Este trabajo fue financiado por la Escuela de Medicina de Yale y el Premio de Investigación en Salud Infantil de la Fundación Charles Hood.

1. Reprogramación por retrovirus Expresando factores de reprogramación <ol><li> Fibroblastos humanos se cultivaron en medio de fibroblastos (10% de FBS en DMEM con Pen / Strep). </li><li> Un día antes de la infección, la placa de 1x10 5 fibroblastos humanos en un pocillo de una placa de 6 pocillos. </li><li> Aspirar el medio para eliminar las células muertas y añadir 2 ml de medio de fibroblastos fresco. Añadir sulfato de protamina a una concentración final de 5 mg / ml. </li><li> Añadir cuidadosamente la cantidad apropiada de cada virus que expresan GFP correspondiente a una multiplicidad de infección (MOI) 5 5. </li><li> Un día después de la infección, separar el sobrenadante viral, lavar tres veces con 2 ml de PBS, a continuación, agregar 2 ml de medio de fibroblastos. </li><li> Tres días después de la infección, consulte la fluorescencia de GFP y reponer el bien con 2 ml de medio de fibroblastos. </li><li> Cuatro días después de la infección, la placa de 1x10 4 / cm 2 de fibroblastos de embriones de ratón irradiados (MEFs) células alimentadoras en medio de fibroblastos sobre una10-cm placa de Petri revestidas con gelatina al 0,1%. Incubar a 37 ° C durante la noche. </li><li> Cinco días después de la infección, separar los fibroblastos humanos infectados con 1 ml de 0,05% typsin / EDTA durante 5 minutos a 37 ° C, y se centrifuga durante 5 minutos a 200 g. Aspirar el medio y resuspender las células con 10 ml de medio de fibroblastos. Transferir las células en un pre-recubiertas 10-cm placa. </li><li> Después de 24 horas, vuelva a colocar el medio con un medio de hESC la cultura (20% Knock-Out reemplazo de suero, DMEM/F12, 0,1 ml no aminoácidos esenciales, de 4 ng / ml de bFGF, Pen / Strep / glutamato, el beta-merceptoethanol). Cambiar el medio día. ESC-como colonias comenzarán a aparecer durante el día 20-27 después de la infección. </li></ol> 2. El aislamiento y la expansión de iPSCs <ol><li> Bajo un microscopio de fluorescencia, comprobar la ausencia de la fluorescencia de GFP en una colonia que muestra una morfología similar a hESCs. </li><li> Con una pipeta 10 l, escoja colonias individuales IPSC y colocarlos en un pocillo de una gelatina y el MEF-CoATed de 12 pocillos y se completará con medio de hESC. Cambiar medio diario. </li><li> Para los pases, se lava la placa con 1 ml de DMEM/F12, a continuación, agregar 0,5 ml de colagenasa, y se incuba durante 10 minutos a 37 ° C. </li><li> Se lavan las células dos veces con DMEM/F12. </li><li> Añadir 2 ml de medio de hESC fresco. El uso de un elevador de la célula, romper las colonias en pequeños pedazos y separar las células restantes de la placa. </li><li> Transferir los trozos de colonias resuspendidas en un pocillo de una gelatina y MEF recubierto-6-así placa. </li></ol> 3. El análisis de inmunofluorescencia de marcadores pluripotentes <ol><li> Se lavan las células tres veces con PBS y fijar con paraformaldehído al 4% durante 20 min a temperatura ambiente. </li><li> Lave suavemente las células tres veces con PBS y permeabilizar con 0,2% de Triton X-100 en PBS durante 30 min. </li><li> Bloque no específicos de unión mediante la incubación de células con 3% de BSA en PBS durante dos horas. </li><li> Se incuban las células con el anticuerpo primario durante la noche a 4 ° C. </li> &lt;li&gt; Se lavan las células tres veces con PBS y se incuban las células con anticuerpo secundario específico durante una hora a temperatura ambiente, protegiendo de la luz. </li><li> Se lavan las células tres veces con PBS y DAPI complemento durante el último lavado, seguido de incubación a temperatura ambiente durante 5 minutos. </li><li> Detectar la tinción con un microscopio de fluorescencia. </li></ol> 4. Cuantitativo en tiempo real PCR para los marcadores pluripotentes <ol><li> Aislar ARN total de las iPSCs humanos derivados de fibroblastos humanos utilizando RNeasy kit de Qiagen. </li><li> Sintetizar el cDNA primer capítulo de la transcriptasa reversa utilizando SuperScript II. </li><li> Realizar qPCR para la detección de genes usando cebadores pluripotencia se informó anteriormente. 6 </li></ol> 5. Los resultados representativos <ol><li> El cambio morfológico durante la reprogramación Estamos infectados fibroblastos humanos BJ1 y Detroit 551 con un cóctel de los retrovirus que llevan Oct4, Sox2, Klf4 y MYC,y fueron capaces de detectar cambios morfológicos durante la reprogramación (Figura 1). Veintiún días después de la infección, reconocemos pequeñas colonias de IPSC por su hESC-como la morfología. Además, reconocemos iPSCs por la fluorescencia de GFP. Las células madre pluripotentes, como los CES e IPSCs, expresan la maquinaria molecular para reprimir la expresión del gen proviral 7-9. Nuestro único vector retroviral expresa GFP junto con los genes reprogramación por LTR retroviral. Así, las células que expresan GFP de forma continua se consideran para expresar transgenes, sin silenciamiento génico proviral. Fielmente reprogramadas colonias IPSC que adquieren la red molecular pluripotencia muestran la ausencia de expresión de las buenas prácticas agrarias (Figura 2) 10. </li><li> Caracterización de la pluripotencia de iPSCs humanos Se analizaron colonias derivadas de Detroit-551 fibroblastos a través de inmunohistoquímica con Tra-1-81, Tra-1-60, SSEA-4, 3-SSEA, Oct4 y anticuerpos NANOG ( <strong&gt; Tabla 2). IPSCs con éxito reprogramadas expresar todos estos marcadores (Figura 3). También se analizó la expresión génica a través de RT-PCR cuantitativa análisis. Hemos observado que la expresión de Oct4, Sox2, Klf4, MYC y NANOG se incrementó significativamente en comparación con las células de fibroblastos de los padres, sino a la de las H9 hESCs (Figura 3B). </li></ol><img alt="Figura 1" src="/files/ftp_upload/3804/3804fig1.jpg" /> Figura 1. Los cambios morfológicos de los infectados por retrovirus fibroblastos humanos. (AD), el cambio progresivo morfológica en las colonias de Detroit-551 fibroblastos infectados con factores de reprogramación. Día 5 (A), el día 10 (B), el día 14 (C), el día 21 (D). Las células muestran la morfología hESC-al igual que después de 21 días. <img alt="Figura 2" src="/files/ftp_upload/3804/3804fig2.jpg" /> Figura 2. Representante de las buenas prácticas agrarias fluorescente de expresión en las células sometidas a una reprogramación. </strong&gt; BJ y Detroit 551 fibroblastos fueron infectadas con retrovirus que expresa cuatro factores de reprogramación 4, y se incubaron en medio hCME durante cuatro semanas. Fibroblastos BJ (A, B) y Detroit 551 (C, D) muestran similares morfológica. Desde el día 21, las colonias de las buenas prácticas agrarias negativos comienzan a formarse, que representan las iPSCs de buena fe 10. (E, F) muestran transformado Detroit 551 células que no se han sometido a una reprogramación adecuada. (A, C, E) colonias bajo punto de vista de contraste de fase. (B, D) adecuadamente reprogramado las células que muestran el silenciamiento de las buenas prácticas agrarias. (F) la expresión de GFP luminosa de la colonia transformada. <img alt="Figura 3" src="/files/ftp_upload/3804/3804fig3.jpg" /> Figura 3. Caracterización de las células madre pluripotentes inducidas (A). Humanos 551-iPS-K1 colonias de células expresan marcadores comunes a las células pluripotentes. DAPI indica el contenido total de células por campo. (B) cuantitativa en tiempo real PCR (RT-qPCR) para la expresión de Oct4, Sox2, Klf4, MYC en f los padresibroblast, 551-IPS-K1 iPSCs y H9 células madre embrionarias humanas (hESCs). Los datos se normalizaron contra limpieza de genes β-actina y trazan en relación con el nivel de expresión en las células de fibroblastos parentales 4.

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<p class="jove_content"&gt; No tenemos nada que revelar.</p

Expresión de los cuatro factores de transcripción reprograma fibroblastos humanos a iPSCs. Muchos intentos se hicieron para generar iPSCs humanos utilizando los enfoques no integrar o no genéticos para generar iPSCs clínicamente seguros. Hasta ahora, estos métodos presentan una eficiencia extremadamente baja y requieren una mayor optimización para mejorar la reproducibilidad 11-14. Métodos de retro-o lentiviral son fácilmente utilizados para obtener y aplicar iPSCs para personas en modelos de...

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		</tr>
		<tr>
			<td colspan="4">hESC medium</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>80%</td>
			<td>Invitrogen</td>
			<td>11330057</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>20%</td>
			<td>Invitrogen</td>
			<td>10828-028</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>2 mM</td>
			<td>Invitrogen</td>
			<td>25030081</td>
		</tr>
		<tr>
			<td>Nonessential Amino Acids (10 mM)</td>
			<td>0.1 mM</td>
			<td>Invitrogen</td>
			<td>11140050</td>
		</tr>
		<tr>
			<td>&beta;-Mercaptoethanol (14.3 M) or MTG</td>
			<td>0.1 mM</td>
			<td>Invitrogen</td>
			<td>M-6250</td>
		</tr>
		<tr>
			<td>bFGF-2 10 &mu;g/ml</td>
			<td>4 ng/ml</td>
			<td>GIBCO/BRL</td>
			<td>GF003AF</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>1%</td>
			<td>Millipore</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td colspan="4">Fiboblasts Medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>90%</td>
			<td>Invitrogen</td>
			<td>11965118</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>10%</td>
			<td>Invitrogen</td>
			<td>10407028</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>1%</td>
			<td>Millipore</td>
			<td>15140-122</td>
		</tr>
	</tbody>
</table>
Table 1. Culture Medium
<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		</tr>
		<tr>
			<td colspan="4">Antibodies</td>
		</tr>
		<tr>
			<td>OCT4</td>
			<td>1:500</td>
			<td>Abcam</td>
			<td>Ab19857</td>
		</tr>
		<tr>
			<td>SSEA3</td>
			<td>1:100</td>
			<td>Milipore</td>
			<td>MAB4303</td>
		</tr>
		<tr>
			<td>SSEA4</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560218</td>
		</tr>
		<tr>
			<td>Tra-1-81</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560173</td>
		</tr>
		<tr>
			<td>Tra-1-60</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560174</td>
		</tr>
		<tr>
			<td>NANOG</td>
			<td>1:500</td>
			<td>Abcam</td>
			<td>Ab21624</td>
		</tr>
		<tr>
			<td>Alexa-Flur 488</td>
			<td>1:1000</td>
			<td>Invitrogen</td>
			<td>A11008</td>
		</tr>
		<tr>
			<td>Alexa-Flur 555</td>
			<td>1:1000</td>
			<td>Invitrogen</td>
			<td>A21422</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>1:5000</td>
			<td>Invitrogen</td>
			<td>D1306</td>
		</tr>
		<tr>
			<td colspan="4">Plasmids</td>
		</tr>
		<tr>
			<td>pMIG-OCT4</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17225</td>
		</tr>
		<tr>
			<td>pMIG-SOX2</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17226</td>
		</tr>
		<tr>
			<td>pMIG-KLF4</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17227</td>
		</tr>
		<tr>
			<td>pMIG-MYC</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>18119</td>
		</tr>
		<tr>
			<td colspan="4">Other Materials</td>
		</tr>
		<tr>
			<td>Collagenase type IV</td>
			<td>1mg/ml</td>
			<td>Invitrogen</td>
			<td>17104019</td>
		</tr>
		<tr>
			<td>Gelatin, Porcine</td>
			<td>0.1%</td>
			<td>Sigma</td>
			<td>G 1890</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>0.2%</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>4%</td>
			<td>Sigma</td>
			<td>47608</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>3%</td>
			<td>American Bioanalytical</td>
			<td>AB01800</td>
		</tr>
		<tr>
			<td>MEF feeder cells</td>
			<td>&nbsp;</td>
			<td>Millipore</td>
			<td>PMEF-N</td>
		</tr>
		<tr>
			<td>Cell Lifter</td>
			<td>&nbsp;</td>
			<td>Corning</td>
			<td>3008</td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td colspan="4">Fluorescent microscopy: inverted microscope with GFP filter</td>
		</tr>
	</tbody>
</table>
Table 2. Reagents and equipment.

reprogramming human somatic cells into induced pluripotent stem cells (ipscs) using retroviral vector with gfp

Las células madre embrionarias (hESCs) son pluripotentes y una fuente inestimable para celulares en el modelado de la enfermedad in vitro y la medicina regenerativa 1. Se ha demostrado previamente que las células somáticas humanas pueden ser reprogramadas para pluripotencia por la expresión ectópica de cuatro factores de transcripción (Oct4, Sox2, Klf4 y Myc) y se convierten en células madre pluripotentes inducidas (iPSCs) 2-4. Al igual que hESCs, iPSCs humanos son pluripotentes y una fuente potencial de células autólogas. A continuación se describe el protocolo de reprogramar las células de fibroblastos humanos con los cuatro factores de reprogramación clonados en las buenas prácticas agrarias que contienen columna vertebral de retrovirales 4. Utilizando el protocolo siguiente, generamos iPSCs humanos en 3-4 semanas bajo condiciones de la cultura humana ESC. Humanos colonias IPSC se parecen mucho a hESCs en la morfología y mostrar la pérdida de la fluorescencia de GFP como resultado de silenciamiento de transgenes retrovirales. colonias aisladas IPSC mecánicamente bajo una fluorescencia microscópicaPE se comportan de una manera similar como hESCs. En estas células, se detecta la expresión de múltiples genes pluripotencia y marcadores de superficie.

Watch this Scientific Journal Video about Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP at JoVE.com

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

Un método para generar células madre pluripotentes inducidas (iPSCs) a través de retrovirus mediada por la expresión ectópica de Oct4, Sox2, Klf4 y MYC se describe. Una forma práctica para identificar las colonias de humanos IPSC basadas en la expresión de GFP también se discute.

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 ...

reprogramming-human-somatic-cells-into-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

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

20.3K vistas.  Yale School of Medicine. Un método para generar células madre pluripotentes inducidas (iPSCs) a través de retrovirus mediada por la expresión ectópica de Oct4, Sox2, Klf4 y MYC se describe. Una forma práctica para identificar las colonias de humanos IPSC basadas en la expresión de GFP también se discute.

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

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

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

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

Generación de células madre pluripotentes inducidas por reprogramación de fibroblastos embrionarios de ratón con un factor de transcripción Cuatro, doxiciclina inducible sistema de transducción de lentivirales

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

Investigación

Biología

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 ...

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 ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Preparación de las células embrionarias de fibroblastos de ratón adecuados para el cultivo de células estaminales embrionarias humanas pluripotentes inducidas, y

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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, ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

En Reprogramación vivo de células somáticas de adultos a Pluripotencia por la sobreexpresión de factores de Yamanaka

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

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 ...

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.

Reprogramación episomal de fibroblastos dérmicos para generar células madre pluripotentes inducidas por humanos (hiPSCs)

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.

Generación y mantenimiento de células madre pluripotentes inducidas por primates derivadas de la orina

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular ...

Caracterización de los antígenos de superficie y la capacidad de proliferación en las MSC que impulsan hiPSC

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

Autor destacado: Avances en iPSCs e investigación de enfermedades genéticas 

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

Comparación de dos métodos representativos para la diferenciación de células madre pluripotentes inducidas humanas en células estromales mesenquimales

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...