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

Dette arbejde blev støttet af et stipendium for melanom forskere fra Joanna M. Nicolay Foundation og National Institutes of Health i henhold Ruth L. Kirschstein National Research Service Award F31. Dette arbejde blev yderligere understøttet gennem tilskud fra NYSTEM og Tri-institutionelle stamcelle-initiativet (Starr Foundation).

BEMÆRK: melanocyt proceduren skitseret her blev først påvist af Mica et al. 1. Forberedelse af Culture Medium, Coated Tallerkener og vedligeholdelse af hPSCs <ol><li> Medium Forberedelse Bemærk: Opbevar alle ved 4 ° C i mørke i op til 2 uger. Filter alle medium for sterilisation. <ol><li> Forbered DMEM / 10% FBS. Bland 885 ml DMEM, 100 ml FBS, 10 ml Pen / Strep og 5 ml L-glutamin. Filter til sterilisering. </li><li> Forbered hESC-medium. Bland 800 ml DMEM / F12, 200 ml KSR, 5 ...

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For en vellykket differentiering af melanocytter fra hPSCs bør tages hensyn til følgende forslag. Først og fremmest er det vigtigt at arbejde under sterile dyrkningsbetingelser på alle tidspunkter. Desuden er det vigtigt at starte med pluripotent, fuldt udifferentierede hPSCs; hvis startpopulationen indeholder differentierede celler udbyttet vil uvægerligt falde som forureningerne ikke kan rette sig mod melanocytter og kan endda yderligere forstyrre korrekt differentierende celler. For ...

Humane pluripotente stamceller (hPSCs) skabe en platform til at efterligne normal differentiering i en skalerbar mode for sygdom modellering, narkotika screening, og celle udskiftning behandlingsformer 1-6. Af særlig interesse, hPSCs åbner muligheder for at studere vanskeligt at isolere eller sjældne / forbigående celletyper hvor patientprøver er knappe. Endvidere inducerede pluripotente stamceller (iPSCs) gøre det muligt for forskerne at studere udviklingen og sygdom modellering i en patient specifik måde at optrævle unikke mekanismer 1,2,7-11. Den tidligere offentliggjort protokol til differentiering af melanocytter fra hPSCs kræver op til 6 ugers opdelinger og involverer dyrkning af celler med konditioneret medium fra L-Wnt3a celler 12. Protokollen først præsenteret af Mica et al. Og beskrevet her producerer pigmenterede celler i tre uger, og fjerner den uklarhed og uoverensstemmelser i forbindelse med konditioneret medium. melanocytter are afledt af crista neuralis, en vandrende population af celler unikke for hvirveldyr. Den neurale crest er defineret under gastrulation og repræsenterer en population af celler på kanten af ​​det neurale plade, der grænser mellem neurale og ikke-neurale ektoderm. Under neurulation, nervevævet udvikler sig fra et neuralt plade til dannelse af neurale folder, der løber sammen i den dorsale midtlinje resulterer i neuralrøret 13,14. Crista neuralis celler forlader toppladen af ​​det neurale rør, overfor rygstrengen, og undergår en epitelial til mesenkymale overgang før strømme væk for at give anledning til en forskelligartet population af differentierede celler. Skæbner de kamceller er defineret delvist af den anatomiske placering af tagpladen langs legemet akse af embryonet. Neuralkam cellederivater omfatter slægter karakteristiske for både mesoderm (glatte muskelceller, osteoblaster, adipocytter, chondrocytter) og ektoderm celler (melanocytter, Schwann calen, neuroner) 14. Neurale crest stamceller opregulerer transskriptionsfaktoren SOX10 og kan isoleres ved fluorescensaktiveret cellesortering med antistoffer til p75 og HNK1. De neurale kamceller skæbnesvangre at blive melanocytter passere gennem en melanoblast scene og opregulere KIT og MITF (mikroftalmia-associeret transkriptionsfaktor) 6,21 MITF er en master regulator af melanocyt udvikling og er en transkriptionsfaktor ansvarlig for kontrollen meget af melanocyt udvikling 22- 24. Humane melanoblasts migrere til det basale lag af epidermis, hvor de bor enten i håret bule eller omgivet af keratinocytter i epidermis (dannende pigmentering enheder) til at tjene som forløbere for de modne, pigmenterede melanocytter. Differentieringen og modning af melanoblasts i pigmenterede melanocytter sker samtidig med kolonisering af hår pære og udtryk for melanin produktionen vejen (TYRP1, TYR, OCA2 ogPmel) 25,26. Isolering humane melanocytter og melanoblasts fra patienter er dyrt, vanskeligt og begrænsende mængde. Denne protokol giver forskerne til at differentiere hPSCs (induceret eller embryonale) i melanocytter eller melanocyt forstadier i et veldefineret, hurtig, reproducerbar, skalerbar og billig metode uden celle sortering. Protokollen blev brugt tidligere til at identificere sygdomsspecifikke defekter når differentiere iPSCs fra patienter med pigmentering lidelser.

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			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
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		<tr>
			<td>apo human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></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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		<tr>
			<td>B27 (B27 Supplement)</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
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		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco-Life Technologies</td>
			<td>21985-023</td>
			<td>10 mg/ml</td>
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			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-bp</td>
			<td></td>
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		<tr>
			<td>CHIR99021</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>4423</td>
			<td>6 mM</td>
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		<tr>
			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>50 mg/ml</td>
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		<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>
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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>
		</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>
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		<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>
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			<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>
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		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
			<td></td>
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			<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>
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		<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>
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		<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.

Denne protokol giver en fremgangsmåde til afledning fuldt pigmenterede, modne melanocytter fra hPSCs i en in vitro feeder-fri, omkostningseffektiv, og reproducerbar måde. I modsætning til den tidligere fastsatte Fang et al. Protokol for hPSC-afledte melanocytter, er den skitserede protokol ikke kræver konditioneret medium og reducerer tidsforbrug. Fang et al. Protokol anvendt konditioneret medium fra en Wnt3a-producerende murin cellelinie og tog op til 6 ug...

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-fri Udledning af melanocytter fra menneskelige pluripotente stamceller

Dette arbejde beskriver en in vitro differentiering protokol til at producere pigmenterede, modne melanocytter fra humane pluripotente stamceller via en neural crest og melanoblast mellemtrin ved hjælp af en feeder-fri, 25 dage protokol.

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

Afledning af Cardiac progenitorceller af embryonale 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 Udledning af retinale pigmentepitel Celler fra 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

Udledning af højt oprenset Cardiomyocytter fra menneskeskabte pluripotente stamceller Brug lille molekyle-moduleret Differentiering og efterfølgende Glucose Sult

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

Tre-dimensionel Kvantificering af dendritiske Spines fra pyramideneuroner Afledt af menneskeskabte pluripotente 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 af induceret pluripotente stamceller fra humane perifere T-celler Brug af Sendai Virus i Feeder-fri Betingelser

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

Defineret og skalerbar Generation af hepatocyt-lignende celler fra menneskelige pluripotente 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

Differentierende chondrocytter fra perifert blodafledte humane inducerede pluripotente 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 afledning af humane inducerede pluripotente stamceller ved anvendelse af 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 af humane pluripotente stamceller i 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 af Somite derivater fra menneskelige induceret celler pluripotente stamceller

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