Os autores agradecem Andrea Axtell, Satoshi Itoh, MD, Jeff Velotta, MD, Grant Hoyt, Robert C. Robbins, MD, Jin Yu, MD, Tim Doyle, PhD, e Stanford Núcleo de Imagem de Pequenos Animais para assistência técnica. Os autores também agradecem AM Bickford, Inc. para suporte de equipamentos veterinários. Esta pesquisa foi suportada por concessões de pesquisa do National Institutes of Health (R01 HL-75774, R01 CA098303, R21 HL085743, 1K12 HL087746), o Programa de Pesquisa do Tabaco Califórnia Related Disease da Universidade da Califórnia (15IT-0257 e 1514RT-0169) , e do Instituto de Medicina Regenerativa da Califórnia (RS1-00183). NH é apoiado por uma bolsa da American Heart Association. pode Heart Association.

1. Diferenciação dos CES murino em células endoteliais <ol><li> O protocolo para a diferenciação em células endoteliais CES é descrito em outros lugares e não é o foco deste protocolo 2,3. Resumidamente, as células podem se diferenciar, e as células que são positivas para os marcadores endoteliais, tais como CD31 ou caderina endotelial vascular (VE-caderina) são purificados por separação de células ativadas por fluorescência (FACS). </li></ol> 2. Construção da Double Fusion Gene Reporter e Transdução Lentivirus <ol><li> Bioluminescência pode ser usado para rastrear as células que são modificados para expressar genes repórter, como vaga-lume luciferase (flutuações). Para esta aplicação, as nossas células contêm flutuações e aprimorados fluorescência verde da proteína do gene de fusão (EGFP) sob o controle de um promotor interno ubiquitina. , A fim de modificar as células, o vetor de transgene lentiviral contendo a chave otimizado elementos genéticos para cumprir os critérios de biossegurança, bem como a eficiência de transdução de aumento, UFP-FG vector desenvolvidos em nosso laboratório carregando a flutuações de eGFP gene repórter de fusão podem ser usadas para estável transdução de células após a diferenciação. Esta construção de fusão permite tanto a bioluminescência e rastreamento de fluorescência das células transplantadas. O procedimento para a geração de partículas virais, transdução e produção de células marcadas que os genes repórter expressar é descrito em outra parte 4. </li></ol> 3. Transplante de ESC-derivados de células endoteliais ao hindlimb isquêmica <ol><li> Iniciar o procedimento, preparando um rato que sofreu isquemia hindlimb para o transplante de células. Para fazer isso, coloque o mouse na câmara de indução anestésica contendo 1-3% de isoflurano em oxigênio a 100% a taxa de fluxo de 1l/min. </li><li> Deixe o mouse na câmara de indução até que ele não responde a estímulos externos. Em seguida, retire o animal da câmara de indução. </li><li> Em seguida, coloque o animal em decúbito dorsal sobre a mesa de operação e conectá-lo a um fluxo contínuo de 1-3% isoflurano em oxigênio a 100% a taxa de fluxo de 1l/min. </li><li> Limpe a pele do membro posterior, com três esfrega alternando betadine e álcool. </li><li> Quando a pele é limpa, obter um milhão ESC derivados de células endoteliais em 30 mL de tampão fosfato (PBS). Carregar essas células em uma agulha de calibre 28. </li><li> Quando as células estão prontos, levante com cuidado e ampliar a hindlimb para melhor visualizar a localização do músculo gastrocnêmio. Enquanto a perna é estendida, insira a agulha através da pele até o músculo subjacente. Tome cuidado para não abordar o osso. Suavemente e injecte lentamente a mistura de células 30 mL para o gastrocnêmio. Por via intramuscular, 30 mL está perto do limite de volume que pode ser injetado. Portanto, calibre 28 seringas de insulina são preferidas porque, em nossa experiência, eles eliminam o volume de perda de agulhas de seringa. </li><li> Após a injeção é completa, devolva o mouse para a gaiola de recuperação e monitorá-lo continuamente até que desperto. Permitir que o animal se recuperar durante várias horas e depois prosseguir com a imagem de bioluminescência vivo das células transplantadas. </li></ol> 4. Imagem de bioluminescência de ESC-derivados de células endoteliais in vivo <ol><li> O transplante de células-ESC derivados endothelials foram modificados para expressar tanto flutuações eo gene de fusão eGFP sob o controle de um promotor interno ubiquitina. Portanto, a bioluminescência pode ser usado para rastrear as células dentro do hindlimb isquêmica. </li><li> Para começar esta etapa, ligue o sistema de imagem de bioluminescência e software Vivendo aquisição de imagens. Em seguida, inicializar o sistema de aquisição e especificar as dimensões do campo de visão. </li><li> Em seguida, coloque papel preto fosco para a caixa de imagem para absorver emissões de fundo. </li><li> Uma vez que a caixa de imagem está pronta, coloque o mouse para dentro da câmara de indução anestésica contendo 1-3% de isoflurano em oxigênio na saída de 1l/min. Deixe o mouse na câmara de indução até que ele não responde a estímulos externos. Em seguida, retire o animal da câmara de indução. </li><li> Remover os pêlos de ambos os membros posteriores usando um barbeador elétrico, se necessário. </li><li> Injectar 10 mL de D-luciferina por grama de peso corporal para o peritônio. A D-luciferina é preparado com antecedência em soluções estoque filtrada de 15 mg / mL em PBS. </li><li> Uma vez que a luciferina é injetado, coloque o animal na caixa de imagem sobre o papel preto em decúbito dorsal, ligada a um fluxo contínuo de isoflurano. </li><li> Colocando o animal em caixa de imagem e conexão com isoflurano. </li><li> Começar a adquirir imagens de 10-60 segundos a fim de determinar um tempo de exposição ideal para que a imagem não está saturado. Se a imagem se torna saturado, reduzir o tempo de exposição. Se o sinal de bioluminescência é muito fraco, aumente o tempo de exposição. </li><li> Using o tempo de exposição ideal, continue aquisição de imagens a cada 1-3 minutos até que o sinal atinge o máximo e depois se desvanece. Quando a aquisição estiver concluída, salve o arquivo. </li><li> Para analisar os dados, selecionar regiões de interesse (ROIs) que cobrem o local da injeção. Como controle negativo, um ROI semelhante pode ser selecionado para a perna não-operada. Usando o software, medir o brilho total, que é expressa em unidades de fótons / segundo / cm 2 / esterradiano (p / s / cm 2 / sr), para a ROIs para cada ponto no tempo. O valor máximo deve ser usado nos dados finais. Os dados também podem ser exportados para planilha Excel para uso futuro. </li><li> Uma vez que todos os dados são adquiridos, devolver o animal para a gaiola de recuperação e monitorar continuamente até que o desperta animal. </li><li> Repita este procedimento para rastrear as células ao longo do tempo. </li><li> Em um ponto de tempo desejado, o animal pode ser sacrificado para avaliação da função do tecido. </li></ol> 5. Resultados representante <img src="/files/ftp_upload/old/1034_2009_1_22_4/figure 1.png" alt="Figura 1" width="600" height="600" /> Uma imagem de bioluminescência representante de células transplantadas na hindlimb deixou isquêmica é mostrado na Figura 1. Durante a aquisição de bioluminescência, a intensidade vai aumentar com o tempo, eo valor máximo obtido durante o curso do tempo deve ser relatado como o valor final.

<ol>
	<li>Niiyama, H., Huang, N. F., Rollins, M., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+hindlimb+ischemia.">Murine model of hindlimb ischemia.</a> JoVE. , (2008).</li><li>Levenberg, S., Golub, J. S., Amit, M., Itsakovitz-Eldor, J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+derived+from+human+embryonic+stem+cells.">Endothelial cells derived from human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 4391-4396 (2002).</li><li>Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Naito, M., Nakao, K., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flk1-positive+cells+derived+from+embryonic+stem+cells+serve+as+vascular+progenitors.">Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors.</a> Nature. 408, 926-926 (2000).</li><li>De, A., Yaghoubi, S. S., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+lentiviral+vectors+in+noninvasive+molecular+imaging.">Applications of lentiviral vectors in noninvasive molecular imaging.</a> Methods Mol Biol. 433, 177-202 (2008).</li><li>Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., Imaizumi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+endogenous+monocyte+chemoattractant+protein-1+in+ischemia-induced+neovascularization.">Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization.</a> J. Am. Coll. Cardiol. 44, 661-666 (2004).</li><li>Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomy+of+the+laboratory+mouse.">The anatomy of the laboratory mouse.</a> , (1976).</li><li>Contag, P. R., Olomu, I. N., Stevenson, D. K., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+indicators+in+living+mammals.">Bioluminescent indicators in living mammals.</a> Nature Med. 4, 245-247 (1998).</li><li>Ray, P., De, A., Min, J. J., Tsien, R. Y., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tri-fusion+multimodality+reporter+gene+expression+in+living+subjects.">Imaging tri-fusion multimodality reporter gene expression in living subjects.</a> Cancer Res. 64, 1323-1330 (2004).</li><li>Huang, N. F., Lee, R. J., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+physical+regulation+of+stem+cells+and+progenitor+cells:+potential+for+cardiovascular+tissue+engineering.">Chemical and physical regulation of stem cells and progenitor cells: potential for cardiovascular tissue engineering.</a> Tissue Eng. 13, 1809-1823 (2007).</li><li>Cao, F., Lin, S., Xie, X., Ray, P., Patel, M., Zhang, X., Drukker, M., Dylla, S. J., Connolly, A. J., Chen, X., Weissman, I. L., Gambhir, S. S., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualization+of+embryonic+stem+cell+survival,+proliferation,+and+migration+after+cardiac+delivery.">In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery.</a> Circulation. 113, 1005-1114 (2006).</li><li>Wilson, K., Yu, J., Lee, A., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+bioluminescence+reporter+gene+imaging+of+human+embryonic+stem+cells.">In vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells.</a> J Vis Exp. , (2008).</li></ol>

CES são uma fonte de células promissora para o tratamento da isquemia tecidual devido à sua plasticidade de diferenciação e sua capacidade de dar origem a linhagens de células compreendendo todas as três camadas germinativas, incluindo células endoteliais. Para superar as preocupações éticas associadas com os CES, as células-tronco pluripotentes induzidas (iPSCs) pode ser uma alternativa de células-tronco pluripotentes fonte que supera as preocupações éticas. Além CES, as células-tronco adultas, como as células progenitoras endoteliais (EPCs) e células-tronco hematopoéticas (HSCs) também podem ser usados, mas estes tipos de células podem ter efeito terapêutico limitado em pacientes com DAP. Entrega intramuscular de células é minimamente invasivo e fácil de realizar, e este tipo de parto também é propícia para a entrega de fatores solúveis ou plasmídeos. No entanto, para facilitar o acesso das células transplantadas para a entrega, vasculatura sistêmica na artéria femoral ou veia da cauda pode ser usado em vez de injeções intramusculares. Imagem de bioluminescência oferece a vantagem de realizar rastreamento de alto rendimento e não-invasivo de sobrevivência da célula. Quando combinado com os ensaios funcionais, tais como laser Doppler perfusão sanguínea ou análise histológica de neovascularização, essas técnicas em conjunto pode permitir aos pesquisadores avaliar o efeito terapêutico do transplante de células na recuperação de isquemia hindlimb. Em conclusão, nós demonstramos um método simples e reprodutível para a entrega e acompanhamento de ESC-derivados de células endoteliais para o tratamento da isquemia dos membros posteriores.

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<td>Surgical tools</td>
<td>Tool</td>
<td>Fine Science Tools</td>
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<td>Syringe needle</td>
<td>Tool</td>
<td>BD</td>
<td>&nbsp;</td>
<td>28G insulin syringe is preferred</td>
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<td>Phosphate Buffered Saline </td>
<td>Reagent</td>
<td>Invitrogen</td>
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<td>D-luciferin</td>
<td>Reagent</td>
<td>Biosynth International, Inc</td>
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<td>Prepare D-luciferin in advance into filtered stock solutions of 15 mg/mL in PBS</td>
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<td>IVIS 200 Bioluminescence imaging system and acquisition software </td>
<td>Equipment</td>
<td>Xenogen Corporation</td>
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embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia

Doença arterial periférica (DAP) resulta de estreitamento das artérias periféricas que fornecem o sangue oxigenado e nutrientes para as pernas e pés, esta patologia provoca sintomas tais como claudicação intermitente (dor com a caminhada), dolorosos ulcerações isquêmicas, ou mesmo gangrena dos membros em risco. Acredita-se geralmente que o endotélio vascular, uma monocamada de células endoteliais que reveste a superfície luminal de todos os vasos sangüíneos e linfáticos, desempenha um papel dominante na homeostase vascular e regeneração vascular. Como resultado, a base de células-tronco a regeneração do endotélio pode ser uma abordagem promissora para o tratamento da PAD. Neste vídeo, demonstramos o transplante de células-tronco embrionárias (ESC) derivados de células endoteliais para o tratamento da isquemia hindimb unilateral como um modelo de PAD, seguido por não-invasiva de rastreamento de celular homing e sobrevivência por imagem de bioluminescência. Os materiais e procedimentos específicos para entrega de células e de imagem serão descritas. Este protocolo segue a outra publicação, ao descrever a indução de isquemia por hindlimb Niiyama et al 1.

Watch this Scientific Journal Video about Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia at JoVE.com

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

O procedimento cirúrgico para a entrega de células-tronco embrionárias derivadas de células endoteliais para hindlimb isquêmica é demonstrada, com não-invasivo de monitoramento por imagem de bioluminescência.

Embrionárias com células-tronco derivadas de células endoteliais para tratamento da isquemia dos membros posteriores

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

embryonic-stem-cell-derived-endothelial-cells-for-treatment-hindlimb

Nanyang Technological University

Research

JoVE Journal

Biology

11.7K visualizações.  Stanford University. O procedimento cirúrgico para a entrega de células-tronco embrionárias derivadas de células endoteliais para hindlimb isquêmica é demonstrada, com não-invasivo de monitoramento por imagem de bioluminescência.

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

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Propagação de-tronco embrionárias humanas (ES) Células

Biologia

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivação de Células-Tronco Hematopoéticas murino de Células-Tronco Embrionárias

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

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 ...

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 ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Cromatina imunoprecipitação de células estaminais embrionárias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

Modelo murino de isquemia dos membros posteriores

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Preparação Spread cromossômica de células estaminais embrionárias humanas para Cariotipagem

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

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 ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Mapeamento de destino de células estaminais embrionárias humanas por formação de teratomas

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

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

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

Diferenciação eficiente de células-tronco embrionárias em neurônios motores

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