Menschliche embryonale Stammzellen-Studien in der Teitell Labor werden von einem California Institute for Regenerative Medicine (CIRM) Saatgut gewähren RS1-00313 unterstützt. Wir danken Mitglieder der Broad Center of Regenerative Medicine and Stem Cell Research an der UCLA, vor allem Dr. Amander Clark, Dr. Jerome Zack, und die Mitglieder des UCLA Broad Institute Stem Cell Core Facility für ihre Unterstützung unserer Studien.

Splitting humanen embryonalen Stammzellen (hES) am embryonalen Maus-Fibroblasten (MEFs) ausplattiert Normalerweise wird eine konfluente hESC Platte kann von 1.06 bis 01.10 Uhr aufgeteilt werden, abhängig von der jeweiligen hESC Linie. Die Split-Platte wird konfluenten wieder 5-7 Tage nach der Trennung. <ol><li> Zwei Tage vor der Spaltung, verkleistern-Platten mit einer 0,1% igen Gelatinelösung. Für eine 6-Well-Platte, fügen 2ml in jede Vertiefung und inkubieren Platte in einem 37 ° C, 5% CO 2 Gewebekultur-Inkubator über Nacht. </li><li> Platte MEFs auf verkleistert 6-well Platten am Tag, bevor Sie planen Aufspaltung der HES. (HINWEIS: MEFs können bis zu 3 Tage vorher bei Bedarf beschichtet werden nach ~ 3 Tage, MEFs flacher und spread-out und kann nicht aufrechterhalten hESCs sowie frischer MEFs werden kann..) Nehmen Sie ein Fläschchen mit g-bestrahlt oder Mitomycin C behandelt CF1 MEFs, mit 5-6 x 10 6 Zellen, aus flüssigem Stickstoff und Auftauen für 2 min in einem 37 ° C Wasserbad. Wash Zellen einmal mit warmem MEF Nährmedien in einem 50ml Falcon-Röhrchen und resuspendieren in das endgültige Volumen des warmen MEF Culture Media. Bei 5-6 x 10 6 Zellen, das ist genug, um Platte zwei oder drei 6-well Platten. </li><li> Während die MEFs gewaschen werden, nehmen Sie die verkleistert Platten, entfernen Sie alle Lösungen aus den Vertiefungen und 2,5 ml des MEF Kultur Medien pro Vertiefung. </li><li> 0,5 ml Suspension pro MEF gut zu erreichen 3ml als letzte Band in jedes Well. Stellen Sie sicher, MEFs gleichmäßig verteilt sind und legen Platten wieder in den Inkubator über Nacht zu begleichen. </li><li> Am Tag der hESC Splitting, bereiten Sie frisches oder &lt;2 Wochen alte sterile Kollagenase IV Lösung 1mg/ml. Entfernen Sie alle Medien aus dem Brunnen hESC Sie teilen möchten, einmal abwaschen mit 2ml pro well warm 1 × PBS, pH 7,4, und fügen Sie 1 ml Collagenase IV-Lösung. Bei 37 ° C für 5-10 min. Nehmen Sie die 6-well hESC Platte aus dem Inkubator und 1ml von ES-Medien (ohne bFGF) in jede Vertiefung. Mit der Lösung in jede Vertiefung, mit einer 1 ml Pipette saugen die Medien und blasen die Stammzellen aus der Platte. Für jeden gut das dauert ca. 5 bis 10 Wiederholungen. Dann überweisen Sie den suspendierten hESCs in ein 50ml Falcon-Röhrchen. Tun Sie dies für alle Brunnen, die geteilt und kombinieren in ein 50ml-Falcon-Röhrchen. Pellet der hESCs bei Raumtemperatur bei 200g für 5 min und waschen einmal mit ES Medien fehlt bFGF. </li><li> Während die hESCs gewaschen werden, nehmen Sie die MEF-Platten, entfernen Sie alle Medien aus dem Brunnen und waschen einmal mit steriler, warm 1 x PBS, pH 7,4 fügen Sie dann 2,5 ml ES Medien (jetzt mit 10 ng / ml bFGF ergänzt) pro gut. </li><li> Resuspendieren der pelletierten hESCs in einem geeigneten Volumen ES Medien, mit 10ng/ml bFGF ergänzt. (Hinweis:.. Wenn die hESCs resuspendiert sind, sollten sie von rund einheitlichen Kolonie Größe und Form werden die Resuspension Volumen hängt vom Teilungsverhältnis) vorsichtig pipettieren hESC Aufhängung oben und unten ein paar Mal um die Kolonien kleiner und gleichmäßiger, aber nicht so viel, dass einzelne Zellen oder sehr kleine Kolonien erzeugt werden. 0,5 ml Suspension pro hESC gut an die MEF-Platten bis 3 ml als ein Endvolumen in jeder Vertiefung zu erreichen. Sichtprüfung sicherstellen, dass die hESCs gleichmäßig, bevor die Platten wieder in den Inkubator über Nacht absetzen verteilt. Nach dem Plattieren, wird es in der Regel ein paar Tage dauern um die Kolonien auf ihre charakteristische Form und Grenze Aussehen. </li></ol>

<ol>
	<li>Thomson, J. a. m. e. s. A., Itskovitz-Eldor, J. o. s. e. p. h., Shapiro, S. a. n. d. e. r. S., Waknitz, M. i. c. h. e. l. l. e. A., Swiergiel, J. e. n. n. i. f. e. r. J., Marshall, V. i. v. i. e. n. n. e. S., Jones, J. e. f. f. r. e. y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Stem+Cell+Lines+Derived+from+Human+Blastocysts.">Embryonic Stem Cell Lines Derived from Human Blastocysts.</a> Science . 282, 1145-1147 (1998).</li><li>Xu, C. h. u. n. h. u. i., Inokuma, M. a. r. g. a. r. e. t. S., Denham, J. e. r. r. o. d., Golds, K. a. t. h. a. l. e. e. n., Kundu, P. r. a. t. i. m. a., Gold, J. o. s. e. p. h. D., Carpenter, M. e. l. i. s. s. a. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nature Biotechnology . 19, 971-974 (2001).</li></ol>

Dieses Video zeigt, wie menschliche embryonale Stammzellen (hES) am embryonalen Maus-Fibroblasten (MEF) Feeder-Zellen wachsen. In den letzten Schritt vor dem Plattieren, wenn die hESCs resuspendiert sind, sollten sie von rund einheitlichen Kolonie Größe und Form sein. Sorgfältig Pipette die hESC Aufhängung oben und unten ein paar Mal um die Kolonien kleiner und gleichmäßiger, aber nicht so viel, dass einzelne Zellen oder sehr kleine Kolonien generated.Immunofluorescence Färbung und Mikroskopie oder Durchflusszytometrie für hESC Plur...

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from mefs to matrigel i: passaging hescs in the presence of mefs

Dieses Video zeigt, wie menschliche embryonale Stammzellen (hES) am embryonalen Maus-Fibroblasten (MEF) Feeder-Zellen wachsen.

Watch this Scientific Journal Video about From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs at JoVE.com

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

Von MEFs auf Matrigel I: Die Passage hESCs in Gegenwart von MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

from-mefs-to-matrigel-i-passaging-hescs-in-the-presence-of-mefs

Research

JoVE Journal

Biology

12.6K Aufrufe.  University of California, Los Angeles. Dieses Video zeigt, wie menschliche embryonale Stammzellen (hES) am embryonalen Maus-Fibroblasten (MEF) Feeder-Zellen wachsen.

Serie: From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

Passagierung humaner neuraler Stammzellen

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Forschung

Biologie

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Labeling hESCs und hMSCs mit Eisenoxid-Nanopartikel für Non-Invasive in vivo-Tracking mit MR-Bildgebung

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Generieren von iPS-Zellen aus MEFS durch Forced Expression von Sox-2, Oct-4, c-Myc und Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

Von MEFs auf Matrigel 2: Splitting hESCs aus MEFs auf Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

Von MEFs auf Matrigel 3: Die Passage hESCs von Matrigel auf Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Reinigung von Mitochondrien aus Hefezellen

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Ein Matrigel-Based Rohr Formation Assay zur vaskulären Aktivität von Tumorzellen Beurteilen

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generierung von Myospheres Von hESCs durch epigenetische Reprogrammierung

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Isolierung von Mitochondrien von minimalen Mengen an Maus-Skelettmuskel für die Hochdurchsatz-Mikroplatten-Atemmessungen

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

Hochauflösender Respirometrie, mitochondriale Funktion intakt Beta-Zellen in Anwesenheit von Naturstoffen zu messen

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

Methods Article

Von MEFs auf Matrigel I: Die Passage hESCs in Gegenwart von MEFs