Agradecemos a Laura Clarke pelo seu inestimável apoio. Este trabalho é suportado pelo NCE: Rede de Células-Tronco, CIHR e NIH.

1. Preparar as soluções Dissecting e soluções enzimáticas <ol><li> Faça o fluido cerebral Artificial Spinal (ACSF) eo soro de mídia livre antes do tempo e leve à geladeira. </li><li> Pesar o ácido cinurênico (0,2 mg / mL) e dissolver em 10 mL de hilo ACSF em um banho-maria 37 ° C antes do tempo, uma vez que não se dissolve prontamente. </li><li> Pesar a tripsina (1,33 mg / mL) e hialuronidase (0,67 mg / mL) e coloque em um tubo de 15 mL e mantê-lo a -20 ° C até ser necessário. Adicionar a solução de ácido cinurênico a este tubo e filtrar através de um filtro de seringa de 22 M apenas antes de usar. </li><li> Pesar inibidor de tripsina (Ovamucoid: 1 mg / mL) e dissolver em água morna soro livre de Mídia e filtro com filtro de seringa de 22μm. </li><li> Fazer a media chapeamento: Soro sem mídia contendo FGF2 (10 ng / mL) e heparina (2 mg / mL). </li></ol> 2. Isolar células estaminais da retina do olho do rato <ol><li> Isolamento das células-tronco da retina é realizado em uma sala específica estéril dedicada a experimentos de cultura primária. Antes de iniciar este procedimento, configure o microscópio de dissecação e de luz fria de origem e, em seguida, estabelecer os instrumentos estéreis de dissecação. Cada capa tem um esterilizador quente talão para a esterilização dos instrumentos entre as etapas. </li><li> Vamos isolar células-tronco na retina dos olhos que foram colocados imediatamente em uma placa de Petri estéreis, contendo Fluid Cerebral Artificial Spinal (ACSF) após a sua retirada de camundongos sacrificados de acordo com a ética protocolos animais aprovados. </li><li> Sob o microscópio de dissecação, limpar o olho com uma pinça: livrar-se do cabelo e do tecido conjuntivo que é anexado à córnea / border escleral. Em seguida, transferir o olho a um novo prato com ACSF. </li><li> Enquanto delicadamente segurando o olho parado com uma pinça serrilhada, use ângulo micro-dissecando tesouras para remover os músculos oculares. Remover o nervo óptico, bem como se ele ainda está ligado ao olho. Tente não esmagar o olho durante este processo. Transferência de olhos para um novo prato com ACSF. </li><li> Use próxima curva micro-dissecando tesoura para cortar o olho ao meio: começar no nervo óptico e passou no meio da córnea, reunião de volta ao nervo óptico onde o corte foi iniciado. O ideal é que as duas partes do olho são aproximadamente equivalentes em tamanho, porque isso vai dar o próximo passo mais fácil. </li><li> Segurando as córneas utilizando duas pinças, descascar delicadamente o olho duas metades separadas. Remova e descarte a lente, vísceras, ea retina neural das conchas dos olhos. Transferir as cascas para um novo prato com ACSF. </li><li> Agora estamos prontos para isolar o epitélio ciliar. Para começar, orientar shell o olho para que a córnea está à sua direita eo epitélio pigmentado da retina (EPR) é à esquerda. Suavemente o pino shell olho para baixo com uma pinça reta do lado RPE. </li><li> Em seguida, use um bisturi para cortar a córnea ea íris longe do epitélio ciliar do olho empurrando levemente para baixo sobre o bisturi ao invés de usar movimentos de corte. </li><li> Depois disso, corte o epitélio ciliar longe do RPE. Uso não-serrilhada fórceps para transferir a faixa de epitélio ciliar de um prato de 35 mm contendo novas ACSF. </li><li> Da mesma forma, isolar o epitélio ciliar da shell outro olho. </li><li> Uma vez que ambas as tiras ciliar epiteliais foram recolhidos, transferir as tiras em um prato de 35 mm contendo 2 ml de Dispase. Coloque o prato com as tiras em uma incubadora de 37 ° C por 10 minutos. </li><li> Após 10 minutos, transferir as faixas a partir do Dispase em um prato contendo 2 ml de tripsina filtrado, hialuronidase e ácido cinurênico. Coloque o prato a 37 ° C por 10 minutos. </li><li> Agora retorne ao microscópio de dissecação. Enquanto mantém a esclera para baixo com uma pinça reta, use a parte inferior da curva não-serrilhada pinça para delicadamente raspar o epitélio ciliar longe da esclera. </li><li> Remova a esclera do prato. Tudo o que deve ser deixado no prato agora são as células de interesse e da solução enzimática. </li><li> Usando um fogo-polido algodão ligado de pipeta, transferir essa solução para um tubo de 14 ml. Triturar esta solução 30 vezes para quebrar as células epiteliais delicadamente forçando a solução dentro e fora da pipeta. </li><li> Centrifugar o tubo por 5 minutos a 1500 RPM. Quando a centrifugação estiver pronto, retire o tubo da centrífuga cuidadosamente vez que as células são propensas a saindo do fundo do tubo nesta fase. </li><li> Aspirar cuidadosamente a maioria do sobrenadante com uma pipeta-fogo polido, e em seguida, adicione 1 mL de inibidor de tripsina em soro livre de mídia para as células. Use um pequeno furo de algodão ligado de pipeta para triturar a amostra de cerca de 50 vezes até que é uma suspensão de uma única célula. </li><li> Centrifugar o tubo novamente por 5 minutos a 1500 RPM. </li><li> Remover o sobrenadante e substituí-la por 1 mL de seu meio de placas. Triturar delicadamente usando uma pipeta de vidro polido-fogo para ressuspender as células. </li><li> Depois que a contagemção das células da placa, eles na densidade desejada em uma placa de 24 cavidades. Nós placa geralmente 10 células / microlitro, preenchendo cada cavidade primeiro com o meio e depois da adição da suspensão celular para obter um volume final de 500 mL de mídia. </li><li> Coloque a placa em um 37 ° C incubadora de CO 2, onde não será movido até você contar as esferas que surgem após 7 dias. </li></ol> 3. Resultados de isolamento de células-tronco da retina <ol><li> Quando este procedimento é realizado com sucesso, o dissecados ciliar células epiteliais deve ficar assim depois de ter sido dissociadas e banhado em baixa densidade (Figura 1). </li><li> Após 7 dias em cultura, as esferas de células-tronco da retina que surgem são contados. A esfera deve ser superior a 75 m de diâmetro e livre-flutuante para ser contada como uma célula-tronco derivadas esfera. (Figura 2). No entanto, algumas células terão esferóides limitada proliferação e de formulário que não atender critério de tamanho e não será contado como esferas de células-tronco derivadas; um exemplo de um esferóide como é mostrado aqui (Figura 2). </li></ol> 4. Resultados representante <img src="/files/ftp_upload/2209/2209fig1.jpg" alt="Figura 1" /> Figura 1. <img src="/files/ftp_upload/2209/2209fig2.jpg" alt="Figura 2" /> Figura 2.

<ol>
	<li>Tropepe, V., Coles, B. L., Chiasson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+stem+cells+in+the+adult+mammalian+eye.">Retinal stem cells in the adult mammalian eye.</a> Science. 287, 2032-2036 (2000).</li><li>Ahmad, I., Tang, L., Pham, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+progenitors+in+the+adult+mammalian+eye.">Identification of neural progenitors in the adult mammalian eye.</a> Biochem Biophys Res Commun. 270, 517-521 (2000).</li><li>Coles, B. L., Angenieux, B., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+isolation+and+the+characterization+of+human+retinal+stem+cells.">Facile isolation and the characterization of human retinal stem cells.</a> Proc Natl Acad Sci U S A. 101, 15772-15777 (2004).</li><li>Cicero, S. A., Johnson, D., Reyntjens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+previously+identified+as+retinal+stem+cells+are+pigmented+ciliary+epithelial+cells.">Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells.</a> Proc Natl Acad Sci U S A. 106, 6685-6690 (2009).</li><li>Lamba, D., Karl, M., Reh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+regeneration+and+cell+replacement:+a+view+from+the+eye.">Neural regeneration and cell replacement: a view from the eye.</a> Cell Stem Cell. 2 (6), 538-549 (2008).</li><li>Coles, B. L., Horsford, D. J., McInnes, R. R., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+retinal+progenitor+cells+leads+to+an+increase+in+the+retinal+stem+cell+population+in+vivo.">Loss of retinal progenitor cells leads to an increase in the retinal stem cell population in vivo.</a> Eur J Neurosci. 23, 75-82 (2006).</li><li>Ang&#233;nieux, B., Schorderet, F. D., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+is+a+neuronal+differentiation+factor+for+retinal+stem+cells+in+vitro.">Epidermal growth factor is a neuronal differentiation factor for retinal stem cells in vitro.</a> Stem Cells. 24 (3), 696-706 (2006).</li><li>Xu, S., Sunderland, M. E., Coles, B. 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F., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+stem+cells+transplanted+into+models+of+late+stages+of+retinitis+pigmentosa+preferentially+adopt+a+glial+or+a+retinal+ganglion+cell+fate.">Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate.</a> Invest Ophthalmol Vis Sci. 48 (1), 446-4454 (2007).</li><li>Canola, K., Arsenijevic, Y. <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+cells+committed+towards+the+photoreceptor+fate+for+retinal+transplantation.">Generation of cells committed towards the photoreceptor fate for retinal transplantation.</a> Neuroreport. 18 (9), 851-855 (2007).</li><li>Djojosubroto, M. 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F., Kooy, D. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+neural+stem+cells+proliferate+in+response+to+EGF+and+FGF+in+the+developing+mouse+telencephalon.+Dev+Biol.">Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol.</a> , 208-201 (1999).</li><li>Coles-Takabe, B. L., Brain, I., Purpura, K. A., Karpowicz, P., Zandstra, P. W., Morshead, C. M., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Don't+look:+growing+clonal+versus+nonclonal+neural+stem+cell+colonies.">Don't look: growing clonal versus nonclonal neural stem cell colonies.</a> Stem Cells. 26 (11), 2938-2944 (2008).</li></ol>

Não há conflitos de interesse declarados.

Este protocolo descreve como isolar células-tronco na retina do epitélio ciliar do mouse ocular 1-3. A metodologia para isolar as tiras de epitélio ciliar pode variar, no entanto as enzimas e metodologia para alcançar uma suspensão de uma única célula foram otimizados neste protocolo. Também é importante que as tiras do epitélio ciliar que foram isolados não contêm grandes quantidades de RPE ou córnea uma vez que estes parecem ter um impacto negativo sobre o número total de esferas que podem ser isoladas de cada olho. Além disso, os meios de comunicação sem soro que fazemos que foi originalmente formulado para o cérebro neurospheres 13, é ideal para o cultivo de células da retina esferas tronco. Deve-se também certificar-se que as células não sejam perturbados depois de terem sido banhado e colocado na incubadora para que as esferas de crescimento não se agregam em conjunto 14. Uma vez que as células-tronco da retina formaram esferas clonal, que pode ser contado para obter um número potencial de células-tronco por olho. As esferas, em seguida, pode ser dissociada em células individuais e várias passagens para verificar auto-renovação ou capacidades diferenciadas em diferentes tipos de células da retina neural e RPE usando diferentes combinações de fatores de crescimento e / ou proteínas.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Stemi 2000 Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cold Light Source</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>KL1500 </td>
<td>dual arm optic light</td>
</tr>
<tr>
<td>Curved Mini Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15000-10</td>
<td>&nbsp;</td>
<tr>
<td>Angled Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15005-08</td>
<td>&nbsp;</td>
<tr>
<td>#7 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11272-30</td>
<td>non-serrated</td>
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<td>#5 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11251-10</td>
<td>straight</td>
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<td>Fine forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11051-10</td>
<td>serrated curved</td>
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<td>#3 Scalpel handle</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2586-M36-10</td>
<td>&nbsp;</td>
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<td>#10 Scalpel blades</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2580-M90-10</td>
<td>&nbsp;</td>
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<td>Hot Bead Sterilizer</td>
<td>&nbsp;</td>
<td>FST</td>
<td>18000-45</td>
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<td>VWR/BD </td>
<td>CACB354235</td>
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<td>T1005</td>
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<td>Hyaluronidase</td>
<td>&nbsp;</td>
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<td>H6254</td>
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<td>K3375</td>
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<td>Trypsin Inhibitor</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10109878001</td>
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<td>35 mm dishes</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA354235</td>
<td>&nbsp;</td>
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<td>24-well plate</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA73521-004</td>
<td>&nbsp;</td>
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<td>Cotton-plugged pipettes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14672-400</td>
<td>fire-polish</td>
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<tr>
<td>14 mL Tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352057</td>
<td>Polystyrene</td>
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<td>Serum-free Media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ref# 13 for formulation</td>
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<td>FGF2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0291</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3149</td>
<td>100 IU</td>
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isolation of retinal stem cells from the mouse eye

O adulto do rato com células-tronco da retina (RSC) é uma célula quiescente rara encontrada no epitélio ciliar (CE) do olho de mamífero 1,2,3. A CE é composta por não-pigmentada interior e camadas de células pigmentadas exterior, e as colônias clonais RSC que surgem a partir de uma única célula pigmentada da CE são feitas de ambas as células pigmentadas e não pigmentadas que podem ser diferenciadas para formar todos os tipos de células da retina neural e RPE. Existe alguma controvérsia sobre se todas as células dentro das esferas contêm pelo menos cerca de 4 pigmento, porém as células ainda são capazes de formar os diferentes tipos de células encontradas na retina neural 1-3. Em algumas espécies, como anfíbios e peixes, seus olhos são capazes de regeneração após a lesão 5, no entanto, o olho dos mamíferos não mostra tais propriedades regenerativas. Procuramos identificar as células-tronco in vivo e para compreender os mecanismos que mantêm a retina de mamíferos as células-tronco quiescentes 08/06, mesmo após a lesão, bem como usá-los como uma fonte potencial de células para ajudar a reparar os modelos físico ou genético de lesão ocular através do transplante 9-12. Aqui nós descrevemos como isolar as células ciliares do epitélio do olho do rato e cultivá-las em cultura, a fim de formar a esferas clonal das células da retina-tronco. Como não existem marcadores conhecidos da célula-tronco in vivo, estas esferas são a única maneira conhecida para prospectivamente identificar a população de células-tronco dentro do epitélio ciliar do olho.

Watch this Scientific Journal Video about Isolation of Retinal Stem Cells from the Mouse Eye at JoVE.com

Isolation of Retinal Stem Cells from the Mouse Eye

Neste vídeo, vamos demonstrar como isolar células-tronco na retina do epitélio ciliar do olho do rato e cultivá-las em cultura para dar forma clonal esferas da retina. As esferas que estão isolados possuem as propriedades cardeal de células-tronco: a auto-renovação e multipotencialidade.

Isolamento de Células-Tronco da retina do olho do rato

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

isolation-of-retinal-stem-cells-from-the-mouse-eye

Nanyang Technological University

Research

JoVE Journal

Biology

24.7K visualizações.  University of Toronto. Neste vídeo, vamos demonstrar como isolar células-tronco na retina do epitélio ciliar do olho do rato e cultivá-las em cultura para dar forma clonal esferas da retina. As esferas que estão isolados possuem as propriedades cardeal de células-tronco: a auto-renovação e multipotencialidade.

Vídeo: Isolation of Retinal Stem Cells from the Mouse Eye

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Isolamento e Análise de Células-Tronco Hematopoéticas da Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Biologia

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Isolamento e derivação de células embrionárias de rato Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Isolamento de Células-Tronco do rato das Glândulas Salivares

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

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 ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolamento e Cultura da Dental epiteliais células-tronco do Adulto Rato Incisivo

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-interno pilha injeção maciça de células-tronco embrionárias leva a taxas mais elevadas de quimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

Culturas de células primárias do Mouse epithelium Retinal do Pigment

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolamento, caracterização e diferenciação de células-tronco cardíacas do coração de rato adulto

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Dissecção eficiente e cultura de células epiteliais de pigmento de retina do rato primário

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

Cultura Primária de Células Epiteliais do Pigmento da Retina Suína

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...