Este trabalho foi apoiado pela Aliança de Friedreich Ataxia Research e uma subvenção piloto da Família Arnold Foundation e O Centro de Células-Tronco e Biologia do Desenvolvimento no MD Anderson Cancer Center.

1. Produção de vírus e de transdução <ol><li> Plate Phoenix células ampho a uma densidade de 8x10 ~ 7-6 por placa de 10 cm em 10 ml de meio DMEM (DMEM glucose elevada, 10% de FBS inactivado calor, 2 mM de L-glutamina, sem antibióticos). Lugar na incubadora e da cultura durante a noite a 37 ° C, 5% de CO 2. </li><li> No dia seguinte, transfectar células Phoenix, utilizando 12 ug de um vector de codificação quer OCT3 / 4, Sox2, KLF4, c-myc, ou GFP gene (Addgene plasmídeos 17.217, 17.218, 17.219, 17.220) e 35 uL Fugene 6. Preparar a mistura de transfecção em 500 uL de meio DMEM (DMEM glucose elevada, 2 mM de L-glutamina, sem FBS e antibióticos). Incubar 20 min. Suavemente pipetar complexos de DNA para as placas de 10 cm contendo 70-80% de células confluentes Phoenix. </li><li> Substituir meios 6-8 h pós-transfecção com meio DMEM (DMEM glucose elevada, 10% de FBS inactivado calor, 2 mM de L-glutamina) contendo penicilina e estreptomicina. </li><li> Subsequentemente, recolher-vírus contendo meios 4 vezes em 12 hintervalos, combinar todas as porções e filtrou-se através de um filtro de 0,45 para remover as células destacadas e detritos (meio contendo partículas virais podem ser mantidos no frigorífico durante 2 semanas sem perder a actividade infecciosa, no entanto congelação não é recomendada). </li><li> Para retrovirais de transdução, de placas fibroblastos humanos adultos (passagem 10, Coriell Laboratories) a partir de um estoque congelado h 24 anteriores ao último dia da coleção de mídia retroviral. De sementes as células em 6-bem gelatina placas cobertas, na densidade de 1x10 5 células por poço em DMEM glucose elevada, 10% de FBS, 2 mM de L-glutamina, penicilina, estreptomicina e não-aminoácidos essenciais. </li><li> O substituto dia seguinte DMEM mídia com a mídia contendo partículas virais obtidos a partir de células de Phoenix. Adicionar meios virais (1 ml de cada Oct4, Sox2, KLF4, e c-myc meios de comunicação, 4 ml no total) suplementado com 6 ug / ml de polibreno em cada poço da placa de 6 poços das culturas de fibroblastos e centrifugar a 1600g para 1h a 20 ° C. Aproximadamente 12 h after a transdução, substituir os meios de comunicação viral com meio de cultura de fibroblastos. Realizar infecção viral e centrifugação 3 vezes em intervalos de 24 h. </li><li> Fibroblastos Cultura em meio DMEM por 48 horas após a infecção passada. Verificar a eficiência da infecção por transduções paralelas com um meio de retrovirais que expressam GFP. A Figura 1 ilustra a eficiência de infecção-centrifugação facilitada retroviral de fibroblastos humana. </li></ol> 2. Reprogramação <ol><li> Prepare 6 placas de poços com γ irradiados camadas alimentadoras MEF por sementeira de células MEF, a uma densidade de aproximadamente 2x10 5 células por poço de 6 a gelatina tratada placa bem em meio de crescimento de fibroblastos. No dia seguinte dividir infectadas fibroblastos humanos usando 0,05% de tripsina / EDTA e de sementes de los na densidade de ~ 1.2x10 4 por poço em meio de crescimento de fibroblastos. </li><li> No dia seguinte, substituir a mídia com CTeh mídia (DMEM/F12, substituição de soro de 20% Knockout, não-aminoácidos essenciais, penicillin / estreptomicina, 2 mM de L-glutamina, 0,1 mM de β-mercaptoetanol, 20 ng / Factor de Crescimento de Fibroblasto ml basal (bFGF), suplementado com 0,5 mM de butirato de sódio para os primeiros 7 dias após a reprogramação. Alterar media diária. </li><li> Monitorizar as alterações morfológicas nas células transduzidas diária. As colónias de células semelhantes HIPS vai começar a emergir aproximadamente 5 - 10 dias após a transferência dos fibroblastos transduzidas para células de alimentação. As colónias hiPSC está pronto para ser isolado cerca de 14 - 28 dias após plaqueamento das células alimentadoras MEF. </li></ol> 3. Isolamento de colónias hiPSC <ol><li> Um dia antes da colheita das colónias hiPSC preparar placas de 24 poços com γ irradiados células MEF alimentador (4x10 ~ de 4 células por poço) em meio de crescimento de fibroblastos. Nesta fase colónias hiPSC também podem ser transferidos para a cultura alimentador livre usando Matrigel e médio mTeSR1. </li><li> Antes de isolamento de colónias hiPSC preparar placas MEF através da remoção de fibroblastos crescimento medium e enxaguamento MEFs com PBS para remover vestígios de FBS. Subsequentemente adicionar 0,5 ml CTeh médio (ou mTeSR1 médio no caso de Matrigel poços revestidos) com 10 uM ROCHA inibidor Y27632 a cada 7,8 poço. </li><li> Se necessário, remova os fibroblastos ao redor das colônias hiPSC com uma agulha de calibre 21 sob um microscópio montado em uma capela de fluxo laminar (Figura 2A - C). Lavam-se as placas com PBS e adicionar meios frescos TEh contendo o anticorpo Tra-1-81 StainAlive específico (1:200, Stemgent). Depois de 30 min., Substituir meios que contêm o anticorpo com meios frescos TEh suplementadas com 10 inibidor ROCHA uM. Examinar as placas sob o microscópio fluorescente e marca Tra-1-81 colónias positivas usando um marcador objectivo (Figura 2D). </li><li> Corte os Tra-1-81 colónias positivas hiPSC sob um microscópio em uma capa laminar, em vários pequenos pedaços usando uma agulha de calibre 21, como mostrado na Figura 2E e F. </li><li> Usando uma pipeta automática fragmentos (P200) de transferência de colônias hiPSC em indpoços ividual da placa de 24 poços com MEFs ou Matrigel. Evitar a transferência de células não-HIPS. Colocar as placas em um 37 ° C, 5% de CO 2 incubadora e permitir que as colónias hiPSC para anexar para 24-36 h. </li><li> Mudança CTeh ou mTeSR1 (para colônias cultivadas em Matrigel) media diária. As colónias hiPSC com a morfologia HES-correcta como será visível 48 h após a transferência inicial para uma placa de 24 poços. </li><li> Manualmente passagem colónias hiPSC cada 6 - 8 dias para um 12-bem e, subsequentemente, numa placa de 6 poços. </li><li> Após a expansão clonal e que estabelece as linhas hiPSC, analisar a expressão de marcadores de pluripotência TRA-1-60, SSEA-4, OCT3 / 4, Sox2 e Nanog utilizando imunocitoquímica como mostrado na Figura 3. Avaliar a integridade genômica e potencial de diferenciação das linhas obtidas utilizando o cariótipo e análises teratoma formação 6,9. </li></ol> 4. Os resultados representativos Transdução eficiente com retrovirus contendo mídia is crítico para a reprogramação. Recomenda-se para conduzir o procedimento de transfecção / infecção inteiro usando um GFP expressando vírus cada experiência única reprogramação para monitorar a eficácia, como mostrado na Figura 1. O título do vírus expressando GFP determinado, como descrito em 10, através da transdução de fibroblastos humanos utilizando meios não-concentrados virais foi tipicamente na gama de 0,5-5 x 10 7 partículas virais por ml (VP / ml). Fibroblastos alterar a morfologia tão cedo quanto 2 dias após a última infecção. Tripsinizadas fibroblastos humanos devem ser cuidadosamente contadas antes, semeando-os nas células alimentadoras do MEF. Recomenda-se a células de semente menos 3 densidades diferentes (6x10 3, 4 1.2x10, 2.5x10 4 por único poço de uma placa de 6 poços) uma vez que cada linha de células demonstra característica de crescimento diferente e densidade de semeadura é crítica para a eficiência de reprogramação. Butirato de sódio utilizado para ainicial de 7 - 14 dias de reprogramação aumenta a eficiência de formação de hiPSC aproximadamente 5 vezes. Frequentemente, especialmente quando fibroblastos infectados foram semeadas a densidade mais elevada, as células de fibroblasto-like pode overgrow um prato de cultura e cobrir colónias hiPSC como mostrado na Figura 2A. Neste caso, a camada de fibroblastos podem ser cuidadosamente levantado e retirado para descobrir colónias hiPSC (Figura 2B). Subsequentemente, as colónias hiPSC deve ser lavado com HES meios de comunicação e coradas com Tra-1-81 de anticorpos. Dependendo das células de fibroblasto, cerca de 20 - 40% de colónias demonstrando com iPSC-like morfologia não mancham com Tra-1-81 de anticorpos. Identificados colónias hiPSC pode ser transferido a partir de uma placa de dentro 12 próxima - 24h. Incubação prolongada irá resultar em diferenciação rápida de hiPSCs. As colónias pode ser manualmente passadas para tanto células alimentadoras MEF ou Matrigel placas revestidas. Após a expansão estabelecido clones hiPSC devem ser testados usando imunocitoquímica (ICC) para a expressão de pluripotency marcadores como demonstrado na Figura 3. Além disso, uma caracterização molecular detalhada das geradas linhas de células iPS deve incluir: análises da expressão do gene pluripotência utilizando RT-PCR, na demonstração da desmetilação DNA com os promotores de genes pluripotência e análises dos transgenes silenciamento 9. <img alt="A Figura 1" src="/files/ftp_upload/3416/3416fig1.jpg" /> Figura 1. A eficiência de transdução virai determinada utilizando retrovírus GFP expressando. (A, B) fibroblastos humanos adultos derivados de paciente Friedreich ataxia (GM03665, Coriell Repository) foram visualizadas após duas infecções consecutivos com meios GFP retrovirais. (C, D) fibroblastos humanos foram infectadas com o mesmo lote de os meios de comunicação GFP retrovirais seguido por centrifugação das células directamente sobre as placas de 6 poços durante 1 hora à 1600g. As imagens foram capturadas 48h após a infecção. <img alt= &quot;Figura 2&quot; src = &quot;/ files/ftp_upload/3416/3416fig2.jpg&quot; /&gt; Figura 2. Identificação e isolamento de colónias hiPSC. (A) da imagem de contraste de fase de uma placa contendo hiPSCs rodeadas por fibroblastos. As células foram cultivadas durante 21 dias em CTeh meios de comunicação. (B, C) colónia O mesmo hiPSC após a remoção da camada de fibroblastos circundante. (D) A correcta reprogramadas colónias hiPSC são identificados através de coloração vivo com Tra-1-81 superfície anticorpo marcador, cortados com uma agulha estéril (E, F) e transferidos para poços separados de uma placa de 24 cavidades. <img alt="A Figura 3" src="/files/ftp_upload/3416/3416fig3.jpg" /> Figura 3. Expressão dos marcadores específicos pluripotência OCT3 / 4, Nanog, Sox2, SSEA4 e Tra-1-60 em hiPSCs foi determinada por imunocitoquímica.

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Os autores não têm nada a revelar.

O estudo de doenças humanas, especialmente neurológicas e neurodegenerativas, foi particularmente difícil devido à falta de acesso aos humanos adequados modelos de celulares. A capacidade de reprogramar células somáticas de fácil obtenção em células tronco pluripotentes induzidas e potencial para diferenciá-las em diversos tipos celulares abriu uma possibilidade de criar modelos celulares de doenças genéticas. Além disso, iPSCs realizar uma grande promessa para o futuro da medicina regenerativa. Portanto, ...

<table border="1"><tbody><tr><td> Reagente </td><td> Companhia </td><td> Número de Catálogo </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 11330 </td></tr><tr><td> KSR </td><td> Invitrogen </td><td> 10828 </td></tr><tr><td> Aminoácidos não essenciais </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Butirato de sódio </td><td> Sigma </td><td> B5887 </td></tr><tr><td> Y27632 </td><td> Stemgent </td><td> 04-0012 </td></tr><tr><td> bFGF </td><td> Stemgent </td><td> 03-0002 </td></tr><tr><td> Tra-1-81 anticorpo </td><td> Stemgent </td><td> 09-0069 </td></tr><tr><td> OCT3 / 4 anticorpo </td><td> Santa Cruz </td><td> sc-8628 </td></tr><tr><td> Nanog anticorpo </td><td> Cell Technology Sinalização </td><td> 4903S &lt;/ Td&gt; </tr><tr><td> Tra-1-60 anticorpo </td><td> Millipore </td><td> MAB4360 </td></tr><tr><td> Sox2 anticorpo </td><td> Cell Technology Sinalização </td><td> 3579S </td></tr><tr><td> SSEA4 </td><td> Millipore </td><td> MAB4304 </td></tr><tr><td> CF1 MEFs </td><td> Globalstem </td><td> GSC-6201G </td></tr><tr><td> Marcador objetivo </td><td> Nikon </td><td> MBW10010 </td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354277 </td></tr><tr><td> mTeSR1 </td><td> StemCell Technologies </td><td> 05850 </td></tr><tr><td> β-mercaptoetanol </td><td> Sigma </td><td> M7522 </td></tr><tr><td> Fugene 6 </td><td> Roche </td><td> 11814443001 </td></tr><tr><td> polibrene </td><td> Sigma </td><td> H9268 </td></tr><tr><td> Marcador de objeto </td><td> Nikon </td><td> MBW10010 </td></tr></tbody></table>

selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts

Apresentamos aqui um protocolo de reprogramação fibroblastos humanos adultos em humanos células pluripotentes induzidas estaminais (hiPSC) utilizando vectores retrovirais que codificam OCT3 / 4, Sox2, KLF4 e c-myc (OSKM) na presença de butirato de sódio 1-3. Usamos este método para reprogramar passagem tardia (&gt; p10) fibroblastos humanos adultos provenientes de paciente ataxia de Friedreich (GM03665, Coriell Repository). A abordagem de reprogramação inclui protocolo de transdução altamente eficiente utilizando centrifugação repetitiva de fibroblastos na presença de vírus que contêm meios de comunicação. As colónias hiPSC reprogramadas foram identificadas utilizando imunomarcação vivo para Tra-1-81, um marcador de superfície de células pluripotentes, separados a partir de fibroblastos não-reprogramadas e manualmente passadas 4,5. Estes hiPSC foram então transferidas para placas de Matrigel e cultivadas em condições isentas de alimentação, directamente a partir da placa de reprogramação. A partir da primeira passagem, as colónias hiPSC demonstrar característica HES-lmorfologia ike. Usando este protocolo mais do que 70% das colónias seleccionadas pode com sucesso ser expandido e estabelecido em linhas celulares. As linhas estabelecidas hiPSC exibido característicos marcadores de pluripotência incluindo marcadores de superfície TRA-1-60 e SSEA-4, bem como marcadores nucleares OCT3 / 4, Sox2 e Nanog. O protocolo apresentado aqui tenha sido estabelecida e testadas usando fibroblastos adultos obtidos a partir de pacientes com AF e indivíduos de controlo 6, fibroblastos humanos recém-nascidos, bem como queratinócitos humanos.

Watch this Scientific Journal Video about Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts at JoVE.com

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

Nós apresentamos um protocolo para a reprogramação eficiente de células somáticas humanas em células-tronco pluripotentes induzidas (hiPSC) usando vetores retrovirais codificando OCT3 / 4, Sox2, KLF4 e c-myc (OSKM) e identificação de hiPSC corretamente reprogramado pela coloração ao vivo com tradi- 1-81 anticorpo.

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 ...

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19.8K visualizações.  University of Texas M.D. Anderson Cancer Center. Nós apresentamos um protocolo para a reprogramação eficiente de células somáticas humanas em células-tronco pluripotentes induzidas (hiPSC) usando vetores retrovirais codificando OCT3 / 4, Sox2, KLF4 e c-myc (OSKM) e identificação de hiPSC corretamente reprogramado pela coloração ao vivo com tradi- 1-81 anticorpo.

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

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus 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 the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

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 ...

Biologia

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolamento e Cultura de Células-Tronco Adultas epitelial da pele humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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 ...

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 ...

Usando biópsias de pele para gerar podócitos derivados de pacientes

Generation of Patient-Derived Podocytes from Skin Biopsies

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes.&#160;Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients&#39; mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

Autor em destaque: Geração de podócitos derivados de pacientes a partir de biópsias de pele  

This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

Geração de Podócitos Derivados do Paciente a partir de Biópsias de Pele

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...