Gostaríamos de agradecer aos membros do laboratório Lee pelas valiosas discussões sobre o manuscrito. Trabalho no laboratório Lee foi apoiada por doações de Robertson Investigator Award of New York Stem Cell Foundation e de Maryland Stem Cell Research Fund (Tedco).

1. Preparação de uma placa de 96 poços <ol><li> Misture 1 ml de Single Cell DNase1 a 9 mL Single Cell Lysis Solution. </li><li> Coloque a solução mista de 10 mL em cada poço da placa de 96 poços de PCR. </li></ol> 2. Detaching hESCs para FACS Purificação <ol><li> Separar OCT4 :: linha de células ES a partir de EGFP o prato de 60 mm e 1 ml de Accutase durante 20 min, a 37 ° C, as quais foram neutralizadas com meio ES humano. </li><li> Prepare a população de células em 1 ml de tampão de FACS e ajustar o celular para 1 x 10 6 células / ml. </li><li> Passar a amostra de células através de um coador de células de 35 mM tampão tubo. </li><li> Armazenar o tubo em gelo antes de separação de células. </li></ol> 3. Lise de FACS purificado célula única em cada poço da placa de 96 poços <ol><li> Classificar a amostra para células positivas EGFP em um classificador de células com um operador treinado. Coloque a única célula em solução Lysis/DNase1 única célula em placa de 96 poços PCR. Se necessário, a 96 poços da placa com a amostra pode ser armazenada num congelador a -80 ° C de profundidade inferior a um mês. </li><li> Incubar as amostras 5 min à temperatura ambiente para a lise celular. </li><li> Adicionar 1 ml de solução de paragem para parar reação de lise. </li><li> Incubar 2 min à temperatura ambiente. </li></ol> 4. Transcrição reversa <ol><li> Adicione a cada uma alíquota de 0,5 mL de 20 mM SMA-T15, SMA-A. </li><li> Adicionar tampão 4 uL de 5X, 2 uL de DTT, 1 ul de transcriptase reversa, e 1 ul de dNTP em cada poço. </li><li> Realizar a transcrição reversa num termociclador. <ol><li> Definir o programa térmico a 42 ° C x 90 min e inactivar transcriptase reversa a 85 ° C x 5 min. </li></ol></li></ol> 5. Amplificação <ol><li> Adicionar 4 mL de reagente ExoSAP-IT para cada amostra transcrita reverter. <ol><li> Incubar as amostras a 37 ° C durante 15 min e 80 ° C durante 15 min para inactivar o reagente ExoSAP-IT. </li></ol></li><li> Preparar a mistura de reacção de PCR wom SMA-p2 (2 nM) </li><li> Adicionam-se 10 ul de mistura de reacção de PCR para cada uma das amostras de transcrição reversa. </li><li> Realizar a amplificação, consistindo em 20 ciclos de desnaturação (94 ° C durante 30 seg), emparelhamento (57 ° C durante 30 seg) e extensão (68 ° C durante 10 min). </li></ol> 6. Desempenho de qRT-PCR <ol><li> Adicionar 10 ul de 2X SYBR Green PCR Master Mix, 1 ul de amplificado de ADNc, os iniciadores 2 nM, e 7 mL de água a cada poço. <ol><li> Definir o programa seguido por 95 ° C durante 3 seg, 60 ° C durante 30 seg x 40 ciclos. </li></ol></li><li> Realiza-se em duplicado por erros técnicos. </li></ol>

<ol>
	<li>Miyanari, Y., Torres-Padilla, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+ground-state+pluripotency+by+allelic+regulation+of+Nanog.">Control of ground-state pluripotency by allelic regulation of Nanog.</a> Nature. 483, 470-473 (2012).</li><li>Leitch, H. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+germ+cells+from+mice+and+rats+exhibit+properties+consistent+with+a+generic+pluripotent+ground+state.">Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state.</a> Development. 137, 2279-2287 (2010).</li><li>Ramskold, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full-length+mRNA-Seq+from+single-cell+levels+of+RNA+and+individual+circulating+tumor+cells.">Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells.</a> Nat Biotechnol. 30, 777-782 (2012).</li><li>Stewart, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+isolation+of+hESCs+reveals+heterogeneity+within+the+pluripotent+stem+cell+compartment.">Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment.</a> Nat Methods. 3, 807-815 (2006).</li><li>O'Malley, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+analysis+with+novel+cell-surface+markers+identifies+routes+to+iPS+cells.">High-resolution analysis with novel cell-surface markers identifies routes to iPS cells.</a> Nature. 499, 88-91 (2013).</li><li>Nichols, J., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+and+primed+pluripotent+states.">Naive and primed pluripotent states.</a> Cell Stem Cell. 4, 487-492 (2009).</li><li>Kim, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-371-3+expression+predicts+neural+differentiation+propensity+in+human+pluripotent+stem+cells.">miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells.</a> Cell Stem Cell. 8, 695-706 (2011).</li><li>Mortazavi, A., Williams, B. A., Mccue, K., Schaeffer, L., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+quantifying+mammalian+transcriptomes+by+RNA-Seq.">Mapping and quantifying mammalian transcriptomes by RNA-Seq.</a> Nat Methods. 5, 621-628 (2008).</li><li>Pan, Q., Shai, O., Lee, L. J., Frey, J., Blencowe, 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=Deep+surveying+of+alternative+splicing+complexity+in+the+human+transcriptome+by+high-throughput+sequencing.">Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.</a> Nat Genet. 40, 1413-1415 (2008).</li><li>Wang, E. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+isoform+regulation+in+human+tissue+transcriptomes.">Alternative isoform regulation in human tissue transcriptomes.</a> Nature. 456, 470-476 (2008).</li><li>Hockemeyer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+human+pluripotent+cells+using+TALE+nucleases.">Genetic engineering of human pluripotent cells using TALE nucleases.</a> Nat Biotechnol. 29, 731-734 (2011).</li><li>Lee, G., Chambers, S. M., Tomishima, M. J., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+neural+crest+cells+from+human+pluripotent+stem+cells.">Derivation of neural crest cells from human pluripotent stem cells.</a> Nat Protoc. 5, 688-701 (2010).</li><li>Zhu, Y. Y., Machleder, E. M., Chenchik, A., Li, R., Siebert, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+transcriptase+template+switching:+A+SMART+(TM)+approach+for+full-length+cDNA+library+construction.">Reverse transcriptase template switching: A SMART (TM) approach for full-length cDNA library construction.</a> Biotechniques. 30, 892-897 (2001).</li><li>Pan, X. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+methods+for+full-length+RNA+sequencing+for+low+quantities+of+cells+and+single+cells.">Two methods for full-length RNA sequencing for low quantities of cells and single cells.</a> P Natl Acad Sci USA. 110, 594-599 (2013).</li><li>Tang, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq+analysis+to+capture+the+transcriptome+landscape+of+a+single+cell.">RNA-Seq analysis to capture the transcriptome landscape of a single cell.</a> Nat Protoc. 5, 516-535 (2010).</li></ol>

Os autores não têm nada a revelar.

Profiling gene célula única pode ser uma ferramenta importante para prever a funcionalidade de uma única célula ou uma população inteira. Devido a uma limitação técnica, análise de perfis gene inteiro tenha sido restrita às médias populacionais. As variações nos padrões de expressão dos genes e os níveis de entre as células individuais e as subpopulações foram propostos para fazer com que a interpretação errada. Tais aspectos celulares diferentes podem ser encontrados em hESCs e sua heterogeneidade faz com que a capacidade sutilmente diferente para a manutenção de pluripotência e processo de especificação destino. Foi desenvolvido e validado para quantitativa única célula de RT-PCR, o que proporciona um método simples, mas robusta para estudar a expressão de genes em células individuais. Para validar o nosso protocolo, lisados ​​de célula única FACS-purificada foi dividido em metade e aplicada a este método para verificar precisões. Nosso resultado com células individuais foi corroborada com amostras de RNA total diluídas em série e encontramos resultados consistentes bntre cada metade de lisado de células (Figura 4). No entanto, com o nosso protocolo, existem algumas limitações. Podemos detectar o nível de expressão de mais de 40 genes, mas informações específicas de transcrição (por exemplo, mRNAs não poliadenilados, miRNA, transcrições desconhecidos, etc) pode não ser detectável. Além disso, existem algumas oportunidades para otimizar os primers para os genes-alvo. O comprimento cartilha aceito é geralmente 18-22 pares de bases. As temperaturas de emparelhamento de primers trabalham geralmente na gama de 50-60 ° C. O conteúdo de GC dos iniciadores pode ser afectada pela capacidade de hibridação do iniciador. Neste trabalho, nós removemos a etapa de recozimento em qPCR para reduzir o erro de primer recozimento. Nós apenas mostrou o gene GAPDH como um controlo, mas o outro house keeping genes pode ser útil como um controlo. Atualmente, estamos otimizando nosso protocolo para RNA abordagem de seqüenciamento, que vai descobrir transcriptoma mais detalhada das células individuais isoladas no futuro próximo. A outra limitação é accuatrevido da FACS purificação para isolar células individuais. Máquina FACS principalmente aloca células individuais em cada poço, mas não podemos excluir a possibilidade de que cada poço pode conter mais de uma célula, que pode ser descoberto com o avanço técnico de sistema de purificação de FACS. No entanto, o nosso protocolo pode ser diretamente aplicável a qualquer laboratório com esforços mínimos e rentável forma de perfis de expressão gênica de células individuais. Como mostrado na Figura 5, OCT4 :: EGFP células individuais positivas por FACS classificadas manter nível similar de expressão do gene OCT4, mas NANOG mostrou diversificada padrão de expressão, o que pode sugere o status diferente de pluripotência de hESCs ou processo de reprogramação <a href="#_ENREF_1" title="Miyanari, 2012 N º 26">1</a> . Pode-se outro perfil de expressão gênica em células de pluripotência únicas de hESCs utilizando diferentes marcadores de superfície celular (TRA-1-60, SSEA3, ICAM1, CD44) e / ou sistema de repórter genética (NANOG, REX1, STELLA, ESRRB, etc) <a href="#_ENREF_5" title="O&#39;Malley, 2013 N º 28">5-7</a> . Ao utilizar este protocolo, a abordagem única expressão génica em células é um método fácil e poderosa para populações heterogêneas, como hESCs ou outros tipos de células-tronco.

Populações eucariotos maioria superiores são heterogêneos, assim, com a análise da amostragem populacional, muitas vezes é difícil de interpretar as suas características celulares. As células individuais dentro de uma população pode ser sutilmente diferente, e essas diferenças podem ter consequências importantes para a propriedade ea função de toda a população 1, 2. Especialmente, as células-tronco embrionárias humanas (hESCs) são conhecidos por ser heterogêneo, o que provoca diferentes níveis de pluripotência e diversas potencialidades para especificação das linhagens em delicadamente e distintas formas 3, 4. Por exemplo, os antigénios de superfície de células diferentes podem ser usados ​​para classificar as células estaminais pluripotentes indiferenciadas, 5 e o grupo Austin Smith propostos diferentes níveis de pluripotência em mastócitos embrionários de ratinho, com base na sua morfologia, propensão diferenciação e dependência da via de sinalização 6. Este fenômeno foi levantada a hipótese de embrião humanoAs células estaminais nic 7. Considerando que os estudos gerais foram realizadas entre as diferentes linhas de células estaminais, e não células-tronco único indivíduo, que poderia ser muito interessante para analisar diferentes níveis de pluripotência no nível da célula única, o que potencialmente afeta as suas capacidades de diferenciação em relação a todas as linhagens de células somáticas. Heterogeneidade celular e molecular pode ser ditada pela transcrição de perfil, que é chamado de &#39;transcriptoma única célula&#39; e destaca novas abordagens para quantificar os níveis de expressão do gene 8-10. Para a análise dos níveis de expressão de genes em células individuais, foi desenvolvido um protocolo simples, mas robusta de uma única célula RT-PCR quantitativo. Foi confirmada a eficácia e viabilidade do nosso protocolo, comparando cada metade de lisados ​​de células individuais, bem como RNAs totais diluídas em série de hESCs, resultando em variações técnicas mínimas e diferenças. Além disso, foi utilizada uma linha repórter genética para isolar homogênea população de hESCs utilizando sistema de metas de gene. O vector dador de segmentação OCT4 lócus (OCT4-2A-EGFP-PGK-Puro construção) e um par de plasmídeos talen foram usadas 11. O vetor doador e um par de plasmídeos Talen foram introduzidos hESCs (H9, WA09) usando nosso nucleofection e protocolo de seleção clonal e manutenção de hESCs foi realizada com base no nosso protocolo de rotina 12. Nós confirmamos esta linha repórter genética expressar EGFP para a expressão OCT4 em OCT4 :: EGFP hESCs. Nosso resultado demonstra que hESCs individuais (classificadas por OCT4 :: EGFP células fortemente positivas) manter altos níveis de expressão OCT4, mas diferentes níveis de expressão NANOG. Então, o nosso ensaio de expressão único gene de células deve ser útil para estudar as heterogeneidades da população de células-tronco pluripotentes.

<table border="0" cellpadding="0" cellspacing="0" width="807">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">96 wll PCR Plate</td>
			<td style="width:123px;">USA scientific</td>
			<td style="width:119px;">1402-8900</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Ambion Cell Lysis Kit</td>
			<td style="width:123px;">Life Technologies</td>
			<td align="right" style="width:119px;">4458235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">SMARTScribe Reverse Transcriptase</td>
			<td style="width:123px;">Clonetech</td>
			<td align="right" style="width:119px;">639536</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ExoSAP-IT</td>
			<td style="width:123px;">USB</td>
			<td align="right" style="width:119px;">78200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Platinum Taq DNA polymerase High Fidelity</td>
			<td style="width:123px;">Invitrogen</td>
			<td align="right" style="width:119px;">11304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">10mM dNTP Mix, PCR Grade</td>
			<td style="width:123px;">Invitrogen</td>
			<td align="right" style="width:119px;">18427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">SYBR Universal 2X Master Mix</td>
			<td style="width:123px;">Kapa biosystem</td>
			<td style="width:119px;">KR0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Accutase</td>
			<td>Innovative Cell Tech</td>
			<td>S-1100-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">FACS buffer</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td>45 ml PBS, 5 ml a-MEM, 100 ul DNase, filter sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">35 &#956;m cell strainer cap tubes</td>
			<td style="width:123px;">BD Biosciences</td>
			<td align="right" style="width:119px;">352235</td>
			<td></td>
		</tr>
	</tbody>
</table>

profiling individual human embryonic stem cells by quantitative rt-pcr

A heterogeneidade da população de células-tronco dificulta compreensão detalhada da biologia de células-tronco, tais como a sua propensão para a diferenciação diferentes linhagens. Um ensaio de transcriptoma única célula pode ser uma nova abordagem para dissecar variação individual. Desenvolvemos o método de uma única célula de qRT-PCR, e confirmaram que o método funciona bem em vários perfis de expressão de gene. Em nível de uma única célula, cada célula-tronco embrionárias humanas, classificado por OCT4 :: células positivas EGFP, tem alta expressão no OCT4, mas um nível diferente de expressão NANOG. Nosso ensaio de expressão único gene da célula deve ser útil para interrogar heterogeneidades da população.

Amplificação de RNA única célula eficiente e robusta Para minimizar a variação transcricional entre hESCs, usamos OCT4 :: EGFP hESC clone para FACS purificação. Após a separação de células positivas Oct4 :: EGFP em uma placa de 96 poços, cada célula é lisadas em tampão de lise e convertido poli (A) + RNA para cDNA de comprimento completo utilizando o SMA-T15 (GACATGTATCCGGATGTTTTTTTTTTTTTTTT) e iniciador de ancoragem com o SMA-A (ACATGTATCCGGATGTGGG ) usando a tecnologia Smart modelo de comutação. Os oligonucleótidos foram digeridas com excesso de reagente ExoSAP-IT, seguida por 18-20 ciclos de amplificação por PCR de cDNA com o SMA-p2 (GACATGTATCCGGATGT) 13. Utilizou-se o ADNc amplificado para fazer o molde de qRT-PCR (Figura 1). Existem vários estudos para o pleno seqüenciamento RNA comprimento e medição da variabilidade da RNA usando baixas quantidades de células e células individuais 3, 14, 15. Para our conhecimento, diluído RNA total de hESCs (valores microgramas) para níveis nano e pico-gram e aplicado nosso protocolo para avaliar a variabilidade técnica e detecção de diferença em baixas quantidades de RNA total. Foi determinada a reprodutibilidade dos níveis de expressão de genes gerados a partir de RNA diluído e as células individuais. Análise do ARN diluída serialmente mostra correlação entre cada uma das amostras e os resultados de qRT-PCR com várias células individuais mostram os valores de Ct semelhantes no gene GAPDH (Figura 2). Avaliação quantitativa de perfis único gene celular Para validar a consistência entre diferentes lotes de reações de transcrição reversa, dividimos lisados ​​unicelulares FACS purificadas em metade, e aplicado o nosso protocolo separadamente para cada metade de lisados ​​para comparações de lote. Nós classificados células positivas para EGFP fortes usando FACS classificador (Figura 3), de modo nível de expressão OCT4 foi elevada em EGFPcélulas positivas, mas o nível de expressão NANOG é vária. O resultado mostra correlação significativa entre o valor de Ct OCT4 de cada metade de lisados ​​de células individuais (Figura 4). Análise de perfis de genes de células-tronco embrionárias Separamos hESCs individuais e analisaram seu nível de expressão de genes usando o nosso protocolo, que mostra o nível consistente de expressão OCT4, em seguida, fizemos o check outro NANOG gene marcador de células-tronco em hESCs. Como resultado, o nível de expressão do gene OCT4 foi elevada em todas as células, mas NANOG mostra diferentes padrões (Figura 5). <img alt="Figura 1" fo:content-width="6in" src="/files/ftp_upload/51408/51408fig1highres.jpg" width="600" /> Figura 1. Visão esquemática de única célula-tronco embrionária humana qRT-PCR após FACS purificação. </&gt; de células positivas individuais EGFP fortes são classificadas em cada poço de uma placa de 96 poços contendo tampão de lise celular. Única célula lisada passou por transcrição reversa com RTase. Nucleotídeos restantes são limpos com SAP / EXO, em seguida, o produto é amplificado pela reação em PCR com Taq DNA polimerase. <img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51408/51408fig2highres.jpg" width="500" /> Figura 2. Em tempo real de RT-PCR de GAPDH utilizando diluída em série de mRNA de células estaminais embrionárias humanas reunidas. Cada ponto mostra o valor Ct do ARNm diluídos em série e de ARNm de célula única classificadas por FACS. Os ARNs totais foram diluídos a partir de 10 ng / ul de 0,1 pg / ul. Repetimos mesmos experimentos para a comparação e enredo linear feita. <img alt="Figura 3" fo:content-width="6in" src="/files/ftp_upload/51408/51408fig3highres.jpg" width="600" /><br/&gt; A Figura 3. Análise FACS de células positivas outubro. Nós classificados célula positiva EGFP usando um classificador de SCAF. OCT4 :: EGFP linha de células estaminais embrionárias humanas repórter mostra população positiva de cerca de 60% ​​em comparação com a população de EGFP negativo (B). Nós selecionamos fortes células positivas EGFP (A). <img alt="Figura 4" fo:content-width="5in" src="/files/ftp_upload/51408/51408fig4highres.jpg" width="500" /> Figura 4. Comparação do nível de expressão de genes em cada metade de lisados ​​de células individuais. Para validar a consistência entre lotes diferentes da transcrição reversa e de amplificação de reacções, dividiu-se os lisados ​​de células individuais em meio e aplicadas nosso protocolo para comparação de lotes. <img alt="Figura 5" fo:content-width="6in" src="/files/ftp_upload/51408/51408fig5highres.jpg" width="600" /> Figura 5.60;. Análise de expressão gênica de células mapa de calor apresentação da expressão do gene de células-tronco embrionárias humanas Individual purificado por FACS mostra a expressão robusta em OCT4, mas diferentes níveis de expressão em NANOG.

Watch this Scientific Journal Video about Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR at JoVE.com

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Ensaio única expressão gênica de células é necessária para a compreensão da heterogeneidade de células-tronco.

Profiling Individual células estaminais embrionárias humanas por RT-PCR quantitativo

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

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11.5K visualizações.  Johns Hopkins University School of Medicine.  Ensaio única expressão gênica de células é necessária para a compreensão da heterogeneidade de células-tronco. 

Vídeo: Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

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

Propagação de-tronco embrionárias humanas (ES) Células

Biologia

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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

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

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

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

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivação de células estaminais embrionárias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Sondagem para a Atividade Complexo mitocondrial em células estaminais embrionárias humanas

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

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

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

Cromatina imunoprecipitação de células estaminais embrionárias humanas

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

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Na rotulagem in vitro de células estaminais embrionárias humanas para Ressonância Magnética

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Cultura e Manutenção de células estaminais embrionárias humanas

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

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

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

Preparação Spread cromossômica de células estaminais embrionárias humanas para Cariotipagem

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

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelamento e descongelamento, células estaminais embrionárias humanas

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Diferenciação de células-tronco embrionárias em precursores oligodendroglial

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...