Étiquetage de manganèse de cellules souches embryonnaires humaines <ol><li> Trypsiniser les tiges embryonnaires humaines qui ont été cultivées dans des conditions exemptes d&#39;alimentation pendant 5 minutes. Neutraliser la trypsine en ajoutant des milieux de culture. </li><li> Isoler les cellules par centrifugation à 800 rpm pendant 5 minutes à 20 ° Celsius. Après remise en suspension du culot cellulaire, compter le nombre de cellules par coloration au bleu trypan. Basé sur le nombre de cellules obtenues, diviser les cellules en quatre parties aliquotes de 3 millions de cellules dans des tubes coniques. Pellet nouveau les cellules par centrifugation. </li><li> Juste avant de commencer l&#39;étiquetage de manganèse, de dissoudre le chlorure de manganèse fraîche avec une solution de chlorure de sodium 0,9% pour faire une solution de chlorure de 0,1 mM de manganèse. </li><li> Afin d&#39;observer l&#39;activité des canaux calciques, quatre échantillons ont été préparés. Le premier échantillon est de notre contrôle, qui contient des cellules incubées dans du chlorure de sodium 0,9% seulement. Le deuxième échantillon contient des cellules incubées dans 0,1 mM de MnCl2. Exemple 3 et 4 contiennent des cellules incubées dans 0,1 mM de 2 MnCl soit avec 5 uM de Ca 2 +-canal agoniste (s )-(-)- Bay K8644, ou 250 M de Ca 2 +-canal antagoniste, le vérapamil. Incuber les quatre échantillons à 37 ° Celsius avec 5% de CO 2 pendant 30 minutes. </li><li> Après 30 minutes, ralentit les cellules à 800 rpm pendant 5 minutes à 20 ° Celsius. Puis aspirer le surnageant et laver les cellules marquées à deux reprises avec du PBS. Après le lavage, remettre en suspension chaque culot avec 200 ul de PBS et le transfert dans des tubes PCR. Ces pastilles sont généralement blanches. </li></ol> Etiquetage Fer de cellules souches embryonnaires humaines <ol><li> Mix de qualité clinique du sulfate de protamine à l&#39;eau distillée pour obtenir une solution à 1 mg / ml. </li><li> Dans un tube contenant embryonnaires humaines supports de culture de cellules souches, ajouter ferumoxides à 100 pg / mL suivie par l&#39;addition de 12 pg / ml de sulfate de protamine. Mélanger la solution vigoureusement pendant cinq minutes. </li><li> Ajouter solution mixte d&#39;un montant égal du milieu de culture cellulaire pour obtenir une concentration finale de 50 pg / ml de ferumoxides et 6 pg / ml de sulfate de protamine l&#39;étiquette pour les cellules. Incuber les cellules dans cette solution pendant 12 heures. </li><li> Laver les cellules deux fois avec PBS, avec le dernier lavage contenant de l&#39;héparine 10U/ml de dissoudre le complexe de sulfate extracellulaire ferumoxides-protamine. Après lavage, les cellules trypsiniser pour obtenir une suspension cellulaire unique. </li><li> Compter les cellules et dans la partie aliquote nombre requis d&#39;échantillons. Après le lavage, remettre en suspension chaque culot avec 200 ul de PBS et le transfert dans des tubes PCR. Ces pastilles doivent être orange foncé de couleur marron. </li></ol> Exécution Cellulaire Imagerie par Résonance Magnétique <ol><li> Faire un fantôme afin de stabiliser les tubes contenant les pastilles de cellules et aussi pour éviter des artefacts de l&#39;air ambiant lors de la numérisation. Pour ce faire, mélangez 0,8% d&#39;agar-agar et 1% de sulfate de cuivre dans l&#39;eau distillée et porter à ébullition par des micro-ondes pendant 5-7 minutes. Refroidir le mélange pendant au moins 1 heure avant de la numérisation pour atteindre un gel. </li><li> Les tubes contenant les pastilles de cellules étiquetées sont placées dans le fantôme pour la numérisation. Cellulaire in vitro IRM est réalisée à Signa HDx IRM 3T (GE Medical Systems) en utilisant la bobine au genou clinique. </li><li> Balayage des cellules de manganèse étiquetés pour chacun des trois échantillons en utilisant une séquence d&#39;écho de spin: TR, temps de répétition = 800 ms. TE, le temps d&#39;écho = minimum, FOV; champ de vision = 12 x 12 cm; matrice = 256x256, 1 NEX. </li><li> Balayage des cellules fer marqué en utilisant une séquence d&#39;écho de gradient. Nous utilisons les paramètres suivants pour cette imagerie: TR = 100 ms; TE = 10 ms, FA; angle de bascule = 30 °; FOV = 12 x 12 cm; matrice = 256 x 256, 1 NEX. </li></ol> Analyser les résultats de l&#39;IRM <ol><li> Analyser des images de l&#39;IRM à partir de 4 échantillons différents de cellules de manganèse-étiqueté: <ul><li> Exemple # 1 est de notre contrôle, qui contient des cellules incubées dans du chlorure de sodium 0,9% seulement. </li><li> Exemple n ° 2 contient des cellules incubées dans 0,1 mM de MnCl2. </li><li> Exemple n ° 3 contient des cellules incubées dans 0,1 mM de 2 à 5 uM MnCl de Ca 2 +-canal agoniste (s )-(-)- Bay K8644. </li><li> Exemple n ° 4 contient des cellules incubées dans 0,1 mM de 2 MnCl avec 250 uM de Ca 2 +-canal antagoniste, le vérapamil. </li></ul></li><li> Comme prévu, les différences dans l&#39;intensité du signal du manganèse cellules marquées sont visibles. Comparé aux cellules marquées purement avec le manganèse, l&#39;intensité du signal augmente nettement dans la présence de l&#39;agoniste des canaux calciques et diminue en présence d&#39;un antagoniste des canaux calciques. La zone où l&#39;intensité du signal a augmenté pour le manganèse cellules marquées sont mesurés en utilisant Image J (NIH, Bethesda, MD, USA). Le même logiciel est utilisé pour analyser la zone d&#39;obscurité hors résonance du signal à partir du fer cellules marquées. La taille du signal sombres du fer cellules marquées dépend du nombre de cellules ou le nombre de particules de fer contenue par les cellules. Dans ce cas,, Un million de cellules montre une zone plus petite des signaux sombres par rapport au signal des trois millions de cellules. </li></ol>

<ol>
	<li>Bruvold, M., Nordhoy, W., Anthonsen, H. W., Brurok, H., Jynge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-calcium+interactions+with+contrast+media+for+cardiac+magnetic+resonance+imaging:+a+study+of+manganese+chloride+supplemented+with+calcium+gluconate+in+isolated+Guinea+pig+hearts.">Manganese-calcium interactions with contrast media for cardiac magnetic resonance imaging: a study of manganese chloride supplemented with calcium gluconate in isolated Guinea pig hearts.</a> Invest Radiol. 40, 117-125 (2005).</li><li>Aoki, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+labeling+for+magnetic+resonance+imaging+with+the+T1+agent+manganese+chloride.">Cell labeling for magnetic resonance imaging with the T1 agent manganese chloride.</a> NMR Biomed. 19, 50-59 (2006).</li><li>Arbab, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+magnetic+cell+labeling+with+protamine+sulfate+complexed+to+ferumoxides+for+cellular+MRI.">Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI.</a> Blood. 104, 1217-1223 (2004).</li><li>Arbab, A. S., Frank, 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=Cellular+MRI+and+its+role+in+stem+cell+therapy.">Cellular MRI and its role in stem cell therapy.</a> Regen Med. 3, 199-215 (2008).</li></ol>

Ces résultats indiquent que le manganèse ne pénètrent par le canal calcique voltage-dépendants, et le manganèse peut être utilisé comme agent de contraste IRM, qui peut également montrer la viabilité cellulaire. Par conséquent, nous suggérons que le manganèse-guidée par IRM peut être utile de surveiller la viabilité des cellules après la transplantation de cellules souches embryonnaires humaines dans le myocarde.

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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>Manganese chloride tetrahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M8054</td>
<td>&nbsp;</td>
<tr>
<td>(S)-(-)-Bay K8644 </td>
<td>Reagent</td>
<td>Sigma</td>
<td>B133</td>
<td>&nbsp;</td>
<tr>
<td>(+)-Verapamil hydrochloride, minimum 99.0% titration </td>
<td>Reagent</td>
<td>Sigma</td>
<td>V4629</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride 0.85-0.90% W/V</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW 3257-1</td>
<td>&nbsp;</td>
<tr>
<td>Feridex I.V. </td>
<td>Reagent</td>
<td>Berlex Laboratories</td>
<td>NDC 59338-7035-5</td>
<td>ferumoxides injectable solution 11.2mg iron/mL</td>
</tr>
<tr>
<td>Protamine sulfate</td>
<td>Reagent</td>
<td>American Pharmaceutical Partners</td>
<td>NDC 63323-229-05 </td>
<td>50 mg (10 mg/mL)</td>
</tr>
<tr>
<td>Knockout SR (Serum replacement for ES cells)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>10828</td>
<td>&nbsp;</td>
<tr>
<td>DMEM/F12 (1:1)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11330</td>
<td>&nbsp;</td>
<tr>
<td>2- mercaptoethanol 98+%</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M 3148</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine 200mM 100x</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomyocin 10,000 units/ml</td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>MEM Non-Essential Amino Acids 100x solution</td>
<td>Reagent</td>
<td>Gibco </td>
<td>11140</td>
<td>&nbsp;</td>
<tr>
<td>Signa HDx 3T MRI</td>
<td>Tool</td>
<td>GE Medical Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>clinical Knee coil</td>
<td>Tool</td>
<td>GE Medical Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>Reagent</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in vitro labeling of human embryonic stem cells for magnetic resonance imaging

Cellules souches embryonnaires humaines (CSEh) ont démontré la capacité de restaurer les blessés du myocarde. Imagerie par résonance magnétique (IRM) a émergé comme l&#39;une des modalités d&#39;imagerie prédominante pour évaluer la restauration de la blessés myocarde. Par ailleurs, des agents de marquage ex vivo, tels que l&#39;oxyde de fer nanoparticules, ont été employés pour suivre et localiser les cellules souches transplantées. Cependant, cette méthode ne permet pas de surveiller une propriété fondamentale de biologie cellulaire quant à la viabilité des cellules transplantées. Il est connu que le chlorure de manganèse (MnCl2) pénètre dans les cellules via voltage-dépendants du calcium (Ca 2 +) des canaux lorsque les cellules sont biologiquement actifs, et s&#39;accumule intracellulairement pour générer l&#39;effet T shortening 1. Par conséquent, nous suggérons que le manganèse-guidée par IRM peut être utile de surveiller la viabilité des cellules après la transplantation de CSEh dans le myocarde. Dans cette vidéo, nous allons montrer comment étiqueter CSEh avec MnCl2 et comment ces cellules peuvent être clairement vus en utilisant l&#39;IRM in vitro. Dans le même temps, l&#39;activité biologique de Ca 2 +-canaux seront modulées utilisant à la fois de Ca 2 +-canal agonistes et antagonistes pour évaluer les changements du signal concomitant.

Watch this Scientific Journal Video about In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging at JoVE.com

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

Dans cette vidéo, nous montrons comment étiqueter cellules souches embryonnaires humaines (CSEh) avec du chlorure de manganèse (MnCl<sub&gt; 2</sub&gt;), Qui peuvent pénétrer dans les cellules via les canaux calciques voltage-dépendants lorsque les cellules sont biologiquement actives. De plus, nous montrons l&#39;utilisation de MnCl<sub&gt; 2</sub&gt; Comme agent de contraste IRM cellulaire pour déterminer la viabilité in vitro des CSEh.

Marquage in vitro des cellules souches embryonnaires humaines pour l&#39;imagerie par résonance magnétique

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

in-vitro-labeling-human-embryonic-stem-cells-for-magnetic-resonance

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10.1K vues.  Stanford University. Dans cette vidéo, nous montrons comment étiqueter cellules souches embryonnaires humaines (CSEh) avec du chlorure de manganèse (MnCl 2), Qui peuvent pénétrer dans les cellules via les canaux calciques voltage-dépendants lorsque les cellules sont biologiquement actives. De plus, nous montrons l'utilisation de MnCl 2 Comme agent de contraste IRM cellulaire pour déterminer la viabilité in vitro des CSEh.

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

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagation des souches embryonnaires humaines (ES) Cellules

Recherche

Biologie

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.

Dérivation des cellules souches embryonnaires humaines par 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 ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Étiquetage des cellules souches avec des colorants fluorescents pour les non-invasive de détection avec l&#39;imagerie optique

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

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.

Palpage pour activité complexe mitochondrial dans les cellules souches embryonnaires humaines

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

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

In vitro et in vivo en bioluminescence gène rapporteur imagerie des cellules souches embryonnaires humaines

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

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.

Immunoprécipitation de la chromatine des cellules souches embryonnaires humaines

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.

Culture et entretien des cellules souches embryonnaires humaines

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.

Préparation Étaler chromosomique des cellules souches embryonnaires humaines pour le caryotype

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.

Congélation et de décongélation cellules souches embryonnaires humaines

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 ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vidéo analyse bioinformatique des droits de croissance de la tige colonie de cellules embryonnaires

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...