Os autores gostariam de agradecer às seguintes concessões do NIH para apoiar esta pesquisa: K08DK082783 (AR), P30DK56465 (BTS), U01HL099993 (BTS), T32HL00715036 (SKS) e K12HL0806406 (SKS); ea Fundação McCarthy JP (AR).

Consentimento informado, aprovado pelos Conselhos de Revisão Institucional da Cancer Research Center Fred Hutchinson e as crianças &quot;s Hospital of Philadelphia, foi obtido de pacientes antes de coletar amostras de sangue periférico. Foram observadas Todas as diretrizes institucionais. Todos os experimentos com animais, incluindo geração e MEF formação de teratomas foram aprovados pelo Comitê de Uso e Cuidado Animal Institucional. 1. Ficoll separação de PBMC e Expansão da Erythroblasts - Dia 0 <ol><li> Dilui-se sangue periférico de 1: 1 com Solução Salina Tamponada com Fosfato de Dulbecco (DPBS). Em um tubo de poliestireno de fundo redondo de 15 ml, a camada cuidadosamente 7 ml de sangue diluído em 3 mL de temperatura ambiente Ficoll-Hypaque. </li><li> Centrifuga-se a amostra a 400 xg durante 30 min. </li><li> As PBMC irão ter resolvido para a interface entre o plasma e o Ficoll-Hypaque, visto como uma camada branca turva. Usando uma pipeta estéril, recolher o PBMCs em um tubo de 15 ml. Levar o volume até 10 ml, utilizando DPBS. </li><li> Usando um hemocitômetro, contar as PBMC. </li><li> Pipeta de 2 x 10 6 PBMC em um tubo de 15 ml separado e centrifugar a 300 xg durante 5 min. Girar as restantes células e congelamento a uma concentração de 2 x 10 6 PBMC / ml em 90% de soro fetal bovino (FBS) a sulfóxido / 10% de dimetilo (DMSO). </li><li> Prepare meio de expansão: meio isento de soro QBSF-60 (protegido da luz) + penicilina / estreptomicina (1%) + ácido ascórbico (50 ng / ml) + factor de células estaminais (SCF, 50 ng / ml) + a interleucina 3 (IL 3, 10 ng / ml) + eritropoietina (EPO, 2 U / ml) + insulin-like growth factor 1 (IGF-1, 40 ng / mL) + dexametasona (1 uM; proteger da luz; descongelar uma alíquota fresca cada um dos meios alterações). </li><li> Ressuspender 2 × 10 6 PBMC em 2 ml de meio de expansão acabado de fazer e colocar em um poço de uma placa de cultura de tecido de 12 poços e incubar numa incubadora a 37 ° C humidificado com uma atmosfera de 5% De O 2, 5% de CO 2, e 90% de N2. </li><li> No dia 3 eo dia 6, substituir a mídia. <ol><li> Recolher as células e lava-se a bem com 2 ml de QBSF-60 para recolher quaisquer células restantes. </li><li> Centrifuga-se a suspensão de células a 300 xg durante 5 min e aspirar o sobrenadante. </li><li> Ressuspender as células em 2 ml de meio de expansão e nova devolvê-las para a mesma placa de 12 poços. </li></ol></li></ol> 2. reprogramar células por Nucleofection - Dia 9 <ol><li> Pipetar os plasmídeos de reprogramação para um tubo de 1,5 ml estéril Eppendorf (Tabela 1). </li><li> Misture a solução nucleofection com suplemento (fornecido pelo fabricante) e pipeta em um estéril de 1,5 ml tubo Eppendorf. </li><li> Pipetar 2 ml de meio de expansão em um poço de uma placa de 12 bem e coloque numa atmosfera humidificada de 37 ° C incubadora (5% de O2, 5% de CO 2, e 90% de N2) por razões de equilíbrio antes da adição de células. </li><li> Recolher océlulas de cultura e lavar o poço com 2 ml de QBSF-60 para recolher as células restantes. </li><li> Centrifuga-se as células a 300 xg durante 5 min e aspirar o sobrenadante. </li><li> Lavam-se as células por ressuspensão em 5 mL de DPBS e centrifugar a 300 xg durante 5 min. </li><li> Aspirar o sobrenadante. </li><li> Ressuspender o pellet celular em 100 ml de solução nucleofection + Suplemento, tendo o cuidado de evitar a geração de bolhas. </li><li> Pipeta a suspensão de células para dentro do tubo contendo os plasmídeos de reprogramação, pipeta-se para baixo e, uma vez, e pipetar a amostra para o fundo da cubeta nucleofection. </li><li> Coloque a cuvete na máquina nucleofection e executar Programa de T-019. </li><li> Transferir 100 ul da expansão pré-aquecido médio (a partir da placa de equilíbrio no Passo 2.3) na cuvete nucleofection. </li><li> Recolher imediatamente a totalidade da amostra e depósito para o poço de pré-aquecido médio Expansão. Evite recolher o, flutuando restos de células mortas branco. </li&gt; <li> Colocar a placa num incubador humidificado a 37 ° C incubadora (5% de O2, 5% de CO 2, e 90% N 2) para o crescimento. </li></ol> 3. chapeamento células alimentadoras - Dia 10 ou 11 <ol><li> No dia 10, as células da placa de alimentação. <ol><li> Brasão uma placa de 6 poços de cultura de tecidos com gelatina. <ol><li> Pipeta de 1 m de gelatina a 0,1% em Solução Salina Tamponada de Hank (HBSS) para cada poço da placa. </li><li> Colocar a placa num incubador humidificado a 37 ° C incubadora durante pelo menos 5 min. </li><li> Imediatamente antes da utilização, aspirar a solução de gelatina a partir da placa. </li></ol></li><li> Descongelar um frasco de 2 x 10 6 de fibroblastos de rato embrionários irradiadas a 3000 cGy em 10 ml de pré-aquecido MEF Meios (Meio Eagle Modificado por Dulbecco (DMEM) + FBS (10%) + penicilina / estreptomicina (1%)). </li><li> Centrifuga-se as células a 300 xg durante 5 min e aspirar o sobrenadante. Ressuspender as células em 12 ml de MEF Mídia e pipeta de 2 ml into de cada poço revestido com gelatina a placa de 6 poços. Colocar a placa num incubador humidificado a 37 ° C incubadora (5% de O2, 5% de CO 2, e 90% N 2) até o dia 12. </li></ol></li></ol> 4. cultivo de células em células alimentadoras e Alterando Médio - Dia 12 <ol><li> Coletar as células nucleofected em um tubo de 15 ml e recolher todas as células restantes lavando as bem com 2 ml de QBSF-60. Centrifuga-se a amostra a 300 xg durante 5 min. </li><li> Prepare reprogramação Meio: Iscove Modificado por Dulbecco (IMDM) + FBS (10%) + não animal de L-glutamina (1 mM) + ácidos aminados não essenciais (NEAA, 1%) + penicilina / estreptomicina (1%) + β -mercaptoetanol (0,1 mM) + factor de crescimento de fibroblastos básico (bFGF, adicionado na alimentação fresca, 10 ng / ml) + ácido ascórbico (adicionado fresco na alimentação, 50 ug / ml). </li><li> Aspirar o sobrenadante e ressuspender as células em 12 ml de Meio A reprogramação. Pipeta de 2 ml por cavidade da suspensão de células ema placa de 6 poços de células alimentadoras (como foi preparada na secção 3). Centrifugar a placa a 50 xg durante 30 min à temperatura ambiente. </li><li> Colocar a placa num incubador humidificado a 37 ° C incubadora (5% de O2, 5% de CO 2, e 90% N 2) para o crescimento. Altere a Reprogramação Médio em dias alternados até colônias IPSC começam a aparecer (1-2 semanas). </li><li> Preparar células estaminais embrionárias humanas Meio: DMEM / F12 1: 1 + KO substituição Serum (20%) + penicilina / estreptomicina (1%) + NEAA (1%) + não animal de L-glutamina (1 mM) + piruvato de sódio (1%) + bicarbonato de sódio (0,12%) + β-mercaptoetanol (0,1 mM) + bFGF (adicionado fresco na alimentação, 10 ng / ml) </li><li> Prepare inibidores de HDAC: butirato de sódio (adicionado fresco em alimentação, 100 M), ácido suberanilohidroxï¿½ico (SAHA, acrescentou fresco na alimentação, 200 nM). Uma vez colônias IPSC aparecer, começam a alimentar as células com inibidores de células estaminais embrionárias humanas Médio + HDAC, mudando médio a cada dia. NOTA: o butirato de sódio é uma pequena molécula que é adsorvido para os tubos de plástico, tor conseguinte armazená-lo em um tubo de vidro de borosilicato, a 4 ° C. </li></ol> 5. Clones Isolando IPSC <ol><li> Uma vez colônias IPSC são grandes o suficiente para ser isolado (20-25 células), buscá-las com uma pipeta P20. </li><li> Preparar os inibidores de HDAC: butirato de sódio (adicionado fresco a alimentação, 100 uM), SAHA (adicionado fresco a alimentação, 200 nM) </li><li> Coloque as colônias isoladas para iMEFs em pratos individuais mm 35 contendo inibidores de HDAC hESC Médio + + Clonagem &amp; Recovery suplemento. </li><li> O dia após a colheita das colônias, mudar o meio de inibidores de células estaminais embrionárias humanas Médio + HDAC. </li><li> Mudar o meio todos os dias até as colónias IPSC estão prontos para passaging. </li><li> Retirar os inibidores de HDAC do meio uma vez colônias estabilizar em passagem 2-3. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Para a geração de iPSC bem sucedido ao usar este protocolo, existem várias advertências importantes que devem ser considerados. Durante a fase de expansão de eritroblastos, o cronograma de mudança de mídia devem ser rigorosamente seguidas, como desvios podem levar à estimulação ineficiente da população de células progenitoras alvo e uma menor eficiência de geração da IPSC. É importante fazer novo meio de expansão com dexametasona fresco com cada mudança de meios de comunicação; e o meio de base QBSF...

iPSCs são derivados a partir de tecidos somáticos via expressão ectópica de um conjunto mínimo de genes de pluripotência. Esta técnica foi inicialmente demonstrado por transdução retroviral de fibroblastos humanos com OCT4, SOX2, KLF4, e cMyc, que são altamente expressos no estado pluripotente 1. Estes transitoriamente expressas &quot;fatores de reprogramação epigenética&quot; alterar perfil de expressão de paisagem e gene da célula-alvo análogo ao de células-tronco embrionárias humanas 2. Uma vez criado, iPSCs podem potencialmente ser diferenciadas em qualquer tipo de tecido para posterior investigação. Assim, eles representam uma promessa para o uso em medicina regenerativa, doença de modelagem, e aplicações de terapia génica. No entanto, interrompendo o genoma do vírus com integrando tem o potencial de alterar a expressão do gene endógeno, influencia o fenótipo celular e, finalmente, viés resultados científicos. Além disso, as integrações aleatórias virais pode levar a deletéria e celularffects, incluindo a possibilidade de transformação maligna 3 ou re-expressão dos transgenes oncogénicos 4. Aplicações clínicas futuras exigirá geração iPSC não-integrantes. Epissomas, que são moléculas de DNA circular cromossômicas extra, oferecem uma estratégia para gerar custo efetivo, iPSCs livre de integração 5. A combinação dos vectores epissomais mostrados na Tabela 1 Factores de expressar a reprogramação OCT4, SOX2, KLF4, MYCL, e LIN28A. O plasmídeo pCXLE_hOCT3 / 4-shp53-F também contém uma p53 shRNA para a supressão temporária de TP53 para melhorar a reprogramação celular 6. A replicação deficiente vetor pCXWB-EBNA1 promove amplificação de fatores de reprogramação e aumento da eficiência de reprogramação, proporcionando um aumento transitório na expressão EBNA1 7. O plasmídeo pCXLE_EGFP pode ser adicionado à mistura nucleofection com a finalidade de determining a eficiência de transfecção ou para aplicações de separação de células. Com a excepção de p-CXWB EBNA1, os plasmídeos epissomais utilizados neste protocolo contêm a origem do vírus de Epstein-Barr de replicação viral e o gene de EBNA 1, que medeiam a replicação e partição do epissoma durante a divisão da célula hospedeira 8. Os epissomas são espontaneamente perdido com expansão sucessiva iPSC 7. A subclonagem e caracterização de iPSCs com perda vector epissomal, que pode ser inferida a partir da perda de expressão eGFP, pode conduzir a integração iPSCs completamente livres para aplicações clínicas futuras. Inerente ao processo de geração iPSC é a supressão de genes específicos de linhagem e reactivação de genes associados à pluripotência. Regulação da expressão gênica ocorre em vários níveis dentro do núcleo, incluindo a modificação de DNA e da cromatina para permitir que fatores de transcrição, elementos de DNA reguladoras e acesso RNA polimerase ao alvogenes. A remodelação da paisagem epigenética através de modificações da cromatina globais é um componente chave para a re-expressão do programa genético pluripotência. Uma modificação da cromatina específico que é importante na regulação da expressão génica é a acetilação das histonas em determinados resíduos de lisina, que permite o acesso aos genes-alvo através da diminuição da tensão da bobina de histona-ADN. Inibidores de HDAC são pequenas moléculas que têm sido mostrados para melhorar iPSC reprogramação e hESC auto-renovação 9,10, provavelmente devido ao apoio do Estado acetilada 11. O protocolo descrito abaixo, adaptado a partir de uma publicação anterior, utilizando os lentivírus integrando 12, proporciona um método passo-a-passo para a geração de iPSC optimizado a partir de sangue periférico utilizando epissomas e inibidores de HDAC. As concentrações de inibidor de HDAC aqui utilizados são metade daqueles descritos por Ware et al. 9, e foram rotineiramente levou a um aumento de 2 vezes na colónias IPSC completamente reprogramadas padrão maisprotocolos de reprogramação epissómicos sem inibidores de HDAC. Este nível de reprogramação está a par com a eficiência que observamos com os métodos de lentivírus. Usando este protocolo, temos gerado de forma eficiente iPSC de indivíduos como velhos como 87 anos de idade.

<table border="0" cellpadding="0" cellspacing="0" width="1100">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Name of Reagent/ Equipment</td>
			<td style="width:151px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:503px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Ficoll-Paque PLUS</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">45-001-750</td>
			<td>Store at 4&#176;C. Warm to room temperature before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DPBS</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">14190-250</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">RPMI 1640</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11875-093</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">fetal bovine serum (FBS)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">10437028</td>
			<td>Store at -20&#176;C until needed. Thaw, aliquot, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">dimethyl sulfoxide (DMSO)</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">154938</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">QBSF-60 serum-free medium</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">50-983-234</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">penicillin/streptomycin</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">15140122</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Cell Line Nucleofector Kit V</td>
			<td style="width:151px;">Lonza</td>
			<td style="width:119px;">VCA-1003</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2% gelatin solution</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1393</td>
			<td>Store at 4&#176;C, liquify in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Hank&#39;s Balanced Saline Solution (HBSS)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">14175-103</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:329px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">12440-061</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">L-glutamine, 200 mM</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">non-essential amino acids (NEAA), 100X</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td>Store at 4&#176;C, away from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DMEM/Ham&#39;s F12, 1:1</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">SH30023.02</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">KnockOut Serum Replacement</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium pyruvate, 100 mM</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11360-070</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium bicarbonate, 7.5%</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">25080094</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">basic fibroblast growth factor (bFGF)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">PHG0263</td>
			<td>Make 10 mg/mL stock in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium butyrate&#160;</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">B5887-1G</td>
			<td>Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">hES Cell Cloning &#38; Recovery Supplement</td>
			<td style="width:151px;">Stemgent</td>
			<td style="width:119px;">01-0014-500</td>
			<td>Store at -20&#176;C until needed. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">ESC-qualified BD matrigel</td>
			<td style="width:151px;">BD Biosciences</td>
			<td style="width:119px;">35-4277</td>
			<td>Thaw overnight on ice at 4&#176;C, aliquot into pre-chilled tubes using pre-chilled pipette tips. Store at -20&#176;C until needed. Thaw at 4&#176;C, use immediately.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">StemSpan SFEM&#160;</td>
			<td style="width:151px;">STEMCELL Technologies</td>
			<td style="width:119px;">9650</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">ascorbic acid, powdered</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4403-100MG</td>
			<td>Make 5 mg/mL stock in DPBS, sterile filter, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">recombinant human stem cell factor (SCF)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">255-SC-010</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">recombinant human interleukin 3 (IL-3)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">203-IL-010</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">erythropoietin (EPO)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">287-TC-500</td>
			<td>Make 1000 U/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">recombinant human insulin-like growth factor 1 (IGF-1)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">291-G1-200</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">&#946;-mercaptoethanol</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7522</td>
			<td>Make 1000X stock (100 mM) in DPBS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">dexamethasone</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4902-25MG</td>
			<td>Make 50X stock (50 &#956;M) in DPBS, sterile filter, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">SAHA (vorinostat)</td>
			<td style="width:151px;">Cayman Chemical</td>
			<td style="width:119px;">149647-78-9</td>
			<td>Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">12 well tissue culture plate</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">08-772-29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">15 mL conical tube</td>
			<td style="width:151px;">Sarstedt</td>
			<td style="width:119px;">62553002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">1.5 mL Eppendorf tube</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">6 well tissue culture plate</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">08-772-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">35 mm tisue culture plates</td>
			<td style="width:151px;">BD Biosciences</td>
			<td style="width:119px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">10 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">5 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">20 &#956;L pipette tips, barrier tips</td>
			<td style="width:151px;">Genessee</td>
			<td style="width:119px;">24-404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">glass Pasteur pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-678-20D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pipette aid</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-681-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hOCT3/4-shp53-F</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hSK</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hUL</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-EGFP</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXWB-EBNA1</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">37624</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient ips cell generation from blood using episomes and hdac inhibitors

Este manuscrito ilustra um protocolo para a criação de células de forma eficiente sem integração humana induzida tronco pluripotentes (iPSCs) do sangue periférico, utilizando plasmídeos epissómicos e inibidores da histona deacetilase (HDAC). As vantagens desta abordagem incluem: (1) o uso de uma quantidade mínima de sangue periférico como fonte de materiais; (2) nonintegrating vetores de reprogramação; (3) um método de custo eficaz para gerar vetores iPSCs livres; (4) uma única transfecção; e (5) a utilização de moléculas pequenas para facilitar a reprogramação epigenética. Resumidamente, as células mononucleares do sangue periférico (PBMC) são isoladas a partir de amostras de flebotomia de rotina e, em seguida, cultivadas em factores de crescimento definidos para se obter uma população de células progenitoras de eritrócitos altamente proliferativo que é extremamente passíveis de reprogramação. Nonintegrating, plasmídeos epissómicos não transmissíveis expressando OCT4, SOX2, KLF4, MYCL, LIN28A, e uma p53 short hairpin (sh) RNA são introduced nos eritroblastos derivados através de um único nucleofection. A cotransfecção de um epissoma que expressa a proteína verde fluorescente melhorada (eGFP) permite a fácil identificação de células transfectadas. Um plasmídeo deficiente na replicação separada expressando de Epstein-Barr antigénio nuclear 1 (EBNA 1), também é adicionado à mistura de reacção para aumento da expressão de proteínas epissomais. As células transfectadas são então plaqueadas sobre uma camada de fibroblastos embrionários de ratinho irradiado (iMEFs) para a reprogramação continua. Assim que colónias-iPSC como aparece a cerca de 12 dias após nucleofection, os inibidores de HDAC são adicionados ao meio para facilitar a remodelação epigenética. Descobrimos que a inclusão de inibidores de HDAC rotineiramente aumenta a geração de colónias de IPSC totalmente reprogramado por 2 vezes. Uma vez colônias IPSC exibem morfologia típica de células-tronco embrionárias humanas (hESC), eles são cuidadosamente transferida para IMEF revestidas de placas individuais de cultura de tecidos para o crescimento contínuo e expansão.

Três dias após nucleofection e antes do plaqueamento das células em nucleofected iMEFs, a eficiência de nucleofection bem sucedida deve ser estimado por microscopia de fluorescência para eGFP. A Figura 1 mostra uma experiência típica nucleofection com aproximadamente 5-10% da população total de células que expressam eGFP. Colónias IPSC reprogramado vão começar a aparecer cerca de duas semanas após nucleofection. As colônias são geralmente circular, com bordas...

Watch this Scientific Journal Video about Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors at JoVE.com

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Aqui nós descrevemos um protocolo para gerar células-tronco pluripotentes induzidas pelo homem a partir do sangue periférico usando uma estratégia de reprogramação episome base e inibidores da histona deacetilase.

Eficiente Geração células iPS a partir de sangue, utilizando epissomas e HDAC Inibidores

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

efficient-ips-cell-generation-from-blood-using-episomes-hdac

Research

JoVE Journal

Biology

12.7K visualizações.  Fred Hutchinson Cancer Research Center.  Aqui nós descrevemos um protocolo para gerar células-tronco pluripotentes induzidas pelo homem a partir do sangue periférico usando uma estratégia de reprogramação episome base e inibidores da histona deacetilase. 

Vídeo: Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Coleta de sangue do caranguejo em ferradura americano, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Biologia

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Geração de Single-Cell Tissue Suspensões de rato Neural

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e cultura de células endoteliais pulmonares de camundongos Neonatal

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Geração de Células-tronco pluripotentes induzidas a partir de sangue periférico usando o STEMCCA Lentivirus Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Geração rápida e eficiente de neurônios a partir de células-tronco pluripotentes humanas, em um formato de placa Multititre

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

Avaliação Geração isométrica e excêntrica Força dos músculos esqueléticos Isolado de modelos murinos de Distrofias Musculares

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Rápida e eficiente Zebrafish Genotipagem Usando PCR com alta resolução Análise Melt

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolamento de Blood-derivado do navio Multipotentes Precursores do músculo-esquelético humano

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Eficiente Geração induzida pelo homem células-tronco pluripotentes a partir de células somáticas humanas com Sendai-vírus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

Perfis de expressão de microRNA iPS Human Cells, Retinal Pigment Epithelium Derivado de Ips, e Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...