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

Questo lavoro è stato sostenuto da una borsa di studio per i ricercatori di melanoma dal Joanna M. Nicolay Foundation e dal National Institutes of Health sotto Ruth L. Kirschstein Nazionale Premio Research Service F31. Questo lavoro è stato ulteriormente supportato attraverso sovvenzioni dal NYSTEM e l&#39;iniziativa di cellule staminali Tri-istituzionale (Starr Foundation).

NOTA: Il protocollo di melanociti qui delineato è stato dimostrato da Mica et al. 1. Preparazione del terreno di coltura, patinata Piatti e manutenzione di hPSCs <ol><li> Media Preparazione Nota: Conservare tutto media a 4 ° C al buio fino a 2 settimane. Filtro di medie per la sterilizzazione. <ol><li> Preparare DMEM / 10% FBS. Mescolare 885 ml DMEM, 100 ml FBS, 10 ml Pen / Strep e 5 ml di L-glutammina. Filtro per la sterilizzazione. </li><li> Preparare hESC-media. Mescolare 800 m...

<ol>
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Per la differenziazione di successo dei melanociti da hPSCs i seguenti suggerimenti dovrebbero essere presi in considerazione. Innanzitutto, è essenziale lavorare in condizioni di coltura sterili in ogni momento. Inoltre, è importante iniziare con pluripotenti, hPSCs completamente indifferenziate; se la popolazione di partenza contiene cellule differenziate del rendimento invariabilmente cadere come gli agenti inquinanti non possono essere indirizzate verso i melanociti e può anche distruggere ulteriormente le celle ...

Le cellule staminali pluripotenti umane (hPSCs) forniscono una piattaforma per imitare la differenziazione normale in un modo scalabile per la modellazione della malattia, lo screening di stupefacenti, e le terapie di sostituzione cellulare 1-6. Di particolare interesse, hPSCs aprono strade per studiare difficili da isolare o tipi di cellule rare / transitori in cui i campioni dei pazienti sono scarse. Cellule staminali pluripotenti Inoltre, indotte (iPSCs) permettono ai ricercatori di studiare lo sviluppo e la modellazione della malattia in modo specifico paziente a svelare i meccanismi unici 1,2,7-11. Il protocollo precedentemente pubblicato per la differenziazione dei melanociti da hPSCs richiede fino a 6 settimane di differenziazioni e coinvolge le cellule di coltura con medium condizionato dalle cellule L-Wnt3a 12. Il protocollo prima presentata da Mica et al. E qui descritto produce cellule pigmentate in tre settimane e rimuove l&#39;ambiguità e incoerenze associate a mezzo condizionato. I melanociti are derivati ​​dalla cresta neurale, una popolazione di cellule migratoria unici per vertebrati. La cresta neurale è definita durante gastrulation e rappresenta una popolazione di cellule sul bordo della piastra neurale, al confine tra la ectoderma neurale e non neurale. Durante neurulazione, il tessuto nervoso evolve da una piastra neurale per formare pieghe neurali, che convergono sulla linea mediana dorsale, con conseguente 13,14 tubo neurale. Le cellule della cresta neurale emergono dalla piastra tetto del tubo neurale, di fronte alla notocorda, e subiscono una transizione epitelio mesenchimale prima di migrare via per dare luogo ad una popolazione eterogenea di cellule differenziate. I destini delle cellule della cresta sono definite in parte dalla posizione anatomica della lamiera del tetto lungo l&#39;asse del corpo dell&#39;embrione. Derivati ​​di cellule della cresta neurale includono linee caratteristiche di entrambi mesoderma (cellule muscolari lisce, osteoblasti, adipociti, condrociti) e le cellule ectoderma (melanociti, Schwann cells, neuroni) 14. Le cellule staminali della cresta neurale upregulate il fattore di trascrizione SOX10 e possono essere isolate da cellule di fluorescenza-attivato l&#39;ordinamento con anticorpi anti p75 e HNK1. Le cellule della cresta neurale destinato a diventare melanociti passare attraverso una fase melanoblast e upregulate KIT e MITF (fattore di trascrizione microftalmia-associato) 6,21 MITF è un regolatore Master di Sviluppo melanociti ed è un fattore di trascrizione responsabile del controllo gran parte dello sviluppo melanociti 22- 24. melanoblasts umani migrano allo strato basale dell&#39;epidermide dove risiedono sia nel ringrosso capelli o circondato da cheratinociti nell&#39;epidermide (unità formanti pigmentazione) da utilizzare come precursori delle maturi, melanociti pigmentati. La differenziazione e la maturazione delle melanoblasts in melanociti pigmentati avviene in concomitanza con la colonizzazione del bulbo pilifero e l&#39;espressione della filiera di produzione di melanina (TYRP1, TYR, OCA2 ePMEL) 25,26. Isolare melanociti umani e melanoblasts da pazienti è costoso, difficile e limitante nella quantità. Questo protocollo consente ai ricercatori di differenziare hPSCs (indotta o embrionali) in melanociti o precursori melanociti in un metodo ben definito, rapida, riproducibile, scalabile ed economica, senza l&#39;ordinamento delle cellule. Il protocollo è stato utilizzato in precedenza per identificare i difetti malattie specifiche quando differenziare iPSCs da pazienti con disturbi della pigmentazione.

<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.

Questo protocollo fornisce un metodo per derivare completamente pigmentate, melanociti adulte hPSCs in un alimentatore-libera, economicamente efficiente e riproducibile modo in vitro. In contrasto con il precedentemente stabilito Fang et al. Protocollo per melanociti HPSC-derivati, il protocollo descritto non richiede terreno condizionato e diminuisce il tempo richiesto. Il protocollo Fang et al. Utilizzato mezzo condizionato da una linea di cellule murine Wnt3...

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

Derivazione senza alimentatore di melanociti da cellule staminali umane pluripotenti

Questo lavoro descrive un protocollo in vitro la differenziazione di produrre pigmentate, i melanociti maturi da cellule staminali pluripotenti umane tramite una cresta neurale e melanoblast fase intermedia utilizzando un protocollo senza alimentatore 25 giorni.

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

Derivazione di cellule cardiache progenitrici di cellule staminali embrionali

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

Ricerca

Biologia dello sviluppo

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Derivazione efficiente di cellule retiniche epitelio pigmentato da cellule staminali

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

Derivazione di Cardiomyocytes altamente purificato da cellule staminali umane pluripotenti indotte utilizzando piccoli Differenziazione Molecola-modulato e successiva glucosio 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

Quantificazione tridimensionale di spine dendritiche da piramidale neuroni derivate da cellule staminali umane pluripotenti indotte

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

Generazione di pluripotenti indotte cellule staminali da umani periferici T Cellule con Sendai virus in condizioni di libera-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

Definito e Generazione scalabile di cellule epatociti-come da cellule staminali umane pluripotenti

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

Differenziare i condrociti da cellule staminali pluripotenti indotte dall&#39;uomo derivato dal sangue

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

Derivazione libera dell&#39;integrazione delle cellule staminali pluripotenti indotte dall&#39;uomo usando la matrice Laminina 521

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

Differenziazione efficiente delle cellule staminali pluripotenti umane nelle cellule del fegato

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

Generazione in Vitro di Somite derivati da umani indotta da cellule staminali pluripotenti indotte

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