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

Ce travail a été soutenu par une bourse pour les chercheurs de mélanome de la Fondation M. Joanna Nicolay et par les Instituts nationaux de la santé en vertu de Ruth L. Award Research Service national Kirschstein F31. Ce travail a également été soutenu par des subventions de NYSTEM et l&#39;initiative sur les cellules souches Tri-institutionnelle (Starr Foundation).

NOTE: Le protocole de mélanocytes décrit ici a été démontrée par Mica et al. 1. Vaisselle Préparation du milieu de culture, enduit et entretien de hPSCs <ol><li> moyen Préparation Remarque: Conserver tout milieu à 4 ° C dans l&#39;obscurité pendant 2 semaines. Filtrez tous les moyen de stérilisation. <ol><li> Préparer DMEM / 10% de FBS. Mélanger 885 ml de DMEM, 100 ml de FBS, 10 ml Pen / Strep et 5 ml L-glutamine. Filtre pour la stérilisation. </li><li> Préparer CSEh...

<ol>
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Pour la différenciation réussie de mélanocytes de hPSCs les suggestions suivantes devraient être prises en considération. Tout d&#39;abord, il est essentiel de travailler dans des conditions de culture stériles en tout temps. En outre, il est important de commencer par pluripotentes, hPSCs complètement indifférenciées; si la population de départ contient des cellules différenciées, le rendement sera invariablement tomber que les contaminants ne peuvent pas être dirigées vers mélanocytes et peuvent encore ...

Les cellules souches pluripotentes humaines (hPSCs) fournissent une plate - forme pour imiter la différenciation normale de façon évolutive pour la modélisation de la maladie, le dépistage des drogues, et les thérapies de remplacement cellulaire 1-6. D&#39;un intérêt particulier, hPSCs ouvrent des pistes pour l&#39;étude difficile d&#39;isoler ou de types rares / transitoires cellulaires où les échantillons des patients sont rares. Les cellules souches pluripotentes En outre, induites (CSPi) permettent aux chercheurs d&#39;étudier le développement et la modélisation de la maladie d&#39;une manière spécifique au patient de démêler les mécanismes uniques 1,2,7-11. Le protocole précédemment publié pour la différenciation des mélanocytes de hPSCs nécessite jusqu&#39;à 6 semaines de différenciations et implique la culture de cellules avec un milieu conditionné à partir de cellules L-Wnt3a 12. Le premier protocole présenté par Mica et al. Et décrit ici produit des cellules pigmentées en trois semaines et supprime l&#39;ambiguïté et les incohérences associées à un milieu conditionné. mélanocytes are dérivé de la crête neurale, une population migratoire des cellules uniques aux vertébrés. La crête neurale est définie lors de la gastrulation et représente une population de cellules sur le bord de la plaque neurale, en bordure entre le neuronal et ectoderme non-neural. Au cours de la neurulation, le tissu nerveux évolue à partir d&#39; une plaque de neurones pour former des plis neuraux, qui convergent vers la ligne médiane dorsale entraînant la 13,14 du tube neural. Les cellules de la crête neurale émergent de la plaque de toit du tube neural, opposée à la notochorde et subissent une transition épithélio-mésenchymateuse avant de migrer loin de donner lieu à une population diverse de cellules différenciées. Le sort des cellules de la crête sont définies en partie par la localisation anatomique de la plaque de toit le long de l&#39;axe du corps de l&#39;embryon. les dérivés de cellules neurales comprennent des crêtes caractéristiques des deux lignées mésoderme (cellules musculaires lisses, les ostéoblastes, les adipocytes, les chondrocytes) et les cellules ectodermiques (mélanocytes, c Schwannaunes, neurones) 14. Les cellules souches de la crête neurale régulation positive du facteur de transcription SOX10 et peuvent être isolés par des cellules activées par fluorescence de tri avec des anticorps dirigés contre p75 et HNK1. Les cellules de la crête neurale Fated pour devenir mélanocytes passent par un stade de mélanoblaste et régulent positivement KIT et MITF (microphtalmie associée facteur de transcription) 6,21 MITF est un régulateur maître du développement des mélanocytes et est un facteur de transcription responsable du contrôle beaucoup du développement des mélanocytes 22- 24. mélanoblastes humaines migrent vers la couche basale de l&#39;épiderme où ils résident soit dans le renflement de cheveux ou entouré par les kératinocytes de l&#39;épiderme (unités formant pigmentaires) pour servir de précurseurs des mélanocytes, pigmentées matures. La différenciation et la maturation des mélanoblastes dans les melanocytes pigmentés se produit concomitant avec la colonisation du bulbe pileux et l&#39;expression de la filière de production de mélanine (TYRP1, TYR, et OCA2PMEL) 25,26. Isoler mélanocytes humains et mélanoblastes des patients est coûteux, difficile et de limiter en quantité. Ce protocole permet aux chercheurs de différencier hPSCs (induite ou embryonnaire) dans les mélanocytes ou des précurseurs de mélanocytes dans un procédé rapide, reproductible, évolutive et économique bien défini sans tri cellulaire. Le protocole a été précédemment utilisé pour identifier des défauts spécifiques de la maladie lors de la différenciation iPSCs des patients atteints de troubles de la pigmentation.

<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.

Ce protocole fournit un procédé pour dériver, les mélanocytes matures totalement pigmentées à partir hPSCs d&#39;une manière in vitro libre dispositif d&#39; alimentation, rentable et reproductible. Par contraste avec les Fang et al. Protocole précédemment établie pour les mélanocytes HPSC dérivés, le protocole décrit ne nécessite pas de milieu conditionné et diminue l&#39;exigence de temps. Le protocole Fang et al. , Utilise un milieu condition...

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

Dérivation libre Feeder-de mélanocytes de pluripotentes humaines Cellules souches

Ce travail décrit un protocole de différenciation in vitro pour produire pigmentées, mélanocytes matures à partir de cellules souches pluripotentes humaines par une crête neurale et mélanoblaste étape intermédiaire en utilisant un protocole de 25 jours sans alimentation.

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

Dérivation des cellules cardiaques progénitrices de cellules souches embryonnaires

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

Recherche

Biologie du développement

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Dérivation efficace des cellules d&#39;épithélium pigmentaire de la rétine à partir de cellules souches

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

Dérivation des cardiomyocytes hautement purifiée de anthropiques cellules souches pluripotentes en utilisant de petits différenciation de Molecule-modulé et ultérieur glucose famine

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

Quantification tridimensionnelle d&#39;épines dendritiques de Pyramidal neurones humains induits Dérivé de cellules souches pluripotentes

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

Génération de cellules souches pluripotentes induites à partir de cellules T périphériques humains à l&#39;aide de virus Sendai dans des conditions libres Feeder-

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

Définition et génération évolutive des cellules hépatocytes-like de pluripotentes humaines Cellules souches

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

Différenciation des chondrocytes provenant des cellules souches pluripotentes induites par le sang périphérique dérivé du sang

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

Intégration de la dérivation libre de cellules souches pluripotentes induites par l&#39;homme à l&#39;aide de 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

Différenciation efficace des cellules souches pluripotentes humaines en cellules hépatiques

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

Dans Vitro génération du Somite dérivés de Human induit Pluripotent Stem cellules

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