Les auteurs sont très reconnaissants de Mme Haiying Pan et M. Ian Bellayr pour leur aide technique sur l&#39;isolement cellulaire pour les films en cours et M. Kyle Holden et M. Jonathan Lucas pour leur aide sur le montage de diapositives. Un grand merci à la liste de personnes suivantes qui ont été impliqués dans le développement des techniques preplate modifié au cours des dernières années: les Drs. Burhan Gharaibeh, Baohong Cao, Deasy Bridget, Thomas R. Payne, Aiping Lu, Hairong Peng, Hideki Oshima, Bo Zeng, Xiaodong Mu, KIMIMASA Tobita, Ron Jankowski, Makato Ikezawa. Nous sommes reconnaissants de l&#39;assistance technique de James H. Cummins, Ryan Pruchnic, Joseph Feduska, Rebecca C. Schugar, Haiying Pan et Tebbets Jessica. L&#39;auteur souhaite également souligner le soutien financier à ce travail de la Muscular Dystrophy Association (MDA), les National Institutes of Health (NIH), le Department of Defense (DOD), l&#39;Hôpital pour enfants de Pittsburgh de l&#39;UPMC, le ministère de chirurgie orthopédique, et l&#39;Université de Pittsburgh.

En général, la procédure complète preplate modifiés nécessite 1-2 jours de préparation, 1 semaine d&#39;effectuer la procédure technique, 2-3 jours d&#39;identification de la cellule avec l&#39;immunocytochimie, et un 2-3 semaines supplémentaires de l&#39;expansion de la population de cellules souches. L&#39;équipement requis pour la culture de cellules souches sont similaires à celles des autres systèmes de culture cellulaire qui inclut une centrifugeuse de paillasse, incubateur à CO 2, à flux laminaire de culture de tissus capot et d&#39;un microscope inversé à fluorescence et équipé d&#39;un appareil photo numérique. Les cellules souches isolées peuvent également être clonés et peuvent être stockées à long terme dans l&#39;azote liquide pour le 1,2 actuelles ou futures études. Tous les réactifs et les matériaux utilisés pour cette procédure doivent être stériles et manipulés de façon aseptique. Étape 1: Préparation <ol><li> Collagène plats manteau de culture de tissus et de flacons: collagène de type I dérivé de peau de veau (0,1 g dans 1 litre; Sigma-Aldrich). Le matériel en plastique pour recouvrir comprend: T-25 flacons, 6 ou 12 plaques à puits, des plats (60 et 100 mm, BD Falcon) comme décrit précédemment 1,2. Secouez la matériel en plastique chaque demi-heure, pendant 4 heures pour assurer une couche de collagène. La solution de collagène est ensuite retiré. Et le matériel en plastique enduit est ensuite mis à sécher dans la hotte de culture tissulaire avec la lumière UV et la hotte à la fois sur, pour assurer la stérilité et, enfin, ou remises couvertes pour une utilisation ultérieure. </li><li> Préparer milieu de culture cellulaire: 400ml DMEM (DMEM, le glucose élevé; Invitrogen), 50mL inactivé par la chaleur sérum fœtal bovin (FBS, Invitrogen), 50 ml de cheval (HS, Invitrogen) sériques, et 5 ml d&#39;extrait d&#39;embryon Chick (PECO; Précis Chemical Co. ) seront filtrés sous vide en utilisant un dispositif jetable de 0,22 um de 500 ml système de filtre stérile (Corning). Stérile pénicilline / solution de streptomycine (Invitrogen) sera ajouté, enfin 100 unités / ml. Les solutions préparées peuvent être conservés à 4 ° C pendant au moins un mois. Cette solution est utilisée pour détacher les cellules du ballon lorsque les cultures scission ou la préparation des cellules pour la transplantation. </li><li> Warm up de solutions pour l&#39;isolement cellulaire: Les réactifs suivants doivent être réchauffés jusqu&#39;à 37 ° C avant leur utilisation pour l&#39;isolement cellulaire: solution de Hank saline tamponnée (HBSS, Invitrogen); 0,2% (poids / volume) de la collagénase de type XI (Sigma -Aldrich), avec une moyenne de 3200 unités de digestion du collagène par mL HBSS; Dispase 2,4 units/1mL (Invitrogen), 0,5% (poids / volume) trypsine (Invitrogen). </li><li> Stériliser le matériel chirurgical en préparation: la procédure chirurgicale nécessite instruments chirurgicaux stériles, y compris: de taille différente forceps, les micro-ciseaux, ciseaux à iris et / ou les lames de bistouri. En outre, les 15 et 50 mL tubes à centrifuger en polypropylène (BD Falcon); filtre cellulaire (taille des pores de 70 m) (BD Falcon), 18 -, 23 - et 27 aiguilles de calibre (BD PrecisionGlide) et 10 - ou 20 ml seringues stériles (BD). </li></ol> Etape 2: biopsies tissulaires, l&#39;isolement et dissociation mécanique 1-4 Les biopsies sont réalisées sous une hotte de culture stérile et comprennent fraîchement récoltés tendons et les muscles squelettiques sont obtenus à partir des muscles du tibia ou du soléaire du membre postérieur des souris de type sauvage (Femme C57BL/6J, 4-5 semaines d&#39;âge ou plus jeunes, Jackson de laboratoire). Tout tissu conjonctif visibles, les vaisseaux sanguins et les tissus adipeux sont alors retirés de la biopsie. Les tissus du tendon et du muscle sont ensuite soigneusement identifiés sous un microscope chirurgical de dissection pour enlever les restes de la peau et les os et placés dans un plat contenant froid (4 ° C) HBSS (complétée par ajouter 5% de FBS). Les tissus sont ensuite isolés placés séparément sur ​​des plats revêtues de collagène contenant HBSS froid et sera hachés (&lt;1x1 mm 3) dans une bouillie grossière à l&#39;aide de micro-ciseaux et / ou les lames de bistouri. Etape 3: La digestion enzymatique des tissus: Le tissu est ensuite hachée enzymatiquement dissocié par une série d&#39;étapes 2,4,5 <ol><li> Transférer les pâtes dans les tissus hachés séparée des tubes de 15 ml et centrifuger à ~ 3500 rpm à 4 ° C pendant 5 min. </li><li> Retirer le surnageant, laver avec du HBSS et répéter la centrifugation à nouveau. </li><li> Retirer le surnageant, et digérer ces boues en ajoutant 10 ml de préchauffée 0,2% de la collagénase de type XI (Sigma). Incuber pendant 60 min à 37 ° C tout en continuant doucement bercer les tubes. Sinon, serrer la main toutes les 10 min. </li><li> Centrifugeuse et remettre ces boues dans 10 ml dispase (2,4 unités / ml, Gibco) solution. Incuber pendant 45 min à 37 ° C, en agitant doucement avec les mains ou à bascule toutes les 10 min. </li><li> Centrifugeuse et re-suspendre ces boues dans 10 ml de solution HBSS 0,2% de trypsine. L&#39;incubation varie en fonction de la mesure dans laquelle les cellules individuelles sont libérés des tissus qui peuvent être effectuées en observant la suspension sous microscope. Il n&#39;est pas recommandé de dépasser 30 mn à 37 ° C avec inversant les tubes ou les secouant délicatement toutes les 10 min. </li><li> Centrifuger les cellules résultants et re-suspendre le culot cellulaire dans 10 ml de prolifération moyen (PM). </li><li> Dissocier la suspension cellulaire en passant l&#39;extrait à travers une série d&#39;aiguilles. Les cellules sont ensuite passés à travers une 2 fois 18G, puis un 23G, et enfin une aiguille 27G. </li><li> Passez l&#39;extrait cellulaire à travers un tamis cellulaire de 70 um. </li></ol> Étape 4: Preplating d&#39;isoler des populations de cellules différentes basées sur le taux d&#39;adhésion cellulaire 1-3 <ol><li> Centrifuger et remettre en suspension les culots cellulaires dans le milieu de prolifération. </li><li> Plaque mélanges de la cellule sur un revêtus de collagène T-25 flacon (ou parabole de 60 mm) et la marquer comme PP1. La suspension cellulaire doit être observé comme possédant un grand nombre de noyaux de réfraction et d&#39;autres débris sous le microscope. </li><li> Incuber à 37 ° C dans un incubateur humidifié, 5% de CO 2 incubateur pendant 2 heures. </li><li> Transfert cellules non adhérentes à un nouveau revêtus de collagène T-25 flacon (ou parabole de 60 mm) et la marque est aussi PP2. Ajouter 5 ml de PM dans la plaque étiquetés PP1. Les premières cellules adhérant qui se fixent sur ​​le flacon sont surtout les myofibroblastes 3. </li><li> Retour des flacons à l&#39;incubateur pendant 24 heures supplémentaires, le transfert cellules non adhérentes à un nouveau revêtus de collagène T-25 flacon (ou plat) et la marquer comme PP3. Ajouter 5 ml de milieu de prolifération dans la plaque étiquetés PP2. Ces cellules adhérentes qui se rattachent à ce flacon sont principalement les fibroblastes 2,3. </li><li> Retour flacons à l&#39;incubateur et répétez la procédure dans un autre 24 heures jusqu&#39;à PP6 population est créé. Maintenir chaque groupe de cellules en suspension et leur permettre de proliférer et de les examiner régulièrement à l&#39;aide d&#39;un microscope inversé. PP3-PP4 sont principalement myoblastes alors que les cellules satellites du muscle sont souvent perçus principalement dans PP5 2. </li><li> La plupart des cellules adhérant lentement se fixent habituellement par PP6. A ce stade, les cellules semblent petits, ronds, de réfraction de lumière et sont très clairsemées en nombre et sont considérés comme une population de cellules souches 1,2. </li></ol> Etape 5: identification et l&#39;expansion de cellules souches isolées <ol><li> Le PP6 isolés cellules sont très peu nombreux et doivent être maintenues dans FBS riche prolifération moyen (20% de FBS dans DMEM) qui est changé tous les jours. La plupart des cellules présentes dans la filière PP6 la culture au cours des 1-2 après des semaines de culture, mais un grand, sain PP6 population est généralement créées après une 1-2 semaines supplémentaires de l&#39;expansion. Les cellules souches isolées de souris hautement exprimer l&#39;antigène des cellules souches 1 (SCA1), le CD34, le foie fœtal kinase 1 (FLK1), et des marqueurs autre tige 1,2,6,7 qui peut être visualisé via coloration immunocytochimique. Ces cellules souches présentent également des capacités de différenciation multiples 3,6,8. </li><li> Les cellules isolées sont maintenus et élargis à une faible densité cellulaire en culture (12 puits plats à 50-100 cellules / puits ou la confluence &lt;20-30% dans le ballon ou un plat) afin d&#39;éviter 1,2 différenciation. Très peu de cellules sous forme de clones lors de l&#39;expansion, et ces cellules souches clonées peuvent subir longtemps la prolifération (LTP) pour toute autre compétence d&#39;études. Les cellules souches clonées peut également être stocker dans l&#39;azote liquide en utilisant les procédures générales de stockage de cellules ex moyennes PM avec 10% de DMSO. Faire un certain nombre de stocks de faible passage des cellules souches pour toutes les autres études jusqu&#39;à dos. </li></ol>

<ol>
	<li>Gharaibeh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique.">Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.</a> Nat Protoc. 3, 1501-1509 (2008).</li><li>Qu-Petersen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+population+of+muscle+stem+cells+in+mice:+potential+for+muscle+regeneration.">Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration.</a> J Cell Biol. 157, 851-864 (2002).</li><li>Li, Y. &. a. m. p. ;. a. m. p., Huard, J. <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+muscle-derived+cells+into+myofibroblasts+in+injured+skeletal+muscle.">Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle.</a> Am J Pathol. 161, 895-907 (2002).</li><li>Watt, D. J., Lambert, K., Morgan, J. E., Partridge, T. A. &. a. m. p. ;. a. m. p., Sloper, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+donor+muscle+precursor+cells+into+an+area+of+muscle+regeneration+in+the+host+mouse.">Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse.</a> J Neurol Sci. 57, 319-331 (1982).</li><li>Lu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+myogenic+progenitor+populations+from+Pax7-deficient+skeletal+muscle+based+on+adhesion+characteristics.">Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics.</a> Gene Ther. 15, 1116-1125 (2008).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+isolation+of+muscle-derived+cells+capable+of+enhancing+muscle+regeneration+and+bone+healing.">Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.</a> J Cell Biol. 150, 1085-1100 (2000).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+receptors+in+the+maturation-dependent+adenoviral+transduction+of+myofibers.">The role of receptors in the maturation-dependent adenoviral transduction of myofibers.</a> Gene Ther. 8, 627-637 (2001).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells+differentiate+into+haematopoietic+lineages+but+retain+myogenic+potential.">Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential.</a> Nat Cell Biol. 5, 640-646 (2003).</li><li>Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+based+on+muscle+stem+cells.">Regenerative medicine based on muscle stem cells.</a> J Musculoskelet Neuronal Interact. 8, (2008).</li><li>Gates, C. B., Karthikeyan, T., Fu, F. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+for+the+musculoskeletal+system+based+on+muscle-derived+stem+cells.">Regenerative medicine for the musculoskeletal system based on muscle-derived stem cells.</a> J Am Acad Orthop Surg. 16, 68-76 (2008).</li></ol>

Il a été reconnu que certains tissus adultes contiennent des cellules souches multiples. Au cours des dernières années d&#39;étude, les cellules souches ont été détectées dans les tissus mous musculo-squelettiques, y compris les muscles squelettiques et du tendon. Ce protocole actuel de détails d&#39;une procédure, connue comme la technique preplate modifié, qui a été utilisé avec succès dans notre laboratoire afin d&#39;isoler les cellules souches adultes à partir des tendons et des muscles squelettiqu...

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#11995-073</td>
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<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#10437-028</td>
<td>&nbsp;</td>
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<td>Heat-inactivated horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#26050-088</td>
<td>&nbsp;</td>
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<td>Chick embryo extract</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>#CE650T-10</td>
<td>&nbsp;</td>
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<td>100U ml penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#15140-122</td>
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<td>Dulbecco&#x2019;s PBS</td>
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<td>Invitrogen</td>
<td>#14190-250</td>
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isolating stem cells from soft musculoskeletal tissues

Les cellules souches adultes ont été longuement débattues en ce qui concerne leur application dans la médecine régénérative. Les cellules souches adultes ont suscité un grand enthousiasme pour le traitement des tissus blessés et malades à cause de leurs capacités impressionnantes de subir la différenciation des cellules de lignées multiples et leur capacité d&#39;auto-renouvellement. Plus important encore, ces qualités ont fait de leur avantage pour une utilisation dans les thérapies transplantation de cellules autologues. Le protocole actuel va introduire le lecteur à la technique preplate modifiés lorsque les tissus mous de l&#39;appareil locomoteur, des tendons et des muscles, par exemple, sont une<sup&gt; Er</sup&gt; Enzymatiquement dissocié puis placés dans des flacons de collagène recouvert d&#39;moyennes. Le surnageant, qui est composé du milieu et les cellules restantes flottante, est en série transmises quotidiennement au nouveaux flacons. Les cellules souches sont les plus lents à se conformer aux flacons qui prend habituellement 5-7 jours (transferts serial ou preplates). En utilisant cette technique, les cellules souches adultes présentes dans ces tissus peuvent être facilement exploitées par le biais assez procédures non invasives.

Watch this Scientific Journal Video about Isolating Stem Cells from Soft Musculoskeletal Tissues at JoVE.com

Isolating Stem Cells from Soft Musculoskeletal Tissues

Isoler des cellules souches adultes provenant de tissus mous musculosquelettiques basé sur la vitesse adhésion de la cellule dans le ballon.

Isoler des cellules souches à partir des tissus mous musculo-squelettiques

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

isolating-stem-cells-from-soft-musculoskeletal-tissues

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Biology

13.3K vues.  Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC. Isoler des cellules souches adultes provenant de tissus mous musculosquelettiques basé sur la vitesse adhésion de la cellule dans le ballon.

Vidéo: Isolating Stem Cells from Soft Musculoskeletal Tissues

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.

Dérivation de cellules souches hématopoïétiques de cellules souches embryonnaires murines

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 ...

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Biologie

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 ...

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.

Dérivation de cellules souches de la souris Trophoblast à partir de blastocystes

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

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Isolement des cellules souches de l&#39;homme xénogreffes de cancer du pancréas

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

L&#39;isolement des cellules souches rétiniennes de l&#39;Oeil de la souris

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Colonies sélectionner et isoler de l&#39;homme cellules souches pluripotentes induites à partir de fibroblastes adultes Reprogrammed

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

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 ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

Isoler des cellules mélanocytes comme primaires du Cœur de souris

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

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 ...

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly in tissues that have a high turnover rate such as the intestinal epithelium. However, adult stem cells are also subject to aging processes and the concomitant decline in function. The Drosophila midgut has emerged as an ideal model system to study molecular mechanisms that interfere with the intestinal stem cells&#8217; (ISCs) ability to function in tissue homeostasis. Although adult ISCs can be easily identified and isolated from midguts of young flies, it has been a major challenge to study endogenous molecular changes of ISCs during aging. This is due to the lack of a combination of molecular markers suitable to isolate ISCs from aged intestines. Here we propose a method that allows for successful dissociation of midgut tissue into living cells that can subsequently be separated into distinct populations by FACS. By using dissociated cells from the esg-Gal4, UAS-GFP fly line, in which both ISCs and the enteroblast (EB) progenitor cells express GFP, two populations of cells are distinguished based on different GFP intensities. These differences in GFP expression correlate with differences in cell size and granularity and represent enriched populations of ISCs and EBs. Intriguingly, the two GFP-positive cell populations remain distinctly separated during aging, presenting a novel technique for identifying and isolating cell populations enriched for either ISCs or EBs at any time point during aging. The further analysis, for example transcriptome analysis, of these particular cell populations at various time points during aging is now possible and this will facilitate the examination of endogenous molecular changes that occur in these cells during aging.

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Understanding the endogenous molecular changes in adult stem cells during aging requires isolating the cells of interest. The method described here presents a simple and robust approach to enrich for and isolate Drosophila intestinal stem cells and the enteroblast progenitor cells by FACS at any time point during aging.

Isoler intestinaux cellules souches de l&#39;adulte<em&gt; Drosophila</em&gt; Mésogastres par FACS pour l&#39;étude des cellules souches Comportement au cours du vieillissement

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly ...

Methods Article

Isoler des cellules souches à partir des tissus mous musculo-squelettiques