Embrionali umane studi sulle cellule staminali in laboratorio Teitell sono supportati da una California Institute for Regenerative Medicine (CIRM) sementi concedere RS1-00313. Ringraziamo i membri del Centro di Medicina Rigenerativa Broad e ricerca sulle cellule staminali presso la UCLA, in particolare il Dott. Amander Clark, il Dr. Jerome Zack, e membri del Fondo per la UCLA Stem Cell Institute Broad core per il loro sostegno dei nostri studi.

Dividere le cellule staminali embrionali umane (hESC) placcato al passaggio del mouse fibroblasti embrionali (MEF) Di solito un piatto hESC confluenti possono essere divisi 1:06-1:10, a seconda della linea hESC particolare. Il piatto sarà diviso confluenti ancora 5-7 giorni dopo la divisione. <ol><li> Due giorni prima della scissione, gelatinizzare piatti utilizzando una soluzione 0,1% di gelatina. Per un 6-pozzetti, aggiungere 2 ml in ogni piatto e incubare a 37 ° C, 5% CO 2 colture di tessuti incubatore durante la notte. </li><li> Piastra MEF sul gelatinizzato 6-pozzetti il ​​giorno prima si pensa di dividere la hESC. (NOTA: MEF può essere placcato fino a 3 giorni prima, se necessario, dopo ~ 3 giorni, MEF si appiattiscono e più diffusa-out e non possono sostenere hESC e più fresca MEF può..) Prendere un flacone di g-irradiati o mitomicina C trattati CF1 MEF, contenente 5-6 x 10 6 cellule, da azoto liquido e scongelare per 2 minuti in un bagno d&#39;acqua a 37 ° C. Lavare le cellule una volta con Media caldo Cultura MEF in un tubo da 50 ml falco e risospendere nel volume finale di Media caldo Cultura MEF. A 5-6 x 10 6 cellule, questo è sufficiente per due o tre piastre da 6 pozzetti. </li><li> Mentre il MEF sono stati lavati, togliere i piatti gelatinizzati, rimuovere tutti soluzione dai pozzetti e aggiungere 2,5 ml di Media Cultura MEF per bene. </li><li> Aggiungere 0,5 ml di sospensione MEF per bene per ottenere 3 ml come un volume finale di ogni bene. Assicurarsi MEF non è completamente disperso e piastre posto indietro nella notte per risolvere incubatrice. </li><li> Il giorno della scissione hESC, preparare freschi o utilizzare &lt;2 settimane di età soluzione sterile collagenasi IV a 1mg/ml. Rimuovere tutti i media dai pozzetti hESC si desidera dividere, lavare una volta con 2 ml per pozzetto di caldo 1 × PBS, pH 7,4, e aggiungere 1 ml di soluzione di collagenasi IV. Incubare a 37 ° C per 5-10 min. Prendere il 6-pozzetti hESC fuori dell&#39;incubatore e aggiungere 1 ml di ES media (senza bFGF) in ciascun pozzetto. Utilizzando la soluzione in ogni pozzetto, con una pipetta 1 ml aspirare i media e far saltare le cellule staminali al largo della piastra. Per ogni bene ci vorranno circa 5 a 10 ripetizioni. Poi, il trasferimento hESC sospeso in un tubo da 50 ml falco. Fate questo per tutti i pozzi di essere per tagliare e unire in un unico tubo falcon da 50ml. Agglomerare le hESC a temperatura ambiente a 200 g per 5 minuti e lavare una volta con i media ES manca bFGF. </li><li> Mentre i hESC vengono lavati, togliere i piatti MEF, rimuovere tutti i supporti dai pozzetti e lavare una volta con sterile, caldo 1 × PBS, pH 7,4 quindi aggiungere 2,5 ml di ES media (ora integrato con 10 ng / ml bFGF) per bene. </li><li> Risospendere il pellet hESC in un volume adeguato di mezzi di ES, integrato con 10ng/ml bFGF. (NOTA:.. Quando le hESC sono risospesi, dovrebbero essere di dimensioni della colonia o meno uniforme e la forma, il volume risospensione dipende dal rapporto di splittaggio) pipetta con attenzione la sospensione hESC su e giù per un paio di volte a rendere le colonie più piccole e più uniformi, ma non così tanto che le singole cellule o colonie molto piccole sono generati. Aggiungere 0,5 ml di sospensione hESC per bene le piastre MEF per ottenere 3 ml come un volume finale di ogni bene. Controllare visivamente per assicurarsi che le hESC sono distribuiti in modo uniforme prima di mettere le piastre indietro nell&#39;incubatore durante la notte per risolvere. Dopo la placcatura, di solito necessari alcuni giorni per le colonie ad assumere la loro forma caratteristica e l&#39;aspetto di confine. </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>

Questo video dimostra come far crescere cellule staminali embrionali umane (hESC) sulle cellule dei fibroblasti del mouse (MEF) le cellule di alimentazione. Nell&#39;ultimo passaggio prima della placcatura, quando le hESC sono risospesi, dovrebbero essere di dimensioni della colonia o meno uniforme e la forma. Con attenzione pipetta la sospensione hESC su e giù per un paio di volte a fare le colonie più piccole e più uniformi, ma non così tanto che le singole cellule o colonie sono molto piccole macchie generated.Immunofluorescence e ci...

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

Questo video dimostra come far crescere cellule staminali embrionali umane (hESC) sulle cellule dei fibroblasti del mouse (MEF) le cellule di alimentazione.

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

Questo video dimostra come far crescere cellule staminali embrionali umane (hESC) sulle cellule dei fibroblasti del mouse (MEF) le cellule di alimentazione. Parte 1 di 3.

Dal MEF per Matrigel I: hESC Passaging in presenza di MEF

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

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Research

JoVE Journal

Biology

12.6K Views.  University of California, Los Angeles. Questo video dimostra come far crescere cellule staminali embrionali umane (hESC) sulle cellule dei fibroblasti del mouse (MEF) le cellule di alimentazione. Parte 1 di 3.

Video: 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.

Passaging cellule staminali neuronali

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

Ricerca

Biologia

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.

Etichettatura hESC e hMSCs con nanoparticelle di ossido di ferro per i non-invasiva in vivo inseguimento con la RM

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.

Generare cellule iPS dal MEF attraverso l&#39;espressione forzata di Sox-2, Oct-4, c-Myc e 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.

Dal MEF per Matrigel 2: hESC scissione dal MEF sul 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.

Dal MEF per Matrigel 3: hESC Passaging da Matrigel su 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.

Purificazione dei mitocondri da cellule di lievito

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.

Un Matrigel-Based Formazione Assay tubo per valutare l&#39;attività vascolare delle cellule tumorali

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.

Generazione di Myospheres Da hESCs di epigenetica Riprogrammazione

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.

L&#39;isolamento di mitocondri da minime quantità di muscolo scheletrico del mouse per misurazioni High Throughput micropiastre respiratorie

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.

Respirometry ad alta risoluzione per misurare la funzione mitocondriale di cellule Beta intatte in presenza di composti naturali

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