1. Reprogramação <ol><li> Semente de células BJ a uma densidade de 1 x 10 5 células por poço de uma placa de seis poços. Cultura das células em 2 ml de meio de crescimento (450 ml EMEM suplementado com 50 ml ES FBS qualificado, 5 ml 10 mM não-aminoácidos essenciais, 5 ml de penicilina / estreptomicina, 5 ml 200 mM L-glutamina e 0,9 ml β 55 mm -mercaptoetanol) durante a noite a 37 ° C e 5% CO 2. </li><li> No mesmo dia, começar a preparar pratos de alimentação MEF pela adição de 0,1% de gelatina diluído em água a uma placa de 6 bem e incubar overnight a 37 ° CO C e 5% 2. </li><li> Após a incubação, remova o meio das células BJ e adicionar 2 ml meio de crescimento suplementado com 6 mg / ml de polibrene, 500 mL hOct4-lentivírus, 50 ul hSox2-lentivírus, 50 ul hNanog lentivírus e 50 ul hLin28 lentivírus-a cada poço . Agite levemente o prato para garantir um revestimento uniforme do médium. Incubar overnight a 37 ° CO C e 5% 2. </li><li> No mesmo dia, retire 1 frasco de células CF-1 MEF de nitrogênio líquido e descongelamento. Remover o líquido dos poços revestidos de gelatina (ver passo 1.2) e adicionar células alimentadoras MEF com uma densidade de 0,2 x 10 5 células / poço. O volume total por poço deve ser de 2ml de células no MEF meio de crescimento (450 ml DMEM suplementado com 50 ml e 5 ml FBS não-aminoácidos essenciais). </li><li> No dia seguinte, retire as células BJ com 0,05% de tripsina / EDTA e centrifugar a 200 xg por 5 minutos. Aspirar a médio e ressuspender em meio de crescimento. Remova o meio do prato alimentador MEF (ver passo 1.4) e adicionar 2 ml / poço da suspensão de células BJ. A concentração de células deve ser de aproximadamente 5 x 10 4 células / poço. Incubar overnight a 37 ° CO C e 5% 2. </li><li> Substituir média 24 horas após a re-semeadura com meio de cultura humana ES / iPS de células (400 ml DMEM/F12 suplementada com 100 ml de soro substituição Knockout, 5 ml 10 mM não-aminoácidos essenciais, 5 ml 200 mM L-glutamina, 0,9 ml 55 mM β-mercaptoetanol e 20 ng / ml bFGF humana recombinante). Mudança média a cada 24 horas durante 7 dias. </li><li> Após sete dias, a mudança de médio a MEF meio condicionado (ver secção 2). </li></ol> 2. Preparação do MEF meio condicionado <ol><li> Semente CF-1 células MEF alimentador de 2 x 10 5 células / poço em uma placa de 6 bem no meio de crescimento 2 ml MEF. Incubar overnight a 37 ° CO C e 5% 2. </li><li> Alterar médio e meio de cultura humana ES / iPS de células. Incubar overnight a 37 ° CO C e 5% 2. </li><li> Recolher o sobrenadante a cada 24 horas durante quatro dias. Filtrar o sobrenadante através de um filtro mM 0,22. </li><li> Suplemento sobrenadante com 50 ng / ml de bFGF. </li></ol> 3. iPS Seleção Colony e Passaging <ol><li> Selecione uma colônia ES-like e re-semente em meio de cultura humana ES / iPS suplementado com 10 mM de Stemolecule Y27632 em pratos de cultura de células pré-semeado com FC-1 células alimentadoras MEF. Incubar overnight a 37 ° CO C e 5% 2. </li><li> Mudar o meio de cultura a cada 24 horas durante os primeiros sete dias (sem a suplementação com Stemolecule Y27632). Após sete dias, a mudança de médio a MEF meio condicionado (ver secção 2 para a preparação). Continuamos passaging as células até que eles apresentaram morfologia ES humano típico. </li></ol> 4. Exame imunocitoquímico de marcadores pluripotência <ol><li> Lave suavemente as células três vezes com PBS. </li><li> Fixar as células com 500 mL de fixador por 20 minutos em temperatura ambiente. </li><li> Lave suavemente as células três vezes com PBS. </li><li> Bloco de ligação não específica com 500 mL de buffer de bloqueio para uma hora em temperatura ambiente. </li><li> Incubar as células com 250 mL do anticorpo primário específico durante a noite a 4 ° C (usamos TRA-1-81, TRA-1-60, SSEA-4, SSEA-3, Oct4, Sox2, Nanog e Lin28 a 1:100 diluições). </li><li> Lave suavemente as células três vezes com PBS. </li><li> Incubar as células com 250 mL de anticorpo secundário por 1 hora à temperatura ambiente, mantendo-se longe da luz (usamos cabra anti-camundongo conjugado IgM Cy3, cabra anti-camundongo conjugado IgG Cy3, cabra anti-IgM Rat Cy3 conjugado e de cabra anti- coelho conjugado Cy3 em diluições 1:300). </li><li> Lave suavemente as células três vezes com PBS. </li><li> Adicionar DAPI (concentração final de 1 mg / ml) para a última lavagem e incubar 5 minutos para visualizar núcleos. </li><li> Analisar as células sob a ampliação. </li></ol> Parte 5: Resultados Representante 1. Resultados morfologia: Fibroblastos humanos prepúcio (BJ), as células foram co-transduzidas com Oct4, Sox2, Nanog e Lin28. Alterações morfológicas foram observadas logo dia 4 pós-transdução, eo aglomerado de células se tornou mais denso no dia 17 (Figura 1). Colônias foram manualmente colhidas no dia 25 e cultivadas em FC-1 células alimentadoras MEF. Para facilitar a formação de colônias de células iPS após a reprogramação, usamos Stemolecule Y27632, Um inibidor ROCK, para o plantio inicial de noite durante cada passagem. colônias de células iPS com boa morfologia foram observadas após três rodadas seqüenciais de colônia de colheita e passaging. 2. Expressão de marcadores pluripotência: De melhor caracterizar as colônias isoladas das células iPS, nós olhamos para a presença de marcadores de pluripotência comuns expressos em células ES. As colônias exibiram forte atividade de fosfatase alcalina (AP) (Figura 2). Além disso, imunocitoquímica (ICC) foi realizado nas colônias de células iPS com um painel de anticorpos marcador específico pluripotência, incluindo marcadores de superfície TRA-1-81, TRA-1-60, SSEA-4 e SSEA-3, bem como marcadores nuclear Oct4, Sox2 e Nanog. As colônias isoladas iPS foram positivos para todos os marcadores (Figura 3). Os resultados mostram que o ICC células iPS exibiram o padrão de expressão apropriada pluripotência marcador, demonstrando que essas células iPS se assemelham células indiferenciadas embrionárias humanas. <img src="/files/ftp_upload/1553/1553fig1.jpg" alt="Figura 1" /> Figura 1: As alterações morfológicas de células transduzidas BJ. Imagens de campo claro de uma colônia típica de células iPS formado em (A) 4 dias e (B) 17 dias após a transdução. <img src="/files/ftp_upload/1553/1553fig2.jpg" alt="Figura 2" /> Figura 2: AP atividade de células reprogramadas BJ. Três colônias diferentes corados com Kit de coloração Fosfatase Alcalina Stemgent. <img src="/files/ftp_upload/1553/1553fig3.jpg" alt="Figura 3" /> Figura 3: As células humanas iPS expressam altos níveis dos marcadores celulares específicos ES seguintes: marcadores de superfície TRA-1-81, TRA-1-60, SSEA-4 e SSEA-3, e marcadores nucleares 04 de outubro, Nanog e Sox2.

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., Thompson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (1917).</li><li>Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B., Nishikawa, S., Muguruma, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Koyanagi, M., Takahashi, J., Arakawa, Y., Doi, D., Fukuda, H., Hayashi, H., Narumiya, S., Hashimoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+Rho/ROCK+pathway+reduces+apoptosis+during+transplantation+of+embryonic+stem+cell-derived+neural+precursors.">Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell-derived neural precursors.</a> J. Neurosci. Res. 86, 270-280 (2008).</li></ol>

Os autores deste artigo são empregados por Stemgent que produz reagentes e instrumentos utilizados neste artigo.

Estes resultados demonstram que o conjunto de Lentivirus Stemgent Humanos TF pode ser usado para gerar de forma eficiente colônias iPS, induzindo a expressão ectópica de transduzidas fatores de transcrição em fibroblastos humanos. Ao projetar experimentos reprogramação, diversas variáveis ​​devem ser considerados para otimizar a eficiência de reprogramação. Primeiro, a relação vírus-a-alvo ativa (multiplicidade de infecção MOI) pode precisar ser modificado durante a etapa de transdução primária para alcançar a eficiência de transdução ideal. Em segundo lugar, a condição de crescimento das células-alvo pode ter impacto reprogramação. Células saudáveis ​​e proliferativa são mais suscetíveis de reprogramação. Terceiro, ao modificar o protocolo de número de células diferentes, recomenda-se que o número de células-alvo ser ajustado proporcionalmente à área da superfície da placa de cultura. Por último, os inibidores da ROCHA aplicação, tais como Y27632 devem ser considerados para ajudar a garantir a reprogramação de sucesso como estudos recentes têm demonstrado a sua utilidade na sobrevivência melhorar colônia TEh 4,5.

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<td>Human TF Lentivirus Set</td>
<td>&nbsp;</td>
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<td>Stemolecule Y27632</td>
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generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human tf lentivirus set

Em 2006, Yamanaka e seus colegas demonstraram que primeiro retrovírus mediada entrega e expressão de Oct4, Sox2, c-Myc e KLF4 é capaz de induzir o estado pluripotente em fibroblastos do ratinho. 1 O mesmo grupo também relatou a reprogramação de células somáticas humanas em pluripotentes induzidas da haste (iPS) células usando versões humanas dos fatores de transcrição mesma entregue por vetores retrovirais. 2 Além disso, James Thomson et al. relataram que os lentivírus mediada por co-expressão de um outro conjunto de fatores (Oct4, Sox2, Nanog e Lin28 ) foi capaz de reprogramar as células somáticas humanas em células iPS. 3 As células iPS são similares às células-tronco embrionárias na proliferação, morfologia e a capacidade de se diferenciar em todos os tipos de tecido do corpo. As células iPS humanas têm uma vantagem distinta sobre células-tronco embrionárias como eles apresentam propriedades-chave das células-tronco embrionárias sem o dilema ético de destruição de embriões. A geração de células específicas do paciente iPS contorna um obstáculo importante para terapias personalizadas medicina regenerativa, eliminando a possibilidade de rejeição imunológica de não-autólogo de células transplantadas. Aqui demonstramos o protocolo para a reprogramação de células de fibroblastos humanos usando o Set Lentivirus Stemgent Humanos TF. Também mostramos que as células reprogramadas com este conjunto começar a mostrar iPS morfologia quatro dias pós-transdução. Usando o Y27632 Stemolecule, foram selecionadas para as células iPS e morfologia correta observada após três rodadas seqüenciais de colônia de colheita e passaging. Também demonstram que, após a reprogramação de células exibido a pluripotência marcador AP, marcadores de superfície TRA-1-81, TRA-1-60, SSEA-4, e SSEA-3, e marcadores nuclear Oct4, Sox2 e Nanog.

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set at JoVE.com

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

Nós demonstramos o protocolo para a geração de células-tronco pluripotentes induzidas a partir de células somáticas humanas usando lentivírus mediada entrega dos fatores humanos Oct4, Sox2, Nanog e Lin28. Pluripotência foi confirmado pela morfologia e pela presença de tronco embrionárias (ES) de células específicas marcadores.

Geração de células-tronco pluripotentes induzidas pela reprogramação fibroblastos humanos com o Lentivirus TF Stemgent Humanos Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

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Unit 1

Embryonic and Induced Pluripotent Stem Cells

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29.7K visualizações.  Stemgent. Nós demonstramos o protocolo para a geração de células-tronco pluripotentes induzidas a partir de células somáticas humanas usando lentivírus mediada entrega dos fatores humanos Oct4, Sox2, Nanog e Lin28. Pluripotência foi confirmado pela morfologia e pela presença de tronco embrionárias (ES) de células específicas marcadores.

Vídeo: Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

Culture and Maintenance of Human Embryonic Stem Cells  

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

Culture and Maintenance of Human Embryonic Stem Cells

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

Cultura e Manutenção de células estaminais embrionárias humanas

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

Biologia

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Geração de células-tronco pluripotentes induzidas pela reprogramação fibroblastos de rato embrionárias com um fator de transcrição Quatro, Doxiciclina induzível Sistema de Transdução Lentivirus

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Freezing and Thawing Human Embryonic Stem Cells

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

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

Congelamento e descongelamento, células estaminais embrionárias humanas

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

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Transfectando e Nucleofecting células-tronco pluripotentes induzidas

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

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

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

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

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

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

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Reprogramação de células somáticas humanas em células tronco pluripotentes induzidas (iPSCs) usando vetor retroviral com GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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, ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

Geração de cardiomiócitos Humanos: Um Protocolo de Diferenciação de Feeder livres Humanos células-tronco pluripotentes induzidas

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

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 ...