Ce travail a été soutenu par la Fundação de Amparo une Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Tecnológico e Científico (CNPq) et l&#39;Instituto Nacional de Ciencia e Tecnologia (INCTC). Nous sommes reconnaissants à S. Paulsen Bruna et Aline M. Fernandes pour les images EB.

1. Fixation et cryoconservation Les cellules souches pluripotentes ont été cultivées sur des fibroblastes embryonnaires de souris (MEF) inactivé à la mitomycine C et maintenus en DMEM/F12 complété par le remplacement KO 20% de sérum (KSR) et 8ng / mL de facteur de croissance des fibroblastes (FGF-2). Afin d&#39;induire la formation de cellules H9 EB ont été transférés à des non-adhérents plats et cultivées pendant 7 jours, maintenus dans DMEM/F12 supplémenté avec 15% KSR 8. Note (!): Cette technique peut être utilisée pour EBS provenant de toute cellule souche embryonnaires pluripotentes et induits. <ol><li> Recueillir l&#39;EBS de la boîte de culture avec une pipette. </li><li> Transfert EBS pour un tube de 15 ml conique et attendre jusqu&#39;à ce que le matériel tombe au fond du tube. </li><li> Retirer moyennes et fixer EBS avec une paraformaldéhyde à 4% (PFA) solution dans du PBS pendant 30 minutes à température ambiante. Note (!): Pour la récupération hybridation in situ d&#39;antigène doit être fait afin d&#39;éviter une réaction croisée avec des anticorps lien en raison de l&#39;utilisation de la PFA 6,7. </li><li> Retirer solution de PFA et laver avec du PBS pendant 5 min. </li><li> EB doit être placée dans de dilution en série de PBS tamponné solutions de saccharose (10, 20 et 30%, dans l&#39;ordre) à 25-28 ° C, chaque solution doit être remplacée toutes les 30 min. Le matériau peut ensuite être stocké dans la solution de saccharose 30% à 4 ° C jusqu&#39;à ce que l&#39;étape d&#39;enrobage avec octobre </li></ol> 2. Enrobage des tissus, d&#39;orientation et de congélation <ol start="6"><li> Recueillir soigneusement l&#39;EBS du tube. Solution de saccharose à décharge, autant que possible. Note (!): Il est important de mouiller la paroi interne de la pointe avec une solution de saccharose avant de recueillir l&#39;EBS. Cette procédure permet d&#39;éviter EB à joindre à la pointe. </li><li> Placez EB dans le moule et retirer la solution de saccharose restant avec du papier filtre. Note (!): Il est important de ne pas remplir le moule avec EBS pour éviter les chevauchements. </li><li> Remplir le moule avec octobre lentement, en prenant soin de ne pas remettre en suspension EBS. Après cela, placez tous EB au centre du moule et retirer les bulles avec la pipette. </li><li> Légèrement agiter pendant 15 minutes. </li><li> Entourez le moule avec la glace sèche écrasée de geler l&#39;échantillon. Octobre devrait être complètement gelé. A ce point blocs peuvent être conservés à -70 ° C, pendant au moins un an. </li></ol> 3. Cryosectioning Technique <ol start="11"><li> Retirez le bloc congelé de -70 ° C et les placer sur cryostat préalablement refroidi à une température comprise entre -18 et -21 ° C. Note (!): Températures en dehors de cette plage peut provoquer des troubles comme les sections de curling, de fusion ou de fissuration. </li><li> Détachez le bloc PTOM de la moisissure et la placer dans le support en ajoutant plus de cryostat octobre </li><li> Il est important d&#39;orienter correctement et le bloc de l&#39;aligner parallèlement au bord de la lame. Note (!): Comme EBS sont plus petites que d&#39;autres échantillons de tissu, cette étape est extrêmement importante pour minimiser la perte de matériel. </li><li> Blocs octobre doit être sectionnée superficiellement jusqu&#39;à ce que le plan de la surface est obtenue. Les lames minces (10 microns) de l&#39;échantillon de tissu doivent être prises et montées sur lames de verre préalablement enduite avec 200 mg / ml de poly L lysine (10 uL par diapositive) 9,10. Note (!): Pour éviter sections torned attention à tout dommage à la lame. </li><li> Toutes les sections doivent être séchés à l&#39;air pendant 1 heure à température ambiante et ensuite être utilisés ou stockés à -70 ° C jusqu&#39;à utilisation. </li></ol>

<ol>
	<li>Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-117 (1998).</li><li>Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H., Benvenisty, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+embryonic+stem+cells+into+embryoid+bodies+compromising+the+three+embryonic+germ+layers.">Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.</a> Mol Med. 6, 88-95 (2000).</li><li>Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+inducing+embryoid+body+formation:+in+vitro+differentiation+system+of+embryonic+stem+cells.">Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells.</a> J Biosci Bioeng. 103, 389-398 (2007).</li><li>Conley, B. J., Young, J. C., Trounson, A. O., Mollard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+propagation+and+differentiation+of+human+embryonic+stem+cells.">Derivation propagation and differentiation of human embryonic stem cells.</a> Int J Biochem Cell Biol. 36, 555-567 (2004).</li><li>Moon, I. S., Cho, S. J., Jin, I., Walikonis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+combined+fluorescence+in+situ+hybridization+and+immunocytochemistry.">A simple method for combined fluorescence in situ hybridization and immunocytochemistry.</a> Mol Cells. 24, 76-82 (2007).</li><li>Daneshtalab, N., Dor&#233;, J. J., Smeda, 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=Troubleshooting+tissue+specificity+and+antibody+selection:+Procedures+in+immunohistochemical+studies.">Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies.</a> J Pharmacol Toxicol Methods. 61, 127-135 (2009).</li><li>Krenacs, L., Krenacs, T., Stelkovics, E., Raffeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-induced+antigen+retrieval+for+immunohistochemical+reactions+in+routinely+processed+paraffin+sections.">Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections.</a> Methods Mol Biol. , 588-5103 (2010).</li><li>Nones, J., Spohr, T. C., Furtado, D. R., Sartore, R. C., Paulsen, B. S., Guimar&#227;es, M. Z., Rehen, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+modulate+cell+survival+in+embryoid+bodies.">Cannabinoids modulate cell survival in embryoid bodies.</a> Cell Biol Int. 12, 399-408 (2010).</li><li>Huang, W. M., Gibson, S. J., Facer, P., Gu, J., Polak, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+section+adhesion+for+immunocytochemistry+using+high+molecular+weight+polymers+of+L-lysine+as+a+slide+coating.">Improved section adhesion for immunocytochemistry using high molecular weight polymers of L-lysine as a slide coating.</a> Histochemistry. 77, 275-279 (1983).</li><li>Kuenzi, M. J., Sherwood, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+specific+relaxin-binding+cells+in+thecervix,+mammary+glands,+and+nipples+of+pregnant+rats.">Immunohistochemical localization of specific relaxin-binding cells in thecervix, mammary glands, and nipples of pregnant rats.</a> Endocrinology. 136, 1367-1373 (1995).</li></ol>

La méthode décrite ici fournit un facile à suivre le protocole pour obtenir PFA fixes sections de cryostat mince de corps embryoïdes utiles pour l&#39;immunofluorescence et dans des essais d&#39;hybridation in situ. Le cryocoupes résultant permettre l&#39;étude des aspects cellulaires et moléculaires de cellules souches humaines différenciation embryonnaire, tout en préservant leur structure et leur organisation en tant que granulats.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>D (+) Sucrose</td>
<td>Reagent</td>
<td>Vetec</td>
<td>228</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Rieden-de Ha&euml;n</td>
<td>16005</td>
<td>&nbsp;</td>
<tr>
<td>PBS solution</td>
<td>Reagent</td>
<td>LGC Biotecnology</td>
<td>13-30259.05</td>
<td>&nbsp;</td>
<tr>
<td>Poly-L-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P2636</td>
<td>200mg/mL in water</td>
</tr>
<tr>
<td>Tissue-Tek O.C.T. Compound</td>
<td>Reagent</td>
<td>Sakura</td>
<td>P2636</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose Solution</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10% Sucrose Solution in PBS w/v</td>
</tr>
<tr>
<td>Sucrose Solution</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>20% Sucrose Solution in PBS w/v</td>
</tr>
<tr>
<td>Sucrose Solution</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>30% Sucrose Solution in PBS w/v</td>
</tr>
<tr>
<td>Conic Tube</td>
<td>Tool</td>
<td>TPP</td>
<td>91015</td>
<td>15mL conic tube</td>
</tr>
<tr>
<td>Plate shaker</td>
<td>Tool</td>
<td>Biomixer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cryostat</td>
<td>Tool</td>
<td>Leica</td>
<td>CM 1850</td>
<td>&nbsp;</td>
<tr>
<td>Mold for OCT platform</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Plastic mold plataform</td>
</tr>		</tbody>
		</table>
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analysis of pluripotent stem cells by using cryosections of embryoid bodies

Les cellules souches embryonnaires (ES) sont des cellules pluripotentes dérivées de la masse cellulaire interne de blastocyste-stade précoce des embryons de mammifères 1. Une étape cruciale dans la différenciation des cellules ES est la formation de corps embryoïdes (EB) des agrégats 2, 3. La formation EB est basée sur l&#39;agrégation spontanée lorsque les cellules ES sont cultivées en plaques non adhérentes. Récapitule EB tridimensionnelle de nombreux aspects de l&#39;embryogenèse chez les mammifères début et se différencient en trois feuillets embryonnaires: ectoderme, mésoderme et endoderme 4. Immunofluorescence et hybridation in situ sont largement utilisés des techniques pour la détection des protéines cibles et de l&#39;ARNm dans les cellules d&#39;une section de 5 tissus, 6, 7. Nous présentons ici une technique simple pour générer cryocoupes haute qualité de corps embryoïdes. Cette approche repose sur l&#39;orientation spatiale d&#39;intégrer EB en OCT suivie par la technique cryosection. Les sections qui en résulte peut être soumis à une grande variété de procédures analytiques afin de caractériser les populations de cellules contenant certaines protéines, ARN ou ADN. En ce sens, la préparation de cryocoupes EB (10 microns) sont des outils essentiels pour l&#39;analyse histologique coloration (par exemple hématoxiline et l&#39;éosine, DAPI), l&#39;immunofluorescence (par exemple Oct4, nestine) ou l&#39;hybridation in situ. Cette technique peut aussi aider à comprendre les aspects de l&#39;embryogenèse à l&#39;égard du maintien de la structure tri-dimensionnelle sphériques d&#39;EBS.

Watch this Scientific Journal Video about Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies at JoVE.com

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Les cellules souches pluripotentes croissante en suspension se différencier en corps embryoïdes (EBS). Ici, nous montrons comment obtenir de haute qualité cryocoupes EB utile pour étudier les aspects cellulaires et moléculaires de l&#39;embryogenèse, tout en préservant leur organisation en tant que granulats.

Analyse de cellules souches pluripotentes en utilisant cryocoupes de corps embryoïdes

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

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13.8K vues.  Universidade Federal do Rio de Janeiro (UFRJ), Brazil. Les cellules souches pluripotentes croissante en suspension se différencier en corps embryoïdes (EBS). Ici, nous montrons comment obtenir de haute qualité cryocoupes EB utile pour étudier les aspects cellulaires et moléculaires de l'embryogenèse, tout en préservant leur organisation en tant que granulats.

Vidéo: Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious approach of pipetting an arbitrary number of cells into well plates. The interactions between the stem cells forced into close proximity of one another promotes the generation of the EBs. Because the media in each of the wells has to be manually exchanged every day, this approach is manually intensive.
Moreover, because environmental parameters including cell-cell, cell-soluble factor interactions, pH, and oxygen availability can be functions of EB size, cell populations obtained from traditional hanging drops can vary dramatically even when cultured under identical conditions. Recent studies have indeed shown that the initial number of cells forming the aggregate can have significant effects on stem cell differentiation. We have developed a simple, rapid, and scalable culture method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development. Finally, these cells are easily accessible for further analysis and experimentation. This method is amenable to any lab and requires no dedicated equipment. We demonstrate this method by creating embryoid bodies using a red fluorescent mouse cell line (129S6B6-F1).

We show a simple and rapid method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development.

Shrinky-gouttes pendantes Dink: une façon simple de formulaire et de la Culture corps embryoïdes

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious ...

Recherche

Biologie

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell sources due to their limitless self-renewal capabilities and their potential to differentiate into multiple cell types. The pluripotent state of hPSCs is typically assessed by techniques such as qPCR, immunocytochemistry, and by other in vitro and in vivo differentiation strategies into multiple cell types. Among these, immunocytochemical techniques have been developed for routine characterization of the undifferentiated state of hPSCs based on analysis of candidate intracellular and cell-surface biomarkers. Given the fact that hPSCs grow as colonies, problems arise in quantifying the expression of these markers at the individual cell level on a routine basis. Flow cytometry analyses serve to address this issue but require cell numbers and use of reagents that are not normally conducive for routine quality control assessment of hPSC cultures. Thus, the development of practical and reproducible means of creating monolayer cell samples with preserved integrity for marker evaluation has many advantages in stem cell research. This greatly benefits immunocytochemical analysis because individual cells from the monolayer can be easily observed and quantified for the expression of specific markers. Towards this goal, a self-made cytospin apparatus was constructed and optimized for use with immunocytochemical staining. Two cell-surface markers (SSEA3/SSEA4) expression were analyzed in a variant BG01 stem cell line for the purpose of this protocol.

Suspension immunocytochemical staining of human pluripotent stem cells (hPSCs) for cell-surface markers (SSEA-3/SSEA-4) was achieved based on use of a self-made cytospin apparatus to create a monolayer of cells for observation and quantification.

Analyse immunocytochimique des cellules souches pluripotentes humaines en utilisant un appareil Self-Made Cytospin

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

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

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

Transfection et Nucleofecting homme cellules souches pluripotentes induites

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

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Reprogrammation des cellules somatiques humaines dans cellules souches pluripotentes induites (CISP) Utilisation vecteur rétroviral avec la GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Génération de souris dérivées de cellules souches pluripotentes induites

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induction et analyse des épithéliale à mésenchymateuses transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

Haut différenciation de l&#39;efficacité de pluripotentes humaines des cellules souches à des cardiomyocytes et caractérisation par cytométrie en flux

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Rétroviral infection de cellules souches embryonnaires murines dérivées de cellules du corps embryoïdes pour l&#39;analyse de la différenciation hématopoïétique

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analyse des suspensions de cellules isolées de tissus solides par Spectral cytométrie en flux

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...