We would like to thank Patrick C. Lee, Cyril Ramathal, and Saravanan Karumbayaram (SK) for their assistance in performing the iPSC derivation and characterization experiments; Aaron Cooper for performing the iPSC analysis experiments; Vittorio Sebastiano and Renee A. Reijo Pera for directing the initial reprogramming efforts; SK, William E. Lowry, Jerome A. Zack, and Donald B. Kohn for directing the establishment of the UCLA GMP facilities permitting the conversion and characterization of clinical-grade iPSCs; Gustavo Mostoslavsky for providing us with the STEMCCA polycistronic reprogramming vector. This work is based on a research collaboration with Fibrocell Science and the Clinical Investigations for Dermal Mesenchymally Obtained Derivatives (CIDMOD) Initiative to generate safe personalized cellular therapeutics. This work was supported by funding from the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, The Phelps Family Foundation, Fibrocell Science, Inc., and the UCLA CTSI Scholar&#8217;s Award to JAB.

NOTA: Este método se utilizó en la investigación publicada en Awe et al 10.. 1. Reprogramación Humana Adulto fibroblastos dérmicos con STEMCCA <ol><li> Fibroblastos dérmicos Descongele adultos humanos (HUFS), paso 4 o inferior, en un 37 ° C baño de agua durante 2 minutos, teniendo cuidado de no sumergir la parte superior del vial con agua. </li><li> Coloque la suspensión 1 ml de fibroblastos humanos en un vial cónico de 15 ml y colocar lentamente 4 ml de medios estándar HUF, pre-calentado a 37 ° C, de una forma gota a gota, para diluir a cabo sulfóxido de dimetilo (DMSO) y volver a suspender las células. </li><li> Centrifugar el vial a 200 xg durante 10 min a temperatura ambiente. Aspirar el sobrenadante y volver a suspender las células en un volumen adecuado de los medios de comunicación para crear una suspensión de 100 000 células / ml y a continuación, añadir 2 ml en un pocillo de una placa de seis pocillos, pre-recubierto durante 20 minutos con 0,2% de gelatina. NOTA: Una hermana bien con un número igual de células por línea y el estado necesita ser sembrado for el recuento de células / multiplicidad de infección (MOI) de cálculo. </li><li> Rock the lado de la placa a lado y lado a otro para distribuir uniformemente las células. Incubar durante la noche a 37 ° C, 5% de CO2 en un incubador humidificado. </li><li> En el día de la transducción, obtener el recuento de células (s) de la hermana así (s) de aproximadamente 24 hr después de la siembra inicial HUF. </li><li> Descongelar 2 x 10 8 TU, o, concentrado lentiviral equivalente (STEMCCA) y diluir a 1 x 10 8 TU / ml en medio HUF. </li><li> Transducir células en una proporción 10-MOI en medios HUF suplementado con agente de transfección 8 mg / ml. Mezclar pozos transducidas de manera uniforme y se incuba durante la noche a 37 ° C, 5% de CO2 en un incubador humidificado. </li><li> Aproximadamente 24 horas después de la transducción lentiviral, cambiar los medios de comunicación HUF a células madre pluripotentes humanas (PSC) medio estándar. </li><li> En los días 2 al 6, cambiar los medios de comunicación todos los días con medio PSC humano. </li><li> El día 6, la placa dos 0,2% recubierto de gelatina placas de 10 cm de1,5 a 1,75 x 10 6 MEFs derivados de ratones CF1 en HUF medios 12. </li><li> El día 7, con cuidado de no centrifugar más de 100 xg, uso tripsina temperatura ambiente para levantar los días 7 fibroblastos reprogramados de un pocillo de la placa de seis pocillos y el paso a una relación 01:16, en placas de todas las células de manera uniforme entre dos placas de 10 cm, con los medios de comunicación PSC humana fresca, que se sembraron con MEFs el día anterior. Distribuir los agregados de células en un movimiento en espiral hacia la derecha y colocar en un 37 ° C, 5% de CO 2 humidificado incubadora durante la noche. </li><li> Aproximadamente 48 horas después de la siembra de las células reprogramadas en MEFs y todos los días a partir de entonces, cambie a cabo gastados medios PSC humanos por medio fresco. </li><li> Después de 3 a 4 semanas, recoger las colonias individuales con la típica morfología ESC como con una aguja de calibre 21 y subclonar a cabo en una placa de 24 pocillos mediante la colocación de aproximadamente 8 a 10 piezas individuales de colonias específicas en pocillos individuales en un 0,2% de gelatina recubierta placa de 24 pocillos pre-semillaed con MEFs derivados de ratones CF1 en medio PSC humano. NOTA: Después de la expansión de las colonias escogidas, las colonias tienen que caracterizarse como pluripotentes de una formación corporal embrioide y análisis de la expresión de marcadores de pluripotencia a nivel de proteínas. </li></ol> 2. Integración de análisis vectorial de Sitio y STEMCCA Excision <ol><li> Analizar el sitio de integración STEMCCA por amplificación lineal no restrictiva PCR como se ha descrito 10. </li><li> Después de someter a ensayo para garantizar una integración en un área deseada del genoma, STEMCCA especial mediante la aspiración del viejo medio ESC humana. Entonces, se combinan 45 l de virus adeno-Cre-PuroR concentrado con 8 g agente de transfección / ml en 3 ml de medio PSC estándar durante 24 h. </li><li> Después del período de incubación de 24 horas, aspirar el sobrenadante viral mixta y se lavan las células dos veces con los medios de comunicación del PSC humanos. Coloque frescas medios PSC humanos junto con 2 mg / ml de puromicina por un período de 5 días, cambiando el ingenio diaria mediosh medios de comunicación y los antibióticos frescas. </li><li> Después de 5 días, subclón colonias con una aguja de calibre 21 restante sobre 0,2% pozos recubiertos de gelatina, en una placa de 12 pocillos pre-recubierto con MEFs derivados de CF1mice y ampliar como se detalló anteriormente. NOTA: Después de una expansión suficiente para obtener una solución madre congelada, se requiere conversión en condiciones libres de alimentador. Espere al menos el 95% de muerte de colonias de células madre como la mayoría de las células no se transducidas con éxito y sucumbirán a los antibióticos durante el período de 5 días de incubación. Aunque adeno-Cre-PuroR integra con una eficiencia de 0,001 a 1% en los cromosomas de acogida de las células infectadas, reexposing una muestra de las colonias factor libre de puromycin para asegurar la muerte celular 100% confirmará ninguna integración se ha producido 13. </li><li> Descongelar un vial de matriz de membrana basal pre-alícuota en hielo, durante un período de aproximadamente 2 hr (recomendaciones del fabricante) hasta que la matriz es un líquido y se diluye a una concentración final de 1:30 de medios basales frío. Coen el número deseado de pozos en una placa de seis pocillos con 1 ml por pocillo. Deje reposar a temperatura ambiente durante 1 hora. </li><li> Cross-Hatch (usando una aguja de calibre 18, o una &quot;punta&quot; de vidrio o de plástico) al menos 20 a 30 colonias de un pozo previamente recubierto con MEFs. Tenga cuidado para reducir MEF edificante y traslado del plato. <ol><li> Generar el dispositivo de sombreado cruzado a través de una serie de enfoques diferentes, desde el calentamiento más complicado, tirando, y acabado de una pipeta Pasteur de vidrio sobre una llama abierta, al simple uso de una punta de pipeta de plástico estéril o, como en nuestro caso, 21- y / o calibre 18 agujas. </li></ol></li><li> Aspirar la matriz desde el pocillo recubierto después de la incubación. </li><li> Retire piezas de las colonias por raspado suave de la placa de edad con una pipeta de 200 l y se coloca cuidadosamente en 3 ml de los medios de comunicación combinados frescas 1 en la placa de matriz recubiertos. Incubar toda la noche en un 37 ° C, 5% de CO 2 humidificado incubadora. Cambiar medios 48 horas después de los pases. </li><li> Extraer el ADN genómico de hiPSCs convertidos post extirpado y libres de alimentador, en más del 80% de confluencia, usando kits de extracción de ADN comercial. </li><li> Analizar por escisión adecuada con primers específicos para las integraciones exógenas del lentivirus STEMCCA: gDNA-hendo-MycS-forward, 5&#39;-acgagcacaagctcacctct-3 &#39;; gDNA-hWPRE-inverso, 5&#39;-tcagcaaacacagtgcacacc-3 &#39;. Reconstituir iniciadores liofilizados con agua de calidad para PCR a una solución madre 10-M. </li><li> Ejecutar una PCR con el siguiente protocolo de cinco pasos: desnaturalización inicial a 95 ° C durante 3 min; 35 ciclos de cada uno de los siguientes: desnaturalización a 98 ° C durante 20 segundos, hibridación del cebador a 62 ° C durante 15 seg, y extensión a 72 ° C durante 15 s; seguido de un único ciclo de extensión final a 72 ° C durante 3 min. </li><li> Hacer un gel de agarosa al 3% pesando 1,5 gramos de agarosa y colocándolo en tampón 1x 50 ml de Tris-acetato-EDTA. Horno de microondas hasta agarosa se disuelve y el lugar en la mancha de gel de ADN en pdilución Roper, mezclar y dispensar un volumen apropiado en casete de gel para solidificar durante 1 hora a temperatura ambiente. </li><li> Cargar 12 l de producto de PCR se mezcla con 3 l de colorante de carga en un gel de agarosa al 3%. Ejecutar el gel durante 30 minutos a 80 V. </li><li> Visualizar con luz ultravioleta para la ausencia de una banda que indica la escisión adecuada. </li></ol> 3. La cuantificación de la expresión génica diferencias mediante el uso de PCR cuantitativa de pre y post hiPSCs extirpados- <ol><li> Extraer el ARN total de hiPSCs convertidos post extirpado y libres de alimentador usando kits de extracción de ARN comerciales. </li><li> Reverse-transcribir hasta 1 g de ARN total mediante el uso de kits comerciales de acuerdo con las instrucciones del fabricante. Use-oligo anclado (dT) 18 y cebadores hexámeros aleatorios. NOTA: Asegúrese de que el protocolo de PCR se adapta para la transcripción de genes de más o menos de 4 kb. Tendrá que ser hecho para que sea i gen STEMCCA integrado originalmente primers específicosnto. </li><li> Reconstituir la imprimación liofilizado con agua de calidad para PCR a una solución de 20 mM. </li><li> Utilice 5 ng de muestra por cada 20 l de reacción que consta de sonda UPL 10 M, 2x LightCycler 480 Sondas Maestro, y 20 mM adelante y atrás primers. </li></ol> 4. La conversión en condiciones GMP grado <ol><li> En el primer día de la transición de los hiPSCs post extirpado a condiciones GMP grado, hacer una mezcla de medios de comunicación que consiste en PSC humano combinado de medios de comunicación (1 medios combinado 1) y grado clínico buenas prácticas de fabricación (GMP) de medios de comunicación que pueden utilizarse (medios combinados 2 ) en una relación de 80:20. </li><li> Aspirar los viejos medios combinados 1 y colocar 3 ml de los nuevos medios de comunicación combinados 1 y 2, previamente calentado, en la relación de 80:20. Cambie los medios día durante 3 días con esta relación específica. Coloque las células de nuevo en un 37 ° C, 5% de CO2. </li><li> En el día 4, hacer que los medios de comunicación combinado 1 y 2 en una proporción de 50:50. Aspirar la edad media y Replace con los medios de pre-calentado, con los medios de comunicación combinados 1 y 2 en la relación de 50:50. Cambie los medios durante 3 días con esta relación específica. Coloque las células de nuevo en un 37 ° C, 5% de CO2. </li><li> El día 7, hacer los medios de comunicación combinada 1 y 2 en una proporción 20:80. Aspirar el medio viejo y reemplazarlo con los medios de comunicación pre-calentado, con los medios de comunicación combinados 1 y 2 en la proporción 20:80. Cambie los medios durante 3 días con esta relación específica. Coloque las células de nuevo en un 37 ° C, 5% de CO2. </li><li> El día 10, hacer los medios de comunicación combinados 1 y 2 a una 0: relación de 100. Aspirar el medio viejo y reemplazarlo con los medios de comunicación pre-calentado, con los medios de comunicación combinados 1 y 2 en el 0: relación de 100. Cambie los medios todos los días a esta relación específica. Las células se encuentran ahora en los medios de comunicación-xeno libre completamente definido. Coloque las células de nuevo en un 37 ° C, 5% de CO2. NOTA: Durante este proceso de conversión, se necesita pases de rutina con una aguja de calibre 18 para mantener tamaño de la colonia y la densidad apropiados. Plan en la passaging células cada 4 a 5 días sobre la base de tamaño de las colonias, la confluencia, y la diferenciación. </li><li> Escudo un pocillo de una placa de seis pocillos con un sustrato-xeno libre definido según lo recomendado por el fabricante. </li><li> Pasaje Mecánicamente un confluente bien, ya convertida en Xeno-libre condiciones de comunicación, a partir de una placa de seis pocillos con una aguja de calibre 18. Es importante destacar que colocar nuevos medios de comunicación (medios combinados 2) en las células con inhibidores ROCA 1x durante 1 hora, antes de que pases a pre-condición los medios de comunicación. </li><li> Aspirar la solución de sustrato sintético de la nueva placa que las células están siendo transferidos a. Quite todos los medios que contienen los grupos de células madre de la placa vieja y la transferencia a la nueva placa. </li><li> Coloque las células de nuevo en un 37 ° C, 5% de CO2 durante 2 días con perturbación física mínima (idealmente placas, una vez dentro de la incubadora, no se tocan directamente en modo alguno) para permitir la unión máxima. NOTA: Las células requerirán cambios de medio día. Passagin Continuag cada 4 días serán importantes. Usando una aguja de calibre 21, de paso sólo las partes de colonias con morfología ESC-como óptima (alto cociente nucleocytoplasmic). Esto asegurará la selección de colonias hiPSC adecuadas que crecerán bien y eventualmente producir colonias homogéneos. El tiempo total de derivación de ESC como colonias es entre 15 a 20 días después del cambio inicial medios combinado. </li><li> Congele por las hiPSCs posteriores a extirpado GMP grado por primera trama cruzada todas las colonias utilizando una aguja de calibre 18. Levantar todas las piezas fuera mediante el uso de una pipeta de 200 l y transferir todos los medios que contienen las células a un vial de 15 ml cónico y centrifugar a 200 xg durante 5 min a 4 ° C. </li><li> Mientras que las células están centrifugación, hacer una congelación medios de comunicación que consiste en volúmenes iguales de medios combinados fríos 2 y medio de congelación con una concentración final de DMSO del 7,5%. </li><li> Después las células se sedimentan, aspirar fuera el medio viejo y gota a gota vuelva a suspender el sedimento con el medio de congelación. Inmediatamente transfer a viales de congelación y poner en un congelador a -80ºC. </li></ol> 5. hiPSCs Caracterización GMP grado Publicar extirpados- <ol><li> Para asegurar la adecuada expresión de los marcadores de pluripotencia después de la conversión, utilice QPCR para cuantificar la expresión de marcadores de pluripotencia clave (SOX2, OCT4, y NANOG) usando los cebadores listados en la Tabla 1 con los mismos pasos como se ha mencionado en el paso 3. metodologías QPCR seguir el mismos pasos que se indican en el paso 3. </li><li> El uso de citometría de flujo para detectar el ácido siálico no humano Neu5Gc (ácido -glycolylneuraminic N) de acuerdo con las condiciones generales que se indican en el kit según lo publicado previamente 10. </li><li> Lave suavemente las placas de cultivo celular con células mediante el uso de solución salina tamponada con fosfato de 1x (PBS). </li><li> Disociar colonias mediante el uso de reactivo de disociación 1x durante 5 min a temperatura ambiente. </li><li> Durante la 5-min tiempo de incubación de la etapa 5.4, preparar la solución de bloqueo (proporcionado enkit) haciendo un 0,5% Agente bloqueador en PBS enfriado con hielo. </li><li> Rotula el siguiente conjunto de tubos para citometría de flujo: no teñida; 4 &#39;, 6-diamidino-2-fenilindol (DAPI) DAPI; El anticuerpo de control 1: 200; Primaria de anticuerpos de 1: 200; Anticuerpo secundario 1: 200 de IgG anti-pollo de burro (H + L); MEFs muestra; Células post extirpado muestra en matriz; y la Muestra células en sustrato sintético puesto extirpada. </li><li> Agitando suavemente los agregados de células de la etapa 5.4 mediante la adición de 2 ml de solución de bloqueo y de transferencia de contenido a un tubo cónico de 15 ml. Centrifugar a 80 xg durante 5 min a 4 ° C. </li><li> Lavar las células dos veces con solución de bloqueo y se centrifuga como se ha indicado en el paso 5.7. </li><li> Suavemente volver a suspender aproximadamente 1 x 10 6 células en 100 l de solución de bloqueo con el volumen de dilución correspondiente de cada anticuerpo. Incubar con suave balanceo a 4 ° C durante 1 hora. </li><li> Repita lavados tres veces como en el paso 5.8. </li><li> Vuelva a suspender el sedimento celular en 400 l de diluyente de Buffer (del kit) con 1: 100 de DAPI para tubos de 2, 6, 7, y 8 como se indica en el paso 5.6. </li><li> Células de la cepa a través de un colador de 40 micras y se ejecutan a través de citómetro de flujo. </li></ol>

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James A. Byrne (JAB) and Agustin Vega-Crespo receive research funding from Fibrocell Science, Inc. JAB is a scientific consultant for Fibrocell Science, Inc. No other authors have any competing interests to disclose.

Se describe una metodología de derivar hiPSCs factor libre y hacerlos clínicamente relevante mediante la conversión de estas células en condiciones GMP grado de diferenciación celular aguas abajo en futuras terapias humanas. Aunque este protocolo es ampliamente aplicable a una variedad de tipos de células, que elegimos para reprogramar fibroblastos dérmicos humanos, debido a la facilidad de extracción de la paciente y su aplicabilidad a la terapéutica humana personalizados. Una vez que se subsanen las limitacio...

Adult human cells have been shown to be capable of undergoing epigenetic remodeling and reprogramming, as a result of lentiviral-based expression of four key transcription factors4,5. An important advancement in the reprogramming field was the use of a single excisable lentiviral stem cell cassette (STEMCCA), which housed all four reprogramming transcription factors that allowed a precise stoichiometric ratio of protein expression6. Additionally, when transduced in specific multiplicity of infection ranges, STEMCCA can lead to predominantly single genomic integration events during the reprogramming process7. The introduction of an excisable version of STEMCCA, which utilizes Cre/loxP technology followed by excision of the reprogramming vector after derivation of the stem cell line, enabled factor-free human induced pluripotent stem cell (hiPSC) lines to be derived8. Additionally, in order to enhance therapeutic applications of hiPSCs, a novel, quick, and readily applicable methodology for good manufacturing practice (GMP)-grade cell line conversion, from xeno-containing to xeno-free conditions, needed to be implemented. Here, we discuss a relevant methodology that more precisely assesses integrated gene expression differences, specifically when integrated into an intron, and clinical-grade cell conversion into putative GMP conditions. 
Previous research has used only relatively insensitive microarray transcriptional analysis to analyze gene expression differences in integrated genes after STEMCCA transduction3,9. Here, we introduce the methodology of sensitive quantitative polymerase chain reaction (PCR) analysis, to further examine integrated gene expression differences. Importantly, current safe-harbor criteria discard hiPSCs that have genes with viral integrations, thus limiting the applicability of these cells for downstream human cellular therapeutics9. We propose that the status quo may change with the use of fully characterized and transgene-free intronically reprogrammed hiPSCs. Additionally, we introduce a robust GMP-grade cell conversion protocol that can be readily applied to a variety of different cell types, which were originally derived under xeno-containing conditions10. This provides significant opportunities for the development of future cell reprogramming experiments, which require clinical-grade conditions to maintain human therapeutic relevance. 
These methodologies provide a foundation upon which current safe-harbor criteria may be expanded to include characterized STEMCCA reprogrammed hiPSC lines that maintain a normal gene expression profile after STEMCCA excision from the integrated intron. Also, full conversion into clinical-grade conditions, free from non-human animal antigens, will help to incorporate many more cell types, which have previously been reprogrammed and characterized only in xeno-containing conditions. These methodologies combined, are persuasive grounds for the US Food and Drug Administration (FDA) to consider expanding their limited approval from human embryonic stem cell (ESC)-based therapeutics to hiPSC-based therapeutics11. 
We recently detailed the derivation of a factor-free hiPSC line that was fully characterized and converted into putative clinical-grade conditions10. Here, we detail the protocol for hiPSC derivation by utilizing the STEMCCA lentivirus. These stem cells then undergo an excision process followed by gene expression characterization. Finally, the hiPSCs are converted over into GMP-grade conditions by a slow conversion methodology.

<table border="0" cellpadding="0" cellspacing="0" width="521">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:135px;">Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:521px;">Media and reagents</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">DMEM/F12 (basal media)</td>
			<td style="width:111px;">Invitrogen (Carlsbad, CA, USA)</td>
			<td style="width:136px;">11330057</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Fetal bovine serum</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">16000044</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:140px;">Minimum essential medium (MEM) non-essential amino acids (NEAA), 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">11140050</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Glutamax, 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">35050-061</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">PenStrep, penicillin-streptomycin, 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15140-122</td>
			<td style="width:135px;">Thaw at 4 &#176;C, aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Knockout serum replacement</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">10828028</td>
			<td style="width:135px;">Thaw at 4 &#176;C, aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Trypsin/EDTA, 0.5%</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15400-054</td>
			<td style="width:135px;">Dilute stock out to 0.05% in 1x PBS</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">Basic fibroblast growth factor</td>
			<td style="width:111px;">GlobalStem (Rockville, MD, USA)</td>
			<td style="width:136px;">GSR-2001</td>
			<td style="width:135px;">Reconstitute to 10 &#181;g/ml stock in 0.1% bovine serum albumin dissolved in 1x PBS and store at &#8722;80 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">&#946;-mercaptoethanol</td>
			<td style="width:111px;">Millipore (Billerica, MA, USA)</td>
			<td style="width:136px;">ES-007-E</td>
			<td></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">Matrigel (basement membrane matrix)</td>
			<td style="width:111px;">BD Biosciences (San Jose, CA, USA)</td>
			<td style="width:136px;">356231</td>
			<td style="width:135px;">Dilute stock Matrigel vial with 10 ml of DMEM/F12 while on ice for a 1:2 dilution. Aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">CELLstart (Synthetic Substrate)</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1014201</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Stemmolecule Y27632</td>
			<td style="width:111px;">Stemgent (Cambridge, MA, USA)</td>
			<td style="width:136px;">04-0012-02</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Puromycin</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1113802</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">LightCycler 480 Probes Master</td>
			<td style="width:111px;">Roche (Basel, Switzerland)</td>
			<td style="width:136px;">4707494001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">ProFreeze-CDM Medium/freezing medium</td>
			<td style="width:111px;">Lonza (Basel, Switzerland)</td>
			<td style="width:136px;">12-769E</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Dimethyl sulfoxide</td>
			<td style="width:111px;">Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td style="width:136px;">D8418</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">PBS</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">14190-250</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">100 BP DNA Ladder</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15628019</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">SYBR Safe DNA Gel Stain 10000x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">S33102</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="89">
			<td height="89" style="height:89px;width:140px;">Agarose</td>
			<td style="width:111px;">Bio-Rad Laboratories, Inc. (Hercules, CA, USA)</td>
			<td style="width:136px;">161-3101</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Gelatin, from porcine skin</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:136px;">G1890-100G</td>
			<td style="width:135px;">Make stock at 0.2% in PBS, autoclave and store at room temperature</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">mTeSR1</td>
			<td style="width:111px;">StemCell Technologies (Vancouver, BC, Canada)</td>
			<td style="width:136px;">5850</td>
			<td style="width:135px;">Combine Supplement 5X with the basic medium, aliquot and store at 4 &#176;C for up to 2 weeks.&#160;</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Stemedia NutriStem XF/FF Culture Medium</td>
			<td style="width:111px;">Stemgent</td>
			<td style="width:136px;">05-100-1A</td>
			<td style="width:135px;">Thaw at 4 &#176;C O/N, aliquot and store at 4 &#176;C for up to 2 weeks.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Primocin</td>
			<td style="width:111px;">InvivoGen (San Diego, CA, USA)</td>
			<td style="width:136px;">ant-pm-1</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Accutase (Dissociation Reagent)</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1110501</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Donkey anti-Chicken IgG AlexaFluor 488</td>
			<td style="width:111px;">Jackson ImmunoResearch (West Grove, PA, USA)</td>
			<td style="width:136px;">703-546-155</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Polybrene/transfection agent</td>
			<td style="width:111px;">Millipore</td>
			<td style="width:136px;">TR-1003-G</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Plasticware</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">12-well plates</td>
			<td style="width:111px;">VWR (West Chester, PA, USA)</td>
			<td style="width:136px;">29442-038</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">6-well plates</td>
			<td style="width:111px;">VWR</td>
			<td style="width:136px;">29442-042</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">10-cm plates</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:136px;">Z688819</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">18-gauge needle</td>
			<td style="width:111px;">Fisher Scientific (Pittsburgh, PA, USA)</td>
			<td style="width:136px;">148265D</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">21-gauge needle</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;">14-829-10D</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Equipment</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">BD LSRII Flow Cytometer</td>
			<td style="width:111px;">KSystem by Nikon (Tokyo, Japan)</td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">BD FACSDiva Version 6.1.3 Software</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Kits</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">PureLink Genomic DNA Mini Kit</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">K182000</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">KAPA HiFi Hotstart ReadyMix PCR Kit</td>
			<td style="width:111px;">KAPA Biosystems (Wilmington, MA, USA)</td>
			<td style="width:136px;">KK2601</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">High Pure RNA Isolation Kit</td>
			<td style="width:111px;">Roche</td>
			<td style="width:136px;">11828665001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:140px;">Transcriptor First Strand cDNA Synthesis Kit</td>
			<td style="width:111px;">Roche</td>
			<td style="width:136px;">4379012001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:140px;">Sialix anti-Neu5Gc Basic Pack Kit</td>
			<td style="width:111px;">Sialix (Newton, MA, USA)</td>
			<td style="width:136px;">Basic Pack</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:140px;">Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Combined media 1</td>
			<td style="width:111px;">StemCell Technologies and Stemgent</td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Consists of equal parts mTeSR1 and Nutristem</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Combined media 2</td>
			<td style="width:111px;">StemCell Technologies and Stemgent</td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Consists of equal parts TeSR2 and Nutristem</td>
		</tr>
		<tr height="190">
			<td height="190" style="height:190px;width:140px;">HUF Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Dulbecco&#8217;s modified Eagle&#8217;s medium/F12 [DMEM/F12] supplemented with 10% fetal bovine serum, 1x non-essential amino acids, 1x Glutamax, and 1x Primocin</td>
		</tr>
		<tr height="253">
			<td height="253" style="height:253px;width:140px;">Human Pluripotent Stem Cell Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;">DMEM/F12 supplemented with 20% knockout serum replacement, 1x Glutamax, 1x non-essential amino acids, 1x Primocin, 1x &#946;-mercaptoethanol, and 10 ng/ml basic fibroblast growth factor</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;">DMEM/F12, Dulbecco&#8217;s modified Eagle&#8217;s medium/F12; PBS, phosphate-buffered saline.</td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
	</tbody>
</table>

derivation and characterization of a transgene-free human induced pluripotent stem cell line and conversion into defined clinical-grade conditions

Las células madre pluripotentes inducidas humanas (hiPSCs) se pueden generar con las metodologías de reprogramación a base de lentivirus. Sin embargo, los rastros de genes potencialmente oncogénicos restantes en regiones activamente transcrito del genoma, limitan su potencial para su uso en aplicaciones terapéuticas humanas 1. Además, los antígenos no humanos derivados de la reprogramación de células madre o la diferenciación en derivados terapéuticamente relevantes se oponen a estas hiPSCs se utilicen en un contexto clínico humano 2. En este vídeo, se presenta un procedimiento para la reprogramación y analizar hiPSCs factoriales libre libres de transgenes exógenos. Estos hiPSCs se pueden analizar para detectar anomalías de expresión génica en el intrón específico que contiene el lentivirus. Este análisis puede llevarse a cabo utilizando la reacción en cadena de la polimerasa cuantitativa sensible (PCR), que tiene una ventaja sobre técnicas menos sensibles utilizado anteriormente para detectar diferencias de expresión génica 3. La conversión total enbuenas prácticas de fabricación de grado clínico (GMP), permite relevancia clínica humana. Nuestro protocolo ofrece otra metodología, siempre que los criterios actuales de salvaguarda ampliarán e incluir los derivados basados ​​en hiPSC caracterizados factoriales libre para aplicaciones fines terapéuticos humanos derivadas hiPSCs GMP-grado, que debe eliminar cualquier riesgo inmunogenicidad debido a antígenos no humanos. Este protocolo es ampliamente aplicable a lentiviral reprogramado células de cualquier tipo y proporciona un método reproducible para la conversión de células reprogramadas en condiciones GMP grado.

Se presenta un protocolo para derivar hiPSCs factor libre de grado clínico utilizando el enfoque basado reprogramación lentiviral-STEMCCA. La figura 1A muestra una imagen representativa de tres diferentes líneas hiPSC previamente extirpada, después de la reprogramación con el enfoque STEMCCA sobre una capa de MEFs. La principal ventaja del enfoque de reprogramación STEMCCA radica en el éxito reprogramación consistente alcanzado por múltiples científicos, en diferentes grupos y las ubicaciones ...

Watch this Scientific Journal Video about Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions at JoVE.com

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

Derivación y caracterización de un transgen libre Humano madre pluripotentes inducidas Línea Celular y Conversión en condiciones definidas de calidad clínica

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

derivation-characterization-transgene-free-human-induced-pluripotent

Research

JoVE Journal

Biology

7.8K vistas.  University of California, Los Angeles (UCLA). We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions. 

Video: Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivación de células estaminales embrionarias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Investigación

Biología

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

El aislamiento y la derivación de células embrionarias de ratón Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 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 ...

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 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 ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

Alta Eficiencia Diferenciación de células madre pluripotentes humanas a cardiomiocitos y Caracterización por citometría de flujo

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

Extracción, etiquetado y purificación de células específicas del linaje de folículos antrales humanos

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...