Name: feeder-free derivation of melanocytes from human pluripotent stem cells
Uploaded: 2016-03-03T15:00:00
Description: Watch this Scientific Journal Video about Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells at JoVE.com

Este trabajo fue apoyado por una beca de investigación para los investigadores del melanoma de la Fundación Joanna M. Nicolay y por los Institutos Nacionales de Salud en virtud F31 Ruth L. Kirschstein Premio al Servicio Nacional de Investigación. Este trabajo fue apoyado además por subvenciones del NYSTEM y la iniciativa de células madre Tri-institucional (Fundación Starr).

NOTA: El protocolo de melanocitos esbozado aquí se demostró por primera vez por Mica et al. 1. Preparación del medio de cultivo, recubierto Platos y Mantenimiento de hPSCs <ol><li> Preparación medio Nota: tienda de todo medio a 4 ° C en la oscuridad durante un máximo de 2 semanas. Filtrar todo el medio para la esterilización. <ol><li> Preparar DMEM / 10% de FBS. Mezclar 885 ml de DMEM, FBS 100 ml, 10 ml de penicilina / estreptomicina y 5 ml de L-Glutamina. Filtro para la este...

<ol>
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B. C., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogrammed+Cells+for+Disease+Modeling+and+Regenerative+Medicine.">Reprogrammed Cells for Disease Modeling and Regenerative Medicine.</a> Annu Rev Med. 64, 277-290 (2013).</li><li>Inoue, H., Nagata, N., Kurokawa, H., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPS+cells:+a+game+changer+for+future+medicine.">iPS cells: a game changer for future medicine.</a> Embo J. 33, 409-417 (2014).</li><li>Kim, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+arrhythmogenic+right+ventricular+dysplasia+with+patient-specific+iPSCs.">Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs.</a> Nature. 494, 105-110 (2013).</li><li>Fang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+conditions+for+the+generation+of+melanocytes+from+human+embryonic+stem+cells.">Defining the conditions for the generation of melanocytes from human embryonic stem cells.</a> Stem cells. 24, 1668-1677 (2006).</li><li>Huang, X., Saint-Jeannet, J. 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Para la diferenciación de los melanocitos éxito de hPSCs las siguientes sugerencias deben ser tomados en consideración. Primero y ante todo, es esencial para trabajar en condiciones de cultivo estériles en todo momento. Además, es importante comenzar con pluripotentes, hPSCs totalmente indiferenciadas; si la población de partida contiene células diferenciadas el rendimiento invariablemente dejar que los contaminantes no pueden ser dirigidas hacia los melanocitos e incluso pueden perturbar aún más las células a...

Las células madre pluripotentes humanas (hPSCs) proporcionan una plataforma para imitar diferenciación normal de una manera escalable para el modelado de la enfermedad, la detección de drogas y terapias de reemplazo celular 1-6. De particular interés, hPSCs abren nuevas vías para el estudio difícil de aislar o tipos de células raras / transitorios en los cuales las muestras de pacientes son escasos. Las células madre pluripotentes Además, inducidas (células iPS) permiten a los investigadores para estudiar el desarrollo y modelado de la enfermedad de una manera específica del paciente para desentrañar los mecanismos únicos 1,2,7-11. El protocolo previamente publicado para la diferenciación de melanocitos de hPSCs requiere hasta 6 semanas de diferenciaciones e implica el cultivo de células con medio condicionado de células L-Wnt3a 12. El protocolo presentado primero por Mica et al. Y descrito aquí produce células pigmentadas en tres semanas y elimina la ambigüedad e inconsistencias asociadas con medio condicionado. ar melanocitose derivado de la cresta neural, una población migratorio de las células únicas de los vertebrados. La cresta neural se define durante la gastrulación y representa una población de células en el borde de la placa neural, en la frontera entre el neural y ectodermo no neural. Durante tubo neural, el tejido nervioso se desarrolla a partir de una placa neural para formar pliegues neurales, que convergen en la línea media dorsal, dando como resultado el 13,14 tubo neural. Las células de la cresta neural emergen de la placa de techo del tubo neural, frente a la notocorda, y se someten a una transición epitelial a mesenquimal antes de migrar de distancia para dar lugar a una población diversa de células diferenciadas. El destino de las células de la cresta se definen en parte por la localización anatómica de la placa de techo a lo largo del eje del cuerpo del embrión. derivados de células de la cresta neural incluyen linajes característicos tanto de mesodermo (células de músculo liso, osteoblastos, adipocitos, condrocitos) y las células del ectodermo (melanocitos, Schwann cells, neuronas) 14. las células madre de la cresta neural upregulate el factor de transcripción SOX10 y se pueden aislar por fluorescencia de células activadas por la clasificación con anticuerpos frente a p75 y HNK1. Las células de la cresta neural predestinadas a convertirse en melanocitos pasan por una etapa melanoblasto y regular al alza KIT y MITF (factor de transcripción asociado con microftalmia) 6,21 MITF es un regulador maestro de desarrollo de melanocitos y es un factor de transcripción responsable de controlar gran parte del desarrollo de melanocitos 22- 24. melanoblastos humanos migran a la capa basal de la epidermis donde residen ya sea en el bulto de pelo o rodeado por los queratinocitos en la epidermis (unidades formadoras de pigmentación) para servir como precursores de los melanocitos maduros, pigmentadas. La diferenciación y la maduración de melanoblastos en melanocitos pigmentados se produce concomitante con la colonización del bulbo piloso y la expresión de la vía de la producción de melanina (TYRP1, TYR, OCA2 yPMEL) 25,26. El aislamiento de melanocitos humanos y melanoblastos de pacientes es caro, difícil y limitando en cantidad. Este protocolo permite a los investigadores para diferenciar hPSCs (inducido o embrionaria) en los melanocitos o precursores de melanocitos en un método bien definido, rápido, reproducible, escalable y de bajo costo y sin la clasificación de células. El protocolo fue utilizado anteriormente para identificar defectos específicos de la enfermedad, cuando la diferenciación de células iPS de pacientes con trastornos de la pigmentación.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>apo human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid (L-AA)</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>B27 (B27 Supplement)</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco-Life Technologies</td>
			<td>21985-023</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-bp</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>4423</td>
			<td>6 mM</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>50 mg/ml</td>
		</tr>
		<tr>
			<td>cAMP (cyclicAMP)</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D2915-100MG</td>
			<td>50 &#956;M</td>
		</tr>
		<tr>
			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco-Life Technologies</td>
			<td>11985-092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 - Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12</td>
			<td>Gibco-Life Technologies</td>
			<td>1133--032</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 powder</td>
			<td>Invitrogen</td>
			<td>12500-096</td>
			<td></td>
		</tr>
		<tr>
			<td>EDN3 (Endothelin-3, human)</td>
			<td>American Peptide Company</td>
			<td>88-5-10B</td>
			<td>100 &#956;M</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>356008</td>
			<td>200 &#956;g/ml</td>
		</tr>
		<tr>
			<td>gelatin (PBS without Mg/Ca)</td>
			<td>in house</td>
			<td></td>
			<td>0.1% in PBS</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Human insulin</td>
			<td>Sigma</td>
			<td>I2643</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS+ Universal Culture Supplement Premix&#160;</td>
			<td>BD Biosciences</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>KSR (Knockout Serum Replacement)</td>
			<td>Gibco-Life Technologies</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco-Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>Invitrogen</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Dissolve 1:20 in DMEM/F12</td>
		</tr>
		<tr>
			<td>MCDB201 Medium</td>
			<td>Sigma</td>
			<td>M6770</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM minimum essential amino acids solution</td>
			<td>Gibco-Life Technologies</td>
			<td>11140-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblasts (7 million cells/vial)</td>
			<td>GlobalStem</td>
			<td>GSC-8105M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Laminin-I</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Poly-L Omithin hydrobromide</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td>15 mg/ml&#160;</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>0.032g in 100ml 100% ethanol</td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2 (Recombinant human FGF basic)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-001MG/CF</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1814</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>SCF (Stem Cell Factor, recombinant Human)&#160;</td>
			<td>Peprotech Inc.</td>
			<td>300-07</td>
			<td>50 &#956;g/ml</td>
		</tr>
		<tr>
			<td>TYRP1 (G-17) Antibody</td>
			<td>Santa Cruz</td>
			<td>10443</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>TYRP2 Antibody</td>
			<td>Abcam</td>
			<td>74073</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Gibco-Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1254</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>

feeder-free derivation of melanocytes from human pluripotent stem cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

Este protocolo proporciona un método para derivar, melanocitos maduros completamente pigmentadas de hPSCs en una manera libre de alimentador, costo eficiente y reproducible in vitro. En contraste con los Fang protocolo previamente establecido et al. Para melanocitos derivados de HPSC, el protocolo descrito no requiere medio acondicionado y reduce el requisito de tiempo. El protocolo de Fang et al. Utilizada medio condicionado de una línea celular murina Wnt3a...

Watch this Scientific Journal Video about Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells at JoVE.com

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Derivación sin alimentador de melanocitos partir de células madre pluripotentes

Este trabajo describe un protocolo de diferenciación in vitro para producir, melanocitos maduros pigmentadas a partir de células madre pluripotentes humanas a través de una cresta neural y melanoblasto etapa intermedia usando un protocolo sin alimentador de 25 días.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Research

JoVE Journal

Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Derivación de células cardiacas progenitoras a partir de células madre embrionarias

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Investigación

Biología del Desarrollo

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Derivación eficiente de células de la retina epitelio pigmentario de Células Madre

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Derivación de cardiomiocitos altamente purificada de células madre humanas pluripotentes inducidas Uso Pequeño Diferenciación molécula modulada y posterior glucosa inanición

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

La cuantificación tridimensional de espinas dendríticas de piramidal neuronas derivadas de células madre humanas pluripotentes inducidas

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Generación de pluripotentes inducidas células madre a partir de células T periféricas Humanos Uso de Sendai virus en Condiciones-Feeder gratuita

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Definido y escalable Generación de células hepatocitos como las de las células madre pluripotentes

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

Diferenciación de los condrocitos de las células madre pluripotentes humanas inducidas por la sangre periféricas derivadas de la sangre

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Integración Derivación Gratuita de Células Madre Pluripotentes Inducidas Humanas Usando Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Diferenciación eficiente de células madre pluripotentes humanas en células hepáticas

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

En la generación de Vitro del Somite derivados humanos inducida por células madre pluripotentes

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...