Grazie a Tim Doyle, PhD e il Centro di Stanford per imaging in vivo per l&#39;assistenza con l&#39;imaging bioluminescenza. Grazie anche a Ngan Huang, PhD per condividere la sua tecnica su cellule staminali co-iniezione con soluzione matrice. Infine, grazie a Steve Feltri, Ph.D. assistenza per la cura degli animali veterinari.

<ol><li> Costruzione di Gene Doppia Reporter Fusion <ol><li> Al fine di eseguire l&#39;imaging bioluminescenza delle cellule staminali embrionali umane, è necessario prima di ottenere cellule che esprimono stabilmente un gene luciferasi giornalista come luciferasi lucciola guidata da un promotore costitutivo come ubiquitina o EF1a. </li><li> Il focus di questo protocollo è sulle applicazioni gene reporter, in modo dettagliato le procedure non sono forniti qui. Tuttavia, la strategia generale del nostro laboratorio è quello di utilizzare un doppio vettore costruire fusione che contiene lucciola luciferasi (fluttuazioni) e migliorato proteina verde fluorescente (EGFP) separate da un distanziale all&#39;interno pCDNA 3.1 +. </li><li> In breve, il nostro gene di fusione doppio era originariamente situato a valle del promotore del citomegalovirus in pCDNA 3.1 (+), quindi il 3,3 kbp frammento è stato asportato con la digestione e NdeI Noti e smussato-end legatura nel sito di clonazione multiplo di un auto-inattivazione (SIN ) vettori lentivirali, sotto il controllo di un promotore Ubiquitina. </li></ol></li><li> Trasduzione lentivirali <ol><li> hESC possono essere trasdotte 3-5 giorni dopo il passaggio ad una molteplicità di infezione (MOI) di 10 (titolo virale di circa 107 incubati con 106 celle). </li><li> Lo stock scongelato virale possono essere aggiunti direttamente ai media hESC fresca. </li><li> Aggiornare i media 12 e 24 ore dopo. </li><li> Dopo 48 ore, efficienza di trasduzione in grado di valutare qualitativamente usando la microscopia a fluorescenza. Successivamente, FACS può essere utilizzato per isolare le cellule infette. </li></ol></li><li> HESC coltura e Introduzione alla Imaging Set-up <ol><li> Cultura tuo hESC luciferasi positivo alimentatore senza condizioni. Noi di solito seguono il protocollo standard WiCell e utilizzare le piastre a 6 e rivestito in fattore di crescita ridotta Matrigel, utilizzando sia i media condizionata o commerciali, senza alimentatore mezzi di comunicazione. </li><li> Per rilevare le cellule luciferasi positivo, è necessario avere la sonda giornalista di D-luciferina già preparato. Per fare questo, luciferina aliquota a 1-1,5 ml preparati ad una concentrazione finale di 45 mg / ml. Mantenere le aliquote a -20 ° C, quando non in uso ed evitare l&#39;esposizione alla luce da coprire con un tovagliolo di carta, quando non in deposito. </li><li> Al fine di visualizzare le cellule luciferasi positivo, usiamo IVIS 50 e 200 IVIS Imaging Systems (Xenogen Corporation, Alameda, CA). Quest&#39;ultimo sistema comprende un apparato integrato isoflourane e la camera di induzione di anestesia temporanea di piccoli animali. </li></ol></li><li> Nell&#39;imaging bioluminescenza in vitro di hESC <ol><li> Per nell&#39;imaging bioluminescenza in vitro, è importante mantenere condizioni di sterilità. Pertanto, il sistema di imaging devono essere sterili, preferibilmente in una stanza di coltura cellulare. </li><li> Prima di imaging, rimuovere il supporto delle cellule e aggiungere quel tanto che basta PBS per coprire le cellule. Ad esempio, aggiungere 1 ml di PBS a ciascun pozzetto di un 6-pozzetti contenenti vostre culture hESC. </li><li> Il rapporto tra D-Luciferin di PBS dovrebbe essere 1:100, quindi aggiungere 10 ml di scongelato D-Luciferin in ciascun pozzetto della piastra ben 6. </li><li> Aspettate un minuto, quindi scattare una foto con un tempo di esposizione di 1 secondo. Se il segnale è debole, aumentare l&#39;intervallo di esposizione e riprovare. </li><li> Il segnale bioluminescente riflette numero di cellulare, in modo da saggi di quantificazione può essere effettuata che correlano segnale con il numero di cellule differenti. </li></ol></li><li> Preparazione del mouse e l&#39;iniezione di cellule per imaging in vivo <ol><li> Reporter gene fornisce un metodo di imaging ad alta velocità, poco costoso e sensibile per le cellule di inseguimento dopo il trapianto per giorni, settimane e anche mesi. Ciò è particolarmente importante in quanto le cellule staminali trapiantate frequente forma teratomi, sono rigettate dal sistema immunitario dell&#39;ospite, o semplicemente morire. </li><li> Il metodo più semplice per la formazione di teratomi l&#39;imaging in vivo è quello di iniettare hESC che esprimono il gene reporter luciferasi lucciola nella schiena di topi SCID e di acquisire immagini bioluminescenti con il sistema IVIS. </li><li> Quando sei pronto per trapiantare le cellule, l&#39;uso dispasi o collagenasi per allentare le cellule, lavare più volte, e sospendere in una miscela 1:1 di fattore di crescita ridotto matrigel e DMEM. Di solito abbiamo prima mescolare le cellule con DMEM refrigerato e poi aggiungere nel matrigel. Per ciascun sito di iniezione sottocutanea, iniettiamo 200.000 cellule sospese in un volume di 50-100 ul del matrigel / DMEM miscela. Il numero di cellule può essere regolata a seconda dell&#39;applicazione in uso. Ricordate di tenere tutto in ghiaccio prima dell&#39;iniezione. </li><li> Una volta che le cellule sono pronte, anestetizzare il mouse inserendo nella casella di induzione con isofluorano flusso acceso. Dopo 1-5 minuti, il mouse è addormentato e può essere posizionato sul tavolo operatorio con isoflurano continuo. </li><li> Perché pelliccia naturale auto-fluorescente e può oscurare l&#39;immagine bioluminescente, avremo bisogno di togliere il pelo dalla parte posteriore del mouse. Il modo più semplice per farlo è di utilizzare un rasoio elettrico, ma è anche possibile utilizzare depilazione gel. Pulire con alcool per sterilizzarela pelle dopo </li><li> Successivamente, elaborare il vostro sospensione di cellule in una siringa. 23-27 aghi calibro funzionano meglio in quanto non si intasano con le cellule. Se l&#39;ago è troppo grande (&lt;23 gauge) del tratto dell&#39;ago può lasciare un grande buco attraverso il quale le cellule possono refluire fuori. </li><li> Iniettare le cellule sotto la pelle nella schiena del mouse. Perché la pelle è piuttosto ampio, con il pollice e l&#39;indice per pizzicare e allungare la zona da iniettare. Iniettare sotto la pelle, facendo attenzione a non forare troppo in profondità. Inoltre, cercare di mantenere l&#39;ago di scivolare fuori del sito di iniezione mentre lo stantuffo: questo impedirà la creazione di un foro nella pelle attraverso i quali le cellule possono perdita se si tenta di iniettare di nuovo. </li><li> Dopo che le cellule vengono iniettate, attendere qualche ora per permettere al mouse per svegliarsi e correre prima di ri-anestesia per l&#39;imaging bioluminescenza. In questo modo evita di tossicità isoflurano. </li></ol></li><li> Nell&#39;imaging bioluminescenza in vivo delle cellule trapiantate <ol><li> Per l&#39;imaging intero bioluminescenza animali, facciamo un&#39;iniezione intraperitoneale di D-luciferina a 375 mg / kg di peso corporeo. Pesare l&#39;animale per calcolare il dosaggio prima dell&#39;iniezione. </li><li> Iniettare il D-luciferina nel peritoneo, poco lontano dalla linea mediana. Fare attenzione a non andare troppo in profondità o si può danneggiare gli organi interni. Se il mouse comincia a svegliarsi, metterlo nella scatola eliminazione diretta e attendere un minuto o due. Per i topi, la bioluminescenza massimo si verifica di solito 15-40 minuti dopo l&#39;iniezione. </li><li> Ricordati di inserire carta nera opaca nella casella di imaging che aiutano ad assorbire qualsiasi luce non emessa dal hESC. Posizionare il mouse (fino retro) nella camera di imaging con isoflurano, in cima alla carta nera. Inizia con un tempo di esposizione di 10 secondi. Se il segnale è saturo, provare a diminuire il tempo di esposizione, se troppo debole, aumentare l&#39;esposizione. </li><li> Una volta che il tempo di esposizione del segnale è stato ottimizzato per il mouse, cominciare a prendere le immagini ogni minuto fino a quando il segnale raggiunge un massimo. Questo è fatto meglio utilizzando il software di imaging per selezionare &quot;regioni di interesse (ROI)&quot; che coprono le sedi di iniezione. Attraverso il monitoraggio della intensità del segnale in ogni ROI si può facilmente determinare quando il segnale comincia a diminuire, indica che l&#39;intensità del segnale massimo è stato raggiunto. Il segnale di massima dovrebbero essere usati come dati finali. </li><li> Quando si è soddisfatti con le vostre immagini, togliere il mouse da isoflurano e permettergli di svegliarsi nella sua gabbia. Di solito, il mouse dovrebbe svegliarsi in 15 minuti. </li><li> Giornalista di imaging bioluminescenza gene può essere ripetuta ogni giorno, settimanali e mensili per tutto il tempo la hESC sopravvivere l&#39;animale. Segnale da un teratoma sviluppo aumenterà in modo esponenziale nel corso del tempo, tipicamente dell&#39;ordine di settimane, come il hESC cominciano a proliferare. </li><li> Dopo il periodo di tempo desiderato di immagini bioluminescenza è acquisito, l&#39;animale può essere sacrificata e sezioni di tessuto utilizzato per l&#39;esame istologico. </li></ol></li></ol>

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Rispetto ad altre modalità come la PET e risonanza magnetica, la bioluminescenza è limitata risoluzione spaziale e di penetrazione nel tessuto ridotto a causa della relativamente debole energia dei fotoni emessi (2-3 eV), per queste ragioni che finora non è stato applicabile in animali di grandi dimensioni. Tuttavia, bioluminescenza ha il vantaggio di essere a basso costo, ad alta produttività, e non invasivo, il che rende altamente auspicabile che in cellule staminali in vivo inseguimento nei piccoli animali. Non bioluminescenza geni r...

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		<thead>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BD Matrigel&#x2122; Basement Membrane Matrix</td>
<td>Growth factor reduced (optional: phenol-red free)</td>
<td>BD Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>mTeSR1 Maintenance Medium for Human Embryonic Stem Cells</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-Luciferin Firefly, potassium salt</td>
<td>&nbsp;</td>
<td>Biosynth AG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Dissolve 30 mg Collagenase Type IV in 30 mL DMEM-F12 media. Sterile filter and store at 4 degrees (Celsius).</td>
</tr>
<tr>
<td>Baked Pasteur pipets</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6-well tissue culture-treated plates</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>92006</td>
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in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells

La scoperta delle cellule staminali embrionali umane (hESC) ha notevolmente aumentato gli strumenti a disposizione degli scienziati medici interessati alla medicina rigenerativa. Tuttavia, l&#39;iniezione diretta di hESC, e le cellule differenziate da hESC, in organismi viventi è stata finora ostacolata dalla morte delle cellule significativi, la formazione di teratoma, e il rifiuto immune. Comprensione del comportamento hESC vivo dopo trapianto richiede nuove tecniche di imaging di monitorare longitudinalmente localizzazione hESC, la proliferazione e la vitalità. L&#39;imaging molecolare ha dato un mezzo investigatori high-throughput, poco costoso, e sensibile per il monitoraggio della proliferazione cellulare in vivo per giorni, settimane e anche mesi. Questo progresso ha notevolmente aumentato la comprensione della spazio-temporale cinetica di attecchimento hESC, la proliferazione, e teratoma-formazione in soggetti viventi. Una delle principali innovazioni nel campo dell&#39;imaging molecolare è stata l&#39;estensione del test non invasivo gene reporter di biologia molecolare e cellulare in vivo in modalità multi-piattaforme di imaging. Questi geni reporter, sotto il controllo di promotori ingegnerizzati e stimolatori che sfruttano macchinari trascrizionale della cellula ospite s, vengono introdotte nelle cellule utilizzando una varietà di metodi vettoriali e non vettoriale. Una volta nella cellula, i geni giornalista può essere trascritto sia costitutivamente o solo in particolari condizioni biologiche o cellulare, a seconda del tipo di promotore utilizzato. Trascrizione e traduzione di geni reporter in proteine ​​bioattive viene poi rilevato con sensibili, strumentazione non invasiva (ad esempio, fotocamere CCD), utilizzando il segnale che generano sonde come la D-luciferina. Per evitare la necessità di luce eccitatoria per monitorare le cellule staminali in vivo, come è richiesto per l&#39;imaging di fluorescenza, la bioluminescenza giornalista sistemi di imaging gene richiedono solo una sonda esogena somministrata per indurre emissione di luce. Firefly luciferasi, derivato dal Fotino lucciola pyralis, codifica un enzima che catalizza D-luciferina al otticamente metabolita attivo, oxyluciferin. Attività ottica può quindi essere monitorato con una fotocamera esterna CCD. Trasdotte stabilmente cellule che trasportano il giornalista costruire nel loro DNA cromosomico passerà il giornalista costruire DNA alle cellule figlie, consentendo il monitoraggio longitudinale di sopravvivenza hESC e la proliferazione in vivo. Inoltre, poiché l&#39;espressione del prodotto del gene reporter è richiesto per la generazione del segnale, solo le cellule vitali genitore e figlia creerà segnale di bioluminescenza; cellule in apoptosi o morte no. In questo video, i materiali e metodi specifici necessari per la proliferazione delle cellule staminali il monitoraggio e la formazione di teratomi, con immagini bioluminescenza saranno descritti.

Watch this Scientific Journal Video about In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells at JoVE.com

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

Con il crescente interesse per le terapie con cellule staminali, le tecniche di imaging molecolare sono ideali per il monitoraggio del comportamento delle cellule staminali dopo il trapianto. Geni reporter luciferasi hanno permesso non invasiva, la valutazione ripetitivo della sopravvivenza cellulare, la posizione e la proliferazione in vivo. Questo video dimostra come monitorare la proliferazione hESC in un topo vivo.

In vitro e in Imaging bioluminescenza gene reporter in vivo di cellule staminali embrionali umane

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

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Research

JoVE Journal

Biology

21.7K Views.  Stanford University School of Medicine. Con il crescente interesse per le terapie con cellule staminali, le tecniche di imaging molecolare sono ideali per il monitoraggio del comportamento delle cellule staminali dopo il trapianto. Geni reporter luciferasi hanno permesso non invasiva, la valutazione ripetitivo della sopravvivenza cellulare, la posizione e la proliferazione in vivo. Questo video dimostra come monitorare la proliferazione hESC in un topo vivo.

Video: In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

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.

Derivazione di cellule staminali embrionali umane da 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 ...

Ricerca

Biologia

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Sondaggio per mitocondriale complessa attività di cellule staminali embrionali umane

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Etichettatura in vitro di cellule staminali embrionali umane per la Risonanza Magnetica

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Cultura e manutenzione di cellule staminali embrionali umane

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelamento e scongelamento cellule staminali embrionali umane

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

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 Bioinformatica Analisi delle embrionali umane crescita Colony cellule staminali

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.

Differenziazione delle cellule staminali embrionali in precursori degli oligodendrociti

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

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.

Derivazione efficiente dei diritti dell&#39;uomo e precursori cardiaci e cardiomiociti pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

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 ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Riprogrammare cellule somatiche umane in cellule staminali pluripotenti indotte (iPSCs) Utilizzo vettore retrovirale con GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...