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 trabalho foi apoiado por uma bolsa para pesquisadores de melanoma do M. Nicolay Fundação Joanna e pelos Institutos Nacionais de Saúde sob Ruth Service Award L. Kirschstein National Research F31. Este trabalho foi ainda apoiada por meio de doações de NYSTEM ea iniciativa de células estaminais Tri-institucional (Starr Foundation).

NOTA: O protocolo de melanócitos descrito aqui foi demonstrada pela primeira vez por Mica et al. 1. Preparação de meio de cultura, revestido Pratos e manutenção de hPSCs <ol><li> médio Preparação Nota: Armazenar todas meio a 4 ° C no escuro, durante até 2 semanas. Filtrar todo o meio para esterilização. <ol><li> Prepare DMEM / FBS a 10%. Misturar 885 mL de DMEM, 100 ml de FBS, 10 ml de Pen / Strep e 5 ml de L-Glutamina. Filtro para esterilização. </li><li> Prepare hESC...

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Para a diferenciação de melanócitos bem sucedida de hPSCs as seguintes sugestões deve ser tomado em consideração. Em primeiro lugar, é essencial para trabalhar sob condições de cultura estéreis em todos os momentos. Além disso, é importante para iniciar com pluripotentes, hPSCs totalmente indiferenciadas; se a população começando contém células diferenciadas o rendimento será invariavelmente cair como os contaminantes não pode ser dirigida para melanócitos e pode mesmo perturbar ainda mais as célula...

Células-tronco pluripotentes humanas (hPSCs) fornecer uma plataforma para imitar diferenciação normal em uma forma escalável para modelagem de doença, o rastreio de drogas e terapias de substituição celular 1-6. De particular interesse, hPSCs abrir caminhos para o estudo difícil isolar ou tipos de células raras / transitórios, onde amostras de pacientes são escassos. Células-tronco pluripotentes Além disso, induzidas (iPSCs) permitir aos investigadores estudar o desenvolvimento e modelagem de doença de uma maneira específica paciente para desvendar os mecanismos exclusivos 1,2,7-11. O protocolo previamente publicado para a diferenciação dos melanócitos a partir hPSCs requer até 6 semanas de diferenciações e envolve a cultura de células com meio condicionado de células L-12 Wnt3a. O protocolo apresentado pela primeira vez por Mica et al. E descrito aqui produz células pigmentadas em três semanas e remove a ambiguidade e inconsistências associados com meio condicionado. ar melanócitose derivados da crista neural, uma população de células migratórias únicas para os vertebrados. A crista neural é definido durante a gastrulação e representa uma população de células na borda da placa neural, na fronteira entre a ectoderme neural e não neurais. Durante neurulação, o tecido nervoso se desenvolve a partir de uma placa neural para formar dobras neurais, que convergem na linha média dorsal, resultando no tubo neural 13,14. As células da crista neural emergir da placa de tecto do tubo neural, em frente da notocorda, e submetido a uma epitelial para mesenquimal antes de migrar para longe para dar origem a uma população diversa de células diferenciadas. O destino das células da crista são definidas em parte por a localização anatómica da placa para telhado, ao longo do eixo do corpo do embrião. derivados de células da crista neural incluem linhagens característicos de ambos mesoderme (células do músculo liso, os osteoblastos, os adipócitos, condrócitos e células da ectoderme) (melanócitos, C Schwannells, neurônios) 14. células estaminais da crista neural regulam positivamente o factor de transcrição SOX10 e pode ser isolado por meio de células activada por fluorescência, com anticorpos para p75 e HNK1. As células da crista neural fadado a tornar-se melanócitos passar por uma fase melanoblasto e upregulate KIT e MITF (fator de transcrição associada à microftalmia) 6,21 MITF é um regulador mestre do desenvolvimento de melanócitos e é um fator de transcrição responsável por controlar grande parte do desenvolvimento de melanócitos 22- 24. melanoblastos humanos migram para a camada basal da epiderme, onde residem quer no bojo cabelo ou rodeado pelos queratinócitos na epiderme (unidades de formação de pigmentação) para servir como precursores para os melanócitos maduros, pigmentadas. A diferenciação e maturação de melanoblastos em melanócitos pigmentados ocorre concomitante com a colonização do bulbo capilar e expressão da via de produção de melanina (TYRP1, Tyr, e OCA2PMEL) 25,26. Isolamento de melanócitos humanos e melanoblastos de pacientes é cara, difícil e limitando a quantidade. Este protocolo permite aos pesquisadores para diferenciar hPSCs (induzido ou embrionária) em melanócitos ou precursores de melanócitos em um método bem definido, rápida, reprodutível, escalável e de baixo custo, sem separação de células. O protocolo foi usado anteriormente para identificar defeitos de doenças específicas ao diferenciar iPSCs de pacientes com distúrbios de pigmentação.

<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 fornece um método para derivar melanócitos maduros, totalmente pigmentadas de hPSCs em um livre-alimentador, eficiente, e reprodutível modo in vitro. Em contraste com o fang et ai. Protocolo previamente estabelecido para os melanócitos HPSC-derivados, o protocolo descrito não requer meio condicionado e reduz a exigência de tempo. O protocolo fang et al. Utilizaram meio condicionado de uma linha celular murino de produção de Wnt3a e levou ...

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

Derivação sem alimentador de melanócitos a partir de células-tronco pluripotentes humanas

Este trabalho descreve um protocolo in vitro de diferenciação para produzir pigmentadas, melanócitos maduros a partir de células-tronco pluripotentes humanas através de uma crista neural e melanoblasto estágio intermediário usando um protocolo sem alimentador de 25 dias.

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

Derivação de Cardiac células progenitoras de células estaminais embrionárias

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

Biologia do Desenvolvimento

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Derivação eficiente do epitélio pigmentar da retina células de células-tronco

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

Derivação de Cardiomyocytes altamente purificada de induzidas pelo homem pluripotentes células estaminais usando pequenos Diferenciação modulada-Molecule e Subsequente Glucose Fome

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

A quantificação tridimensional das espinhas dendríticas de Piramidal neurônios derivados pluripotentes induzidas pelo homem Células-Tronco

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

Geração de pluripotentes induzidas a partir de células-tronco periférico humano T células usando Sendai Virus em condições livres de Alimentador

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 e Geração de Scalable de células de hepatócitos-like de células-tronco pluripotentes humanas

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

Condrócitos diferenciadores de células-tronco pluripotentes induzidas por humanos derivadas de sangue periférico

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

Derivação livre de integração de células-tronco pluripotentes induzidas por seres humanos com 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

Diferenciação eficiente de células-tronco pluripotentes humanas em 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

Na geração de Vitro do Somite derivados de humanos induzida por células tronco pluripotentes

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