Os autores são muito satisfeito com a Sra. Haiying Pan e Ian Mr. Bellayr por sua ajuda técnica sobre o isolamento de células para os filmes atuais e Kyle Sr. Holden eo Sr. Jonathan Lucas pela ajuda na edição de slide. Muito obrigado a seguinte lista de pessoas que estiveram envolvidas com o desenvolvimento das técnicas preplate modificado ao longo dos últimos anos: os drs. Burhan Gharaibeh, Baohong Cao, Deasy Bridget, Thomas R. Payne, Aiping Lu, Hairong Peng, Hideki Oshima, Bo Zeng, Xiaodong Mu, KIMIMASA Tobita, Ron Jankowski, Makato Ikezawa. Estamos gratos pela assistência técnica de James H. Cummins, Pruchnic Ryan, Feduska Joseph, C. Rebecca Schugar, Pan Haiying e Tebbets Jessica. O autor também gostaria de agradecer o apoio financeiro para este trabalho a partir da Muscular Dystrophy Association (MDA), o National Institutes of Health (NIH), o Departamento de Defesa (DOD), Hospital Infantil de Pittsburgh de UPMC, o Departamento de Cirurgia Ortopédica, e da Universidade de Pittsburgh.

Em geral, o procedimento completo preplate modificada exige 1-2 dias de preparação, uma semana de realizar o procedimento técnico, 2-3 dias de identificação de células com imunocitoquímica, e um adicional de 2-3 semanas de tronco expansão da população celular. Os equipamentos necessários para a cultura de células-tronco são semelhantes ao de sistemas de célula de outra cultura, que inclui uma Centrífuga de bancada, incubadora de CO 2, câmara de fluxo laminar de cultura de tecidos e um microscópio invertido equipado com fluorescência e uma câmera digital. As células-tronco isoladas também podem ser clonados e pode ser armazenado a longo prazo em nitrogênio líquido para os 1,2 estudos atuais ou futuras. Todos os reagentes e materiais utilizados para este procedimento deve ser estéril e manuseados assepticamente. Etapa 1: Preparação <ol><li> Colágeno casaco pratos de cultura de tecidos e frascos: colágeno tipo I derivado do bezerro de pele (0,1 g em 1 litro; Sigma-Aldrich). O plasticware para revestir inclui: T-25 frascos, 6 ou 12 pratos bem, pratos (60 e 100 mm, BD Falcon) como descrito anteriormente 1,2. Agite suavemente o plasticware cada meia hora, durante 4 horas para garantir até mesmo revestimento do colágeno. A solução de colágeno é então removido. E o plasticware revestido é então deixada para secar na capa de cultura de tecidos com a luz UV eo capô tanto, para garantir a esterilidade e, finalmente, recapeadas ou cobertas para uso posterior. </li><li> Prepare meio de cultura celular: 400mL DMEM (DMEM, glicose alta, Invitrogen), 50 ml de calor de soro fetal bovino inativado (FBS, Invitrogen), 50 ml soro de cavalo (HS, Invitrogen), e 5 mL Extrato de embrião de galinha (CEE; Accurate Chemical Co. ) será filtrada a vácuo através de um descartáveis ​​0,22 M-500-ml sistema de filtro estéril (Corning). Estéril de penicilina / estreptomicina solução (Invitrogen) será adicionado finalmente 100 Unidades / mL. As soluções preparadas podem ser armazenadas a 4 ° C durante pelo menos um mês. Esta solução é usada para separar as células do frasco quando as culturas divisão ou preparar as células para transplante. </li><li> Warm up soluções para isolamento de células: Os reagentes a seguir precisam ser aquecidos a 37 ° C antes da sua utilização para o isolamento de células: Solução de Hank Sal Buffered (HBSS, Invitrogen); 0,2% (wt / vol) colagenase do tipo XI (Sigma -Aldrich), com uma média de 3.200 unidades de digestão de colágeno por mL HBSS; Dispase 2,4 units/1mL (Invitrogen), 0,5% (wt / vol) de tripsina (Invitrogen). </li><li> Esterilizar equipamentos cirúrgicos em preparação: O procedimento cirúrgico requer instrumentos cirúrgicos estéreis, incluindo: diferentes tamanhos fórceps, micro-tesouras, tesoura de íris e / ou lâminas de bisturi. Além disso, 15 e 50 mL tubos de polipropileno de centrifugação (BD Falcon); filtro Cell (tamanho dos poros 70 m) (BD Falcon); 18 -, 23 - e 27-gauge agulha (BD PrecisionGild) e 10 - ou 20 ml seringas esterilizadas (BD). </li></ol> Passo 2: biópsias de tecidos, isolamento e dissociação mecânica 04/01 As biópsias de tecido são realizados sob uma capa de cultura estéreis e incluem recém-colhidas tendões e músculos esqueléticos são obtidos a partir dos músculos sóleo tíbia ou do membro posterior de camundongos selvagens (C57BL/6J Feminino, 4-5 semanas de idade ou mais jovens, Jackson laboratório). Qualquer tecido conjuntivo visíveis, vasos sanguíneos e tecido adiposo são então removidas das amostras de biópsia. Os tecidos de tendão e músculo são, então, cuidadosamente identificados sob um microscópio cirúrgico de dissecação para remover restos de pele e osso e colocada em um prato contendo frio (4 ° C) HBSS (suplementado com acrescentar 5% de FBS). Os tecidos isolados são então colocados separadamente em pratos de colágeno revestido contendo HBSS frio e será picada (&lt;milímetros 1x1 3) em uma pasta grossa usando micro-tesoura e / ou lâminas de bisturi. Passo 3: A digestão enzimática dos tecidos: O tecido é então enzimaticamente picada dissociado através de uma série de passos 2,4,5 <ol><li> Transferência de suspensões de tecido picada em separado, 15 ml tubos e centrifugar a 3.500 rpm ~ a 4 ° C por 5 minutos. </li><li> Remover o sobrenadante, lavar com HBSS e repita a centrifugação novamente. </li><li> Remover o sobrenadante, e digerir essas pastas, adicionando 10 ml de pré-aquecida 0,2% de colagenase tipo XI (Sigma). Incubar por 60 minutos a 37 ° C, enquanto continuamente balançando suavemente os tubos. Alternativamente, agitar a mão a cada 10 minutos. </li><li> Centrifugar e ressuspender essas suspensões em 10 ml dispase (2,4 Unidades / mL, Gibco) solução. Incubar por 45 minutos a 37 ° C, agitando delicadamente com as mãos ou balançar a cada 10 minutos. </li><li> Centrífuga e re-suspender estas lamas em 10 ml de solução de tripsina 0,2% HBSS. De incubação varia de acordo com a medida em que as células individuais são liberados a partir de tecidos que podem ser realizados através da observação da suspensão ao microscópio. Não é recomendável ultrapassar 30 minutos a 37 ° C com invertendo os tubos ou balançando suavemente a cada 10 minutos. </li><li> Centrífuga da célula, resultandos e re-suspender o pellet celular em 10 ml de Proliferação Médio (PM). </li><li> Dissociar a suspensão de células, passando o extrato através de uma série de agulhas. As células são então passadas duas vezes através de um 18G, em seguida, uma 23G e, finalmente, uma agulha 27G. </li><li> Passe o extrato celular através de um filtro de células 70 mícrons. </li></ol> Passo 4: Preplating para isolar populações de células diferentes com base em taxa de adesão celular 03/01 <ol><li> Centrifugar e ressuspender o pellets de células em meio de proliferação. </li><li> Misturas placa da célula em um colágeno revestido frasco T-25 (ou 60 prato mm) e marcá-la como PP1. A suspensão de células devem ser observados como possuindo um grande número de núcleos de refração e outros detritos sob o microscópio. </li><li> Incubar a 37 ° C em um umidificado, 5% incubadora de CO 2 por 2 horas. </li><li> Transferência de células não-aderentes a um novo colágeno revestido T-25 frasco (ou 60 prato mm) e marcar é tão PP2. Adicionar 5 ml da PM na placa rotulados PP1. As células iniciais aderindo, que atribuem ao balão são na sua maioria miofibroblastos 3. </li><li> Devolver os frascos para a incubadora por mais 24 horas, a transferência de células não-aderentes a um novo colágeno revestido T-25 frasco (ou prato) e marcá-la como PP3. Adicionar 5 ml de meio de proliferação na placa rotulados PP2. Estas células aderentes, que atribuem a este frasco são principalmente fibroblastos 2,3. </li><li> Frascos de retorno para a incubadora e repita o procedimento em mais 24 horas até PP6 população é criada. Manter cada grupo de células em suspensão e permitir que eles se proliferam e examiná-los regularmente usando um microscópio invertido. PP3 PP4-são principalmente os mioblastos, enquanto as células musculares satélite são muitas vezes vistos principalmente no PP5 2. </li><li> A maioria das células lentamente aderindo tipicamente anexar por PP6. Nesta fase, as células parecem pequenos, refração, redondos de luz e são muito escassos em número e são considerados uma haste 1,2 população celular. </li></ol> Passo 5: Identificação e expansão de células-tronco isoladas <ol><li> O isolado PP6 células são muito poucos em número e precisam ser mantidos em meio de proliferação FBS ricos (20% FBS em DMEM), que é trocada diariamente. A maioria das células presentes na cultura PP6 morrer durante os seguintes 1-2 semanas de cultivo, mas um grande PP6 população saudável é geralmente criado após um adicional de 1-2 semanas de expansão. As células-tronco isoladas de camundongos altamente expressar antígenos de células-tronco 1 (SCA1), CD34, quinase fígado fetal 1 (Flk1), e marcadores-tronco outros 1,2,6,7 que pode ser visualizado através de coloração imuno-histoquímico. Essas células-tronco também apresentam capacidades de diferenciação múltiplas 3,6,8. </li><li> As células isoladas são mantidas e expandidas com uma densidade baixa de células em cultura (12-bem pratos a 50-100 células / poço ou &lt;confluência de 20-30% no frasco ou prato) para evitar 1,2 diferenciação. Muito poucas células, formam clones durante a expansão, e essas células-tronco clonadas podem sofrer proliferação tempo (LTP) para quaisquer estudos competência outros. As células-tronco também podem ser clonados loja em nitrogênio líquido através de procedimentos gerais de armazenamento de células p.ex PM com DMSO 10%. Fazer uma série de unidades populacionais de baixa passagem das células-tronco para os estudos de outros para trás. </li></ol>

<ol>
	<li>Gharaibeh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique.">Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.</a> Nat Protoc. 3, 1501-1509 (2008).</li><li>Qu-Petersen, Z. <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+a+novel+population+of+muscle+stem+cells+in+mice:+potential+for+muscle+regeneration.">Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration.</a> J Cell Biol. 157, 851-864 (2002).</li><li>Li, Y. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+muscle-derived+cells+into+myofibroblasts+in+injured+skeletal+muscle.">Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle.</a> Am J Pathol. 161, 895-907 (2002).</li><li>Watt, D. J., Lambert, K., Morgan, J. E., Partridge, T. A. &. a. m. p. ;. a. m. p., Sloper, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+donor+muscle+precursor+cells+into+an+area+of+muscle+regeneration+in+the+host+mouse.">Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse.</a> J Neurol Sci. 57, 319-331 (1982).</li><li>Lu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+myogenic+progenitor+populations+from+Pax7-deficient+skeletal+muscle+based+on+adhesion+characteristics.">Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics.</a> Gene Ther. 15, 1116-1125 (2008).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+isolation+of+muscle-derived+cells+capable+of+enhancing+muscle+regeneration+and+bone+healing.">Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.</a> J Cell Biol. 150, 1085-1100 (2000).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+receptors+in+the+maturation-dependent+adenoviral+transduction+of+myofibers.">The role of receptors in the maturation-dependent adenoviral transduction of myofibers.</a> Gene Ther. 8, 627-637 (2001).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells+differentiate+into+haematopoietic+lineages+but+retain+myogenic+potential.">Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential.</a> Nat Cell Biol. 5, 640-646 (2003).</li><li>Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+based+on+muscle+stem+cells.">Regenerative medicine based on muscle stem cells.</a> J Musculoskelet Neuronal Interact. 8, (2008).</li><li>Gates, C. B., Karthikeyan, T., Fu, F. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+for+the+musculoskeletal+system+based+on+muscle-derived+stem+cells.">Regenerative medicine for the musculoskeletal system based on muscle-derived stem cells.</a> J Am Acad Orthop Surg. 16, 68-76 (2008).</li></ol>

Não há conflitos de interesse declarados.

Foi reconhecido que alguns tecidos adultos contêm células-tronco múltiplas. Ao longo dos últimos anos de estudo, células-tronco têm sido detectadas nos tecidos moles músculo-esqueléticas, incluindo o músculo esquelético e tendão. Este protocolo atual detalhes de um procedimento, conhecido como a técnica preplate modificado, que tem sido utilizado com sucesso em nosso laboratório para isolar células-tronco adultas a partir de tendões e músculo esquelético. Usando a técnica preplate modificado descrito a...

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#11995-073</td>
<td>&nbsp;</td>
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<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#10437-028</td>
<td>&nbsp;</td>
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<td>Heat-inactivated horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#26050-088</td>
<td>&nbsp;</td>
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<td>Chick embryo extract</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>#CE650T-10</td>
<td>&nbsp;</td>
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<td>100U ml penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#15140-122</td>
<td>&nbsp;</td>
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<td>Dulbecco&#x2019;s PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#14190-250</td>
<td>&nbsp;</td>
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<td>Hank&#x2019;s Buffered Salt Solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#24020-117</td>
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<td>Collagenase type XI 0.2%(wt/vol)</td>
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<td>Sigma-Aldrich</td>
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<td>&nbsp;</td>
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<td>Dispase2.4U ml</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#17105-041</td>
<td>&nbsp;</td>
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<td>Trypsin-EDTA0.5%(wt/vol) (10x;5g liter trypsin (1:250)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#15400-054</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#C-9791</td>
<td>&nbsp;</td>
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isolating stem cells from soft musculoskeletal tissues

Células-tronco adultas têm sido discutidas em relação à sua aplicação na medicina regenerativa. Células-tronco adultas têm gerado uma grande emoção para o tratamento de tecidos lesionados e doentes, devido à sua capacidade impressionante de se submeter multi-linhagem diferenciação celular e sua capacidade de auto-renovação. Mais importante ainda, essas qualidades fizeram-lhes vantajoso para uso em terapias de transplante autólogo de células. O protocolo atual irá introduzir os leitores à técnica preplate modificado onde os tecidos moles do sistema músculo-esquelético, por exemplo, tendão e músculo, são um<sup&gt; St</sup&gt; Enzimaticamente dissociada e depois colocadas em frascos de colágeno revestido com o meio. O sobrenadante, que é composto por médias e as células restantes flutuante, é em série transferidos diariamente para frascos de novo. As células-tronco são as mais lentas a aderir aos frascos que é normalmente leva 5-7 dias (transferências serial ou preplates). Usando esta técnica, células-tronco adultas presentes nesses tecidos podem ser facilmente colhidas através bastante procedimentos não invasivos.

Watch this Scientific Journal Video about Isolating Stem Cells from Soft Musculoskeletal Tissues at JoVE.com

Isolating Stem Cells from Soft Musculoskeletal Tissues

Células adultas isolando-tronco de tecidos moles músculo-esqueléticas com base na velocidade da célula a adesão ao frasco.

Isolar células-tronco de tecidos moles músculo-esqueléticas

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

isolating-stem-cells-from-soft-musculoskeletal-tissues

Research

JoVE Journal

Biology

13.3K visualizações.  Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC. Células adultas isolando-tronco de tecidos moles músculo-esqueléticas com base na velocidade da célula a adesão ao frasco.

Vídeo: Isolating Stem Cells from Soft Musculoskeletal Tissues

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

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

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

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

Cromatina imunoprecipitação de células estaminais embrionárias humanas

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

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 of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Isolamento de células-tronco de xenoenxertos Humano do Câncer de pâncreas

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation of Retinal Stem Cells from the Mouse Eye

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 non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

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 ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Seleção e isolamento de colônias de células-tronco pluripotentes induzidas a partir de fibroblastos adultos reprogramado

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

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 ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

Isolar células de melanócito-like primárias do coração mouse

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells isolamento de culturas primárias de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...