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

Diese Arbeit wurde durch ein Stipendium für Melanom-Forscher von der Joanna M. Nicolay-Stiftung und von den National Institutes of Health unter Ruth L. Kirschstein National Research Service Award F31 unterstützt. Diese Arbeit wurde weiter durch Zuschüsse aus NYSTEM und der Tri-institutionellen Stammzellen Initiative (Starr Foundation) unterstützt.

HINWEIS: Die Melanozyten-Protokoll hier skizziert wurde zuerst von Mica et al. 1. Herstellung von Kulturmedium, beschichtete Platten und Wartung von hPSCs <ol><li> Medium Vorbereitung Hinweis: Speichern Sie alle Medium bei 4 ° C im Dunkeln für bis zu 2 Wochen. Filter alle Medium für die Sterilisation. <ol><li> Bereiten Sie DMEM / 10% FBS. Mischen Sie 885 ml DMEM, 100 ml FBS, 10 ml Pen / Strep und 5 ml L-Glutamin. Filter für die Sterilisation. </li><li> Bereiten Sie hES-Medium. M...

<ol>
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Für die erfolgreiche Differenzierung von Melanozyten aus hPSCs sollten die folgenden Vorschläge in Betracht gezogen werden. In erster Linie ist es wichtig, zu allen Zeiten unter sterilen Kulturbedingungen zu arbeiten. Zusätzlich ist es wichtig, mit pluripotenten zu beginnen, vollständig undifferenzierte hPSCs; wenn die Ausgangspopulation differenzierten Zellen enthält, wird die Ausbeute immer fallen, da die Verunreinigungen nicht in Richtung Melanozyten gerichtet werden und die richtig differenzierenden Zellen noch...

Humanen pluripotenten Stammzellen (hPSCs) bieten eine Plattform normale Differenzierung in einer skalierbaren Art und Weise für Krankheitsmodelle, Drogen - Screening zu imitieren, und Zell - Ersatz - Therapien 1-6. Von besonderem Interesse, öffnen hPSCs Wege bis zur Untersuchung von schwer zu isolieren oder seltene / transient Zelltypen, bei denen Patientenproben knapp sind. Darüber hinaus induzierte pluripotente Stammzellen (iPS - Zellen) können die Forscher die Entwicklung und Krankheitsmodelle bei einem Patienten bestimmte Art und Weise zu studieren 1,2,7-11 einzigartige Mechanismen zu entwirren. Die bisher veröffentlichten Protokoll zur Differenzierung von Melanozyten von hPSCs erfordert bis 6 Wochen von Differenzierungen und werden Zellen mit konditioniertem Medium von L-Wnt3a Zellen 12 kultiviert werden . Das Protokoll zuerst von Mica et al. Und hier beschrieben produziert pigmentierten Zellen in drei Wochen und entfernt die Mehrdeutigkeit und Inkonsistenzen mit konditioniertem Medium verbunden. Melanozyten are von der Neuralleiste abgeleitet, einer wandernden Population von Zellen, die einmalig für Wirbeltiere. Die Neuralleiste wird während der Gastrulation definiert und stellt eine Population von Zellen am Rand der Neuralplatte, an der Grenze zwischen der neuronalen und nicht-neuronalen Ektoderm. Während Neurulation entwickelt sich das Nervengewebe einer neuralen Platte Neuralfalten zu bilden, die an der dorsalen Mittellinie im Neuralrohr 13,14 resultierende konvergieren. Die Neuralleistenzellen ergeben sich aus der Dachplatte des Neuralrohrs, gegenüber dem notochord, und durchlaufen eine epitheliale zu mesenchymale Transition vor der Migration weg zu einer vielfältigen Bevölkerung von differenzierten Zellen zu geben. Die Schicksale der Kammzellen werden zum Teil durch die anatomische Lage der Dachplatte entlang der Körperachse des Embryos definiert. Neuralleistenzellen Derivate umfassen Abstammungslinien charakteristisch sowohl Mesoderm (glatte Muskelzellen, Osteoblasten, Adipozyten, Chondrozyten) und Ektoderm Zellen (Melanozyten, Schwann cells, Neuronen) 14. Neuralleiste Stammzellen upregulate den Transkriptionsfaktor SOX10 und durch fluoreszenzaktivierte Zell mit Antikörpern gegen p75 und HNK1 Sortierung isoliert werden. Die Neuralleistenzellen beschieden zu Melanozyten durch eine Melanoblasten Bühne passieren werden und KIT und MITF (Mikrophthalmie-assoziierter Transkriptionsfaktor) 6,21 MITF upregulate ist ein Hauptregulator der Melanozyten - Entwicklung und ist ein Transkriptionsfaktor , verantwortlich für viel von Melanozyten Entwicklung Controlling 22- 24. Menschliche Melanoblasten wandern in der basalen Schicht der Epidermis, wo sie entweder in den Haaren Ausbuchtung wohnen oder von Keratinozyten in der Epidermis umgeben (Bildung Pigmentierung Einheiten) als Vorläufer für den reifen, pigmentierten Melanozyten zu dienen. Die Differenzierung und Reifung von Melanoblasten in pigmentierten Melanocyten tritt mit der Kolonisierung des Haarzwiebel und die Expression des Melaninproduktion Wegs (TYRP1, TYR, OCA2 Begleit- undPMEL) 25,26. Isolieren von humanen Melanozyten und Melanoblasten von Patienten ist teuer, schwierig und in der Menge zu begrenzen. Dieses Protokoll ermöglicht es Forschern, hPSCs (induziert oder embryonale) in Melanozyten oder Melanozyten-Vorstufen in einem gut definierten, schnelle, reproduzierbare, skalierbare und kostengünstige Methode ohne Zellsortierung zu unterscheiden. Das Protokoll wurde zuvor verwendet, um krankheitsspezifische Defekte zu identifizieren, wenn iPSCs von Patienten mit Pigmentstörungen zu unterscheiden.

<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.

Dieses Protokoll stellt ein Verfahren zur effizienten in einem in vitro - Feeder frei, Kosten vollständig pigmentiert, reife Melanozyten aus hPSCs Ableiten und reproduzierbar. Im Gegensatz zu dem zuvor festgelegten Fang et al. Protokoll für HPSC abgeleitetes Melanozyten, wird das umrissenen Protokoll nicht konditionierten Medium erfordern , und verringert den Zeitbedarf. Der Fang et al. Protokoll konditioniertes Medium aus einer Wnt3a produzierenden Maus - Ze...

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

Feeder freie Ableitung von Melanozyten aus humanen pluripotenten Stammzellen

Diese Arbeit beschreibt ein in vitro Differenzierung Protokoll pigmentiert, reife Melanozyten von menschlichen pluripotenten Stammzellen über einen Neuralleiste und Melanoblasten Zwischenstufe mit einem Zubringer frei, 25 Tage - Protokoll zu erzeugen.

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

Ableitung von Herzvorläuferzellen aus embryonalen Stammzellen

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

Forschung

Entwicklungsbiologie

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Effiziente Ableitung von Retinapigmentepithel-Zellen aus Stammzellen

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

Ableitung von Highly Purified Kardiomyozyten aus Menschen induzierten pluripotenten Stammzellen mit Small Molecule modulierte Differenzierung und anschließende Glucose Starvation

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

Dreidimensionale Quantifizierung der dendritischen Dornen von Pyramidenzellen aus humanen induzierten pluripotenten Stammzellen

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

Erzeugung von induzierten pluripotenten Stammzellen aus menschlichen peripheren T-Zellen Mit Sendai Virus in Feeder-freien Bedingungen

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

Definierte und Scalable Generierung von Leber-ähnlichen Zellen aus humanen pluripotenten Stammzellen

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

Unterscheidung von Chondrozyten aus periphere Blut-abgeleiteten menschlichen induzierten pluripotenten Stammzellen

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

Integration Freie Ableitung von human induzierten pluripotenten Stammzellen mit 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

Effiziente Differenzierung menschlicher pluripotenter Stammzellen in Leberzellen

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

In vitro-Generation der Somiten Derivate aus menschlichen induzierten Zellen pluripotenten Stammzellen

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