AEC è un collega della bara Jane Childs Memorial Fund per la ricerca medica e Merck Research Laboratories. Ringraziamo D. Egli e C. Cowan per la consulenza sperimentali e discussione.

<html> Congelati embrioni umani usati per staminali embrionali umane (HES) derivazione delle cellule sono stati donati per la ricerca seguenti corretto consenso informato in protocolli di ricerca che sono stati esaminati e approvati sia dal comitato per l&#39;uso di soggetti umani e sulle cellule staminali embrionali comitato di sorveglianza di ricerca presso la Harvard University . Protocollo scritto che segue si basa su parametri pubblicato riportato da Cowan et al. 2004, con lievi modifiche. Embrione Cultura Scongelare gli embrioni umani utilizzando Scongelare Quinn Advantage Kit e la cultura in gocce di medio globale integrata con il 15% Plasmanate fino allo sviluppo allo stadio di blastocisti espansa. Immunosurgery basata isolamento della massa cellulare interna Preparazione: <ol><li> Preparare le piastre per la rimozione della zona pellucida e immunosurgery (vedi sotto) attraverso la creazione di gocce di ogni soluzione in lastre di 10 cm. Sovrapposizione con olio minerale per prevenire l&#39;evaporazione. Preparare una singola riga per ogni embrione destinato derivazione. <ul><li> EmbryoMax Tyrodes acida, l&#39;uso è come. </li><li> hES derivazione delle cellule dei media: il 75% KO-DMEM, 10% KO-siero di ricambio, il 10% Plasmanate, il 2,5% delle cellule ES-testato siero fetale bovino (FBS), 2mM Glutamax-I, 1% di aminoacidi non essenziali, 50 unità / ml di penicillina, streptomicina 50μg/ml, 0.055mM b-mercaptoetanolo, 5ng/ml bFGF. </li><li> Coniglio anti-umano globuli rossi anticorpo primario, ricostituito secondo il suggerimento del produttore, diluito 1:10 con mezzi di derivazione delle cellule HES. </li><li> Cavia siero complemento, ricostituito secondo il suggerimento del produttore, diluito 1:10 con mezzi di derivazione delle cellule HES. <img src="/files/ftp_upload/old/574_2008_9_18_6/574_Figure-01.png" alt="574_Figure-01" width="600" height="166" /> </li></ul></li><li> Equilibrare tutte le piastre a 37 ° C in incubatore cultura del tessuto prima dell&#39;uso. </li><li> Tirare pipette capillari 50μl-100μl (VWR) per l&#39;utilizzo in embrioni trasferire. Tagliate le estremità con la penna di diamante e l&#39;uso con tubi in bocca pipetta dotato di filtro 0.2μm. </li></ol> Immunosurgery procedura: Note: <ul><li> In questa procedura, le cellule del trophectoderm sono distrutti da una breve esposizione di anticorpi diretti contro le cellule umane in tandem con l&#39;attività del complemento. Pertanto, il protocollo funziona meglio con alta qualità degli embrioni con un trophectoderm intatto, in quanto solo l&#39;integrità strutturale della blastocisti impedisce l&#39;ICM anche da essere suscettibile alla reazione immunologica. </li><li> Tutte le procedure eseguite utilizzando una portata dissezione dotato di palco riscaldato. </li></ul> Passi: <ol><li> Sciacquare la bocca pipetta da utilizzare per il trasferimento degli embrioni con FBS per evitare che si attacchino gli embrioni. </li><li> Trasferimento di blastocisti piatto da cultura a goccia hES detenzione di AT piatto. </li><li> Iniziare procedura di trasferimento di blastocisti da goccia hES attraverso serie di AT gocce. In calo in terzo luogo, guardare per lo scioglimento della zona pellucida (secondi di solito 5-30). Fare attenzione a non sovra-trattamento o l&#39;integrità dell&#39;embrione può essere compromessa. <img src="/files/ftp_upload/old/574_2008_7_21_2/574_Figure-02.png" alt="574_Figure-02" width="551" height="273" /> </li><li> Blastocisti di trasferimento attraverso serie di mezzi di derivazione delle cellule hES scende a risciacquare AT. Pipetta precarico con i media si raccomanda di neutralizzare immediatamente a reazione. Blastocisti può essere ritenuto qui fino a quando tutti sono stati AT-trattati. </li><li> Trasferimento di blastocisti attraverso serie di gocce anticorpo primario, lasciando in terza goccia per 30 minuti di incubazione a 37 ° C in un tessuto culturale incubatore. </li><li> Blastocisti di trasferimento attraverso serie di mezzi di derivazione delle cellule hES scende a risciacquare primaria. </li><li> Trasferimento di blastocisti attraverso serie di gocce di complemento, lasciando in terza goccia a guardare per la lisi dei globuli trophectoderm. Osservare blastocisti per i primi 30 secondi sul palco di &quot;bolle&quot; di cellule trophectoderm come lisare. Se si osserva la lisi entro questo lasso di tempo, tenere piatto sul palco microscopio per monitorare la reazione fino al completamento. Se non bolle si osserva durante i primi 30 secondi, tornare a incubatore, controllando ogni 5 minuti fino a quando la maggior parte delle trophectoderm ha lisate. Questa operazione può durare 5-15 minuti. Monitorare attentamente la reazione di ridurre al minimo i tempi di reazione e potenziali danni alla massa cellulare interna. <img src="/files/ftp_upload/old/574_2008_7_21_3/574_Figure-03.png" alt="574_Figure-03" width="571" height="273" /> </li><li> Blastocisti di trasferimento attraverso serie di mezzi di derivazione delle cellule hES scende a lavare via l&#39;attività del complemento. </li><li> Utilizzando una pipetta di vetro con un diametro che è leggermente più piccola della blastocisti (tirato da 10μl VWR tubi capillari), disegnare la blastocisti su e giù per spogliare la maggior parte dei trophectoderm lisate. g &quot;alt =&quot; 574_Figure-0 &quot;width =&quot; &quot;height =&quot; 577 270 &quot;/&gt; </li><li> Piastra ICM isolare su cellule feeder di fibroblasti di topo mitoticamente inattivato embrione (MEF). MEF deve essere preparato il giorno prima derivazione e passati a cellule di derivazione hES mezzi giornata di. 4-anche i piatti NUNC funzionare bene per i primi passi di derivazione. </li><li> Non disturbare piastra per i primi 1-2 giorni. Successivamente, controllare ogni giorno per conseguenza di colonie di cellule staminali embrionali (in genere evidente entro 1-2 settimane). Media dovrebbe essere cambiato in volumi di un mezzo ogni inizio altro giorno in giorno 3. </li></ol> ES escrescenza cellulare e passaging Quando la cellula escrescenza hES ha raggiunto circa 0,5 millimetri o più, è pronto per l&#39;espansione di raccolta meccanica. <ol><li> Meccanicamente disperdere la metà dei escrescenza e trasferimento in nuovi piatti contenenti alimentatori fresca. Ammassi di cellule staminali embrionali dovrebbe essere circa 30-50 cellule ciascuno. Lascia altra metà della crescita degli come back-up per il primo passaggio (p1). L&#39;escrescenza (p0) possono essere selezionati da diverse volte in quanto continua ad espandersi. <img src="/files/ftp_upload/old/574_2008_7_21_5/574_Figure-05.png" alt="574_Figure" width="639" height="219" /> </li><li> Colonie secondarie possono essere allo stesso modo disperso e ampliata attraverso i piatti della cultura più grande fino passaggi 3-5, momento in cui le nuove linee cellulari possono essere adattati alle passaging enzimatica con tripsina. Il contenuto siero dei media cultura dovrebbe essere gradualmente ridotta a 1% e 0% nel corso di questi passaggi iniziali per minimizzare la differenziazione delle cellule ES. </li><li> Tutti stabilito linee di cellule staminali embrionali dovrebbe essere incassato da congelamento giù una parte di passaggi presto per future espansioni. </li><li> Inoltre, ogni linea cellulare dovrebbe essere caratterizzata da analisi del cariotipo e validato da studi pluripotenza e la differenziazione in vitro e in vivo. </li></ol>

<ol>
	<li>Cowan, C. A., Klimanskaya, I., McMahon, J., Atienza, J., Whitmyer, J., Zucker, J. P., Wang, S. P., Morton, C. C., McMahon, A. P., Powers, D., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+embryonic+stem-cell+lines+from+human+blastocysts.">Derivation of embryonic stem-cell lines from human blastocysts.</a> N ENGL J MED. 350 (13), 1353-1356 (2004).</li></ol>

The authors have nothing to disclose.

Il protocollo qui presentato fornisce ai ricercatori un conciso, facile da seguire schema di come derivare embrionali umane linee di cellule staminali da immunosurgical isolamento della massa cellulare interna.

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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>Quinn's Advantage Thaw Kit </td>
<td>&nbsp;</td>
<td>Sage</td>
<td>ART-8016</td>
<td>&nbsp;</td>
<tr>
<td>Global medium</td>
<td>&nbsp;</td>
<td>Life Global</td>
<td>GMGB-050</td>
<td>&nbsp;</td>
<tr>
<td>Plasmanate</td>
<td>&nbsp;</td>
<td>Talecris</td>
<td>61325</td>
<td>&nbsp;</td>
<tr>
<td>EmbryoMax Acidic Tyrodes</td>
<td>&nbsp;</td>
<td>Chemicon/Millipore</td>
<td>MR-004-D</td>
<td>&nbsp;</td>
<tr>
<td>KO-DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>10829-018</td>
<td>&nbsp;</td>
<tr>
<td>KO-Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>ES cell-tested fetal bovine serum, Defined</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30070.03</td>
<td>&nbsp;</td>
<tr>
<td>Glutamax-I</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>35050-079</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential amino acids</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>11140-076</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>15070-063</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human basic fibroblast growth factor</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13256-029</td>
<td>&nbsp;</td>
<tr>
<td>Beta-mercaptoethanol </td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit anti-human red blood cell primary antibody</td>
<td>&nbsp;</td>
<td>Rockland</td>
<td>109-4139</td>
<td>&nbsp;</td>
<tr>
<td>Guinea-pig complement serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S-1639</td>
<td>&nbsp;</td>
<tr>
<td>Capillary pipettes in 10 microL, 50 microL, 100 microL sizes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mouth pipettes with tubing</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>supplied with capillary pipettes from VWR</td>
</tr>
<tr>
<td>0.2 microm Acrodisc syringe filter </td>
<td>&nbsp;</td>
<td>Pall Life Sciences</td>
<td>PN4192</td>
<td>&nbsp;</td>
<tr>
<td>0.5% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>25300-120</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dishes</td>
<td>&nbsp;</td>
<td>NUNC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nikon SMZ1500 dissection scope equipped with Nikon Tokai Hit ThermoPlate warming stage </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mouse embryonic fibroblasts (E12.5)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

derivation of human embryonic stem cells by immunosurgery

La capacità di cellule staminali embrionali umane per auto-rinnovarsi e differenziarsi in tutti i tipi di cellule del corpo suggerisce che essi sono molto promettenti per le applicazioni mediche e come strumento di ricerca per affrontare questioni fondamentali per lo sviluppo e la malattia. Qui, mettiamo a disposizione una concisa, passo-passo il protocollo per la derivazione di cellule staminali embrionali umane da embrioni da immunosurgical isolamento della massa cellulare interna.

Watch this Scientific Journal Video about Derivation of Human Embryonic Stem Cells by Immunosurgery at JoVE.com

Derivation of Human Embryonic Stem Cells by Immunosurgery

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 ...

derivation-of-human-embryonic-stem-cells-by-immunosurgery

Research

JoVE Journal

Biology

33.8K Views.  Harvard. La capacità di cellule staminali embrionali umane per auto-rinnovarsi e differenziarsi in tutti i tipi di cellule del corpo suggerisce che essi sono molto promettenti per le applicazioni mediche e come strumento di ricerca per affrontare questioni fondamentali per lo sviluppo e la malattia. Qui, mettiamo a disposizione una concisa, passo-passo il protocollo per la derivazione di cellule staminali embrionali umane da embrioni da immunosurgical isolamento della massa cellu...

Video: Derivation of Human Embryonic Stem Cells by Immunosurgery

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 ...

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 ...

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 ...

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 ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Derivazione di cellule staminali trofoblasto mouse da blastocisti

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...