Nos gustaría dar las gracias a los miembros del laboratorio Lee valiosa para los debates sobre el manuscrito. El trabajo en el laboratorio de Lee fue apoyado por becas de Robertson Investigator Award de la Fundación de Células Madre de Nueva York y de Maryland Stem Cell Research Fund (TEDCO).

1. Preparación de la célula y Medios de Comunicación (Día 1) <ol><li> Cultura y ampliar fibroblastos humanos con medio DMEM que contiene 10% de SFB. </li><li> Placa fibroblastos humanos (Figura 1) en una placa de 24 pocillos a una densidad apropiada por pozo en el día antes de la transducción. NOTA: Se recomiendan las siguientes diluciones en serie (200K, 100K, 50K, 25K, 12.5K y 6,25), ya que los diferentes tipos de células tienen diferente capacidad de unión. </li><li> Se incuban las células durante un día más en un 37 ° C, 5% de CO 2 incubadora, lo que garantiza que las células han cumplido plenamente y extendida. </li></ol> 2. Realizar Transducción (Día 2) <ol><li> En el día de la transducción, comprobar la densidad celular y elegir los pozos de densidad más eficientes (Figura 2). Es mejor escoger tres densidades diferentes: alto (80 ~ 90%), media (50 ~ 70%) y baja (20 ~ 40%). </li><li> Por lo menos 1 hora antes de la transducción, aspirar el fibroblmedios de AST de las células y cambiar nuevos 300 l de medio de fibroblastos. </li><li> Retire un conjunto de 4 tubos de virus Sendai de formas diferentes por -80 ° C de almacenamiento. Descongele cada tubo al mismo tiempo en un 37 ° C baño de agua durante unos segundos, y luego tomar los tubos fuera del baño de agua. Después de eso, descongelar a temperatura ambiente; tubos de centrifugación a 6.000 xg durante 10 segundos y los colocan en hielo hasta su uso. No vuelva a congelar y descongelar el virus ya que los títulos no se mantendrán. </li><li> Agregue los volúmenes indicados de cada uno de los cuatro tubos de virus Sendai (Oct-4, Klf-4, c-Myc y Sox-2) al tubo de microcentrífuga. Asegúrese de que la solución se mezcla bien con la pipeta suavemente. Por ejemplo, si 50K células / un pocillo de 24 pocillos Lookwell placa para la transducción: (Concentración basada en el certificado de análisis de Life Technologies, puede ser diferente de lote a lote) Tube hOct-4 = 6,0 x 10 7 CIU, 3 MOI = 5 l Tube hSox-2 = 6,5 x 10 7 CIU, 3 MOI = 4,6 μ; L Tube hKlf-4 = 6,3 x 10 7 CIU, 3 MOI = 4,8 l Tube hc-Myc = 7,8 x 10 7 CIU, 3 MOI = 3,8 l Total = 18,2 l de cuatro factores de virus / mezcla de un pocillo (50K células) de placa de 24 pocillos </li><li> Dependiendo de las densidades de células; añadir 2X, 1X, 0,5 X y el volumen de la mezcla de virus en el pozo. Agite suavemente la placa frontal a la parte posterior, izquierda y derecha (2X: 36,4 l de mezcla de virus, 1X: 18,2 ly 0.5X: 9,1 l). </li><li> Colocar las células en un 37 ° C, 5% de CO 2 incubadora y se incuba durante la noche. </li></ol> 3. Sustitución del medio de cultivo (día 3 y el día 5) <ol><li> 24 horas después de la transducción, sustituir el medio con 500 l medio de fibroblastos fresco. </li><li> En el Día 5, cambio de medio con los medios de comunicación de fibroblastos fresco. </li></ol> 4. Preparar platos MEF para la Co-cultura (Día 8) <ol><li> Preparar las células MEF en 60 mm placas de cultivo con 10% de FBS contenía medio DMEM uno daY antes de pasar las fibroblastos transducidos en alimentador células MEF. Nota: Estamos utilizando 5 x 10 5 de MEF en cada placa de 60 mm. </li></ol> 5. Start Co-cultura transducidas células con células alimentadoras MEF (Día 9) <ol><li> Antes de chapado células transducidas, aspirado 10% de SFB contenía medio DMEM desde platos MEF alimentador y añadir fresca 10% de SFB contenía medio DMEM. </li><li> Retire el medio de los fibroblastos transducidos que se encuentran en la placa de 24 pocillos y se lavan las células una vez con D-PBS. Añadir 200 l de 0,25% de tripsina / EDTA en las células de fibroblastos enel placa de 24 pocillos. Después de menos de 5 min de incubación con tripsina / EDTA, cuando las células comienzan a desprenderse de la placa, recoger las células con medio de crecimiento en 15 ml de tubo cónico. Entonces centrifúguelas en 6000 × g durante 4 min. NOTA: El agrupamiento de 2X, 1X, 0,5 X y el virus se recomienda fibroblastos transducidos. </li><li> Retire el medio sobrenadante y lavar para resuspenderel sedimento celular con 4-5 ml de medio fresco de fibroblastos para la neutralización de tripsina. Entonces centrifúguelas a 135 g durante 4 min. </li><li> Células de la placa como la densidad de dilución en serie y empezar a co-cultivo con medios de fibroblastos en MEF: tal como 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, y 1/128 por 60 mm plato. Dependiendo de la tasa de muerte celular durante la primera semana de la transducción, uno no puede tener que diluir a 1/2 o 1/128 de la densidad. Dejar a un lado las células restantes para extraer el ARN total como control positivo para el transgen de RT-PCR. </li><li> Coloque placas de 60 mm en 37 ° C, 5% de CO 2 incubadora durante la noche. </li></ol> 6. Alimente Embrionarias Humanas Medio de Células Madre y Monitorear las células (Día 10) <ol><li> El día siguiente, cambiar los medios de comunicación a los medios de fibroblastos ES humano con 10 mM de Y-27632. Después de eso, cambio de medio al día con medio ES humana: 800 ml de DMEM/F12 + 200 ml Knockout suero reemplazo + 10 ml de L-glutamina + 10 ml de 10 aminoácidos no esenciales mínimos mM MEM+ 1 ml de 2-mercaptoetanol + 10 ng / ml de factor de crecimiento básico de los fibroblastos. </li><li> Hacer un cambio de medio día con nuevos medios embrionarias humanas. Después de una semana, revise los platos una vez por 2 ~ 3 días bajo un microscopio para detectar la formación de ES-colonias como grupos de células. Dependiendo del tipo de células, la cultura va a tomar más o menos tiempo antes de que se encontraron colonias. </li></ol> 7. Elegir la madre pluripotentes inducidas colonias de células y expandir las células (Día 20 ~) <ol><li> Tres semanas después de la transducción, las colonias deben aparecer crecido adecuadamente para su recolección. </li><li> El día antes de recoger las colonias, preparar alimentador de MEF en placa de 24 pocillos. NOTA: Estamos utilizando 12,5 x 10 5 de MEF en una placa de 24 pocillos. </li><li> El día siguiente, cambio de medio de platos MEF a partir de medios de fibroblastos a los medios ES humano con 10 mM de Y-27632. Recoger manualmente una colonia en cada momento bajo el microscopio en la cosecha del capó y hacer montones más pequeños con la pipeta y transferirlosen recién preparado MEF dishes.Try para recoger varios clones. </li><li> Pasaje y ampliar la cada pocillo de una placa de 24 pocillos de una placa de 6 inicialmente. Luego de una placa de 6 pocillos a un 60 mm platos antes de seguir propagando. </li></ol> 8. Caracterización de iPSCs Humano (después de 10 pasajes) <ol><li> Inmunofluorescencia <ol><li> Placa iPSCs humanas en placas de 24 pocillos y la cultura durante 4-5 días con cambio de medio día. </li><li> Para comenzar el ensayo de inmunofluorescencia, se lava la placa con D-PBS una vez. Entonces fijar las células inmediatamente en 0,4% de paraformaldehído durante 30 min. Después de que lavar tres veces en D-PBS durante 5 min. </li><li> Tratar las células fijas con 0,1% de Triton X-100 en PBS solución durante 15 min a temperatura ambiente. </li><li> Aspirar la solución de permeabilización (Triton X-100 0,1% solución), a continuación, agregar solución de bloqueo BSA al 0,5% durante 1 hora a temperatura ambiente. </li><li> Lleve a cabo una detección de Nanog, Oct-4, SSEA (antig embrionaria etapa específicaes) y TRA-1-81 (rechazo antígeno tumoral) usando un anticuerpo anti-humano en solución de BSA al 0,5% (comprobar la tabla de materiales para la tasa de dilución de anticuerpo). Incubar anticuerpo primario durante 2 horas a temperatura ambiente. A continuación, lavar tres veces en D-PBS durante 5 minutos cada uno. </li><li> Compruebe mediante microscopía de fluorescencia después de 1 h de incubación con Alexa Fluor 488 cabra anti-ratón anticuerpos IgG como anticuerpo 2 ª, que se diluyó 1:2000 en solución de BSA al 0,5% a temperatura ambiente. Teñir todos los núcleos de cada MEF células alimentadoras y hiPSCs con DAPI. </li></ol></li><li> RT-PCR del transgén Confirmación <ol><li> Extracto total de ARNm de sedimento celular mediante el uso de reactivos de extracción de RNA. </li><li> Hacer una transcripción inversa en partes alícuotas (1 g) de ARN total y el ADNc resultante para la amplificación de PCR por transcriptasa inversa. </li><li> Preparar las mezclas de PCR para contener 2 l de molde de ADNc, 10 l de mezcla maestra de PCR 2X, 2 l de 2 pmol de cebadores(Hacia adelante: 1 l e invertir: 1 l) y 6 l de DW. Para detectar la expresión del gen, hacer la RT-PCR con los cebadores listados en la Tabla 1. </li><li> Realizar la RT-PCR con el gen de GAPDH como control para la eficiencia de las reacciones de amplificación. Visualizar y analizar el producto de PCR en un 1% electroforesis en gel de agarosa. </li></ol></li></ol>

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Los autores no tienen nada que revelar.

La reprogramación de las células somáticas humanas con hiPSCs sostiene promesas sin precedentes en la biología básica, la medicina personal, y el trasplante 12. Anteriormente, las generaciones IPSC humanos necesarios virus de ADN que tiene riesgo de integración en el genoma huésped, que puede crear mutaciones genéticas indeseables que limitan otras aplicaciones clínicas tales como el desarrollo de medicamentos y terapias de trasplante 13. Con este motivo, se ha informado de muchos estudios...

Las células madre embrionarias (hESCs) tienen una capacidad de auto-renovación in vitro y tienen la pluripotencia, lo que podría ser potencialmente útil para el modelado de la enfermedad, para la detección de drogas, y para el desarrollo de terapias basadas en células para tratar lesiones de la enfermedad y del tejido. Sin embargo, hESCs tienen una limitación para la terapia de reemplazo de la célula debido a las barreras inmunológicas, oncológicas y éticas, y para estudiar los genes relacionados con la enfermedad, hESCs específicos de la enfermedad pueden ser aislados a través de pre-implantación diagnóstico genético (PGD) se acerca, pero sigue siendo un reto técnico y las donaciones de embriones son bastante raros. Estos problemas están relacionados con el progreso en la biología de células madre, lo que ha llevado al desarrollo de células madre pluripotentes inducidas (hiPSCs). IPSCs humanos se reprograman genéticamente a partir de células somáticas adultas humanas y albergan características madre pluripotentes similares a células similares a hESCs, que los convierte en una fuente útil para la medicina regenerativa, como ddescubrimiento alfombra, el modelado de la enfermedad y la terapia celular en forma específica para cada paciente 1,2. Hasta ahora, hay varios métodos para generar iPSCs humanos, incluyendo mediada por virus (retrovirus y adenovirus) 3, no el virus mediada (sistema de BAC y vectores de transfección) 4 transducciones de genes, y del sistema de administración de proteínas 5-7. Aunque una entrega de genes mediada por virus puede garantizar un cierto nivel de eficiencia, vectores virales podrían dejar huella genética, debido a que se integran en los cromosomas del hospedador para expresar genes de reprogramación de una manera incontrolada. Incluso cuando la integración viral de factores de transcripción puede activar o inactivar genes de acogida 8, que puede causar una aberración genética inesperado y el riesgo de 5,9 tumorigénesis. Por otra parte, se informó de la introducción directa de proteínas o ARN en las células somáticas, pero tienen algunas desventajas tales como, transf repetida mano de obra intensivaexión, y el bajo nivel de la reprogramación de 7,10. Incluso episomal y no la integración de adenovirus, virus adeno-asociados, y vectores plasmídicos son todavía relativamente menos eficiente 11. Por estas razones, es plausible para elegir métodos de reprogramación no-integración con alta eficacia de generación de IPSC y un menor número de anomalías genéticas. En este estudio, utilizamos una reprogramación basada Sendai-virus. Este método es conocido por ser no integrado en el genoma huésped y produce consistentemente iPSCs humanos sin integraciones transgén.

<table border="0" cellpadding="0" cellspacing="0" width="1109">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">CytoTune-iPS Reprogramming Kit</td>
			<td style="width:195px;">invitrogen</td>
			<td style="width:119px;">A1378002</td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CF-6,MEFs, neomycin-resistant, mitomycin C treated&#160;</td>
			<td>Global stem</td>
			<td>GSC-6105M&#160;</td>
			<td>5X105/6cm or 12.5X105/24 well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA 0.25% Trypsin with EDTA 4Na 1X</td>
			<td style="width:195px;">invitrogen</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12 medium</td>
			<td style="width:195px;">&#160;invitrogen</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Cell Culture Plate, flat-bottom with lid</td>
			<td style="width:195px;">BD</td>
			<td>353935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Y-27632</td>
			<td style="width:195px;">TOCRIS</td>
			<td style="width:119px;">1254</td>
			<td>10 uM (Stock: 10 mM)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">basic fibroblast growth factor</td>
			<td>LIFE TECHNOLOGIES&#160;</td>
			<td>PHG0263</td>
			<td>10 ng (Stock : 100 ug)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Knock-out serum replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle Medium (D-MEM, DMEM) (1X), liquid (high glucose)</td>
			<td style="width:195px;">&#160;invitrogen</td>
			<td>11965118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Fetal bovine serum</td>
			<td style="width:195px;">Thermo Scientific Fermentas</td>
			<td style="width:119px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine-200 mM (100X), liquid</td>
			<td style="width:195px;">GIBCO</td>
			<td>25030-081</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution, 100X</td>
			<td>LIFE TECHNOLOGIES&#160;</td>
			<td>11140050</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol (1,000X), liquid</td>
			<td style="width:195px;">GIBCO</td>
			<td>21985023</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hausser Phase Contrast Hemacytometers</td>
			<td>Hausser Scientific</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EmbryoMax 0.1% Gelatin Solution</td>
			<td style="width:195px;">Millipore</td>
			<td style="width:119px;">ES-006-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">SSEA-4</td>
			<td style="width:195px;">DSHB</td>
			<td>MC-813-70</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Tra-1-81</td>
			<td style="width:195px;">cell signaling</td>
			<td>4745S</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal Oct4 antibody</td>
			<td>Santa Cruz</td>
			<td>SC-5279</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Nanog</td>
			<td>R&#38;D</td>
			<td>AF1997</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Alexa Flouor 488 goat anti-mouse</td>
			<td>invitrogen</td>
			<td>948492</td>
			<td>1/2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS (Dulbecco&#39;s Phosphate-Buffered Saline), 1X without calcium &#38; magnesium</td>
			<td style="width:195px;">cellgro</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:543px;">QuantiTect Reverse Transcription Kit</td>
			<td style="width:195px;">QIAGEN</td>
			<td>205313</td>
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		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">PCR Master Mix [2X]</td>
			<td style="width:195px;">Thermo Scientific Fermentas</td>
			<td style="width:119px;">K0171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol</td>
			<td style="width:195px;">invitrogen</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">picking hood</td>
			<td>NuAire</td>
			<td style="width:119px;">&#160;NU-301&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">dissecting scope&#160;</td>
			<td style="width:195px;">Nikon</td>
			<td>SMZ745</td>
			<td></td>
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efficient generation human induced pluripotent stem cells from human somatic cells with sendai-virus

Hace unos años, la creación de células madre pluripotentes inducidas humanas (iPSCs) marcó el comienzo de una nueva era en la biomedicina. Los usos potenciales de iPSCs humanos incluyen el modelado patogénesis de enfermedades genéticas humanas, la terapia celular autóloga después de la corrección de genes, y la detección de drogas personalizada, proporcionando una fuente de células relevantes y los síntomas específicos del paciente. Sin embargo, hay varios obstáculos que superar, como la eliminación del factor de reprogramación transgén expresión restante después de la producción iPSCs humanos. Más importante aún, la expresión transgénica residual en iPSCs humanas indiferenciadas podría obstaculizar diferenciaciones adecuadas y confundir a la interpretación de los fenotipos in vitro relevantes enfermedad. Con este motivo, se han desarrollado-la integración libre y / o iPSCs humanos transgenes gratis con varios métodos, como el adenovirus, el sistema piggyBac, minicircle vector, vectores episomales, entrega directa de proteínas y ARNm sintetizado. Sin embargo, la eficiencia de reprogramming utilizando métodos de integración libre es bastante bajo en la mayoría de los casos. A continuación, presentamos un método para aislar iPSCs humanos mediante el uso de Sendai-virus (virus RNA) sistema de reprogramación en base. Este método de reprogramación muestra resultados consistentes y de alta eficiencia en forma rentable.

Por lo general, los fibroblastos infectados no muestran cambios morfológicos en varios días después de la transducción de virus Sendai, pero cinco días después, empiezan a tener diferentes formas (Figura 1). Como se describe en un panel de la derecha de la Figura 1, las células no tienen la morfología típica de fibroblastos más. Ellos tienen una forma redonda y el núcleo más grande que el citoplasma. Incluso cuando la transducción se realiza en el 80% de confluencia celular...

Watch this Scientific Journal Video about Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus at JoVE.com

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

A continuación, presentamos nuestro método establecido para reprogramar las células somáticas humanas en transgenes libre iPSCs humanas con virus Sendai, que muestra resultados consistentes y una mayor eficiencia.

Eficiente generación, por actividad humana células madre pluripotentes de células somáticas humanas con virus Sendai-

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 ...

efficient-generation-human-induced-pluripotent-stem-cells-from-human

Research

JoVE Journal

Biology

22.2K vistas.  Johns Hopkins University School of Medicine.  A continuación, presentamos nuestro método establecido para reprogramar las células somáticas humanas en transgenes libre iPSCs humanas con virus Sendai, que muestra resultados consistentes y una mayor eficiencia. 

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

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.

Generación de células madre pluripotentes inducidas por reprogramación de fibroblastos humanos con los Lentivirus Stemgent Humanos TF 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 ...

Investigación

Biología

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.

Transfección y Nucleofecting Humanos células madre pluripotentes inducidas

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

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivación eficiente de los Derechos Humanos precursores cardiacos y cardiomiocitos de pluripotentes células madre embrionarias humanas mediante la inducción de moléculas pequeñas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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.

Selección y aislamiento de las colonias de humanos células madre pluripotentes inducidas 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.

La reprogramación de células somáticas humanas en células madre pluripotentes inducidas (iPSCs) utilizando el vector retroviral con las buenas prácticas agrarias

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.

Generación de ratones derivados de células madre pluripotentes inducidas

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.

Generación de las células madre pluripotentes inducidas a partir de sangre periférica mediante el STEMCCA vector lentiviral

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

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

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

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

Generación rápida y eficiente de las neuronas a partir de células madre pluripotentes en un formato de placa Multititre

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

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.

La generación de cardiomiocitos humanos: Protocolo de diferenciación de células madre pluripotentes inducidas humanas Feeder libres

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

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

To begin, take a flask of cultured fibroblast cells, coat a 10-centimeter cell culture plate suitable for hiPSC culture with four milliliters of cold coating solution per plate, and incubate the plate for one hour at 37 degrees Celsius. Afterward, remove the media from the fibroblasts and wash them with four milliliters of pre-warmed PBS per flask. Aspirate the PBS and associate the fibroblasts with four milliliters of Trypsin-EDTA per flask by incubating for five to seven minutes at 37 degrees Celsius.Monitor cell detachment using a phase contrast microscope, and if needed, tap against the side of the flask to detach the cells from the plastic surface. Transfer the detached cells to a 50-milliliter conical tube and wash the empty flasks with six milliliters of pre-warmed fibroblast medium. Collect and pool the remaining cells in the conical tube.Then centrifuge the cells at 200 g for five minutes at 20 degrees Celsius. Aspirate the supernatant and re-suspend the cell pellet in three milliliters of fresh, pre-warmed PBS. Count the cells, transfer 1.5 times 10 to the sixth fibroblasts into a new conical tube and fill up with PBS to the five millimeter mark.Centrifuge the tube at 200 g for five minutes at 20 degrees Celsius. Discard the supernatant, re-suspend the cell pellet in five milliliters of electroporation medium, and centrifuge again. In the meantime, aspirate the coating solution from the coated 10-centimeter cell culture plate and add seven milliliters of pre-warmed fibroblast medium.At the end of the centrifugation, discard the supernatant and re-suspend the cell pellet in electroporation medium with a concentration of 1.5 times 10 to the sixth cells in 250 microliters. Transfer the 250 microliter cell suspension to an electroporation cuvette with a four millimeter gap distance. Next, prepare a plasma transfection mix by adding four micrograms of each plasmid to a total volume of 50 microliters of electroporation medium.Transfer the transfection mix to the cuvette, mix gently by flicking, and electroporate with one pulse at 280 volts. Finally, transfer 150 microliters of the electroporated fibroblasts to the prepared 10-centimeter cell culture plate. Agitate the plate thrice in all directions to distribute the cells, and incubate overnight.Fibroblasts present in a long, spindle-like phenotype, the size of 150 to 300 nanometers. After reprogramming, colonies with 50 micrometer small hiPSCs occur displaying distinct borders. The hiPSCs are distinguished by a high nuclei to body ratio and increased proliferation rate.