Este trabalho foi financiado por uma seqüência Fronteiras NCCR em Genética prêmio piloto, Juvenile Diabetes Research Foundation Grant 41-2009-775 e Grant 12-126875 de Det Frie Forskningsråd / Sundhed og Sygdom. Os autores agradecem o laboratório Spagnoli por sediar a gravação de vídeo.

Este protocolo destina-se a crescer organoids pancreáticas derivados de E10.5 de murino dissociadas células pancreáticas epiteliais. O protocolo exige aprovação ética para experimentação animal. 1. Dissecção da Dorsal pâncreas Bud de E10.5 embriões de camundongos <ol><li> Sacrifício camundongos cronometrado grávidas no dia embrionário (E) 10,5, abrir o abdômen com uma tesoura, retire os dois cornos uterinos e coloque-os em um prato de 10 centímetros de Petri cheia de fosfato frio salina tampão (PBS) ou Dulbecco modificado meio essencial (DMEM) conservados em gelo. O experimento total a partir do sacrifício a semeadura de células é feito em 60-90 min para evitar danos nas células. </li><li> Separe o útero em segmentos de embriões individuais usando uma tesoura pequena. Transferência de um embrião para um prato de 35 milímetros Petri com PBS frio e visualizá-lo sob um microscópio de dissecação com iluminação de cima. Com uma pinça de dissecção remover o músculo, decídua e gema em torno sac e expor o embrião. Coloque o embrião de volta para o meio de DMEM frio. Os embriões podem ser facilmente transferidas usando um 3 ml de transferência de pipeta de plástico / conta-gotas. Isolar cada embrião indivíduo de uma forma semelhante, antes de continuar. </li><li> Coloque um embrião em um ambiente limpo 35 milímetros placa de Petri em PBS. <ol><li> Use uma pinça fina (largura 0,05 mm) para remover o membro anterior. Suavemente inserir a pinça na abertura para retirar o tracto digestivo da região da medula espinhal (Figura 1). Opcional: Para ver mais convenientemente o estômago, remover a parte superior do corpo do embrião, para baixo para o coração e a região da cauda a seguir a haste de gema. </li><li> Localize o estômago, fígado e intestino. Usando fórceps, isolar o tracto gastrointestinal a partir do estômago para o intestino e colocá-lo em DMEM frio em gelo (Figura 1). O botão de pâncreas dorsal está ligado dorsalmente, posterior ao estômago (Figura 1). </li><li> Isolar cada trato gastrointestinal individual em um similar forma antes de continuar. Se os embriões precisam ser genotipados, recolher a cauda no momento do esvaziamento e manter cada tracto gastrointestinal num indivíduo bem diferente em uma placa de 24 poços com DMEM frio em gelo. </li></ol></li><li> Coloque um trato gastrointestinal em um ambiente limpo 35 milímetros placa de Petri em PBS frio. Agora use iluminação abaixo em campo claro de visualizar e dissecar o dorsal gemas pancreático sob o microscópio de dissecação. Estas condições irão optimizar a visualização de volumes. Usando agulhas de tungstênio eletroliticamente-afiadas ou 20 g de agulhas de seringa, isolar o broto pancreático com tão pouco quanto possível mesenchyme ao redor do epitélio (Figura 1). <ol><li> Transferir o botão isolado em uma placa de Petri contendo uma solução fria de dispase (1,25 mg / ml) durante 2-3 min. A partir deste ponto, a transferência pode ser realizada mais convenientemente com capilares de vidro de 50 mL puxou-chamas ligados a um tubo flexível controlada aftosa. Em alternativa, mas com mais risk de perder pela raiz, use a 10 mL pipeta automática (Pipetman) com pontas de plástico apropriados. </li><li> Realize bud aspiração e ejeção de pâncreas sob controle microscópico. Coloque o broto pancreático volta em PBS. Além disso limpar a gema de pâncreas isolado do mesênquima com as agulhas e aspiração suave usando o capilar de vidro (Figura 1). </li><li> Quando todo o mesênquima é removido, lave o broto pancreático em PBS frio; transferir cada broto de DMEM frio em poços individuais (60 poços mini-bandejas cheias com 10 ml de DMEM frio). É importante não remover o mesênquima em dispase, o que torna o tecido muito pegajosa. </li></ol></li></ol> 2. Chapeamento e Cultura de células dispersas <ol><li> Transfira o epitélio dissecado de todos os embriões com um capilar de vidro, puxado-chama em poços cônicos de 60 poços mini-bandejas cheias de 10 mL PBS para enxaguar. <ol><li> Transferir o botão em 10 ul de tripsina a 0,05% de umnd deixe incubar a 37 ° C durante 4 min. Inactivar a tripsina pela transferência do broto para um poço com 10 mL de DMEM + 10% soro fetal bovino (FCS). </li><li> Dissociar a suspensão de células por aspiração através de um capilar fino puxado com um puxador de pipeta. É importante evitar bolhas nesta fase, enquanto pipetando para cima e para baixo para dissociar as células. Organoids Pâncreas optimamente começar a partir de pequenos grupos de 5-15 células e, portanto, a dissociação parcial é recomendada (Figura 1). </li></ol></li><li> Reunir as células de vários embriões para um tubo Eppendorf, a fim de minimizar as diferenças devidas ao processamento individual. Dilui-se a suspensão de células em Matrigel refrigerados a uma razão de 1:3. Aliquota desta mistura para uma placa de 96 poços, 8 ul / poço ou numa placa optimizado para imagiologia (ver abaixo). </li><li> Incubar a placa a 37 ° C durante 5 minutos, permitindo que o Matrigel para engrossar. Preencha os poços com 70 mL de meio de escolha (organóides ou esfera, consulte Tables 1 e 2) e deixar em um ambiente humidificado contendo 5% de CO 2 e 95% de ar a 37 ° C. </li><li> Substituir o meio de cada 4 º dia. Monitorar as crescentes colônias pancreáticas diário e documentar o processo de geração de imagens. </li><li> As moléculas pequenas ou proteínas de interesse pode ser adicionado ao meio, nesta fase, para as experiências de interferência, como relatado anteriormente 28. </li></ol> Tabela 1: médio organóides. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Nome de Materiais </td><td> Banco de Concentração </td><td> Concentração na mídia final </td><td> Volume de estoque </td></tr><tr><td> Penicilina-Estreptomicina </td><td> 100% </td><td> 1% </td><td> 50 ul </td></tr><tr><td> KnockOut substituição Serum (suplemento) </td><td> 100% </td><td> 10% </td><td> 500 ul </td></tr><tr><td> 2-mercaptoetanol </td><td> 14,3 M </td&gt; <td> 0,1 mM </td><td> 1 ul </td></tr><tr><td> Phorbol Myristate acetato (PMA) </td><td> 16 uM </td><td> 16 nM </td><td> 5 ul </td></tr><tr><td> Y-27632 (inibidor ROCK) </td><td> 50 mM </td><td> 10 uM </td><td> 1 ul </td></tr><tr><td> EGF </td><td> 50 ng / mL </td><td> 25 ng / mL </td><td> 2,5 mL </td></tr><tr><td> Recombinante Humana R-espondina 1 </td><td> 250 ug / ml </td><td> 500 ng / mL </td><td> 10 ul </td></tr><tr><td> - Ou - </td><td></td><td></td><td></td></tr><tr><td> Recombinante do rato R-espondina 1 </td><td> 250 ug / ml </td><td> 500 ng / mL </td><td> 10 ul </td></tr><tr><td> Recombinante FGF1 humano (aFGF) </td><td> 100 ug / ml </td><td> 25 ng / mL </td><td> 1,25 mL </td></tr><tr><td> A heparina (Liquemine) </td><td> 2500 U / ml </td><td> 2,5 U / mL </td><td> 2 ul </td></tr><tr><td> RecomFGF10 Humano binant </td><td> 100 ug / ml </td><td> 100 ng / mL </td><td> 5 ul </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4,412.25 mL </td></tr><tr><td> Total </td><td></td><td></td><td> 5000 mL </td></tr></tbody></table> Tabela 2: médio Sphere. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Nome de Materiais </td><td> Banco de Concentração </td><td> Concentração na mídia final </td><td> Volume de estoque </td></tr><tr><td> Penicilina-Estreptomicina </td><td> 100% </td><td> 1% </td><td> 50 ul </td></tr><tr><td> B27 x50 (suplemento) </td><td> 100% </td><td> 10% </td><td> 100 ul </td></tr><tr><td> Recombinante FGF2 humano (bFGF) </td><td> 100 ug / ml </td><td> 64 ng / mL </td><td> 3,2 mL </td></tr><tr><td> Y-27632 (inibidor ROCK) </td><td> 50 mM </td><td> 10 uM </td><td> 1 ul </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4845,8 mL </td></tr><tr><td> Total </td><td></td><td></td><td> 5000 mL </td></tr></tbody></table> 3. Imagem da Progressão de Desenvolvimento organoide <ol><li> Imagem organoids quer diárias ou por microscopia de lapso de tempo usando um microscópio fluorescente time-lapse. Por lapso de tempo de imagem, use um XY (Z) microscópio de fluorescência invertido automatizado. </li><li> Depósito pequenas gotículas de 3 mL em placas de 4 poços ou placas com fundo de vidro preenchidos com 2-5 ml médio. Imagem com um objetivo de longa distância 10x. Nota: Os ratinhos transgénicos que expressam marcadores fluorescentes podem ser usados. Filme 1 mostra, por exemplo, a expansão inicial de organoids de Pdx1-Ngn3-ER TM-ires-nGFP + camundongos 4. A GFP nuclear permite ao utilizador controlar as células como objectos individuais, mas os mesmos princípios podem ser aplicados para controlar as células com fluorescência de membrana taiscomo MT / MG camundongos 29. </li><li> Comece lapso de tempo de imagem de 3 horas após a semeadura das células a fim de evitar desvios de foco e definir a automação controlar software para tirar uma foto / h por 3 ou mais dias, em posições definidas manualmente. Para cada posição, adquirir uma interferência diferencial contraste da imagem (DIC) bem como o sinal de GFP, a expressão do marcador fluorescente relatórios. </li></ol> 4. Recuperação de Organóides para Histologia <ol><li> Colocar a placa de 96 poços sobre gelo e remover o meio, substitui-lo com PBS gelado. Este despolimeriza parcialmente Matrigel. </li><li> Gentilmente aspirar cada organoide individual, removendo o Matrigel circundante usando uma ponta 1.000 mL, a fim de não perturbar a arquitetura geral. Transferir cada organóide para um poço com PBS gelado. Manter a placa de gelo. Fixação direta em Matrigel também é possível. </li><li> Fixe o organoide por 15 min em 4% paraformaldeído (PFA), criopreservar-lo em sacarose e incorporá-lo em gelatina. Processar cadaorganóide para cryosectioning e histologia, como descrito anteriormente (Johansson et al., 2007) 4. </li></ol> 5. Recuperação de Organóides por PCR e Bioquímica <ol><li> Colocar a placa de 96 poços sobre gelo e remover o meio. Adicionar 60 ul de RNAlater por poço, de modo a estabilizar e proteger o ARN celular. </li><li> Interromper o gel em cada poço mecanicamente por despolimerização que parcialmente em gelo. Ou recuperar organoids individuais usando uma ponta de 1.000 mL, a fim de não perturbar a arquitetura global ou recuperar todo o bem (com Matrigel) por perturbar o gel mecanicamente com um mL ponta 200. Utilizar uma ponta 1000 ul de transferir o conteúdo do poço para um tubo Eppendorf livre de não-pegajoso RNAse mantido em gelo. </li><li> Lavar os poços com 60 ul RNAlater e adicionar o restante conteúdo para o mesmo tubo de Eppendorf. </li><li> Girar os tubos durante 5 min a 500-1000 x g a 4 ° C. </li><li> Remover o sobrenadante, deixando apenas 20-30 mLde RNAlater no tubo juntamente com o pellet. Para bioquímica, armazenar as amostras o mais seco possível. </li><li> Não congelar amostras em RNAlater imediatamente; armazenar a 4 ° CO / N (para permitir RNAlater para penetrar profundamente no tecido). O tecido pode ser armazenada a -20 ° C para armazenamento a longo prazo e pode ser posteriormente processado para a ruptura de tecido e a extracção de pequenas quantidades de ARN. </li></ol>

<ol>
	<li>Pagliuca, F. W., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+functional+beta-cell.">How to make a functional beta-cell.</a> Development. 140, 2472-2483 (2013).</li><li>Percival, A. C., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+pancreatic+development+using+a+cell+lineage+label.">Analysis of pancreatic development using a cell lineage label.</a> Exp Cell Res. 247, 123-132 (1999).</li><li>Attali, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+beta-cell+differentiation+by+the+pancreatic+mesenchyme.">Control of beta-cell differentiation by the pancreatic mesenchyme.</a> Diabetes. 56, 1248-1258 (2007).</li><li>Johansson, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+control+of+neurogenin3+activity+in+pancreas+progenitors+reveals+competence+windows+for+the+generation+of+different+endocrine+cell+types.">Temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types.</a> Dev Cell. 12, 457-465 (2007).</li><li>Speier, S., Rupnik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+in+situ+characterization+of+pancreatic+beta-cells.">A novel approach to in situ characterization of pancreatic beta-cells.</a> Pflugers Arch. 446, 553-558 (2003).</li><li>Golosow, N., Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitheliomesenchymal+interaction+in+pancreatic+morphogenesis.">Epitheliomesenchymal interaction in pancreatic morphogenesis.</a> Dev Biol. 4, 242-255 (1962).</li><li>Miralles, F., Czernichow, P., Scharfmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follistatin+regulates+the+relative+proportions+of+endocrine+versus+exocrine+tissue+during+pancreatic+development.">Follistatin regulates the relative proportions of endocrine versus exocrine tissue during pancreatic development.</a> Development. 125, 1017-1024 (1998).</li><li>Petzold, K. M., Spagnoli, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+ex+vivo+culturing+of+embryonic+pancreas.">A system for ex vivo culturing of embryonic pancreas.</a> J. Vis. Exp. , 3979 (2012).</li><li>Miralles, F., Battelino, T., Czernichow, P., Scharfmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+plays+a+key+role+in+morphogenesis+of+the+pancreatic+islets+of+Langerhans+by+controlling+the+activity+of+the+matrix+metalloproteinase+MMP-2.">TGF-beta plays a key role in morphogenesis of the pancreatic islets of Langerhans by controlling the activity of the matrix metalloproteinase MMP-2.</a> J Cell Biol. 143, 827-836 (1998).</li><li>Hope, K., Bhatia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+interrogation+of+stem+cells.">Clonal interrogation of stem cells.</a> Nat Methods. 8, 36-40 (2011).</li><li>Sugiyama, T., Rodriguez, R. T., McLean, G. W., Kim, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+markers+of+fetal+pancreatic+epithelium+permit+prospective+isolation+of+islet+progenitor+cells+by+FACS.">Conserved markers of fetal pancreatic epithelium permit prospective isolation of islet progenitor cells by FACS.</a> Proc Natl Acad Sci U S A. 104, 175-180 (2007).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Dontu, G., et al. <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+propagation+and+transcriptional+profiling+of+human+mammary+stem/progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.</a> Genes Dev. 17, 1253-1270 (2003).</li><li>Smukler, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+mouse+and+human+pancreas+contain+rare+multipotent+stem+cells+that+express+insulin.">The adult mouse and human pancreas contain rare multipotent stem cells that express insulin.</a> Cell Stem Cell. 8, 281-293 (2011).</li><li>Seaberg, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+identification+of+multipotent+precursors+from+adult+mouse+pancreas+that+generate+neural+and+pancreatic+lineages.">Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.</a> Nat Biotechnol. 22, 1115-1124 (2004).</li><li>Jin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colony-forming+cells+in+the+adult+mouse+pancreas+are+expandable+in+Matrigel+and+form+endocrine/acinar+colonies+in+laminin+hydrogel.">Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel.</a> Proc Natl Acad Sci U S A. 110, 3907-3912 (2013).</li><li>Sugiyama, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+pancreas+development+from+purified+progenitor+cells+reveals+genes+essential+for+islet+differentiation.">Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation.</a> Proc Natl Acad Sci U S A. 110, 12691-12696 (2013).</li><li>Huch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unlimited+in+vitro+expansion+of+adult+bi-potent+pancreas+progenitors+through+the+Lgr5/R-spondin+axis.">Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.</a> Embo J. , (2013).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. , (2009).</li><li>Ootani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+in+vitro+intestinal+epithelial+culture+within+a+Wnt-dependent+stem+cell+niche.">Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.</a> Nat Med. 15, 701-706 (2009).</li><li>Barker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lgr5(+ve)+stem+cells+drive+self-renewal+in+the+stomach+and+build+long-lived+gastric+units+in+vitro.">Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.</a> Cell Stem Cell. 6, 25-36 (2010).</li><li>Huch, M., et al. <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+expansion+of+single+Lgr5++liver+stem+cells+induced+by+Wnt-driven+regeneration.">In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.</a> Nature. 494, 247-250 (2013).</li><li>Lukacs, R. U., Goldstein, A. S., Lawson, D. A., Cheng, D., Witte, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+cultivation+and+characterization+of+adult+murine+prostate+stem+cells.">Isolation, cultivation and characterization of adult murine prostate stem cells.</a> Nat Protoc. 5, 702-713 (2010).</li><li>Rock, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+cells+as+stem+cells+of+the+mouse+trachea+and+human+airway+epithelium.">Basal cells as stem cells of the mouse trachea and human airway epithelium.</a> Proc Natl Acad Sci U S A. 106, 12771-12775 (2009).</li><li>Eiraku, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472, 51-56 (2011).</li><li>Spence, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+pluripotent+stem+cells+into+intestinal+tissue+in+vitro.">Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.</a> Nature. 470, 105-109 (2011).</li><li>Lancaster, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 10, (2013).</li><li>Greggio, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+three-dimensional+niches+deconstruct+pancreas+development+in+vitro.">Artificial three-dimensional niches deconstruct pancreas development in vitro.</a> Development. 140, 4452-4462 (2013).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li></ol>

The authors have nothing to disclose.

A produção em grande escala de células beta funcionais in vitro ainda é ineficaz 1. Neste contexto desafiador, estudos de biologia do desenvolvimento pode ajudar a decifrar os sinais exatos que são necessários para a diferenciação das células beta funcionais. Este protocolo permite a manutenção, expansão e diferenciação de células progenitoras pancreáticas embrionárias in vitro. Isso inclui a formação de células beta produtoras de insulina que não co-expressar outros hor...

Cultura órgão fornece um modelo útil que preenche as lacunas entre o complexo, mas altamente relevante em investigações in vivo ea simulação conveniente, mas aproximado de modelos da linha celular. No caso do pâncreas, não existe uma linha de células perfeitamente equivalente a progenitores pâncreas embora existam linhas de células transformadas que simulam células endócrinas e exócrinas. Todo o pâncreas adulto não podem ser cultivadas; ilhotas isoladas endócrinos pode ser mantida durante algumas semanas, sem proliferação celular e fatias de tecidos podem ser mantidos in vitro durante 5 horas. Cultura pâncreas embrionário tem sido amplamente utilizado, não só para estudar o seu desenvolvimento, mas também para investigar interacções epiteliais mesenquimais 4,6,7, a imagem processa 8 ou para interferir quimicamente com eles 9. Dois métodos de cultura de órgãos são usados ​​principalmente: a primeira consiste na cultura de gomos pancreáticas em placas revestidas com fibronectina 2, que é convenient para fins de imagem; a segunda opção é a cultura dos órgãos de filtros na interface ar-líquido 3,4 que melhor preserva a morfogênese. Apesar de muito úteis, estes métodos levam a um certo grau de achatamento; a expansão de células progenitoras é muito limitado em comparação com o normal desenvolvimento e a população de partida é complexo que compreende todos os tipos de células pancreáticas e células mesenquimais. A capacidade de cultura e expandir células primárias dispersas é valioso para estudar relações de linhagem e descobrir as propriedades intrínsecas dos tipos de células isoladas 10. Sugiyama et al. 11 poderia manter progenitores pâncreas e progenitores endócrinas que retiveram alguns personagens funcionais por 3-5 dias em cultura em camadas alimentadoras. Pancreatospheres, semelhante ao neurospheres 12 e mammospheres 13, foram ampliados a partir de ilhotas adultas e células ductais embora a natureza dos progenitores / células-troncoque geram essas esferas não é clara. Além disso, em contraste com o desenvolvimento fisiológico, os pancreatospheres continha alguns neurónios 14,15. Spheres também foram recentemente produzidos a partir de células progenitoras embrionárias pâncreas 16,17 e regenerar pancreata 18 com boa expansão e diferenciação de células progenitoras subseqüente, mas não conseguiu recapitular morfogênese. Modelos 3D a partir de células dispersas e, muitas vezes definidos que se auto-organizam em órgãos miniaturizados recentemente floresceu e simular desenvolvimento ou adulto o volume de negócios de vários órgãos, como o intestino 19,20, o estômago 21, o fígado 22, a próstata 23 e traquéia 24. Em alguns casos, a morfogénese e diferenciação de desenvolvimento foram recapitulado em 3D a partir de células ES, como é o caso dos copos óptica 25, 26 do intestino ou do cérebro 27. Aqui, desCribe um método para expandir progenitores multipotentes pancreáticas dissociadas em um andaime Matrigel 3D onde podem diferenciar e se auto-organizar.

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			<td>E9644-2MG</td>
			<td>Stock keept at -80&#176;C</td>
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			<td>Recombinant Human R-spondin 1</td>
			<td>R&#38;D</td>
			<td>4645-RS-025/CF</td>
			<td>Stock keept at -80&#176;C</td>
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			<td>&#160;- or -&#160;</td>
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			<td>3474-RS-050</td>
			<td>Stock keept at -80&#176;C</td>
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			<td>Recombinant Human FGF1 (aFGF)</td>
			<td>R&#38;D</td>
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			<td>Stock keept at -80&#176;C- do not include to increase beta cell production</td>
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			<td>Heparin (Liquemin)</td>
			<td>Drossapharm</td>
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			<td>Stock keept at 4&#176;C</td>
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			<td>Recombinant Human FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG-025</td>
			<td>Stock keept at -80&#176;C</td>
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			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
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			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
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			<td>B27 x50 (supplement)</td>
			<td>Gibco</td>
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			<td>Stock keept at -20&#176;C</td>
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			<td>Recombinant Human FGF2 (bFGF)</td>
			<td>R&#38;D</td>
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			<td>Stock keept at -80&#176;C</td>
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			<td>Y-27632 (ROCK inhibitor)</td>
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			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
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			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Stock keept at -20&#176;C</td>
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			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Stock keept at 4&#176;C</td>
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		<tr>
			<td>RNAlater - RNA stabilizing reagent</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>Store at room temperature</td>
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			<td>Dispase&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D4818-2MG</td>
			<td>Stock keept at -20&#176;C</td>
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			<td>BSA for reconstitution</td>
			<td>Milipore</td>
			<td><a href="http://www.millipore.com/catalogue/item/81-068-3">81-068</a></td>
			<td>For reconstituition of cytokines&#160; - Stock keept at -20&#176;C</td>
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			<td>Fetal calf serum (FCS)</td>
			<td>Gibco</td>
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			<td>Stock keept at -20&#176;C</td>
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			<td>60 well MicroWell trays</td>
			<td>Sigma Aldrich</td>
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			<td>4-well plates</td>
			<td>Thermo Scientific</td>
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			<td>95-well plates F bottom</td>
			<td>Greiner Bio</td>
			<td>6555180</td>
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			<td>Glas bottom plates</td>
			<td>Ibidi</td>
			<td>81158</td>
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			<td>Disposal micropittes</td>
			<td>Blaubrand</td>
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in vitro pancreas organogenesis from dispersed mouse embryonic progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

E10.5 dorsais progenitores pancreáticas dissociadas e semeadas em Matrigel 3D recapitular desenvolvimento pâncreas. Progenitores pode ser mais fácil de ser seguido com repórteres fluorescentes. No nosso caso, usamos um camundongo transgênico que expressa uma proteína GFP nuclear controlada por Pdx1 promotor (Pdx1-Ngn3-ER TM-nGFP) (Filme 1), na ausência de tamoxifeno e, portanto, sem ativar Neurog3 4 (Figura 2). Com o meio de org...

Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

O método de cultura tridimensional descrito neste protocolo recapitula o desenvolvimento do pâncreas dispersos progenitores rato pâncreas embrionárias, incluindo a sua expansão substancial, a diferenciação ea morfogênese em um órgão ramificada. Este método é passível de imagiologia, interferência funcional e a manipulação do nicho.

In Vitro Pâncreas Organogenesis de dispersos rato embrionárias Progenitores

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

in-vitro-pancreas-organogenesis-from-dispersed-mouse-embryonic

Research

JoVE Journal

Biology

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

13.7K visualizações.  Swiss Institute for Experimental Cancer Research.  O método de cultura tridimensional descrito neste protocolo recapitula o desenvolvimento do pâncreas dispersos progenitores rato pâncreas embrionárias, incluindo a sua expansão substancial, a diferenciação ea morfogênese em um órgão ramificada. Este método é passível de imagiologia, interferência funcional e a manipulação do nicho. 

Vídeo: In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Na diferenciação in vitro de rato-tronco embrionárias (MES) células usando o método da gota de suspensão

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Biologia

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Gravações de uma única célula de sucção de cones fotorreceptores rato

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo de imagens e tratamentos terapêuticos em um modelo de camundongo ortotópico de câncer de ovário

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purificação de Progenitores do músculo esquelético

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Um Sistema de<em&gt; Ex vivo</em&gt; A cultura de Pâncreas embrionárias

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Rotulagem celular e injeção em Desenvolvimento embrionárias corações do rato

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolamento de RNA Rato Pâncreas: Um tecido rico em Ribonuclease

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Isolamento de proteínas a partir da Embryonic Rato Coração Região Válvula Desenvolvimento

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Procedimento para avaliar a eficiência de floculantes para a Remoção de partículas dispersas de extractos de plantas

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...