Les auteurs tiennent à souligner l&#39;aide inestimable de: Dr Neal First et Kourtney Wilkinson pour le manuscrit d&#39;édition, le Dr Lucy Senter, le Dr Brigitte Willeford et Mike Basett de l&#39;aide pour les soins de formation et d&#39;animaux à l&#39;installation MSU ALAC souris accrédités; Dr Dwayne Wise pour son aide à la microscopie et la capture d&#39;image; Cesar Monroy, Hannah Swoope, et Bobbie Huddleston pour leur aide à la production vidéo à la MSU. Cette recherche a été financée par l&#39;Office de la recherche institutionnelle et le Département des sciences biologiques à la Mississippi State University.

Partie 1: Laparotomie souris enceintes <ol><li> Par dislocation cervicale, euthanasier une souris C57BL6J enceinte femelle à 10/05 au 11/05 DPC. </li><li> Nettoyer l&#39;abdomen avec un savon antimicrobien, et puis, il raser. </li><li> Après le rasage, lavage de l&#39;abdomen avec une solution saline. </li><li> Ensuite, séchez l&#39;abdomen avec une gaze stérile. </li><li> Couvrir la souris avec un nouveau champ stérile. </li><li> Faire une incision ventrale aide de pinces et de ciseaux de dissection. </li><li> Identifier et éliminer tout l&#39;utérus de la cavité abdominale. Embryons de souris sera visible à l&#39;intérieur de l&#39;utérus. </li><li> Transfert de l&#39;utérus dans une boîte de Pétri remplie de D-PBS, et le garder sur la glace. </li><li> L&#39;utilisation d&#39;un scalpel stérile et pince, retirer le placenta et les tissus embryonnaires supplémentaires de chaque embryon. </li><li> Transfert des embryons dans un plat frais de Pétri remplie de D-PBS. </li><li> Mesurer la longueur de l&#39;embryon de souris. Nous avons constaté que les tailles diffèrent en fonction de l&#39;âge de l&#39;embryon. Par exemple 8,5 dpc mesurée à une moyenne de 6 mm, 10,5 jpc mesurée à une moyenne de 11mm, et 12,5 jpc mesurée à une moyenne de 16 mm. </li><li> Ensuite, retirer leurs queues pour l&#39;extraction de l&#39;ADN. </li></ol> Partie 2: Dissection Ridge gonadique Sous stéréomicroscope et Lumière de source Schott Fostec <ol><li> Placez un papier filtre dans une boîte de Pétri. Ensuite, placer l&#39;embryon de souris au-dessus du papier filtre pour sécher l&#39;embryon et immobiliser pour la dissection. </li><li> L&#39;utilisation d&#39;un scalpel stérile, faire une coupe transversale de l&#39;embryon de souris au-dessus de l&#39;origine du cordon ombilical. </li><li> Sous un microscope stéréoscopique, en utilisant une pince fine stérile et une aiguille stériles moqueries fines, enlever les intestins et le foie de sorte que le système urinaire est visible. Les reins sont situés latéralement dans la cavité abdominale et la vessie se situe médiale et caudale par rapport aux reins. L&#39;embryon peut être identifiée comme masculine, car les crêtes gonadiques sont situés de chaque côté de la vessie. Chez la femelle, les crêtes gonadiques sont fermement attachés à l&#39;extrémité caudale latérale du rein. </li><li> Peler les crêtes génitales à en glissant une aiguille derrière elle. La crête gonadique est enlevé en coupant les tissus qui la soutiennent. </li><li> Transférer les crêtes gonadiques d&#39;un nouveau plat de Pétri remplie de fraîcheur D-PBS. Au microscope, la crête gonadique masculine devrait être dépouillé, grandes et de forme ovale. En revanche, la crête gonadiques chez la femme doivent être repérés, ont forme allongée et plus petite en comparaison de la crête gonadique masculine. </li></ol> Partie 3: Digestion Ridge gonadique Sous stéréomicroscope: <ol><li> Dissection mésonéphros: Séparez les gonades à partir du mésonéphros et dorsale à l&#39;aide d&#39;une aiguille fine et pointue. <ul><li> Il est important de retirer le mésonéphros de la crête gonadique car c&#39;est un tissu somatique qui va proliférer dans le processus de dérivation PGC. </li></ul></li><li> Digestion Ridge gonadiques: Recueillir potable crêtes gonadiques par une baisse des frais 0.5ul D-PBS. Ajouter 20 ml de collagénase / dispase solution (concentration finale à 1mg/ml). <ul><li> Les embryons doivent être traités individuellement. Chaque embryon doit avoir son propre plat à part et étiquetés de Pétri. </li></ul></li><li> Gonadique Ridge Mincing: Couper la crête gonadique en petits morceaux à l&#39;aide d&#39;une aiguille stérile et stérile pinces fines n ° 4. </li><li> Incubation: boîtes de Pétri sont transférés à l&#39;étuve à 37 ° C pendant 15 minutes. <ul><li> Le temps d&#39;incubation peut varier entre les tissus. </li></ul></li><li> Pipetage: Après 15 minutes, le tissu peut être dissocié par pipetage. En utilisant des pipettes en verre tiré capillaire avec un diamètre compris entre 40 mm -100 Break up les morceaux par aspiration et refoulement. Ce sera sous forme de petites touffes. Transférer les touffes d&#39;0.5ml d&#39;une pré réchauffé D-PBS baisse pour un lavage final. </li><li> Après lavage, transférer les touffes de tubes Eppendorf. <ul><li> Ne pas surexposer le tissu à la collagénase. </li><li> Ne pas faire une suspension cellulaire unique. Ceci est important pour la formation de colonies. Si c&#39;est une suspension cellulaire unique, PGC ont tendance à se différencier et migrer. </li><li> Ce processus ne devrait pas être plus long que 20 minutes, ou vos cellules vont perdre leur viabilité. </li></ul></li><li> Centrifuger à 2000 rpm pendant 5 minutes à température ambiante. </li><li> Après centrifugation, éliminer le surnageant et resuspendre le culot dans 0,5 ml de knock complétées par les médias (préchauffé à 37 ° C). </li></ol> Partie 4: Primordial Culture Germinal Cellules «Vous devriez effectuer les étapes suivantes rapidement pour préserver la viabilité des PGC pour l&#39;isolement et la dérivation. <ol><li> 24 heures avant ce processus, vous devriez préparer un mitotiquement inactivé MEF-CF1 alimentation couche suivant le protocole de Zhang J. et al 2008. <ul><li> Préparer 20 plats pour un prégnant des femmes de la souris (6-8 embryons). </li><li> FAE perdre la capacité d&#39;être une couche nourricière (promouvoir la croissance et empêcher la différenciation) après 5-6 passages. </li><li> Ne pas utiliser plus de 48 heures FAE. </li></ul></li><li> Immédiatement avant PGC plaquage, le milieu MEF doit être lentement retirée de la couche nourricière MEF. </li><li> Ensuite, ajoutez 0,5 ml / puits de pré-chauffé moyennes KO complété sur la couche d&#39;alimentation du MEF. <ul><li> MEF support contient sérum fœtal bovin qui induit la différenciation des PGC. </li><li> Knockout moyennes doivent être complétées par des facteurs de croissance spécifiques specie immédiatement avant usage afin d&#39;assurer l&#39;activité du facteur de croissance. </li></ul></li><li> En utilisant la distribution symétrique, PGC plaque sur la couche d&#39;alimentation du MEF. <ul><li> Si ces pièces sont trop proches, ils ont tendance à s&#39;agréger et de faire des colonies denses qui s&#39;attachent mal ou commencer différencier. </li><li> Des boîtes de culture doivent être étiquetés avec le nom de lignée cellulaire embryonnaire Germinal, le nombre de passages, et la date. </li></ul></li><li> Soigneusement, le transfert des boîtes de culture dans un incubateur à 37 ° C et 5% de CO 2. </li><li> Surveiller les cellules toutes les 24 heures. </li><li> Après 48 heures, retirer 250 ml de milieux de la haute hiérarchie de la culture pour éviter de perturber l&#39;attachement de la colonie. Ensuite, ajoutez 250 ml de frais complété knock-out des médias (préchauffé à 37 ° C). </li><li> Après l&#39;étape 7, attendre encore 48 heures, et ensuite, enlever complètement tous les médias et le remplacer par Fresh Media complétée knock-out (préchauffé à 37 ° C). Pour ce faire, deux tous les jours pendant 8-10 jours jusqu&#39;à ce PGC forment des colonies. </li></ol> Partie 5: cellules embryonnaires Germinal <ol><li> Après 10 jours d&#39;isolement PGC, embryonnaires colonies cellule germinale sont formées. </li><li> Lignées cellulaires embryonnaires germinales sont maintenues en vie par des passages manuels tous les 8-10 jours. Ils continuent de présenter une morphologie indifférenciée et expriment des marqueurs tels que la pluripotence alkalyne phosphatase, Oct-4, SSEA-1, et SOX-2 </li></ol> Remarques <ul><li> Des conditions stériles / aseptique sont essentiels dans la salle de culture. </li><li> Pour l&#39;élaboration et à la culture, PGC sont traitées dans un cabinet Purificateur classe II de sécurité Bio. </li><li> Les incubations sont dans un 37 humidifié ° C et 5% de CO 2 incubateur. </li><li> Tous les médias et les réactifs sont filtrés avant utilisation dans un filtre de 0,2 um, conservés à 4 ° C, et pré-chauffé à 37 ° C avant utilisation. </li><li> Pendant les étapes de la dissection, tous les embryons et les tissus doivent être conservés sur glace. </li><li> Tous les flacons de réactifs sont décontaminés avec de l&#39;éthanol avant de placer dans une armoire. </li><li> Utilisez des gants, des blouses de laboratoire, une infirmière et casquettes sont obligatoires. </li></ul> Conclusion Nous avons fourni une vidéo qui vous montre comment isoler, dégager, et la culture des cellules embryonnaires germinales à partir de 10.05 à 11.05 crêtes gonadiques embryons de souris DPC. L&#39;isolement et la culture reproductibles à long terme de lignées cellulaires embryonnaires germinales fournit un fondement essentiel pour l&#39;étude du développement embryonnaire précoce, les modèles génétiques et épigénétiques germinales, la formation des gonades, les effets environnementaux des organes environnants pendant le processus de développement de la gamétogenèse chez les embryons mâles et femelles, et la détermination des voies qui mène au cancer et l&#39;infertilité. <img src="/files/ftp_upload/1635/1635figure1.jpg" alt="Figure 1" />

<ol>
	<li>DeFelici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Biology: A laboratory Handbook. 1, 73-85 (1998).</li><li>Donovan, P. J., De Miguel, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+Germ+Cells+into+Stem+Cells.">Turning Germ Cells into Stem Cells.</a> Current Opinion in Genetics and Development. 13, 463-471 (2003).</li><li>Labosky, P. A., Hogan, B. L. M., Sharp, P. T., Mason, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Part+12:+Mouse+Primordial+Germ+Cells:+isolation+and+in+vitro+culture.">Part 12: Mouse Primordial Germ Cells: isolation and in vitro culture.</a> Molecular embryology: methods and protocols. 97, (1999).</li><li>Mc Laren, A., Southee, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+Mouse+Embryonic+Germ+Cells+into+Meiosis.">Entry of Mouse Embryonic Germ Cells into Meiosis.</a> Developmental Biology. 187, 107-113 (1997).</li><li>Yoshimizu, T., Obinata, M., Matsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+tissue+and+cell+interactions+play+key+roles+in+mouse+germ+cell+specification.">Stage-specific tissue and cell interactions play key roles in mouse germ cell specification.</a> Development. 128, 481-490 (2001).</li><li>Zhang, J., Hhvorostov, I., Teitell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+MEFs+to+Matrigel+I:+Passaging+hESCs+in+the+Presence+of+MEFs.">From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs.</a> J Vis Exp. , (2008).</li></ol>

Cette recherche a été menée après examen et institutionnel de protection des animaux et utilisation comité (IACUC) l&#39;approbation de notre protocole de la souris # 08030 (Le rôle de l&#39;réactives de l&#39;oxygène-ROS dans le développement de la lignée germinale et la formation de tumeurs: Holding et l&#39;élevage de protocole) à la Mississippi State University.

Pince n ° 14 et 15, 35 mm bistouri, des ciseaux de dissection, champs chirurgicaux, savon antiseptique, une solution saline à 0,9%, de la gaze, contenant en mousse de glace, lame de rasoir, scalpel 35 mm, une pince fine n ° 4 et 5, l&#39;aiguille fine taquiner avec poignée, filtre à feuilles de papier, et a tiré des pipettes en verre.<br /<br /&gt; Équipement: stéréomicroscope à dissection, source de lumière Schott Fostec, microscope inversé, micropipette 10 et 200ul, centrifugeuse, le Cabinet de sécurité biologique.

isolation and derivation of mouse embryonic germinal cells

La capacité des cellules embryonnaires germinales (EG) de se différencier en cellules germinales primordiales (PGC) et plus tard en gamètes premiers stades de développement est un modèle parfait pour répondre à nos hypothèses sur le cancer et l&#39;infertilité. Ce protocole montre comment isoler des cellules germinales primordiales gonades en développement dans les embryons 10/05 au 11/05 jours post-coïtum souris (DPC). Développer les crêtes gonadiques d&#39;embryons de souris (C57BL6J) ont été dissociées par une rupture mécanique avec de la collagénase, puis étalées en une couche de fibroblastes d&#39;embryon de souris d&#39;alimentation (MEF-CF1) qui était auparavant mitotiquement inactivé par la mitomycine C, en présence des médias à élimination directe et complétée par un inhibiteur de la leucémie Cell Factor Factor (LIF), facteur de croissance basique des fibroblastes (bFGF), et souches (SCF). L&#39;utilisation de ces méthodes optimisées pour l&#39;identification PCG, l&#39;isolement, et l&#39;établissement de conditions de culture permet les cultures à long terme des cellules EG depuis plus de 40 jours. Les lignes de cellules embryonnaires germinales montré phénotype embryonnaire et l&#39;expression des marqueurs utilisés commune de l&#39;état pluripotent. L&#39;isolement et la dérivation de cellules germinales dans la culture de fournir un outil pour comprendre leur développement in vitro et offrent la possibilité de surveiller les dommages cumulatifs aux niveaux génétiques et épigénétiques après l&#39;exposition au stress oxydatif.

Watch this Scientific Journal Video about Isolation and Derivation of Mouse Embryonic Germinal Cells at JoVE.com

Isolation and Derivation of Mouse Embryonic Germinal Cells

La capacité des cellules embryonnaires germinales à se différencier en cellules germinales primordiales durant les stades de développement précoce est un modèle parfait pour répondre à nos hypothèses sur le cancer et l&#39;infertilité. Ce protocole montre comment isoler des cellules germinales primordiales gonades en développement en 10/05 à 11/05 jours post coïtum embryons de souris.

L&#39;isolement et la dérivation de cellules germinales embryonnaires de souris

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

isolation-and-derivation-of-mouse-embryonic-germinal-cells

Research

JoVE Journal

Biology

16.0K vues.  Mississippi State University. La capacité des cellules embryonnaires germinales à se différencier en cellules germinales primordiales durant les stades de développement précoce est un modèle parfait pour répondre à nos hypothèses sur le cancer et l'infertilité. Ce protocole montre comment isoler des cellules germinales primordiales gonades en développement en 10/05 à 11/05 jours post coïtum embryons de souris.

Vidéo: Isolation and Derivation of Mouse Embryonic Germinal Cells

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 ...

Recherche

Biologie

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Dérivation des cellules souches embryonnaires humaines par Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

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 ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultes et embryonnaires Culture squelettique Microexplant musculaire et isolement de cellules souches du muscle squelettique

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Isolement et culture de cellules progénitrices embryonnaires aviaires valvulaire

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolement et culture de cellules de la zone néphrogénique du rein d&#39;embryon de souris

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Dérivation efficace des ressources humaines précurseurs cardiaques et les cardiomyocytes à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Préparation des cellules de souris embryonnaires fibroblastiques conviennent pour la culture embryonnaires humaines et cellules souches pluripotentes induites

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolement des cellules endothéliales embryonnaires ventriculaires

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Protein Isolation du développement embryonnaire Souris Coeur Région Valve

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...