1. Retrait de l&#39;embryon et du placenta de dissection <ol><li> Pour commencer, les embryons doivent d&#39;abord être isolés à partir des barrages enceinte. </li><li> Les animaux sont euthanasiés selon les procédures approuvées - dans notre cas nous allons utiliser une dose létale de l&#39;isoflurane anesthésie </li><li> Tout d&#39;abord, le ventre de pulvérisation avec de l&#39;éthanol et de faire une petite incision avec une paire de ciseaux. </li><li> Arrachez la peau avec les deux mains pour découvrir l&#39;abdomen et à ciel ouvert le péritoine. </li><li> Avec des pinces, ramasser les cornes utérines et de recueillir leur aide de ciseaux à chaque extrémité distale. Placez les cornes utérines dans une boîte de Pétri remplie de PBS et placer sur la glace. Aussi, n&#39;oubliez pas de laver plusieurs fois avec du PBS avant d&#39;isoler le placenta. Nous pouvons maintenant isoler le placenta. </li><li> Avec deux pinces, commencent attentivement retirant le tissu de l&#39;endomètre entourant le conceptus embryon. Il faut être prudent à ne pas percer ou autrement infliger des dommages structurels à des embryons, car cela compliquerait les étapes ultérieures de dissection. </li><li> Placez le conceptus isolés dans un nouveau plat de pétri avec du PBS sur la glace et continuer jusqu&#39;à ce que tous conceptus ont été libérés de l&#39;endomètre. </li><li> Transfert d&#39;une conceptus un nouveau plat de pétri avec du PBS placé sous le microscope et avec deux pinces éplucher les decidua loin de la barrière placentaire. Enlevez autant de la caduque que possible que les cellules decidua va interférer avec une seule cellule de suspension et de cytométrie en flux. Un mouvement continu lisse peeling est recommandé de réaliser les meilleurs résultats. </li><li> Couper le sac vitellin loin de le placenta à la jonction entre les deux organes et tirez doucement le sac vitellin et les vaisseaux vitellins loin de la barrière placentaire. Il faut prendre soin de ne pas perturber la plaque choriale du placenta, surtout avec des embryons précoces. </li><li> Soigneusement tenant la plaque choriale du placenta avec une paire de pinces et le cordon ombilical avec un autre, tirez le cordon ombilical et de l&#39;embryon attachée loin de la barrière placentaire. </li><li> Retirer l&#39;excès de tissu à cellules géantes sur les bords du placenta afin de minimiser les cellules agglutination pendant la préparation de la suspension à cellule unique. </li><li> Recueillir le placenta dans un récipient approprié, tel qu&#39;un tube de 15 ml Falcon, avec PBS% FCS et 5 sur la glace. Faites cela pour tous les placentas disséqués. </li><li> À ce stade, vous pouvez soit procéder à la préparation d&#39;une suspension à cellule unique pour la cytométrie de flux, des tests fonctionnels, tels que la culture in vitro ou la transplantation, ou de préparer le placenta entier pour la fixation des tissus et fixes bloc congelé intégration éventuelle pour l&#39;immunohistochimie. </li></ol> 2. Préparation de unicellulaire suspension de tissu placentaire <ol><li> Nous allons maintenant vous montrer comment générer une suspension cellulaire unique à partir de placenta pour analyse FACS. </li><li> Pour préparer une suspension cellulaire unique du placenta, la mécanique et de la dissociation enzymatique est nécessaire. D&#39;abord préparer une solution de collagénase 0,1% dans PBS avec 10% de SVF et 1% d&#39;une solution de pénicilline / streptomycine. </li><li> Ajouter un volume approprié de solution de collagénase à l&#39;placentas, selon le nombre que vous avez. Un volume qui est le double du volume du placenta, et entre 2-5ml est recommandée. </li><li> Utilisez une aiguille 16 G monté sur une seringue de 5 ml mécaniquement à détruire le tissu par passage de la solution de collagénase et de placenta par l&#39;aiguille 3 fois. Répétez l&#39;opération avec une aiguille 18-G. </li><li> Placer l&#39;échantillon dans un 37 ° C, 5% de CO 2 incubateur pendant 45 minutes. </li><li> Après l&#39;incubation de 45 minutes dans de la collagénase, le passage de la solution de la cellule grâce à une aiguille 20 G, et incuber pendant 45 minutes supplémentaires, à 37 ° C. </li><li> Après les heures d&#39;incubation 1,5 total dans la collagénase, le passage de la solution par le biais de cellules de 22 G et 25 G-aiguilles 3 fois avec chaque aiguille. </li><li> Filtrer l&#39;échantillon à travers un filtre de 50 um à un nouveau 15 ml de tube Falcon, laver avec du PBS 5% SVF et centrifuger à 4 ° C pendant 5 minutes à 300 x g. </li><li> À ce stade, nous avons une suspension cellulaire unique adapté à l&#39;analyse FACS ou des tests fonctionnels. </li></ol> 3. Fixation des tissus pour les coupes congelées <ol><li> Immédiatement après avoir isolé le placenta de chaque embryon, elles sont placées dans du PBS 1X froid. Habituellement une plaque de 96 puits qui fonctionne le mieux pour les placentas jeunes (E8.5-11.5), mais avec plus de placentas, E12.5 et plus, une plaque de 24 puits peut être plus pratique. </li><li> Chaque placenta est transféré dans un nouveau puits rempli de 4% fraîchement dégelé paraformaldahyde ou PFA. Pour la E12.5 placentas, le tissu est déplacé vers un nouveau puits à la main, mais pour le E10.5 vous pouvez retirer le CPE soigneusement avec une pipette et ajoutez les PFA. Il est important que le placenta est complètement immergé pendant 2-4 heures à 4 ° C, selon l&#39;âge et la taille des tissus. </li><li> Transférer le tissu dans 30% de sucrose, ou si les tissus sont très petits et fragiles, aspirer la PFA et ajouter du saccharose directement. A ce stade, les placentas seront flottants en solution. Laissez les tissus nuit à 4 ° C.</li><li> Après l&#39;étape de nuit dans 30% de sucrose, les tissus devrait finalement couler au fond du puits, indicatif de cryoconservation approprié. Retirer la moitié de la solution de saccharose et de remplacer le volume avec les PTOM et placer le tissu de retour à 4 ° C pendant 1-2 heures. Cela facilite la pénétration octobre </li><li> Transfert le tissu à 100% pour octobre 1 heure, après quoi il peut être incorporé. </li></ol> 4. Intégrer le tissu fixe <ol><li> Moules étiquette en plastique avec une identification appropriée des tissus (par exemple la moisissure spectacle). </li><li> Tirez le tissu de la solution PTOM et coupé le placenta dans la moitié en orientant le placenta en forme de disque avec le côté du cordon ombilical vers le bas et soigneusement couper avec une lame de rasoir propre en veillant à déplacer la lame avant et en arrière avant de relâcher la pression pour assurer une même coupe. Le placenta est coupé en deux pour obtenir le meilleur prientation au plus tard, de visualiser les CSH placentaires. </li><li> Moitié du placenta Placer un au fond du moule avec le bord coupé en contact avec la surface du fond du moule. Répétez l&#39;opération avec un nouveau moule pour l&#39;autre moitié. Pour E10.5 placentas, les deux moitiés peuvent être placés dans le même moule en utilisant la même technique. </li><li> Lors du coulage PTOM dans le moule, il peut être difficile de garder le placenta dans le bon sens. Fixez le placenta en place par la tenue de la moitié des tissus avec une pince. Verser lentement la solution PTOM dans le moule et éviter de produire des bulles. Pour E10.5 placentas, maintenez la moitié des tissus par des pinces et le repos de la moitié des autres tissus contre l&#39;extérieur de la pince. Verser lentement la solution PTOM décrit pour E12.5 placentas. </li><li> Une fois le moule est rempli avec des PTOM, de placer rapidement le moule sur la glace sèche, les PTOM se tourneront flash blanc geler le tissu </li><li> Placer le moule dans un petit sac en plastique contenant Drierite et immédiatement dans le magasin de congélateur à -80 ° C. </li></ol>

<ol>
	<li>Gekas, C., Dieterlen-Lievre, F., Orkin, S. H., Mikkola, H. K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+placenta+is+a+niche+for+hematopoietic+stem+cells.">The placenta is a niche for hematopoietic stem cells.</a> Dev Cell. 8, 365-375 (2005).</li><li>Rhodes, K. E., Gekas, C., Wang, Y., Lux, C. T., Francis, C. S., Chan, D. N., Conway, S., Orkin, S. H., Yoder, M. C., Mikkola, H. K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Emergence+of+Hematopoietic+Stem+Cells+Is+Initiated+in+the+Placental+Vasculature+in+the+Absence+of+Circulation.">The Emergence of Hematopoietic Stem Cells Is Initiated in the Placental Vasculature in the Absence of Circulation.</a> Cell Stem Cell. 2, 252-263 (2008).</li></ol>

Les procédures expérimentales décrites dans ce protocole permettra à l&#39;isolement et la visualisation des tissus placentaires et souches hématopoïétiques et les cellules progénitrices. Pour un résumé complet sur le rendement attendu et cellulaires nombre de CSH dans le placenta et les autres organes hématopoïétiques fœtales à travers le développement du fœtus nous nous référons à Gekas, et al. 2005. Pour la localisation des CSH en développement et d&#39;autres cellules hématopoïétiques dans le placenta nous nous r...

Liste de Matériaux: Le glucose 100 mM 10 mM NaN3 15 ml conique tubes (BD) 16-G, 18 G, 20 G, 22-G et 25 G-aiguilles (BD) 30% Solution Sucrée 35mm boîtes de Pétri (BD) Paraformaldéhyde à 4% 50 - Filtres de cellules m (Celltrics) Seringues de 5 ml (BD) Phosphatase alcaline Substrat Kit I (Vector Rouge, Vector) Phosphatase alcaline Substrat Kit III (Blue Vector, Vector) Antibiotiques (pénicilline / streptomycine, P / S) solution de Collagénase de type I (Sigma) Cryomold moules jetables Spécimen de vinyle (10mm x 10mm x 5 mm, Tissue-Tek) Cytokératine anticorps primaire (DakoCytomation) Pinces à dissection, acier inoxydable ou en titane, numéro 5 ou 55 Drierite Sérum fœtal bovin (FBS) Fisherbrand plastique Microscope Glissez Mailer (Fisher) 1000u/ml glucose oxydase Lévamisole (Vector) Montage des médias, à base aqueuse milieu de montage (VectamountTM AQ) Sérum de cheval normal (Vector) Température de coupe optimale (PTOM, Tissue-Tek) Substrat Peroxydase DAB Kit (Vector) Tampon phosphate salin (PBS) avec du calcium / magnésium (Ca 2 + w / Mg 2 +) Tampon phosphate salin (PBS) sans calcium / magnésium (w / o Ca2 + / Mg2 +) Slide Box plastique (détient 100 diapositives) Tubes en plastique (PVC Nalgene 180 autoclavable non toxique) Tube en polypropylène de test à fond rond, 5 ml (12x75 mm, BD Falcon) Protéinase K 20mg/ml Solution (Amresco) Produits de recherche International Corp super Pap Pen HT marqueurs * Slide (2,5 mm, Fisher) Culture tissulaire traitée en U (à fond rond) des plaques de 96 puits ou 24 puits (Falcon BD) Tween 20, SigmaUltra (Sigma) Kit d&#39;amplification tyramide (Invitrogen) Vectastain ABC phosphatase alcaline standard Kit (Vector) Vectastain ABC Standard Elite Kit (Vector)

isolation and analysis of hematopoietic stem cells from the placenta

Les cellules souches hématopoïétiques (CSH) ont la capacité de s&#39;auto-renouveler et de générer tous les types de cellules des lignées de sang à travers la vie d&#39;un individu. Tous les CSH apparaissent pendant le développement embryonnaire, après quoi leurs dimensions de la piscine est maintenue par l&#39;auto-renouvellement des divisions cellulaires. Identifier l&#39;origine anatomique des CSH et les événements critiques de développement régulation du processus de développement HSC a été compliqué car beaucoup de sites anatomiques participer durant l&#39;hématopoïèse fœtale. Récemment, nous avons identifié le placenta comme un organe majeur où les CSH hématopoïétiques sont générées et élargi dans des niches uniques microenvironnementales (Gekas, et al 2005, à Rhodes, et al 2008). En conséquence, le placenta est une source importante de CSH au cours de leur émergence et l&#39;expansion initiale. Dans cet article, nous montrons les techniques de dissection pour l&#39;isolement du placenta de souris E10.5 E12.5 et embryons, ce qui correspond aux étapes de développement de l&#39;initiation des CSH et le pic de la taille de la piscine HSC dans le placenta, respectivement. En outre, nous présentons un protocole optimisé pour la dissociation enzymatique et mécanique du tissu placentaire dans une seule cellule de suspension pour une utilisation en cytométrie en flux ou des tests fonctionnels. Nous avons constaté que l&#39;utilisation de la collagénase pour une seule cellule de suspension du placenta donne des rendements suffisants de CSH. Un facteur important affectant le rendement HSC du placenta est le degré de dissociation mécanique avant, et la durée de traitement enzymatique. Nous fournissons également un protocole pour la préparation de surgelés fixe des coupes de tissus placentaires pour la visualisation de développer CSH par immunohistochimie dans leurs niches précises cellulaire. Comme hématopoïétiques antigènes spécifiques ne sont pas conservés lors de la préparation des sections paraffine, nous utilisent couramment fixes coupes congelées pour localiser les CSH et les progéniteurs placentaire.

Watch this Scientific Journal Video about Isolation and Analysis of Hematopoietic Stem Cells from the Placenta at JoVE.com

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Nous avons identifié le placenta comme un organe majeur lors du développement hématopoïétique. Nous avons constaté que les cellules souches hématopoïétiques (CSH) sont à la fois générées et développées dans le placenta dans des niches uniques microenvironnementales. Ici, nous décrivons des techniques expérimentales nécessaires à l&#39;isolement et la visualisation des CSH dans le placenta de souris.

Isolement et analyse de cellules souches hématopoïétiques à partir du placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

isolation-and-analysis-of-hematopoietic-stem-cells-from-the-placenta

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Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

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20.3K vues.  University of California, Los Angeles. Nous avons identifié le placenta comme un organe majeur lors du développement hématopoïétique. Nous avons constaté que les cellules souches hématopoïétiques (CSH) sont à la fois générées et développées dans le placenta dans des niches uniques microenvironnementales. Ici, nous décrivons des techniques expérimentales nécessaires à l'isolement et la visualisation des CSH dans le placenta de souris.

Vidéo: Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

L&#39;isolement et la transplantation de cellules souches hématopoïétiques (CSH)

Recherche

Biologie

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 ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

L&#39;isolement des premiers cellules souches hématopoïétiques de murin sac vitellin et AGA

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

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 ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviraux médiée Knockdown cours Ex Vivo</emÉrythropoïèse> des cellules souches hématopoïétiques humaines

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Analyse phénotypique et isolement de cellules souches hématopoïétiques murines et Lineage-engagés progéniteurs

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

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 ...

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 ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Identification des facteurs clés de régulation auto-renouvellement et de différenciation dans les cellules hématopoïétiques précurseurs EML par l&#39;analyse de l&#39;ARN-séquençage

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolement, caractérisation et différenciation des cellules souches cardiaques du coeur de souris adulte

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...