The authors wish to thank Harene Ranjithakumaran and Stacey Jackson for technical support. This work was supported in part by grants from the National Health and Medical Research Council (R.C.B. Wong, A. P&#233;bay), the University of Melbourne (R.C.B. Wong), Retina Australia (R.C.B. Wong, S.S.C. Hung, A. P&#233;bay) and the Ophthalmic Research Institute of Australia (R.C.B. Wong, S.S.C. Hung, A. P&#233;bay); Australian Research Council Future Fellowship (A. P&#233;bay, FT140100047), Cranbourne Foundation Fellowship (R.C.B. Wong); intramural funding from the National Institutes for Health (R.C.B. Wong, S.S.C. Hung) and operational infrastructure support from the Victorian Government.

A coleta de amostra de cabelo humano de indivíduos requer aprovação ética pelo comitê de ética em pesquisa em humanos nas instituições de acolhimento e deve ser feito em conformidade com as diretrizes institucionais. 1. Isolamento de queratinócitos a partir de cabelo arrancado <ol><li> Descongelar solução Matriz Extracelular (ECM) (isto é, Matrigel) em gelo, O / N. </li><li> Usando pontas de pipetas pré-refrigerados, adicione 200 mL de solução de ECM 12 ml de meio DMEM / F12 refrigerados. Bata de uma placa de 12 poços com 1 ml de solução diluída de ECM para cada poço. Incubar a placa revestida O / N a 37 ° C. Lavar a placa revestida com o meio DMEM pré-aquecido / F12 antes da utilização. </li><li> Colete cabelo humano primário da cabeça do indivíduo, colocando os dedos perto da raiz do cabelo e arrancar o cabelo em um movimento rápido e suave. Verifique para confirmar que cada cabelo recolhido contém um folículo de cabelo intacto e determinar a fase de crescimento do cabelo under um microscópio de dissecação (Figura 1). <ol><li> Recolha 5-10 cabelos anágenos de cada indivíduo para extrair queratinócitos. Processo arrancado os cabelos tão rapidamente quanto possível para assegurar derivação bem sucedida de queratinócitos. Nota: A Figura 1 ilustra a morfologia de cabelos em diferentes fases do ciclo de crescimento. Os cabelos anágenos está em fase de crescimento, com uma camada exterior visível do epitélio em torno do comprimento do cabelo, conhecidas como as ORS. Por outro lado, o cabelo eflúvio representa cabelo na fase de repouso e não tem uma ORS visíveis, mas tem uma bola de clara de células que cobrem a raiz dos cabelos, conhecido como o bojo telógena. </li></ol></li><li> Coloque as amostras de cabelo para uma placa de Petri contendo 10 ml de mistura de antibióticos para lavar durante 5 min à TA. </li><li> Usando tesouras e pinças esterilizadas, cortar para fora do eixo de cabelo excessiva, deixando um fragmento de cabelo com um folículo de cabelo intacto e em torno de 0,5-1 cm de haste capilar. </li><li> Cuidadosamente transfer fragmento de um cabelo em cada poço da placa revestida com ECM usando uma pinça. </li><li> Usando uma pipeta de 1 ml, adicionar cerca de 3 gotas de KO substituição Serum (KSR) meio (~ 100-200 uL) com as amostras de cabelo. Nota: Mantenha os fragmentos de cabelo em estreito contacto com a superfície da placa para permitir a fixação e posterior crescimento de queratinócitos. Adição excessiva da mídia nesta etapa pode faz com que os fragmentos de cabelo a flutuar e diminui a chance de fixação de cabelo bem sucedida. </li><li> Incubar as amostras de cabelo em um 37 ° C incubadora com 5% de CO 2 e permitir que os folículos pilosos para anexar O / N. No dia seguinte, adicionar mais suavemente 3 gotas de meio KSR (~ 100-200 uL) para manter as amostras de cabelo húmido. </li><li> Observe as amostras de cabelo diariamente para confirmar que os folículos pilosos ter anexado com sucesso. Geralmente fixação dos folículos pilosos é evidente dentro de 1-3 dias. Pêlos grossos são geralmente mais fácil de anexar comparação com cabelos finos, como pêlos finos têm maiortendência a flutuar que retardam o processo de fixação. <ol><li> Uma vez que o cabelo se ligado à placa de revestimento, adicionar 500 uL de meio KSR para o poço. Tome cuidado extra ao adicionar a forma como amostras de cabelo pode não ter ligado com firmeza. A partir deste ponto, mudar de mídia a cada 2 dias. Nota: excrescências queratinócitos deve ser visível a partir da região do fragmento de SRO cabelo após 3-7 dias (Figura 2A-B). Permitir que os queratinócitos a crescer até 14 dias antes da passagem das células, como descrito na próxima seção. </li></ol></li></ol> 2. Manutenção e Passaging de queratinócitos <ol><li> Pré-revestimento de uma placa de 6 poços, utilizando o Kit de revestimento de matriz. Adicionar 680 ul de meio de diluição a partir do kit a cada poço, seguido de 6,8 ml de solução de revestimento de matriz. Agitar a placa para assegurar uma distribuição uniforme da solução de revestimento de matriz na placa. </li><li> Permitir que a placa revestida com a incubar durante 30 min à temperatura ambiente. Removera solução de revestimento de matriz antes do plaqueamento de queratinócitos. </li><li> Dissociar queratinócitos numa suspensão de células individuais com 500 ul de tripsina a 0,25% por poço durante 2-5 min a 37 ° C. Inactivar a tripsina com 500 ul de meio contendo soro (por exemplo, de Soro Fetal Bovino (FBS) meio) e juntar-se em um tubo de 15 mL estéril. Remover o fragmento de cabelo usando fórceps esterilizados. </li><li> Determinar o número de células recolhidas utilizando um hemocitómetro. </li><li> Centrifugar as células durante 3 minutos a 200 x g. Lavam-se as células com PBS e centrifugação repetida. Aspirar o PBS e ressuspender o sedimento de células em meio fresco de queratinócitos. </li><li> Placa de deslocamento 4 × 10 4 queratinócitos para um poço de placa revestida com ECM na presença de meio de queratinócitos. Manter os queratinócitos em uma temperatura de 37 ° C incubadora com 5% de CO 2 (Figura 2C). Mudança de mídia a cada dia e células de passagem quando a cultura é de aproximadamente 80% confluentes, which pode demorar cerca de 2-7 dias dependendo do número de passagens e taxa de proliferação dos queratinócitos. Os queratinócitos derivados deve ser expandido para gerar stocks congelados suficientes usando métodos de criopreservação lento de congelamento padrão 16. Nota: As condições de cultura descritas suporta a expansão da população homogénea de queratinócitos e com a morfologia típica de calçada (Figura 2C). Ocasionalmente, alguns queratinócitos diferenciadas, com morfologia de grande e plana e perderam potencial replicativo, pode ser observado na cultura. Caracterização de queratinócitos pode ser realizada por imunocoloração utilizando anticorpos contra citoqueratina 14, como previamente descrito 17. Linhas celulares de queratinócitos estabelecidas devem também ser testados para micoplasma após 1-3 passagens utilizando o kit disponível comercialmente. </li></ol> 3. Geração de hiPSCs de Queratinócitos Usando vectores epissomais <ol><li> Pré-revestimento de uma placa de 6 poços comGelatina a 0,1% (2 ml / poço). Permitir que a gelatina de revestimento durante pelo menos 20 min à temperatura ambiente. </li><li> Para o experimento de reprogramação, de uso queratinócitos não mais tarde do que a passagem 6. dissociar os queratinócitos em uma única suspensão de células com 1 ml de tripsina a 0,25% por poço, durante 2-5 min a 37 ° C. Inactivar a tripsina com 1 ml de meio contendo soro (por exemplo, meios de FBS) e recolher num tubo de 15 ml. </li><li> Centrifugar as células durante 3 min a 200 xg e ressuspender as células em meio de queratinócitos. Placa 1 × 10 × 10 5 -1,5 5 queratinócitos por cavidade em uma placa gelatinizado O / N. </li><li> No dia seguinte (dia 0), verifique a placa para garantir células são 50-60% confluentes. Nota: Se os queratinócitos são menos de ~ 50% confluentes eles podem não crescer bem após a transfecção. </li><li> No Dia 0, efectuar a transfecção dos vectores epissomais de reprogramação nos queratinócitos, usando uma proporção de reagente de transfecção de 8 ul a 2? G de DNA total de plasmídeo.<ol><li> Tirar 0,5 ug de cada um dos quatro plasmídeos (pCXLE-eGFP, pCXLE-hOct3 / 4-shP53F, pCXLE-HSK, pCXLE-Hul) e dilui-se em 100 ul de meio de soro reduzido (por exemplo, meio OPTI-MEM). Nota: O plasmídeo pCXLE-eGFP é usado para monitorar as eficiências de transfecção em queratinócitos e não é necessário para reprogramação. </li><li> Adicionar 8 mL de reagente de transfecção de ADN diluída. Misture sacudindo o tubo. Permitir que a mistura de reacção contendo o ADN para descansar sem perturbação à temperatura ambiente durante 15 min. </li><li> Substituir o meio de queratinócitos com meio fresco. </li><li> Adicionar cuidadosamente a mistura reaccional para a cultura de queratinócitos de ADN e incubar numa incubadora a 37 ° C com 5% de CO 2 durante 4 horas. Nota: Não é recomendado para executar prolongado transfecção O / N em queratinócitos como eles apresentam baixa sobrevivência pós-transfecção. </li><li> Depois de 4 horas após a transfecção, substituir o meio de queratinócitos com meio fresco. </li></ol></li><li> No Dia 1 (1 dia de pós-transinfecção), alterar a forma de queratinócitos e verificar a eficiência de transfecção pela estimativa da percentagem de células positivas eGFP sob um microscópio fluorescente. Tipicamente&gt; 60-70% de eficiência de transfecção é observado para queratinócitos primários usando esse protocolo. </li><li> No Dia 2, repetir a transfecção como descrito na etapa 3.5. Dois transfecções sucessivas seriam tipicamente atingir&gt; 90% de eficiência de transfecção. Cultura de queratinócitos geralmente atinge confluência até ao final do segundo transfecção. <ol><li> Prepare uma placa de 10 cm de fibroblastos de rato embrionárias (MEF) alimentador para cada reação reprogramação. Prepare alimentadores MEF mitoticamente inactivadas como descrito anteriormente 18. </li><li> Pré-revestimento de uma placa de 10 cm com 5 ml de gelatina a 0,1% durante pelo menos 20 min. Placa 4 × 10 6 MEF mitotically inativado por placa de 10 cm em meio FBS. Permitir que as células assentar e unir O / N em uma temperatura de 37 ° C incubadora com 5% de CO 2. </li></ol></li><li> No Dia 3, harcolete os queratinócitos com 0,25% de tripsina, tal como descrito no passo 3.2 e 3.3. Ressuspender as queratinócitos em meio KSR e placa 90% de queratinócitos a partir de um poço de uma placa de 6 poços num prato de 10 centímetros MEF alimentador com meio KSR. </li><li> A partir do dia 4 em diante, mudar o meio KSR todos os dias. <ol><li> Alternativamente após o Dia 18, substituir o meio de cultura a cada segundo dia. Nota: os queratinócitos não-reprogramado deixará de proliferar, enquanto colônias hiPSC com limite definido que se assemelham a células estaminais embrionárias humanas (hESCs) vão surgir a partir do dia 14-21. </li></ol></li><li> Isolar colônias hiPSC por dissecção manual a partir do dia 21 em diante. Evite colônias hiPSC que estão intimamente agrupadas e apenas escolher colônias hiPSC que estão claramente separados dos outros. Nota: colônias hiPSC pode ser pego mais cedo, mas a colônia com limites definidos pode não ser óbvio em momentos anteriores, dada a dimensão pequena colônia. Eficiências de reprogramação podem variar entre different queratinócitos do paciente, e também é influenciado por outros factores, tais como o número de passagens ou a taxa de proliferação de queratinócitos. <ol><li> Para dissecção manual das colônias hiPSC, use uma agulha de 26G para cortar colônias hiPSC sob um microscópio de dissecação. Cortar uma colónia hiPSC em pequenos pedaços ao redor de 300-600 uM de comprimento. Transferir os pedaços de uma colônia hiPSC em um novo alimentador de placas MEF (2,5 × 10 4 MEF / cm 2) para estabelecer uma linha clonal de hiPSCs. Inicialmente, a cultura de cada linha clonal hiPSC em um poço de uma placa de 12 poços ou de cultura de órgãos pratos, em seguida, expanda a 6 formato de placa bem. </li><li> Manter linhas clonais estabelecidos de hiPSCs em meio KSR sobre alimentadores MEF. Uma vez que a cultura atingir 70 - 80% de confluência com colónias hiPSC ~ 1,5 milímetros de diâmetro, hiPSCs passagem utilizando métodos padrão enzimáticas Passaging (Accutase, dispase ou colagenase IV) de acordo com as instruções do fabricante. </li></ol></li><li> Caracterizar linha hiPSC estabelecidos para confirmar pluripotência e sua capacidade de se diferenciar em células representativas das três camadas germinativas, in vitro e / ou in vivo 13,19. Além disso, linhas hiPSC Testar rotineiramente estabelecidos para micoplasma usando kits disponíveis comercialmente para garantir que eles são livres do patógeno. Rotineiramente monitorar estabilidade genômica de linhas hiPSC por cariótipo ou análise baseada em array variação do número de cópias (CNV). </li></ol>

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The authors have no conflict of interest.

Geração de hiPSCs específicos do paciente oferece uma abordagem única para estudar patogênese nos tipos de células doentes in vitro, e também fornece uma plataforma para a triagem de drogas para identificar novas moléculas que podem resgatar os fenótipos da doença. Esta abordagem de modelagem doença usando hiPSCs tem produzido resultados promissores para uma variedade de doenças, incluindo a síndrome de QT longo, doença de Huntington, doença de Parkinson e esclerose lateral amiotrófica 22.<...

A descoberta de humanos induzida por células estaminais pluripotentes (hiPSCs) revolucionou o campo da medicina regenerativa, proporcionando um método viável para a produção de células estaminais específicos do paciente 1-3. hiPSCs ter sido gerado com êxito a partir de vários tipos de células somáticas, incluindo fibroblastos, células hematopoiéticas 4,5 6,7, células epiteliais renais de urina 8 e queratinócitos 9,10. Até à data, os fibroblastos da pele e células hematopoiéticas representam as fontes de células mais comumente utilizados para gerar iPSCs específicos do paciente. Indiscutivelmente, isso se deve ao fato de que as biópsias de pele e coleta de sangue são procedimentos médicos de rotina e grandes biobancos de sangue ou de pele amostras de pacientes foram estabelecidos em muitos países. Em contraste com as células sanguíneas e de fibroblastos da pele que requerem métodos de extracção invasiva, queratinócitos representam um tipo de células facilmente acessíveis para a geração hiPSC. Keratinocytes são células epiteliais rico em queratina que formam a barreira epidérmica exterior da pele e são também encontrados em unhas e cabelo 11. Em particular, os queratinócitos podem ser encontrados na bainha externa da raiz (ORS) dos folículos pilosos, uma camada celular externa que cobria o eixo do cabelo, juntamente com a bainha radicular interna (IRS), células (12, Figura 1). Como coleta de cabelo é um procedimento simples, que não requer a assistência de pessoal médico, ele fornece uma oportunidade para os pacientes de recolher e enviar suas próprias amostras de cabelo para laboratórios, o que facilitaria muito a recolha de amostras de doentes para a geração de hiPSC. Queratinócitos epidérmicos também têm uma maior eficiência de reprogramação e cinética de reprogramação mais rápidas em comparação com fibroblastos, a adição às vantagens da utilização de queratinócitos como as células de partida para a geração de hiPSC 9,13. Além disso, hiPSCs também pode ser gerado usando outras populações de células dentro do folículo piloso,incluindo as células de papila dérmicas localizadas na base do folículo piloso 14,15. Relatórios anteriores de geração iPSC usando células derivadas de cabelo muitas vezes utilizam métodos de reprogramação retrovirais ou à base de lentivírus 9,14,15. No entanto, estes métodos virais introduzir integração genómica indesejável de transgenes estranhos durante a reprogramação. Em comparação, a utilização de vectores epissomais representa um método viável reprogramação, não-viral para gerar iPSCs livre de integração 4. Nós já desenvolveu um método simples, eficaz e não-viral para reprogramar eficiente queratinócitos em hiPSCs usando vetores epissomais 13. Aqui nós fornecemos um protocolo detalhado para a geração de hiPSCs derivado de queratinócitos, incluindo a derivação de queratinócitos a partir de cabelo arrancado, de expansão e de manutenção dos queratinócitos e reprogramação subsequente para gerar hiPSCs.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic Mix:&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>250 ng/ml Antimycotic amphotericin B</td>
			<td>Sigma</td>
			<td>A2942-20ml</td>
			<td>Antibiotic mix is made up in PBS.&#160;</td>
		</tr>
		<tr>
			<td>1X Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (-)</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement (KSR) medium:&#160;</td>
			<td></td>
			<td></td>
			<td>KSR medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. bFGF is added fresh to the media before use.</td>
		</tr>
		<tr>
			<td>20% knockout serum replacement (KSR)</td>
			<td>Invitrogen</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 with glutamax</td>
			<td>Invitrogen</td>
			<td>10565-042</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#215; MEM non-essential amino acid</td>
			<td>Invitrogen</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;0.5&#215; Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;0.1 mM &#946;-mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;bFGF (10 ng/ml, added fresh)</td>
			<td>Millipore</td>
			<td>GF003</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratinocyte medium:&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;EpiLife with 60&#160;&#181;M Calcium</td>
			<td>Invitrogen</td>
			<td>M-EPI-500-CA</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#215; Human keratinocyte growth supplement (HKGS)</td>
			<td>Invitrogen</td>
			<td>S-001-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS) medium:&#160;</td>
			<td></td>
			<td></td>
			<td>FBS medium is filtered using Stericup (Millipore, #SCGPU05RE)&#160; before use.</td>
		</tr>
		<tr>
			<td>10% fetal bovine serum (FBS)</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Invitrogen</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5&#215; Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mM L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Extracellular Matrix (ECM):</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning&#160;</td>
			<td>354234</td>
			<td>Aliquot Matrigel stock and store in -80&#176;C following manufacturer&#8217;s instructions. Stock concentration of Matrigel varies slightly from batch to batch (~9mg/ml). We recommend to use 200&#181;l matrigel for coating a 12-well plate (~150&#181;g/well).&#160;</td>
		</tr>
		<tr>
			<td>Coating Matrix Kit&#160;</td>
			<td>Invitrogen</td>
			<td>R-011-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids:&#160;</td>
			<td></td>
			<td></td>
			<td rowspan="5">Note that pCXLE-eGFP is only used for monitoring transfection efficiency and is not required for reprogramming.</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-eGFP</td>
			<td>Addgene</td>
			<td>27082</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-hOct3/4-shP53F</td>
			<td>Addgene</td>
			<td>27077</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-hSK</td>
			<td>Addgene</td>
			<td>27078</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-hUL</td>
			<td>Addgene</td>
			<td>27080</td>
		</tr>
		<tr>
			<td>Transfection reagent Fugene HD</td>
			<td>Promega</td>
			<td>E231B</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin (from porcine skin)</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td>Make up 0.1% gelatin in distilled water. Autoclave before use.&#160;</td>
		</tr>
		<tr>
			<td>Reduced Serum medium: OPTI-MEM</td>
			<td>Invitrogen</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma</td>
			<td>A6964-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblast (MEF) feeder</td>
			<td></td>
			<td></td>
			<td>MEF can be inactivated by mitomycin C treatment or irradiation as described previously 16.</td>
		</tr>
		<tr>
			<td>26G needle</td>
			<td>Terumo</td>
			<td>NN2613R</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate (tissue culture treated)</td>
			<td>BD Biosciences</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate (tissue culture treated)</td>
			<td>BD Biosciences</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm dish (tissue culture treated)</td>
			<td>BD Biosciences</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Invitrogen</td>
			<td>17105-041</td>
			<td>Use at 10mg/ml</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Invitrogen</td>
			<td>17104-019</td>
			<td>Use at 1mg/ml</td>
		</tr>
		<tr>
			<td>TRA-160 antibody</td>
			<td>Millipore</td>
			<td>MAB4360</td>
			<td>Use at 5&#181;g/ml</td>
		</tr>
		<tr>
			<td>OCT4 antibody</td>
			<td>Santa Cruz</td>
			<td>SC-5279</td>
			<td>Use at 5&#181;g/ml</td>
		</tr>
		<tr>
			<td>NANOG antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF1997</td>
			<td>Use at 10&#181;g/ml</td>
		</tr>
		<tr>
			<td>MycoAlert Detection kit</td>
			<td>Lonza</td>
			<td>LT07-418</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of integration-free human induced pluripotent stem cells using hair-derived keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

O cabelo passa por três fases diferentes do ciclo de crescimento: anágena (fase de crescimento), catágena (fase de regressão) e telógena (na fase de repouso) 20,21. O folículo piloso anágena contém várias camadas de epitélio; essas camadas incluem os ORS, IRS e do eixo do cabelo (Figura 1). Cabelos anágenos eventualmente sofre a transição para a fase catágena, que é marcado por apoptose das ORS e rescisão de diferenciação eixo do cabelo. Finalmente, a transição de cabelo c...

Watch this Scientific Journal Video about Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes at JoVE.com

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Geração de Integração Induced-livre Human células estaminais pluripotentes usando o cabelo derivado queratinócitos

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

generation-integration-free-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Developmental Biology

11.4K visualizações.  University of Melbourne. This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors. 

Vídeo: Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Geração de pluripotentes induzidas a partir de células-tronco Congelado Buffy Coats usando não-integração epissomais Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Biologia do Desenvolvimento

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Geração de pluripotentes induzidas a partir de células-tronco periférico humano T células usando Sendai Virus em condições livres de Alimentador

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

Geração de células-tronco pluripotentes induzida por Usando sinoviócitos semelhantes a fibroblastos Isolado de articulações de pacientes com artrite reumatóide

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Geração de células-tronco pluripotentes induzidas a partir de melanoma humano linfócitos tumor infiltrantes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

Induzida Geração Stem Cell pluripotentes a partir de células de sangue com o vírus Sendai e centrifugação

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Geração de Integração-livres induzidas células-tronco pluripotentes a partir de células mononucleares de sangue periférico humano Usando epissï¿½ica Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Definido e Geração de Scalable de células de hepatócitos-like de células-tronco pluripotentes humanas

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Derivação livre de integração de células-tronco pluripotentes induzidas por seres humanos com Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

Geração de padronizada e reprodutível Forebrain-tipo Organoids Cerebral de humanos induzida por células-tronco pluripotentes

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

Usar humanos induzida pluripotentes células-tronco derivadas hepatócitos células para descoberta de drogas

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...