Die Autoren möchten die unschätzbare Hilfe zu bestätigen: Dr. Neal First und Kourtney Wilkinson für Manuskript-Bearbeitung; Dr. Lucy Senter, Dr. Brigit Willeford und Mike Basett für die Unterstützung bei Ausbildung und Pflege der Tiere an der MSU ALAC akkreditierten Maus-Anlage, Dr. Dwayne Wise für seine Unterstützung bei der Mikroskopie und Bilderfassung; Cesar Monroy, Hannah Swoope und Bobbie Huddleston für ihre Unterstützung mit Video-Produktion an der MSU. Diese Arbeit wurde vom Office of Institutional Research und dem Department of Biological Sciences an der Mississippi State University gefördert.

Teil 1: Schwangere Maus Laparotomie <ol><li> Mit Genickbruch, euthanize eine schwangere C57BL6J weiblichen Maus bei 10,5-11,5 dpc. </li><li> Reinigen Sie den Bauch mit antimikrobieller Seife, und dann rasieren sie. </li><li> Nach der Rasur, waschen den Bauch mit einer Kochsalzlösung. </li><li> Dann trocknen die Bauchhöhle mit einer sterilen Gaze. </li><li> Decken Sie die Maus mit einem neuen sterilen Bereich. </li><li> Machen Sie eine ventrale Inzision mit einer Pinzette und Schere Dissektion. </li><li> Und entfernen Sie die gesamte Gebärmutter aus der Bauchhöhle. Maus-Embryonen wird sichtbar in der Gebärmutter. </li><li> Übertragen Sie die Gebärmutter in eine Petrischale mit D-PBS gefüllt, und bewahren Sie sie auf Eis. </li><li> Mit einem sterilen Skalpell und Pinzette, entfernen Sie die Plazenta und zusätzliche embryonale Gewebe eines jeden Embryos. </li><li> Transfer-Embryonen in eine frische Petrischale mit D-PBS gefüllt. </li><li> Messen Sie die Länge der Maus-Embryonen. Wir fanden, dass die Größen nach dem Embryo das Alter unterschieden. Beispielsweise 8,5 DPC bei einem Durchschnitt von 6 mm gemessen, gemessen 10,5 dpc bei einem Durchschnitt von 11mm und 12,5 dpc bei einem Durchschnitt von 16 mm gemessen. </li><li> Dann entfernen Sie ihre Schwänze zur DNA-Extraktion. </li></ol> Teil 2: Gonadenmissbildungen Ridge Dissection Unter Stereomikroskop und Lichtquelle Schott Fostec <ol><li> Legen Sie ein Filterpapier in einer Petrischale. Legen Sie dann die Maus-Embryo auf dem Filterpapier in den Embryo trocken und unbeweglich für die Präparation. </li><li> Mit einem sterilen Skalpell, einen Schnitt quer durch die Maus-Embryo über den Ursprung der Nabelschnur. </li><li> Unter einem Stereomikroskop, mit einer sterilen feinen Pinzette und einer sterilen feinen Hänseleien Nadel, entfernen Sie den Darm und Leber, so dass die Harnwege sichtbar ist. Die Nieren sind seitlich in der Bauchhöhle befindet, und die Blase befindet sich medial und kaudal im Vergleich zu den Nieren. Der Embryo kann als männlich identifiziert werden, da die Gonaden Rippen auf beiden Seiten der Blase befinden. In der weiblichen, sind die Gonaden Grate fest an der Schwanzflosse seitlichen Ende der Nieren angebracht. </li><li> Schälen Sie die Gonaden Grat aus, indem Sie eine Nadel dahinter. Die Gonaden Grat ist durch Wegschneiden der Gewebe, die es unterstützen entfernt. </li><li> Übertragen Sie die Gonaden Grate, um eine neue Petrischale mit frischem D-PBS gefüllt. Mikroskopisch, sollte die männlichen Keimdrüsen Grat werden abgestreift, groß und oval. Im Gegensatz dazu die weiblichen Keimdrüsen Grat gesichtet werden sollte, haben längliche Form und kleiner im Vergleich zu den männlichen Keimdrüsen Kamm. </li></ol> Teil 3: Gonadenmissbildungen Ridge Verdauung Unter Stereomikroskop: <ol><li> Mesonephros Dissection: Trennen Sie die Gonade aus dem Mesonephros und Rücken mit einem feinen und spitzen Nadel. <ul><li> Es ist wichtig, die Urniere aus der Gonaden Grat zu entfernen, weil es sich um eine somatische Gewebe, das in der PGC Ableitung Prozess überwuchern Wille ist. </li></ul></li><li> Gonaden Ridge Verdauung: Collect sauber Gonaden Bergrücken in einer 0.5ul Tropfen frischen D-PBS. 20 ml Collagenase / Dispase-Lösung (Endkonzentration an 1mg/ml). <ul><li> Embryonen sollten einzeln bearbeitet werden. Jeder Embryo sollte über eine eigene getrennte und beschriftet Petrischale. </li></ul></li><li> Gonaden Ridge Mincing: Schneiden Sie die Gonaden Grat in kleine Stücke mit einer sterilen Nadel und sterilen feinen Pinzette Nr. 4. </li><li> Inkubation: Petrischalen werden in einen Inkubator bei 37 übertragen ° C für 15 Minuten. <ul><li> Die Inkubationszeit kann zwischen Geweben variieren. </li></ul></li><li> Pipettieren: Nach 15 Minuten kann das Gewebe werden durch Pipettieren dissoziiert. Mit gezapftes Kapillarpipetten mit Durchmessern zwischen 40 -100 mm Break up die Stücke durch Auf-und Abpipettieren. Dies bildet kleine Klumpen. Übertragen Sie die Klumpen zu 0,5 ml einer vorher erwärmt D-PBS Drop für eine endgültige waschen. </li><li> Nach dem Waschen, übertragen Sie die Klumpen auf Eppendorf-Röhrchen. <ul><li> Nicht zu entlarven das Gewebe Kollagenase. </li><li> Machen Sie nicht einen einzigen Zellsuspension. Dies ist für die Kolonie-Bildung wichtig. Wenn es eine einzige Zellsuspension wird, neigen PGCs zu differenzieren und zu migrieren. </li><li> Dieser Vorgang sollte nicht länger als 20 Minuten, oder Ihre Zellen Lebensfähigkeit verlieren. </li></ul></li><li> Zentrifugation bei 2000 rpm für 5 Minuten bei Raumtemperatur. </li><li> Nach der Zentrifugation den Überstand und das Pellet in 0,5 ml ergänzt knock out Medien (bei 37 ° C vorgewärmt). </li></ol> Teil 4: Primordial Keimzellen Kultur &quot;Sie sollten die nächsten Schritte rasch durchzuführen, um die Lebensfähigkeit der PGCs für die Isolierung und die Ableitung zu erhalten. <ol><li> 24 Stunden vor diesem Prozess sollten Sie bereiten mitotisch inaktivierten MEF-CF1 Feeder-Schicht nach dem Protokoll von Zhang J. et al 2008. <ul><li> Es werden 20 Gerichte für eine pregnant weiblichen Maus (6-8 Embryonen). </li><li> MEFs verlieren die Fähigkeit, eine Feeder-Schicht werden (Förderung von Wachstum und Differenzierung zu verhindern) nach 5-6 Passagen. </li><li> Verwenden Sie keine MEFs älter als 48 Stunden. </li></ul></li><li> Unmittelbar vor dem Ausplattieren PGCs, muss der MEF Medium langsam aus dem MEF Feeder-Schicht entfernt werden. </li><li> Dann fügen Sie 0,5 ml / well vorgewärmtes ergänzt Knockout Mediums auf die MEF Feeder-Schicht. <ul><li> MEF Medium enthält Rinderfötenserum dass eine Differenzierung der PGCs induziert. </li><li> Knockout Medium muss mit specie spezifischen Wachstumsfaktoren ergänzt werden unmittelbar vor Gebrauch, um die Aktivität der wachsen-Faktor sorgen. </li></ul></li><li> Mit symmetrische Verteilung, Platte PGCs über die MEF Feeder-Schicht. <ul><li> Wenn diese Stücke zu nahe sind, neigen sie zu aggregieren und bilden dichte Kolonien, die schlecht anbringen oder beginnen differenzieren. </li><li> Kulturschalen muss mit embryonalen Germinal Zelllinie Namen, Durchgang Nummer und Datum beschriftet werden. </li></ul></li><li> Vorsichtig, überweisen Sie den Kulturschalen in einem Brutschrank bei 37 ° C und 5% CO 2. </li><li> Überwachen Sie die Zellen alle 24 Stunden. </li><li> Nach 48 Stunden zu entfernen 250 ml von Medien aus der Spitze der Kultur nicht zu stören der Kolonie Anhang. Dann fügen Sie 250 ml frischem ergänzt Knock-out-Medien (bei 37 ° C vorgewärmt). </li><li> Nach Schritt 7, warten weitere 48 Stunden, und dann vollständig zu entfernen alle Medien und ersetzen Sie es mit frischen ergänzt Knock-out-Medien (bei 37 ° C vorgewärmt). Tun Sie dies alle 2 Tage für 8-10 Tage, bis PGC Kolonien zu bilden. </li></ol> Teil 5: Embryonale Keimzellen <ol><li> Nach 10 Tagen PGC Isolation, sind embryonale Keimzelle Kolonien gebildet. </li><li> Embryonale Keimzelle Linien sind am Leben gehalten durch manuelle Passagen alle 8-10 Tage. Sie fahren fort, eine undifferenzierte Morphologie präsentieren und ausdrücken Pluripotenz Marker wie alkalyne Phosphatase, Oct-4, SSEA-1 und SOX-2 </li></ol> Hinweise <ul><li> Sterile / aseptischen Bedingungen sind unerlässlich in der Kultur Raum. </li><li> Für die Ableitung und Kultur, sind PGCs in einem Luftreiniger Class II Bio Sicherheit Kabinett verarbeitet. </li><li> Inkubationen werden in einem befeuchteten 37 ° C und 5% CO 2-Inkubator. </li><li> Alle Medien und Reagenzien sind vor in einer 0,2 um Filter, Lagerung bei 4 ° C verwenden, filtriert und bei 37 vorgewärmten ° C vor dem Gebrauch. </li><li> Während der Dissektion Schritte müssen alle Embryonen und Gewebe auf Eis gehalten werden. </li><li> Alle Reagenzien Flaschen werden mit Ethanol, bevor sie in einem Schrank dekontaminiert. </li><li> Verwendung von Handschuhen, Kittel und Krankenschwester caps sind Pflichtfelder. </li></ul> Abschluss Wir haben ein Video, das Ihnen zeigt, wie zu isolieren, zu leiten und Kultur embryonalen Keimzellen von Gonaden Grate 10,5-11,5 dpc Maus-Embryonen zur Verfügung gestellt. Die reproduzierbare Isolierung und langfristig Kultur der embryonalen Keimzelle Linien bietet eine entscheidende Grundlage für das Studium der frühen Embryonalentwicklung, genetischen und epigenetischen germinal Muster, Gonade Bildung, Umwelt-Auswirkungen der umliegenden Organe während des Entwicklungsprozesses der Gametogenese in der männlichen und weiblichen Embryonen, und Bestimmung der Wege, die zu Krebs und Unfruchtbarkeit führt. <img src="/files/ftp_upload/1635/1635figure1.jpg" alt="Abbildung 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>

An der Mississippi State University: Diese Arbeit wurde nach Überprüfung und Institutional Animal Care und Verwenden Committee (IACUC) Genehmigung unserer Maus-Protokoll # 08030 (Holding und Zucht-Protokoll Die Rolle von reaktiven Sauerstoffspezies-ROS in der Keimbahn Entwicklung und Tumorbildung) durchgeführt.

Zange Nr. 14 und 15, Skalpell 35 mm, Dissektion Scheren, chirurgischen Bereichen, antimikrobielle Seife, Kochsalzlösung 0,9%, Gaze, Eis-Schaum-Behälter, Rasierklinge, Skalpell 35 mm, feinen Pinzette Nr. 4 und 5, feine Hänseleien Nadel mit Griff-, Filter-Papierbögen, und zog Glaspipetten.<br /<br /&gt; Equipment: Sezieren Stereomikroskop Lichtquelle Schott Fostec, inverse Mikroskop Mikropipette 10 und 200 &amp;mgr; l, Zentrifuge, bio Sicherheit Kabinett.

isolation and derivation of mouse embryonic germinal cells

Die Fähigkeit von embryonalen Keimzellen (EG) in primordialen Keimzellen (PGC) und später in Keimzellen während der frühen Entwicklungsstadien zu unterscheiden, ist eine perfekte Modell, um unsere Hypothese über Krebs und Unfruchtbarkeit Adresse. Dieses Protokoll zeigt, wie Ur-Keimzellen aus Entwicklungsländern Gonaden in 10,5-11,5 Tage nach coitum (DPC) Maus-Embryonen zu isolieren. Die Entwicklung der Gonaden Kämme aus Maus-Embryonen (C57BL6J) wurden durch mechanische Störungen mit Kollagenase, dann in einem Maus-Embryo-Fibroblasten-Feeder-Schicht (MEF-CF1), die zuvor mitotisch mit Mitomycin C wurde in Anwesenheit von Knockout-Medien inaktiviert und ergänzt mit Leukämie Inhibitor vernickelt dissoziiert Factor (LIF), basischen Fibroblasten-Wachstumsfaktor (bFGF) und Stem Cell Factor (SCF). Mit diesen optimierten Methoden für PCG Identifikation, Isolierung und Etablierung der Kulturbedingungen ermöglicht langfristig Kulturen von EG-Zellen für mehr als 40 Tage. Die embryonalen Keimzelle Linien zeigten embryonalen Phänotyp und die Expression von häufig verwendeten Marker der pluripotenten Zustand. Isolation und Ableitung von Keimzellen in der Kultur ein Instrument, um ihre Entwicklung in vitro zu verstehen und bieten die Möglichkeit, kumulative Schäden an genetischen und epigenetischen zu überwachen nach der Exposition gegenüber oxidativem Stress.

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

Die Fähigkeit von embryonalen Keimzellen in primordialen Keimzellen während der frühen Entwicklungsstadien zu unterscheiden, ist eine perfekte Modell, um unsere Hypothese über Krebs und Unfruchtbarkeit Adresse. Dieses Protokoll zeigt, wie Ur-Keimzellen aus Entwicklungsländern Gonaden in 10,5-11,5 Tage nach coitum Maus-Embryonen zu isolieren.

Isolation und Ableitung von embryonalen Maus-Keimzellen

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 Aufrufe.  Mississippi State University. Die Fähigkeit von embryonalen Keimzellen in primordialen Keimzellen während der frühen Entwicklungsstadien zu unterscheiden, ist eine perfekte Modell, um unsere Hypothese über Krebs und Unfruchtbarkeit Adresse. Dieses Protokoll zeigt, wie Ur-Keimzellen aus Entwicklungsländern Gonaden in 10,5-11,5 Tage nach coitum Maus-Embryonen zu isolieren.

Serie: 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.

Ableitung von hämatopoetischen Stammzellen aus embryonalen Stammzellen der Maus

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 ...

Forschung

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.

Gewinnung menschlicher embryonaler Stammzellen durch 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.

Ableitung der Maus Trophoblast Stammzellen aus Blastozysten

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.

Adulten und embryonalen Skelettmuskel Microexplant Kultur und Isolation der Skelettmuskulatur 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 ...

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.

Isolierung und Kultivierung von Avian Embryonale Valvular Progenitorzellen

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.

Isolierung und Kultivierung von Zellen aus dem Nephrogene Zone der embryonalen Maus Niere

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.

Effiziente Ableitung von Human Cardiac Vorstufen und Kardiomyozyten aus pluripotenten humanen embryonalen Stammzellen mit 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 ...

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.

Vorbereitung von embryonalen Maus-Fibroblasten-Zellen zum Kultivieren von humanen embryonalen und induzierten pluripotenten Stammzellen

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

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Ein System für<em&gt; Ex vivo</em&gt; Kultivierung von embryonalen Pankreas

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

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.

Isolierung von embryonalen ventrikuläre Endothelzellen

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