Este trabalho foi financiado pelo New York State Department of Health (C026878). MS foi um New York Stem Cell Foundation Fellow-Druckenmiller. Suportado em parte pela Research Grant No. 5-AF11-571 da March of Dimes Foundation.

1. Preparação de células de alimentação Note-se que todos os reagentes descritos em seguida devem ser preparadas de forma estéril (ver Tabelas 1 e 2). Este protocolo utiliza a linha celular de JK1 (Cell Biolabs, Inc., catálogo # CBA-315) como alimentadores, que é um derivado de transformada de rato adulto células testiculares somáticas e foi descrito em outro lugar 3. Note-se também que todos os procedimentos com animais deve ser realizado de acordo com as diretrizes institucionais e regulamentos. <ol><li> JK1 células podem ser mantidas em cultura e utilizadas com sucesso como alimentadores-se a passagem 39. Cultura JK1 células em uma placa de cultura de 100 milímetros de células (BD Falcon, catálogo # 353003), com 10 ml de meio esterilizado por filtração alimentador de crescimento (DMEM, suplementado com 10% soro fetal de bovino [FBS], 2 mM L-glutamina e antibióticos). Quando a cultura atinge 95% de confluência, as células divididas 1:06 -1:10 ratio. </li><li> Remova o meio de confluente mono célula JK1 alimentadorcamada. </li><li> Lavar camada de células uma vez com PBS sem cálcio e magnésio. </li><li> Adicionar pré-aquecido tripsina / EDTA, 1x (0,05% trypsin/0.53 mM EDTA; catálogo Corning Cellgro, # 25-051-CI), e incubar a 37 ° C durante 5-10 min. </li><li> Inactivar a tripsina com um volume igual de meio de crescimento do alimentador. Recolher as células e centrifugar a 300 xg durante 5 min. Re-suspender as células em meio de crescimento do alimentador. </li><li> Pré-revestimento de poços múltiplos placas de cultura de células por adição de 0,4% de solução suficiente para cobrir a gelatina bem e remover depois de 5 min à temperatura ambiente. </li><li> Placa ~ 250 células por mm2 em gelatina-revestido placa de cultura de células (por exemplo, formato de 48 poços ou de 6 poços). Incubar a cultura a 37 ° C durante 16 a 24 horas. </li><li> Remover o meio de cultura a partir de poços. CUIDADO: Para inactivar o crescimento celular, adicionar uma solução fresca de mitomicina-C a 10 ug / ml diluído em DMEM a cada poço (200 ul, por exemplo / poço de uma placa de 48 poços). A mitomicina-C é tóxico. Manuseie e descarte-a com cuidado. Incubar a37 ° C durante 4 horas. </li><li> Remover a mitomicina C-solução a partir de células. Lavar 3 vezes com DMEM antes de células-tronco do chapeamento. </li><li> Remover DMEM alimentador de células de poços e adicionar meio suficiente de células estaminais (100 ul / poço de uma placa de 48 poços, ver Tabela 3), para evitar a dessecação dos alimentadores durante passaging. Adicionar SSC mesmo dia, como descrito abaixo nas secções 3.1 e 3.4, utilizando os tamanhos indicados de poços contendo alimentadores. </li></ol> 2. Dissociação de Rato tecido testicular para obter células germinativas dos doadores <ol><li> Colheita testículos adultos de mouse em forma estéril e armazenar temporariamente os testículos em uma placa de Petri coberta (sem qualquer líquido acrescentado). O prato tem colocado imediatamente em gelo para evitar a dessecação e manter a viabilidade celular. </li><li> Usando estéreis pinça fina e uma tesoura bem em uma capa de cultura de células, fazer uma incisão transversal na túnica albugínea sem completamente atravessa em testículos (ou seja, corte ~ ½ - ¾ distância through) e utilizar uma pinça para espremer para fora dos túbulos seminíferos em um canto da placa; túnica de descarte e tecido em anexo. Manter em gelo durante placa para manter o tecido refrigerados, mantendo assim a sua integridade e prevenir a evaporação. </li><li> Rapidamente mince túbulos com uma tesoura fina de mola, pelo menos, 3 minutos por testículo, mantendo placa em gelo. </li><li> Recolher os fragmentos dos túbulos num tubo cónico de 50 ml e lava-se em ~ 40 ml de soro de PBS / 1% de albumina bovina, refrigerada. </li><li> Centrifugar a 60 xg durante 10 minutos para separar os fragmentos dos túbulos de espermatozóides e detritos. Elimine o sobrenadante. </li><li> Num tubo cónico de 15 ml, ressuspender o sedimento em 3 ml por testículo de pré-aquecida tampão de dissociação (DMEM, 0,05% de tripsina, colagenase tipo I de 0,03%, 80% U / ml de DNAse I e 0,5 de albumina sérica bovina; Ver Tabela 2 para detalhes mais específicos de dissociação buffer). </li><li> Colocar o tubo cónico horizontalmente num suporte de um conjunto agitador a 150 rpm a 37 ° C durante 15 minutos para maximizar agitation. </li><li> Girar para baixo a 60 xg durante 10 minutos para separar as células individuais a partir de pedaços não dissolvidas. </li><li> Guardar os pedaços de pelotização contendo até a próxima etapa. Recolher o sobrenadante do passo 2.8 e adicionar 3 ml de DMEM/10% de soro fetal bovino para neutralizar tampão de dissociação. Coloque o tubo em gelo temporariamente. </li><li> Repetir o passo 2,6-2,7 com o pellet a partir do passo guardado 2,9 a re-digest pedaços restantes após a etapa 2.8. </li><li> Adicionar um volume igual de DMEM/10% de FBS para neutralizar tampão de dissociação. </li><li> Recombinam suspensão do passo 2,11 com suspensão de células guardadas a partir do passo 2.9. Centrifugar durante 5 minutos a 300 x g. </li><li> Ressuspender o granulado em meio de células estaminais em um volume de 16 ml por cada par de testículos (ver Tabela 3). </li></ol> 3. Chapeamento de suspensão de células de testículo <ol><li> Placa 2 ml de suspensão de células por poço numa placa de 6 poços contendo células alimentadoras mitoticamente inactivadas com mitomicina-C-e o local de uma cultura de células de incubadora a 37 &amp;deg; C em 5% de CO 2. </li><li> Depois de 48 horas de médio, aspirado, adicionar 2 ml de meio fresco e voltar a incubadora. </li><li> Após 48 horas, adicionar 0,5 ml de meio fresco de células estaminais a cada poço e voltar a incubadora. </li><li> Após 48 horas, em seguida alimentar as células três vezes por semana depois disso, como se segue, até grandes colónias discretas (&gt; 50 células) aparece (dentro de 7 a 21 dias). Da primeira e segunda alimentação em cada semana (por exemplo segunda-feira e Sexta-Feira) remover aproximadamente 50% do meio e adiciona de volta a 50% em volume de meio fresco. Para uma alimentação terceiro (por exemplo, sexta-feira), aspirar todo o meio e substituir com 2 ml de meio fresco. Esta abordagem baseia-se no raciocínio de que evitar mudanças de meio completo minimiza as flutuações nas concentrações de sinais parácrinos que são secretadas para o meio de cultura de 9. </li></ol> 4. Picking colônia de primeira passagem <ol><li> Remover médio de colónias em poços contendo a ser passadas e adicionar meio fresco de células estaminais. </li><li> Identificar colônias usando uma objetiva de 10x. Com o microscópio ligeiramente de-concentrado, colónias SSC na borda do poço aparecerá como homogéneas aglomerações luminosas, compostas por muitas células de 11-12 um de diâmetro, em que é difícil ou impossível de distinguir as bordas das células individuais, uma vez que as células aparecem fundidos (Figura 1A). Não-SSC colônias pode parecer mais escuro, mais granular, ou menos homogêneo, com fronteiras discerníveis (Figura 1A, encarte). </li><li> Utilizando uma ponta de pipeta de 200 uL, com o conjunto de pipetteman de 50 ul, em primeiro tomar-se a partir do meio e assim expulsar para molhar a ponta. Em seguida, deslocar o suavemente com a ponta da colónia antes de retirar rapidamente 50 ul de meio para deslocar a colónia por aspiração para dentro da ponta (Figura 1B). </li><li> Expelir o meio contendo a colónia em um poço de uma placa de 48 poços contendo células alimentadoras inactivadas com 100 ul de meio de células estaminais (Figura 1C). </li><li> Repita o passo4,3 e adicionar até 8 colónias para o bem mesmo sem ultrapassar 500 ul por poço. </li><li> Repita os passos 4,3-4,5 para preparar poços da placa de 48 cavidades com colónias de passagem um CCD. </li><li> Wells estará pronto para dividir em 7-14 dias ou depois de grandes aglomerações de até 500 células surgiram (Figura 1D). </li></ol> 5. Expansão subseqüente e passaging de SSCs por trituração <ol><li> Células de passagem deve ser subcultivados para alimentadores frescas após até 14 dias a uma razão de divisão de 1:2 para os primeiros três passagens e, em seguida, 01:04 - 01:06 (a cada 7 dias) depois disso, como se segue. Triturar suavemente colónias fora de um poço semi-confluentes (por exemplo, ~ 300.000 SSC / poço de placa de 6 poços), com uma ponta de pipeta ml por meio de lavagem através das células. As colónias podem ser vistas de extracção. Muita força com o fluxo de líquido causará células alimentadoras indesejados para sair também. </li><li> Recolher as células triturados num tubo cónico e centrifugar a 300 xg durante5 min. Nota: Um passo tripsinização pode ser adicionado com segurança aqui, se desejado pelo investigador. </li><li> Aspirar o sobrenadante e re-suspensão em meio fresco com células-tronco através de uma profunda pipetando para cima e para baixo para interromper colônias. </li><li> SSC placa em células alimentadoras recém-preparados inativadas com mitomicina-C, como descrito na seção 1. </li><li> Alimentar as células três vezes por semana, como se segue. Para a alimentação primeiro e segundo (por exemplo segunda-feira e Sexta-Feira) adicionar 50% em volume de meio fresco. Para uma alimentação terceiro (por exemplo, sexta-feira), aspirar todo o meio e substituir com 2 ml de meio fresco. Placas vai se tornar confluentes em 7-10 dias. As culturas devem ser constituídos por células germinativas&gt; 98% (isto é, &lt;1% de células somáticas contaminantes) após passagens 5-7, com base na coloração imunológica (por exemplo, utilizando o marcador de células germinativas murino GCNA) para distinguir as células germinativas de células somáticas 10. Nota: Estes métodos foram desenvolvidos de acordo com as normas e exigências estabelecidas pela Instituiçãoal Animal Care e do Comitê Uso do Weill Cornell Medical College. </li></ol>

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MS recebe royalties do Weill Cornell Medical College através de uma licença de celular Biolabs, Inc. para distribuir JK1 células.

Este método para derivar SSCs adultos utilizando células adultas derivadas de testículos de alimentação é robusto e tem êxito quando comuns origens genéticas (por exemplo, FVB, C57BL6 e 129SV/C57Bl/6 mista) e diferentes cepas mutantes foram empregados 2,3,7,14. Na verdade, o microambiente criado pelo sistema de cultura é suficiente para superar algumas barreiras genéticas para manter SSC adultos in vivo (por exemplo, no caso de plzf - / - de animais) 7.</su...

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> DMEM </td><td> Corning </td><td> 10-013 </td><td> Diluente para a dissociação de tampão </td></tr><tr><td> Tripsina / EDTA </td><td> Mediatech </td><td> 25-051 CI- </td><td></td></tr><tr><td> Caule meio base celular (StemPro-34) </td><td> Life Technologies </td><td> 10639-011 </td><td> Requer suplementação como por Shinohara et al. (2003) * </td></tr><tr><td> Stem suplementos médio de células </td><td> vário </td><td> ver a Tabela 3 </td><td> Requer suplementação como por Shinohara et al. (2003) * </td></tr><tr><td> JK1 células </td><td> Célula Biolabs, Inc. </td><td> CBA-315 </td><td> Pode substituir com adultos testiculares células do estroma como por Seandel et al. (2007) </td></tr><tr><td> mitomicina-C (CUIDADO) </td><td> Sigma-Aldrich </td><td> M4287 </td><td> Tóxico; Manusear com cuidado. </td></tr><tr><td> Gelatina </td><td> Sigma-Aldrich </td><td> G1890 </td><td> Solução a 0,4% em água </td></tr><tr><td> EVOS xl digitais microscópio invertido </td><td> Grupo de microscopia avançada </td><td> - </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabela 1. Reagentes e equipamentos específicos. * Ver Tabela 3 </td></tr><tr><td> DMEM </td><td> Corning </td><td> 10-013 </td><td> Diluente para a dissociação de tampão </td></tr><tr><td> tripsina (1:250) </td><td> Life Technologies </td><td> 27250-018 </td><td> Tampão de dissociação: Final 0,05% p / v </td></tr><tr><td> colagenase tipo I, 235 U / ml </td><td> Worthington </td><td> CLS1 235 </td><td> Dissociação buffer: 0,03% final p / v </td></tr><tr><td> DNAse I </td><td> Sigma-Aldrich </td><td> DN25 </td><td> Dissociação tampão: Final 80 U / ml </td></tr><tr><td> albumina de soro bovino </td><td> ICP Bio </td><td> ABRE-100g </td><td> Tampão de dissociação: Final 0,5% em peso / vol </td></tr><tr><td></td><td></td><td></td><td> Tabela 2. Dissociação tampão </td></tr><tr><td> StemPro-34 SFM </td><td> Life Technologies </td><td> 10639-011 </td><td></td></tr><tr><td> StemPro-34 Suplemento Nutriente </td><td> Life Technologies </td><td> 10639-011 </td><td></td></tr><tr><td> Suplementos adicionais ** </td><td></td><td></td><td></td></tr><tr><td> Não-aminoácidos essenciais </td><td> Sigma-Aldrich </td><td> M7145 </td><td> 1X </td></tr><tr><td> MEM solução Vitamina </td><td> Life Technologies </td><td> 11120-052 </td><td> 1X </td></tr><tr><td> L-glutamina </td><td> Mediatech </td><td> 25-005 </td><td> 2 mM </td></tr><tr><td> albumina de soro bovino </td><td> ICP Bio </td><td> ABRE </td><td> 0,50% </td></tr><tr><td> Antibiótico-antimicótico Solução </td><td> Mediatech </td><td> 30-004 CI- </td><td> 1X </td></tr><tr><td> D (+) glicose </td><td> Sigma-Aldrich </td><td> G8769 </td><td> 6 mg / ml </td></tr><tr><td> β-estradiol </td><td> Sigma-Aldrich </td><td> E2758 </td><td> 30 ng / ml </td></tr><tr><td> progesterona </td><td> Calbiochem </td><td> 5341 </td><td> 60 ng / ml </td></tr><tr><td> de soro fetal bovino </td><td> variável </td><td> n / a </td><td> 1% </td></tr><tr><td> holo-transferrina bovina </td><td> Sigma-Aldrich </td><td> T1283 </td><td> 100 ug / ml </td></tr><tr><td> insulina </td><td> Gémeos Bio-Products </td><td> 700-112P </td><td> 25 ug / ml </td></tr><tr><td> GDNF humano </td><td> Vida Techngias </td><td> PHC7041 </td><td> 10 ng / ml </td></tr><tr><td> bFGF humana </td><td> Life Technologies </td><td> PHG0023 </td><td> 10 ng / ml </td></tr><tr><td> rato EGF </td><td> Life Technologies </td><td> PHG0313 </td><td> 20 ng / ml </td></tr><tr><td> putrescina </td><td> Research Organics </td><td> 0778P </td><td> 60 uM </td></tr><tr><td> selenito de sódio </td><td> Sigma-Aldrich </td><td> S5261 </td><td> 30 nM </td></tr><tr><td> ácido pirúvico </td><td> Alfa Aesar </td><td> A13875 </td><td> 30 ug / ml </td></tr><tr><td> DL-ácido láctico </td><td> JT Baker </td><td> 0196-04 </td><td> 1 ug / ml </td></tr><tr><td> β-mercaptoetanol </td><td> Life Technologies </td><td> 21985-023 </td><td> 50 uM </td></tr><tr><td> ácido ascórbico </td><td> Sigma-Aldrich </td><td> A4544 </td><td> 100 uM </td></tr><tr> &lt;td&gt; D-biotina </td><td> Sigma-Aldrich </td><td> B4639 </td><td> 10 | ig / ml </td></tr><tr><td></td><td></td><td></td><td> Tabela 3. Stem meio celular </td></tr><tr><td></td><td></td><td></td><td> * Nota: Adicionar suplementos antes de utilizar meio. Filtrar esterilizar e manter a 4 ° C. O meio é estável durante pelo menos 2 semanas. </td></tr><tr><td></td><td></td><td></td><td> ** Nós empregamos diferentes fabricantes, formulações e / ou números de lote de estes reagentes, sem quaisquer efeitos deletérios aparentes. Em geral, os reagentes de grau de cultura de células deve ser empregado. </td></tr></tbody></table>

serial enrichment of spermatogonial stem and progenitor cells (sscs) in culture for derivation of long-term adult mouse ssc lines

Tronco espermatogônias e as células progenitoras (SSCS) dos testículos representam um exemplo clássico de células-tronco adultas de mamíferos e preservar a fertilidade por quase toda a vida do do animal. Embora os mecanismos precisos que controlam a auto-renovação e diferenciação in vivo são difíceis de estudo, vários sistemas têm sido desenvolvidos anteriormente para propagar SSC murinos in vitro, utilizando uma combinação de meios de cultura e as células especializadas do alimentador 1-3. Mais em incursões in vitro sobre a biologia do SSC ter derivado de linhas celulares a partir de recém-nascidos, possivelmente devido à dificuldade na obtenção de linhas de células adultas 4. No entanto, os testículos continua a amadurecer-se até ~ 5 semanas de idade na maioria das estirpes de ratinho. No período pós-natal precoce, mudanças drásticas ocorrem na arquitectura dos testículos e na biologia de ambos células somáticas e de espermatogénese, incluindo alterações nos níveis de expressão de células estaminais numerosos relacionadagenes. Portanto, as linhas-neonatal derivados SSC não pode inteiramente recapitular a biologia do SSC adultos que persistem após o testículo adulto chegou a um estado estacionário. Vários fatores têm dificultado a produção de linhas de adultos SSC historicamente. Em primeiro lugar, a proporção de células-tronco funcionais pode diminuir durante a idade adulta, quer devido a factores intrínsecos ou extrínsecos 5,6. Além disso, como com outras células-tronco adultas, tem sido difícil para enriquecer suficientemente SSC a partir de células adultas totais testiculares sem o uso de uma combinação de estratégias de triagem ou imuno outros 7. Estratégias geralmente utilizadas incluem a utilização de ratinhos criptorquídios como uma fonte de células do doador, devido a uma maior proporção de células estaminais para outros tipos de células 8. Com base na hipótese de que a remoção de células somáticas a partir da cultura inicial perturba as interacções com o nicho de células estaminais que são essenciais para a sobrevivência SSC, que anteriormente desenvolvidos métodos para derivar l adultoines que não necessitam de imuno ou doadores criptorquídios mas sim empregar enriquecimento série de SSC em cultura, a seguir referido como SESC 2,3. O método descrito a seguir envolve um procedimento simples para derivar linhas adultos SSC, dissociando adulto dador túbulos seminíferos, seguido por plaqueamento das células em alimentadores que compreendem uma linha celular de estroma testicular (JK1) 3. Através de série passagem, fortemente aderente, contaminando germinativas células não estão esgotados a partir da cultura de enriquecimento concomitante de SSC. Culturas produzidas desta forma contém uma mistura de espermatogônias em diferentes estágios de diferenciação, as quais contêm, com base em SSC a longo prazo a capacidade auto-renovação. O cerne do método SESC é que ele permite SSC para fazer a transição difícil de auto-renovação in vivo a longo prazo de auto-renovação in vitro num microambiente radicalmente diferente, produz linhas de longo prazo do CCD, livre de contaminaçãocélulas somáticas, e desse modo permite a manipulação experimental subsequente de SSC.

A aparência do tipo passagem de zero selvagem, as colónias adultas SSC depois de 7 dias é mostrado na Figura 1. Tridimensionais colónias são constituídas por uma camada de células planas ligadas aos alimentadores ou matriz extracelular underling depositados pelos alimentadores com múltiplas camadas de SSC que crescem no topo. Enquanto SSC saudáveis ​​são brilhantemente refrangente e uniformemente 11-12 um de diâmetro, as bordas das células são difíceis de distinguir e o tamanho das col...

Watch this Scientific Journal Video about Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines at JoVE.com

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

Um método simples para obter e manter a haste espermatogónias e linhas de células progenitoras a partir de ratos adultos, é aqui apresentada. O método utiliza células de alimentação provenientes do compartimento de células somáticas do testículo do rato adulto. Esta técnica é aplicável a linhagens de camundongos comuns, incluindo transgênico, knock-out, e bater-nos camundongos.

Enriquecimento de série-tronco de esperma e células progenitoras (SSC) em Cultura para Derivação de longo prazo Adultos Linhas rato SSC

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

serial-enrichment-spermatogonial-stem-progenitor-cells-sscs-culture

Research

JoVE Journal

Biology

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

13.7K visualizações.  Weill Cornell Medical College. Um método simples para obter e manter a haste espermatogónias e linhas de células progenitoras a partir de ratos adultos, é aqui apresentada. O método utiliza células de alimentação provenientes do compartimento de células somáticas do testículo do rato adulto. Esta técnica é aplicável a linhagens de camundongos comuns, incluindo transgênico, knock-out, e bater-nos camundongos.

Vídeo: Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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

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

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

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

Biologia

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Isolamento e expansão em grande escala de Adulto Humanos endotelial Colony Células Progenitoras Forming

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

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 ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Derivação de células-tronco do rato trofoblasto de blastocistos

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolamento e Cultura de Células-Tronco Adultas epitelial da pele humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A longo prazo de Teste de toxicidade letal com o crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolamento e Cultura de miofibras individuais e suas células satélites do músculo esquelético

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclastos Derivação do mouse Medula Óssea

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...