Graças a Tim Doyle, PhD e do Centro de Stanford para In Vivo Imaging para assistência com a imagem de bioluminescência. Também graças à Ngan Huang, PhD para compartilhar sua técnica sobre células-tronco co-injeção com uma solução matriz. Por último, graças a Steve Felt, Ph.D. de assistência com cuidados animais veterinária.

<ol><li> Construção de Gene Double Fusion Reporter <ol><li> A fim de realizar imagem de bioluminescência de células estaminais embrionárias humanas, é preciso primeiro obter células que expressam de forma estável um gene repórter luciferase, como vaga-lume luciferase dirigido por um promotor constitutivo como Ubiquitina ou EF1a. </li><li> O foco deste protocolo é em aplicações gene repórter, então os procedimentos detalhados não são fornecidos aqui. No entanto, a estratégia geral do nosso laboratório é usar um vetor de construção de dupla fusão que contém firefly luciferase (flutuações) e proteína verde fluorescente melhorada (EGFP) separados por um espaçador dentro pCDNA 3.1 +. </li><li> Resumidamente, o nosso gene de fusão dupla foi originalmente localizada a jusante do promotor citomegalovírus em pCDNA 3.1 (+), de modo que o fragmento foi retirado 3,3 kbp usando NdeI e NotI digestão e finais blunt ligada no sítio múltiplo de clonagem de uma auto-inativação SIN ( ) vector lentiviral, sob controle de um promotor da ubiquitina. </li></ol></li><li> Transdução lentiviral <ol><li> hESCs pode ser transduzidas 3-5 dias após a passagem de uma multiplicidade de infecção (MOI) de 10 (título viral de cerca de 107 incubadas com 106 células). </li><li> O estoque viral descongeladas podem ser adicionados diretamente à mídia CTEh fresco. </li><li> Atualizar o media 12 e 24 horas depois. </li><li> Após 48 h, a eficiência de transdução pode avaliados qualitativamente por meio de microscopia de fluorescência. Posteriormente, FACS pode ser usada para isolar as células infectadas. </li></ol></li><li> HESCs cultura e Introdução à Imagem Set-up <ol><li> Cultura da sua luciferase hESCs positiva no alimentador sem condições. Normalmente, seguir o protocolo WiCell padrão e utilizar placas de 6 poços revestida em fator de crescimento reduzida Matrigel, usando meios condicionados ou comercial, alimentador de mídia livre. </li><li> De detectar as células luciferase positivo, você precisará ter a sonda repórter D-luciferina já preparado. Para fazer isso luciferina alíquota, em mL 1-1,5 preparações numa concentração final de 45 mg / mL. Manter as alíquotas a -20 ° C, quando não em uso e evitar a exposição à luz, cobrindo com toalha de papel quando não estiver em armazenamento. </li><li> Para visualizar as células luciferase positivo, usamos IVIS 50 e 200 IVIS Imaging Systems (Xenogen Corporation, Alameda, CA). Este último sistema inclui um aparelho integrado isoflourane e câmara de indução para a anestesia temporária de pequenos animais. </li></ol></li><li> Na geração de imagens vitro bioluminescência de hESCs <ol><li> In vitro para a imagem de bioluminescência, é importante manter condições estéreis. Portanto, o sistema de imagem deve ser estéril, de preferência em uma sala de cultura celular. </li><li> Antes de imagem, remova a mídia de células e adicionar PBS suficiente apenas para cobrir as células. Por exemplo, adicione 1 mL de PBS a cada poço de uma placa de 6 poços contendo o seu CTEh culturas. </li><li> A proporção de D-Luciferina a PBS deve ser 1:100, então adicionar 10 mL de descongelado D-Luciferina a cada poço da placa de 6 poços. </li><li> Espere um minuto, em seguida, tomar uma imagem usando um tempo de exposição de um segundo. Se o sinal é fraco, aumentar o intervalo de exposição e tente novamente. </li><li> O sinal bioluminescente reflete o número de células, por isso os ensaios de quantificação pode ser realizada sinal de que se correlacionam com os números de celulares diferentes. </li></ol></li><li> Preparação de rato e células injetando para in vivo de imagens <ol><li> Gene repórter de imagem fornece um método de alto rendimento, barato e sensível para rastreamento de células após o transplante ao longo de dias, semanas e até meses. Isto é especialmente importante uma vez que as células-tronco transplantadas freqüentemente formam teratomas, são rejeitadas pelo sistema imunológico do hospedeiro, ou simplesmente morrer. </li><li> O método mais simples para geração de imagens a formação de teratoma in vivo é injetar hESCs que expressam o gene repórter luciferase vagalume para as costas de camundongos SCID e adquirir imagens bioluminescentes com o sistema IVIS. </li><li> Quando você está pronto para o transplante de células, use dispase ou colagenase para soltar as células, lave várias vezes, e suspendê-las em uma mistura 1:1 de fator de crescimento reduzido Matrigel e DMEM. Normalmente nós primeiro mix as células com DMEM refrigerados e depois adicionar no Matrigel. Para cada local de injeção subcutânea, injetamos 200.000 células suspensas em um volume 5-10 ul da mistura matrigel / DMEM. O número de células pode ser ajustada dependendo da sua aplicação. Lembre-se de manter tudo em gelo antes da injeção. </li><li> Uma vez que as células estão prontas, anestesiar o rato, colocando-o na caixa de indução com isoflurano fluxo ligado. Depois de 1-5 minutos, o mouse está dormindo e pode ser colocado na mesa de operação contínua com isoflurano. </li><li> Porque pele naturalmente auto-fluorescente e pode obscurecer a imagem de bioluminescência, teremos de remover o cabelo do lado de trás do mouse. A maneira mais fácil de fazer isso é usar um barbeador elétrico, mas você também pode usar depilação-géis. Limpe com álcool para esterilizara pele depois </li><li> Em seguida, elaborar a sua suspensão de células em uma seringa. 23-27 agulhas funcionam melhor que elas não irão entupir com as células. Se a agulha é muito grande (&lt;23 gauge) no trato agulha pode deixar um grande buraco através do qual as células podem vazar para fora. </li><li> Injetar as células sob a pele em volta do mouse. Porque a pele é bem solta, use o polegar eo indicador para comprimir e estender a área que deseja injetar. Injetar sob a pele, tomando cuidado para não perfurar muito profundamente. Além disso, tente manter a agulha deslizando para fora do local de injecção enquanto pressiona o êmbolo: isso vai impedir a criação de um buraco na pele através do qual as células podem vazar se você tentar injetar novamente. </li><li> Depois que as células são injetadas, esperar algumas horas para permitir que o mouse para acordar e correr ao redor antes de voltar a anestesiar a imagem de bioluminescência. Fazê-lo evita toxicidade isoflurano. </li></ol></li><li> Em imagem de bioluminescência vivo de células transplantadas <ol><li> Para imagens de toda a bioluminescência de animais, fazemos uma injeção intraperitoneal de D-luciferina em 375 peso corporal mg / kg. Pesar o animal para calcular a dose antes da injeção. </li><li> Injetar o D-luciferina no peritônio, ao largo da linha média. Tenha cuidado para não ir muito fundo ou você pode danificar os órgãos internos. Se o rato começa a acordar, coloque-o novamente na caixa de knockout e esperar um minuto ou dois. Para camundongos, a bioluminescência máximo ocorre geralmente 15-40 minutos após a injeção. </li><li> Lembre-se de colocar papel preto fosco na caixa de imagem para ajudar a absorver toda a luz não é emitida pela hESCs. Posicione o mouse (até backside) na câmara de imagem com isoflurano, em cima do papel preto. Comece com um tempo de exposição de 10 segundos. Se o sinal é saturado, tente diminuir o tempo de exposição, se demasiado fraco, aumentar a exposição. </li><li> Uma vez que o tempo de exposição do sinal foi otimizado para o seu mouse, as imagens começam a tomar a cada minuto até que o sinal atinge um máximo. Este é o melhor feito usando o software de imagem para selecionar &quot;regiões de interesse (ROI)&quot;, que cobrem seus locais de injecção. Ao monitorar a intensidade do sinal em cada ROI você pode facilmente determinar quando o sinal começa a diminuir, indicando que a intensidade máxima do sinal foi atingido. A máxima do sinal deve ser utilizado como seus dados final. </li><li> Quando estiver satisfeito com suas imagens, remova o mouse de isoflurano e permitir que ele acorda em sua gaiola. Normalmente, o mouse deve acordar em 15 minutos. </li><li> Bioluminescência imagem gene repórter pode ser repetida diariamente, semanalmente e mensalmente durante o tempo que o hESCs sobreviver no animal. Sinal de um teratoma desenvolvimento irá aumentar exponencialmente ao longo do tempo, tipicamente da ordem de semanas, como o hESCs começam a proliferar. </li><li> Após o curso de tempo desejado de imagens bioluminescência é adquirido, o animal pode ser sacrificado e secções de tecido usado para histologia. </li></ol></li></ol>

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Em comparação com outras modalidades, tais como PET e RM, a bioluminescência tem resolução espacial limitada penetração nos tecidos e reduzido devido à energia relativamente fraca de fótons emitidos (2-3 eV); por estas razões que até agora não foi aplicável em animais de grande porte. No entanto, a bioluminescência tem a vantagem de ser de baixo custo, alto rendimento, e não-invasivo, tornando-se altamente desejável para in vivo com células-tronco de rastreamento em pequenos animais. Genes não bioluminescência-repórter, ...

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

A descoberta de células estaminais embrionárias humanas (hESCs) aumentou drasticamente as ferramentas disponíveis para cientistas médicos interessados ​​em medicina regenerativa. No entanto, a injeção direta de hESCs e células diferenciadas de hESCs, em organismos vivos até agora tem sido dificultada pela morte celular significativa, a formação de teratoma, e rejeição imune do hospedeiro. Compreensão do comportamento em CTEh vivo após o transplante exige técnicas de imagem inovadora para monitorar longitudinalmente localização CTEh, proliferação e viabilidade. A imagem molecular tem dado investigadores um meio de alto rendimento, baixo custo, e sensível para o rastreamento na proliferação celular vivo ao longo de dias, semanas e até meses. Este avanço tem aumentado significativamente a compreensão da cinética espaço-temporal de enxerto CTEh, proliferação e formação de teratoma, nos indivíduos que vivem. Um grande avanço em imagem molecular tem sido a extensão dos ensaios não-invasivo gene repórter da biologia molecular e celular em plataformas in vivo modalidade multi-imagem. Estes genes repórter, sob controle de promotores e potenciadores de engenharia que se aproveitam da maquinaria transcricional da célula hospedeira s, são introduzidas nas células usando uma variedade de métodos de vetor e vetor não. Uma vez na célula, genes repórter pode ser transcrita ou constitutivamente ou apenas sob determinadas condições biológicas ou celular, dependendo do tipo de promotor usado. Transcrição e tradução de genes repórter em proteínas bioativas é detectada com instrumentação, sensível não-invasiva (por exemplo, câmeras CCD) usando o sinal de geração de sondas, tais como D-luciferina. Para evitar a necessidade de luz de excitação para rastrear as células-tronco in vivo como é necessário para imagens de fluorescência, bioluminescência repórter sistemas de imagem gene exigem apenas uma sonda exogenamente administrada para induzir a emissão de luz. Firefly luciferase, derivado do Photinus firefly pyralis, codifica uma enzima que catalisa D-luciferina ao opticamente metabólito ativo, oxiluciferina. Atividade óptica pode ser monitorada com uma câmera CCD externa. Estavelmente transduzidas células que carregam o repórter construir dentro de seu DNA cromossômico vai passar o repórter construção de DNA para as células filhas, que permitam um acompanhamento longitudinal de sobrevivência e proliferação CTEh in vivo. Além disso, como expressão do produto do gene repórter é necessário para a geração do sinal, apenas as células viáveis ​​pai e filha vai criar sinal de bioluminescência; células apoptóticas ou morto, não. Neste vídeo, os materiais e métodos específicos necessários para a proliferação de rastreamento de células-tronco e formação de teratoma com a imagem de bioluminescência será descrito.

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

Com o crescente interesse em terapias com células-tronco, técnicas de imagem molecular são ideais para monitorar o comportamento de células-tronco após o transplante. Genes repórter luciferase permitiram não-invasivo de avaliação, repetitiva de sobrevivência da célula, localização e proliferação in vivo. Este vídeo vai demonstrar como controlar a proliferação CTEh em um rato vivo.

In vitro e in vivo de imagens Gene Reporter bioluminescência de células estaminais embrionárias humanas

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

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JoVE Journal

Biology

21.7K visualizações.  Stanford University School of Medicine. Com o crescente interesse em terapias com células-tronco, técnicas de imagem molecular são ideais para monitorar o comportamento de células-tronco após o transplante. Genes repórter luciferase permitiram não-invasivo de avaliação, repetitiva de sobrevivência da célula, localização e proliferação in vivo. Este vídeo vai demonstrar como controlar a proliferação CTEh em um rato vivo.

Vídeo: 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.

Derivação de células estaminais embrionárias humanas por 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 ...

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.

Sondagem para a Atividade Complexo mitocondrial em células estaminais embrionárias humanas

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.

Na rotulagem in vitro de células estaminais embrionárias humanas para Ressonância Magnética

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 Manutenção de células estaminais embrionárias humanas

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 descongelamento, células estaminais embrionárias humanas

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.

Vídeo Bioinformática Análise de Crescimento Humano embrionárias Colony Stem Cell

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.

Diferenciação de células-tronco embrionárias em precursores oligodendroglial

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.

Derivação eficiente dos recursos humanos Precursores cardíaca e Cardiomyocytes pluripotentes a partir de células estaminais embrionárias humanas com a indução de moléculas pequenas

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.

Reprogramação de células somáticas humanas em células tronco pluripotentes induzidas (iPSCs) usando vetor retroviral com 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 de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto mRNA

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