This work was supported by a Pilot and Feasibility Grants from the Indiana University School of Medicine Center (IUSM) for Excellence in Molecular Hematology (NIDDK, 5P30DK090948). Additional funding was provided by a Biomedical Enhancement Grant from IUSM. We would like to thank Dr. Karen Cowden Dahl for commenting on the manuscript.

1. Embryoidkörper (EB) Bildung <ol><li> Gelatine-Anpassung WSR, die auf Mausembryo-Fibroblasten (MEF) gehalten wurden. Passage WSR 3 mal am 6. Gewebekulturplatte mit 0,1% Gelatine beschichtet, um MEFs entfernen. <ol><li> Wachsen Zellen im ESC Wartung Medien mit LIF, um die Zellen undifferenziert (Tabelle 1) zu halten. Stellen Sie sicher, Zellen 80% Konfluenz nie überschreiten. Verwenden Sie niedrige Durchgang (10 oder weniger Stellen nach dem Entfernen von MEF-Zellen) für die Differenzierung. </li></ol></li><li> Am Tag vor WSR sind für EB Differenzierung kultiviert, Durchgang die Zellen, so dass sie etwa 50-70% konfluent am nächsten Tag. Weiter Kultivieren von Zellen in Erhaltungsmedium ESC zu Zelle Pluripotenz behalten. </li><li> Am Tag der Differenzierung, absaugen ESC Erhaltungsmedium und Waschen mit 1 ml phosphatgepufferter Salzlösung (PBS: 137 mM NaCl, 2,7 mM KCl, 11,9 mM Phosphatpuffer, pH 7,4) pro Vertiefung einer 6-Well-Platte. <ol><li> Geben Sie 200 ul von 0,25% trypsin / EDTA zu jeder Vertiefung und bei 37 ° C für 3 min. </li><li> Inaktivierung des Trypsin / EDTA mit 800 ul der Differenzierung Medien. Pipette auf und ab in einem 2 ml-Pipette mit einer serologischen p200 Spitze gegen Aufbruch der Zellen in einer Einzelzellsuspension. Sicherzustellen, dass die p200 Spitze passt genau auf das Ende so Medien geht nicht zwischen der Pipettenspitze und Schnittstelle. </li><li> Pipette auf und ab ca. 5-10 mal. Achten Sie darauf, Blasen beim Pipettieren erstellen. </li></ol></li><li> Übertragungszellen zu einem 15 ml konischen Röhrchen. Um das Volumen auf 10 ml mit PBS. Zentrifuge bei 315 × g für 5 min bei Raumtemperatur. </li><li> Überstand entfernen. Zweimal waschen mit 5 ml PBS Reste enthält, LIF entfernen. Zentrifuge nach Schritt 1.4 für jede Waschung. <ol><li> Resuspendieren endgültige Zellpellet in 2 ml Differenzierungsmedium (Tabelle 2). </li></ol></li><li> Zählen von Zellen mit einer Zählkammer oder Zellzähler und Platte 6.000-10.000 Zellen / ml in einer 10 cm Petrischale(Nicht-Gewebekultur-behandelten). <ol><li> Bestimmen Sie die genaue Zellen / ml empirisch für jede Zeile ESC. Sterile Petrischalen in der Regel für die bakterielle Arbeit oder geringe Haftung Platten verwendet. HINWEIS: Die besten Ergebnisse Differenzierung, sollte das EBS Kugeln, die in Suspension bleiben, ohne Befestigung an der Platte zu bilden. Testen Sie mehrere Marken von Platten, um diejenigen mit der geringsten Einhaltung zu finden. </li><li> Die Differen WSR inkubieren bei 37 ° C Brutschrank mit 5% CO 2. </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0"><colgroup><col /><col span="2" /><col /><col /><col /></colgroup><tbody><tr><td> Reagens </td><td> Lager </td><td> Endkonzentration </td><td> Volumen </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> DMEM </td><td> 1x </td><td></td><td> 410 ml </td><td> S<html> igma / Aldrich </td><td> D5796 </td></tr><tr><td> FBS (ES abgeschirmt) </td><td> 100% </td><td> 15% </td><td> 75 ml </td><td> Hyclone </td><td> SH30070.03E </td></tr><tr><td> Nicht-essentielle Aminosäuren </td><td> 100x </td><td> 1x </td><td> 5 ml </td><td> Life Technologies </td><td> 11140 </td></tr><tr><td> L-Glutamin </td><td> 100x </td><td> 1x </td><td> 5 ml </td><td> Life Technologies </td><td> 35050 </td></tr><tr><td> Penncillin / Streptomycin </td><td> 100x </td><td> 1x </td><td> 5 ml </td><td> Life Technologies </td><td> 15070 </td></tr><tr><td> β-Mercaptoethanol </td><td> 14,3 M </td><td> 114 uM </td><td> 4 ul </td><td> Sigma / Aldrich </td><td> M3148 </td></tr><tr><td> Leukemia Inhibitory Factor (LIF) </td><td> 10 7 Einheiten / ml </td><td> 1.000 Einheiten / ml </td><td> 50 ul </td><td> Millipore </td><td> ESG1107 </td></tr></tbody></table>NHALT &quot;&gt; Tabelle 1: ESC Wartung Medien. <table border="0" cellpadding="0" cellspacing="0"><colgroup><col /><col span="2" /><col /><col span="2" /></colgroup><tbody><tr><td> Reagens </td><td> Lager </td><td> Endkonzentration </td><td> Volumen </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> DMEM </td><td> 1x </td><td></td><td> 410 ml </td><td> Sigma / Aldrich </td><td> D5796 </td></tr><tr><td> FBS (User Abgeschirmte für optimale Differenzierung) </td><td> 100% </td><td> 15% </td><td> 75 ml </td><td> Hyclone </td><td> SH30070.03E </td></tr><tr><td> Nicht-essentielle Aminosäuren </td><td> 100x </td><td> 1x </td><td> 5 ml </td><td> Life Technologies </td><td> 11140 </td></tr><tr><td> L-Glutamin </td><td> 100x </td><td> 1x </td><td> 5 ml </td><td> Leben TechnoloGies </td><td> 35050 </td></tr><tr><td> Penncillin / Streptomycin </td><td> 100x </td><td> 1x </td><td> 5 ml </td><td> Life Technologies </td><td> 15070 </td></tr><tr><td> β-Mercaptoethanol </td><td> 14,3 M </td><td> 114 uM </td><td> 4 ul </td><td> Sigma / Aldrich </td><td> M3148 </td></tr></tbody></table> Tabelle 2: Differenzierung Medien. 2. Vorbereitung EB Zellen für Virus-Infektion <ol><li> Bei der gewünschten Ausbaustufe (zwischen Tag 2 und Tag 3), übertragen Sie die EVG aus der Petrischale 10 cm bis 15 ml konischen Röhrchen. Waschen Sie die Platte mit 5 ml PBS und fügen Sie den zu waschen, um die konische Röhre. </li><li> Pellet die EVG bei 315 × g bei Raumtemperatur für 5 min. Mit 5 ml PBS waschen Sie die EB-Zellen. </li><li> 1 ml der aufgetauten Zellablösung Lösung (mit proteolytischen Enzymen und kollagenolytischen) und legen Sie die Röhrchen in einem 37 ° C warmen Wasserbad (oder Inkubator) für 30 min mit occasional Agitation (von schnippen oder Licht Vortex). </li><li> 1 ml Differenzierungsmedium. Pipette auf und ab mit einer 2-ml-Pipette mit einem p200 Pipettenspitze. Das Röhrchen wird in 37 ° C warmen Wasserbad wieder für 30-60 min. </li><li> Spin-Down der Zellen bei 315 xg für 5 min und waschen Sie die Zellen mit PBS. </li></ol> 3. Virus-Infektion <ol><li> Resuspendieren der Zellen in 1,5 ml Differenzierungsmedium. Übertragen der Zellsuspension auf eine 6-Well-Gewebekultur nicht behandelten Platte. </li><li> Bereiten Sie das Retrovirus durch Standardverfahren vor der Zeit. Hinzufügen von 5 bis 100 ul Virusüberstand, um Zellen abhängig von viralen Titer. Sie Polybren nicht hinzufügen, um eine Infektion zu erhöhen, da dies hemmt die spätere Reformation der EVG. </li><li> Spinoculate der Zellen mit Virus durch Zentrifugieren bei 2.120 × g für 90 min bei Raumtemperatur. </li></ol> 4. Differenzierung Viral Infizierte EB Zellen in hängenden Tropfen <ol><li> 2 ml Differenzierungsmedium in jede Vertiefung infected EB Zellsuspension. </li><li> Pipette 3,5 ml EB infizierten Zellsuspension in ein steriles Reagenz Reservoir für multipipettors. Verwenden Sie die multipipettor 15-20 Reihen von acht 20 ul Tropfen auf dem umgekehrten Deckel des 15 cm Petrischale pipettieren. </li><li> 10 ml steriler PBS oder Wasser auf die untere Hälfte der Petrischale. Dann kehren die Deckel mit den Tropfen und Ort auf die Petrischale. Stellen Sie sicher, dass die Tropfen auf den Kopf von Deckel mit PBS oder Wasser unten hängen zu halten, der Kammer befeuchtet. </li><li> Die Schalen werden in den 37 ° C Brutschrank mit 5,0% CO 2. </li><li> Nach 2 Tagen sammeln EBs Tropfen durch Invertieren Deckel und Waschen mit 4,5 ml Differenzierungsmedium im hängenden gebildet. <ol><li> Übertragungsmedien und EBS zu einem neuen 10 cm nicht-Gewebekulturpetrischale. Mit 3 ml Differenzierungsmedium und Transfer zum 10 cm Teller waschen Sie den Deckel wieder. </li></ol></li><li> Ernten der Zellen für die Analyse durch Durchflusszytometrie nach insgesamt 8Tagen Kultur ausgehend von der anfänglichen Übertragung von Zellen auf Differenzierungsmedium (-LIF). </li></ol> 5. Vorbereitung Zellen für Durchflusszytometer-Analyse <ol><li> Übertragungszellen zu einem 15 ml konischen Röhrchen mit einer 10-ml-Pipette. Waschen Sie die Platte mit 5 ml PBS und Transfer zum gleichen konischen Rohr. </li><li> Pellet-Zellen bei 315 x g. Pellets mit 10 ml PBS waschen. </li><li> 1 ml der aufgetauten Zellablösung Lösung und legen Sie die Röhrchen in einem 37 ° C Wasserbad für 30 min mit gelegentlichem Rühren. Für den Nachweis von bestimmten Zelloberflächenantigenen, kann Trypsin stattdessen verwendet werden. </li><li> 1 ml Differenzierung Medien und pipettieren nach oben und unten mit einem 2-ml-Pipette. Verwenden Sie ein p200 Spitze zum Ende der Pipette zu helfen, brechen-up verdaut EB in Einzelzellsuspension. <ol><li> Das Röhrchen wird in ein 37 ° C Wasserbad für 60 min wieder, damit die integrale Membranproteine ​​auf der Zelloberfläche zu recyceln. </li></ol></li><li> Pellet-Zellen bei 315 × g für 5 min. </li><li> Waschen Sie die Zellen with 0,1% Rinderserumalbumin Fraktion V (BSA) in PBS (PBS / BSA) hergestellt und Inkubation der Zellen mit spezifischen fluoreszenzmarkierten Antikörpern. </li></ol> 6. Durchflusszytometrie-Analyse für hämatopoetische Vorläufer <ol><li> Platz 10 6 EB-abgeleiteten Zellen in einem 5 ml 12 x 75 mm Rundrohr unten. Zur Einstellung Durchflusszytometer Korrektureinstellungen verwenden Entschädigung Perlen nach Herstelleranweisungen. </li><li> Pellet-Zellen bei 315 x g. Überstand abgießen und Pellet in Rest PBS / BSA-Lösung in der Röhre. </li><li> Zum Erfassen ESC HPC, Inkubation der Zellen mit fluoreszenzmarkierten Antikörper: anti-Maus-CD41-PE (R-Phycoerythrin) und Anti-Maus-CD117 (CKIT) -APC / CY7 (Allophycocyanin Cyanin 7). <ol><li> Verdünnte Antikörper 1: 100 in PBS / BSA. 100 ul des verdünnten Antikörpers zu jeder Probe. Bestimmen Sie die richtige Verdünnung des Antikörpers für jeden Lieferanten, Klon und viel oder Antikörper. Bestimmen Sie optimale Verdünnung für die Markierung von Zellen einzeln. </li&gt; </ol></li><li> Proben auf Eis inkubieren im Dunkeln für 30 min. </li><li> 2 ml PBS / BSA-und Pellet-Zellen bei 315 × g für 5 min. </li><li> Überstand wird abgesaugt und das Pellet erneut in 300 ul PBS oder einem isotonischen Puffer. </li><li> Filter resuspendierten Zellen durch eine Zelle Sieb Kappe (0,35 uM), um große Zellaggregate zu entfernen. </li><li> Analysieren Zellen mit einem Durchflusszytometer. </li></ol>

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Die Autoren haben keine konkurrierenden finanziellen Interessen offen zu legen.

Wie oben diskutiert, können Klone, die ESC induzierbar ein Gen von Interesse exprimieren mit Doxycyclin Systeme erzeugt werden, jedoch ist die Erzeugung dieser Leitungen zeitaufwendig und arbeitsintensiv. In diesem Protokoll beschrieben ist eine Methode, um ein Gen von Interesse in Einzelzellsuspension von ESC vorbereitet abgeleitet EVG auszudrücken. Diese infizierten Zellen werden dann durch hängenden Tropfen auf nachfolgende Differenzierung zu untersuchen, in EBs reformiert. In dem Beispiel (Figur 3)</stron...

Murine embryonic stem cells (ESCs) are pluripotent, remaining undifferentiated and self-renewing in the presence of the cytokine Leukemia Inhibitory Factor (LIF)1. Upon withdrawal of LIF they will spontaneously differentiate into 3-dimensional (3D) structures called embryoid bodies (EBs)2. The 3D architecture allows for the development of the three germ layers ectoderm, endoderm, and mesoderm, which then later give rise to mature tissue types3. ESCs are an exceptional model for elucidating the molecular mechanisms of cellular differentiation, particularly the investigation of the development of early hematopoietic progenitor cells (HPCs)4. 
Gene expression in ESCs can be manipulated by several techniques that allow for the determination of a role for individual molecules in development. One of the most common techniques is to use homologous recombination to generate ESC lines, which lack a gene of interest5,6. There are also a number of techniques that have been used to overexpress genes. The first technique used to modify gene expression in ESCs was to infect them with recombinant retroviruses7,8. The gene of interest however is often silenced as the ESCs differentiate into progenitor and mature cell types. Use of lentiviruses has been successful in limiting the silencing of virally expressed genes9. Other viral vectors used for overexpression include adenovirus and adeno-associated virus10. In addition standard transfection techniques to stably introduce expression plasmids are widely used for ESC transgene expression11. One difficulty with these systems is that often expression of a specific gene has different effects depending on its temporal expression. For example, the Smad1 protein affects the development of hematopoietic cells differently during different stages of EB development12,13. 
This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest. The most common system for inducibly expressing transgenes in ESCs uses the tetracycline resistance operon from E. Coli (Escherichia coli). Several different tetracycline systems have been developed. One of the more popular strategies was developed by Kyba and colleagues14. They generated an ESC line (Ainv15), which has the reverse tetracycline transactivator gene inserted into the constitutively active ROSA26 locus. A tetracycline response element (TRE), a downstream LoxP site (locus of X-over P1 from bacteriophage P1), and a promoterless neomycin cassette were introduced into the HPRT (Hypoxanthine-guanine phosphoribosyltransferase) locus on the X chromosome. Using a CRE recombination approach a gene of interest along with a eukaryotic promoter to drive the expression of the neomycin resistance gene can be inserted into the LoxP site. Correctly targeted ESCs are isolated by G418 selection. These targeted clones then must be tested for doxycycline (or tetracycline) inducible expression of the transgene. This approach has successfully generated ESC lines that inducibly express HoxB4, Stat5, SCL, and Smad714-17. However, generation of these inducible cell lines is time consuming. Described here is a method to disaggregate ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infect the cell suspensions at different days of development, and then reform the EBs by hanging drop. Downstream differentiation is later evaluated by flow cytometry. In this article an example of how miRNA expression in EB-derived cells effects hematopoietic differentiation is shown. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

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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.

In diesen Studien verkleistert RW4 (abgeleitet von 129X1 / SVJ Mausstamm) WSR verwendet wurden. EBs wurden nach 3 Tagen der Differenzierung isoliert. Für beste Ergebnisse sollte das EBS kugelförmig und nicht haft (Abbildung 1A, B). Nach spinoculation mit Virus co-exprimieren, die Fluoreszenzmarker GFP zusammen mit einem Gen von Interesse wurden EBs des Hängetropfenverfahren reformiert. EBs wurden erfolgreich aus Zellsuspensionen von 2,0, 2,5 und 3,0 Tage EVG vorbereitet reformiert. Allerdings könnte...

Watch this Scientific Journal Video about Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation at JoVE.com

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

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.

Retrovirale Infektion von murinen embryonalen Stammzellen abgeleitete Embryoidkörper Zellen für die Analyse von hämatopoetischen Differenzierung

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

retroviral-infection-murine-embryonic-stem-cell-derived-embryoid-body

Research

JoVE Journal

Biology

8.4K Aufrufe.  Harper Cancer Research Institute. 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. 

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

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Isolierung und Transplantation von hämatopoetischen Stammzellen (HSZ)

Forschung

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.

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 ...

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.

Isolierung von Anfang hämatopoetischen Stammzellen aus Murine Dottersack und AGM

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

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

Embryonale Stammzellen gewonnenen Endothelzellen zur Behandlung von Ischämie Hinterlauf

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video bioinformatischen Analyse von humanen embryonalen Stammzellen Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Differenzierung von embryonalen Stammzellen in Oligodendrozyten Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

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.

Lentivirale-vermittelte Knockdown Während Ex Vivo Erythropoese von humanen hämatopoetischen Stammzellen

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.

Phänotypische Analyse und Isolierung von murinen hämatopoetischen Stammzellen und Lineage-festgelegte Vorläuferzellen

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

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Effiziente Differenzierung von embryonalen Stammzellen der Maus in Motoneuronen

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Ein Video-Protokoll von retrovirale Infektion in der Grunddarm Organoide Kultur

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...