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

Detta arbete stöddes av ett stipendium för melanom forskare från Joanna M. Nicolay Foundation och av National Institutes of Health enligt Ruth L. Kirschstein National Research Service Award F31. Detta arbete stöddes ytterligare genom bidrag från NYSTEM och Tri-institutionella stamcells initiativ (Starr Foundation).

OBS: melanocyt protokoll som beskrivs här visades först av Mica et al. 1. Beredning av odlingsmedium, Belagd Porslin och underhåll av hPSCs <ol><li> Medium Framställning Obs: Lagra alla medium vid 4 ° C i mörker i upp till två veckor. Filtrera all medium för sterilisering. <ol><li> Förbereda DMEM / 10% FBS. Blanda 885 ml DMEM, 100 ml FBS, 10 ml Pen / Strep och 5 ml L-glutamin. Filter för sterilisering. </li><li> Förbered hESC-medium. Blanda 800 ml DMEM / F12, 200 ml KSR, ...

<ol>
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För framgångsrik differentiering av melanocyter från hPSCs följande förslag bör beaktas. Först och främst är det viktigt att arbeta under sterila odlingsbetingelser vid alla tillfällen. Dessutom är det viktigt att börja med pluripotenta, fullt odifferentierade hPSCs; om utgångspopulationen innehåller differentierade celler utbytet kommer alltid sjunka eftersom föroreningarna inte kan riktas mot melanocyter och kan även ytterligare störa korrekt differentierande cellerna. Fö...

Humana pluripotenta stamceller (hPSCs) tillhandahålla en plattform för att efterlikna normal differentiering i en skalbar mode för sjukdom modellering, drug screening och cellersättningsterapi 1-6. Av särskilt intresse, hPSCs öppnar vägar för att studera svåra att isolera eller sällsynta / gående celltyper där patientprover är knappa. Vidare inducerade pluripotenta stamceller (iPSCs) göra det möjligt för forskare att studera utveckling och sjukdom modellering i en patient specifikt sätt att riva unika mekanismer 1,2,7-11. Det tidigare publicerade protokoll för differentiering av melanocyter från hPSCs kräver upp till 6 veckor av differentieringar och involverar odling av celler med konditionerat medium från L-Wnt3a cellerna 12. Protokollet först presenterades av Mica et al. Och beskrivs här producerar pigmenterade celler i tre veckor och tar bort den tvetydighet och inkonsekvenser i samband med konditionerat medium. melanocyter are härrör från neural crest, en vandrande population av celler som är unika för ryggradsdjur. Neurallisten definieras under gastrulation och representerar en population av celler vid kanten av det neurala plattan, som gränsar mellan neurala och icke-neurala ektoderm. Under neurulation, utvecklas nervvävnad från ett neuralt platta för att bilda neurala veck, som konvergerar vid ryggens mittlinje resulterar i neuralröret 13,14. Neurallisten celler ta sig ur takplåten av neuralröret, mittemot notochord, och genomgår en epitelial till mesenkymala övergång innan den vandrar bort för att ge upphov till en mångskiftande population av differentierade celler. Öden de crest celler definieras delvis av den anatomiska lokaliseringen av takplåten längs kroppsaxeln hos embryot. Neurallisten cellderivat inkluderar härstamningar som är karakteristiska för både mesoderm (glatta muskelceller, osteoblaster, adipocyter, kondrocyter) och ektoderm celler (melanocyter, Schwann calnar, neuroner) 14. Neurallisten stamceller uppreglera transkriptionsfaktom SOX10 och kan isoleras genom fluorescensaktiverad cellsortering med antikroppar mot p75 och HNK1. Neurallisten celler ödesbestämd att bli melanocyter passera genom en melanoblast scen och uppreglera KIT och MITF (mikroftalmi-associerad transkriptionsfaktor) 6,21 MITF är en mästare regulator av melanocyt utveckling och är en transkriptionsfaktor som ansvarar för att kontrollera en stor del av melanocyt utveckling 22- 24. Mänskliga melanoblasts migrera till basalskiktet av epidermis där de bor antingen i håret bula eller omgiven av keratinocyter i epidermis (som bildar pigmente enheter) för att tjäna som föregångare till den mogna, pigmenterade melanocyter. Differentieringen och mognad av melanoblasts i pigmenterade melanocyter sker samtidigt med koloniseringen av hårsäcken och uttryck av melanin produktionskedja (TYRP1, TYR, OCA2 ochPMEL) 25,26. Isolera humana melanocyter och melanoblasts från patienter är dyrt, svårt och begränsande i kvantitet. Detta protokoll gör det möjligt för forskare att skilja hPSCs (inducerad eller embryonala) i melanocyter eller melanocytstimulerande prekursorer i en väl definierad, snabb, reproducerbar, skalbar och billig metod utan cellsortering. Protokollet användes tidigare för att identifiera sjukdomsspecifika brister när differentiera iPSCs från patienter med pigmentstörningar.

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			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
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			<td>apo human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
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			<td>Ascorbic Acid (L-AA)</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td>100 mM</td>
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			<td>B27 (B27 Supplement)</td>
			<td>Invitrogen</td>
			<td>17504044</td>
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			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco-Life Technologies</td>
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			<td>10 mg/ml</td>
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			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-bp</td>
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			<td>CHIR99021</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>4423</td>
			<td>6 mM</td>
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			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>50 mg/ml</td>
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			<td>cAMP (cyclicAMP)</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>100 mM</td>
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			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D2915-100MG</td>
			<td>50 &#956;M</td>
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			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco-Life Technologies</td>
			<td>11985-092</td>
			<td></td>
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			<td>DMEM/F12 - Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12</td>
			<td>Gibco-Life Technologies</td>
			<td>1133--032</td>
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			<td>DMEM/F12 powder</td>
			<td>Invitrogen</td>
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			<td>EDN3 (Endothelin-3, human)</td>
			<td>American Peptide Company</td>
			<td>88-5-10B</td>
			<td>100 &#956;M</td>
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			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>356008</td>
			<td>200 &#956;g/ml</td>
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			<td>gelatin (PBS without Mg/Ca)</td>
			<td>in house</td>
			<td></td>
			<td>0.1% in PBS</td>
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			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
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			<td>Human insulin</td>
			<td>Sigma</td>
			<td>I2643</td>
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			<td>ITS+ Universal Culture Supplement Premix&#160;</td>
			<td>BD Biosciences</td>
			<td>354352</td>
			<td></td>
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			<td>KSR (Knockout Serum Replacement)</td>
			<td>Gibco-Life Technologies</td>
			<td>10828-028</td>
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			<td>Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
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			<td>L-Glutamine</td>
			<td>Gibco-Life Technologies</td>
			<td>25030-081</td>
			<td></td>
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			<td>LDN193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td>100 mM</td>
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			<td>Low glucose DMEM</td>
			<td>Invitrogen</td>
			<td>11885-084</td>
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			<td>Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Dissolve 1:20 in DMEM/F12</td>
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			<td>MCDB201 Medium</td>
			<td>Sigma</td>
			<td>M6770</td>
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			<td>MEM minimum essential amino acids solution</td>
			<td>Gibco-Life Technologies</td>
			<td>11140-080</td>
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			<td>Mouse embryonic fibroblasts (7 million cells/vial)</td>
			<td>GlobalStem</td>
			<td>GSC-8105M</td>
			<td></td>
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			<td>Mouse Laminin-I</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td>1 mg/ml</td>
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			<td>Neurobasal medium</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
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		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
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		<tr>
			<td>Poly-L Omithin hydrobromide</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td>15 mg/ml&#160;</td>
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		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>0.032g in 100ml 100% ethanol</td>
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		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
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		<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>
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		<tr>
			<td>SB431542</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1814</td>
			<td>10 mM</td>
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			<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.

Detta protokoll ger en metod för att härleda fullt pigmenterade, mogna melanocyter från hPSCs i en matare fritt, kostnadseffektivt och reproducerbart in vitro sätt. I motsats till den tidigare fastställda Fang et al. Protokoll för hPSC-härledda melanocyter, betyder den skisserade protokollet inte kräver konditionerat medium och minskar tidsåtgången. Protokollet Fang et al. Användes konditionerat medium från en WNT3A-producerande murin cellinje och t...

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-fria Derivering av Melanocyter från Human pluripotenta stamceller

Detta arbete beskriver ett in vitro differentiering protokoll för att producera pigmente, mogna melanocyter från humana pluripotenta stamceller via en neural crest och melanoblast mellansteg med hjälp av en matarfria, 25 dagars protokoll.

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

Härledning av Cardiac progenitorceller från embryonala stamceller

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

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Effektiv Derivering av näthinnans pigmentepitel celler från stamceller

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

Härledning av renat Cardiomyocytes från Human inducerade pluripotenta stamceller Använda småmolekylära-module Differentiering och Efterföljande Glukos Svält

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

Tredimensionell Kvantifiering av Dendritutskotten från Pyramidal nervceller Härstammar från Human inducerade pluripotenta stamceller

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

Generering av inducerade pluripotenta stamceller från humana perifera T-celler med Sendai virus i Feeder fria förhållanden

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

Definierade och skalbar generation av hepatocyte-liknande celler från mänskliga pluripotenta stamceller

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

Differentierande kondrocyter från perifert blod härledda humana inducerade plöripotenta stamceller

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

Integrationsfri avledning av humana inducerade plöripotenta stamceller med användning av 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

Effektiv differentiering av humana pluripotenta stamceller till leverceller

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

I Vitro Generation av Somite derivat från mänskliga inducerade stamceller pluripotenta

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