatante lasciando circa 200 ml. Risospendere il pellet con delicatezza sfogliando il tubo poi aggiungere 5 ml di soluzione di fissativo fresco (metanolo / acido acetico glaciale 3:1), lentamente dalla parete del tubo durante la rotazione del tubo. Ripetere questa fase di lavaggio altre 2 volte per un totale di tre lavaggi. NOTA: La soluzione fissativo dovrebbe essere preparato. </li></ol> Preparare le diapositive <ol start="12"><li> Alla fase di centrifugazione scorso lasciare sufficiente quantità di soluzione per omogeneizzare il pellet ed ottenere una densità sufficiente di cellule nelle slide (vedi figura 1). </li><li> Aggiungere 20-30 ml di sospensione cellulare su un vetrino pulito e asciutto, spostando la pipetta molto vicino e parallelo alla superficie. Come la soluzione evapora fissativo, la superficie di scorrimento diventa granulosa. </li><li> Immediatamente, esporre la diapositiva, a faccia in su, nel vapore di acqua calda (90 °) per 30 secondi per provocare le cellule saltare in aria. NOTA: In questo momento, il metanolo dalla evapora la soluzione fissativa, con un conseguente aumento della concentrazione di acido acetico, che insieme con l&#39;acqua stimola il cromosoma diffusione. </li><li> Guardate un microscopio a contrasto di fase solo per verificare se la concentrazione e la distribuzione delle cellule è buono (vedi figura 1). </li><li> Ora le diapositive sono pronte per essere utilizzato per controllare il set di cromosomi da approcci citogenetica come G-banding (Giemsa), FISH, SKY o CGH. </li></ol> CONSIGLI IMPORTANTI Come scegliere un metafase adeguata da analizzare La scelta di metafasi rotondo e isolato (figura 2D), si può evitare di considerare aneuploidie falso come il guadagno dei cromosomi generata da due o più si diffonde miscelati tra loro o la perdita di cromosomi generato da un cromosoma lanciato. Quindi, evitare di scegliere metafasi vicino nuclei perché alcuni cromosomi possono essere nascoste da loro (figura 2A e 2B). Inoltre, cercare metafasi giro ed essere sicuro che ogni cromosoma è separato dalla metafase (figura 2C). Cellule feeder e cariotipo hESC Per G-banding e analisi SKY, la presenza di cellule di alimentazione nella cultura hESC non interferisce con i risultati, perché le cellule di alimentazione non sono in uno stato che divide (inattivato dalla mitomicina C o irradiazione) e non sarà parte della popolazione metafase. Tuttavia, per l&#39;applicazione della tecnica FISH nei nuclei interfase, si è preferito separare la popolazione hESC a partire da cellule alimentatore dalla raccolta delle colonie manualmente prima tripsinizzazione per l&#39;analisi FISH. <img border="0" src="/files/ftp_upload/1512/1512fig1tmb.jpg" alt="Figura 1" /> Figura 1: Come sapere la appropriata densità delle cellule nelle diapositive. Le immagini del microscopio a contrasto di fase obiettivo 10x. (A) La densità delle cellule è troppo alta. Le frecce indicano metafase si diffonde troppo vicino dei nuclei. (B) Una diapositiva con una buona densità cellulare. I cerchi mostrano diffonde metafasi da nuclei isolati o metafasi altri. Si prega di <a href="https://www.jove.com/files/ftp_upload/1512/1512fig1.jpg">cliccare qui</a> per vedere una versione più grande della figura 1. <img border="0" src="/files/ftp_upload/1512/1512fig2tmb.jpg" alt="Figura 2" /> Figura 2: Come scegliere un metafase adeguata da analizzare. I cromosomi sono colorati con DAPI e le immagini ottenute con un microscopio a fluorescenza a obiettivo 100x. (A) (B) Evitare metafasi vicino a nuclei (indicati dalle frecce) (C) e metafasi non rotondi che i cromosomi presentano separati (cerchio). (D) Un metafase rotonda è una metafase bello per essere analizzati. Si prega di <a href="https://www.jove.com/files/ftp_upload/1512/1512fig2.jpg">cliccare qui</a> per vedere una versione più grande della figura 2.

<ol>
	<li>Thomson, J. A. <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), 1145-1145 (1998).</li><li>Draper, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+gain+of+chromosomes+17q+and+12+in+cultured+human+embryonic+stem+cells.">Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.</a> Nat Biotechnol. 22 (1), 53-53 (2004).</li><li>Maitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+alterations+in+cultured+human+embryonic+stem+cells.">Genomic alterations in cultured human embryonic stem cells.</a> Nat Genet. 37 (10), 1099-1099 (2005).</li><li>Mitalipova, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+the+genetic+integrity+of+human+embryonic+stem+cells.">Preserving the genetic integrity of human embryonic stem cells.</a> Nat Biotechnol. 23 (1), 19-19 (2005).</li><li>Buzzard, J. J., Gough, N. M., Crook, J. M., Colman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotype+of+human+ES+cells+during+extended+culture.">Karyotype of human ES cells during extended culture.</a> Nat Biotechnol. 22 (4), 381-381 (2004).</li><li>Caisander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+integrity+maintained+in+five+human+embryonic+stem+cell+lines+after+prolonged+in+vitro+culture.">Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture.</a> Chromosome Res. 14 (2), 131-131 (2006).</li><li>Imreh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+conditions+favoring+selection+of+chromosomal+abnormalities+in+human+ES+cells.">In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells.</a> J Cell Biochem. 99 (2), 508-508 (2006).</li><li>Robin-Wesselschmidt, J. L., Loring, J. F., Robin-Wesselschmidt, J. L., Robin-Wesselschmidt, S. c. h. w. a. r. t. z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Stem Cell Manual. , 59-59 (2007).</li><li>Rooney, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cytogenetics. , (2001).</li><li>Heslop-Harrison, P., Schwarzacher, T., Heslop-Harrison, P., Schwarzacher, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical in situ hybridization. , 51-51 (2000).</li></ol>

The authors have nothing to disclose.

La preparazione degli spread cromosoma è un passo fondamentale per un&#39;analisi efficace della situazione genetica delle cellule staminali embrionali da tecniche di routine come G-banding, e le tecniche più sofisticate come la FISH, SKY e CGH. Questa procedura può essere applicata per molti tipi di cellule, variando il periodo di incubazione Colcemid, che dipende dalla durata del ciclo cellulare. Per le colonie di cellule staminali embrionali, aspettiamo 3 ore, mentre per corpi embriona...

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<td>208</td>
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chromosomal spread preparation of human embryonic stem cells for karyotyping

Sebbene cellule staminali embrionali umane (hESC) hanno dimostrato di presentare un cariotipo diploide stabile 1, molti studi hanno riportato che in base alle condizioni cultura diventano inclini ad acquisire le anomalie cromosomiche come la aggiunta di interi o parti di cromosomi. Infatti, durante il lungo periodo la cultura, le alterazioni del cariotipo si osservano quando dissociazione enzimatica o chimica sono usati 2,3,4, mentre dissezione manuale di colonie per passaging mantiene un cariotipo stabile 5. Inoltre, cambiamenti nell&#39;ambiente come la rimozione delle cellule alimentatore sembrano anche compromettere l&#39;integrità genetica di hESC 3,6. Una volta che le alterazioni cromosomiche potrebbero influenzare la fisiologia cellulare, la caratterizzazione della integrità genetica di hESC in vitro è di fondamentale importanza hESC considerare come uno strumento essenziale negli studi di embriogenesi e test della droga. Inoltre, per futuri scopi terapeutici alterazioni cromosomiche sono una reale preoccupazione in quanto è frequentemente associata a carcinogenesi. Qui vi mostriamo un metodo semplice e utile per ottenere alta qualità dei cromosomi si diffonde per la successiva analisi del cromosoma fissati dal G-banding, FISH, SKY o tecniche di CGH 7,8. Si consiglia di verificare lo stato cromosomico di routine con intervalli di 5 passaggi al fine di monitorare la comparsa di traslocazioni e aneuploidie Priscila Britto e Rafaela Sartore contribuito in maniera uguale alla carta.

Watch this Scientific Journal Video about Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping at JoVE.com

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Cariotipo è una tecnica semplice e utile ampiamente utilizzato per la rilevazione di alterazioni genetiche. Qui si descrive un passo dal protocollo passo per la preparazione diffusione cromosoma di cellule staminali embrionali umane per monitorare lo stato cromosomico di queste cellule mantenute in coltura.

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 ...

chromosomal-spread-preparation-human-embryonic-stem-cells-for

Research

JoVE Journal

Biology

48.6K Views.  Federal University of Rio De Janeiro-UFRJ. Cariotipo è una tecnica semplice e utile ampiamente utilizzato per la rilevazione di alterazioni genetiche. Qui si descrive un passo dal protocollo passo per la preparazione diffusione cromosoma di cellule staminali embrionali umane per monitorare lo stato cromosomico di queste cellule mantenute in coltura.

Video: Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

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

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

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 ...

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 ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Preparazione del pool lisato piastrinico umani (pHPL) come supplemento efficiente per animali siero-Free Culture sulle cellule staminali umane

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

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 ...