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 arbeidet ble støttet av et fellesskap for melanom forskere fra Joanna M. Nicolay Foundation og av National Institutes of Health i henhold til Ruth L. Kirschstein National Research Service Award F31. Dette arbeidet ble også støttet gjennom tilskudd fra NYSTEM og Tri-institusjonelle stamcelle initiativ (Starr Foundation).

MERK: melanocyte protokollen skissert her ble først demonstrert av Mica et al. 1. Utarbeidelse av dyrkingsmedium, Belagt Retter og vedlikehold av hPSCs <ol><li> medium Forberedelse Merk: Oppbevar alle medium ved 4 ° C i mørke i opptil to uker. Filter alle medium for sterilisering. <ol><li> Forbered DMEM / 10% FBS. Bland 885 ml DMEM, 100 ml FBS, 10 ml Pen / Strep og 5 ml L-Glutamine. Filter for sterilisering. </li><li> Forbered hESC-medium. Bland 800 ml DMEM / F12, 200 ml KSR, 5 m...

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For vellykket differensiering av melanocytter fra hPSCs bør tas i betraktning følgende forslag. Først og fremst er det viktig å arbeide under sterile dyrkningsbetingelser til enhver tid. I tillegg er det viktig å starte med pluripotent, fullt udifferensiert hPSCs; hvis utgangspopulasjon inneholder differensierte celler utbyttet vil alltid falle som forurensningene ikke kan rettes mot melanocytter, og kan til og med ytterligere forstyrre skikkelig differensierende celler. For å sikre ce...

Menneskelige pluripotente stamceller (hPSCs) gi en plattform for å etterligne normal differensiering i en skalerbar mote for sykdom modellering, narkotika screening, og celle erstatning terapi 1-6. Av spesiell interesse, hPSCs åpne opp muligheter for å studere vanskelig å isolere eller sjeldne / forbigående celletyper hvor pasientprøver er knappe. Videre induserte pluripotente stamceller (iPSCs) gjør forskerne å studere utvikling og sykdom modellering i en pasient bestemt måte å løse unike mekanismer 1,2,7-11. Den tidligere utgitt protokoll for differensiering av melanocytter fra hPSCs krever opp til 6 uker med differensiering og innebærer dyrking celler med kondisjonert medium fra L-Wnt3a celler 12. Protokollen først presentert av Mica et al. Og beskrevet her produserer pigmenterte celler i tre uker, og fjerner tvetydighet og uoverensstemmelser i forbindelse med kondisjonert medium. melanocytter are avledet fra neural crest, en vandrende populasjon av celler som er unike for virveldyr. Neural crest er definert under gastrulation og representerer en populasjon av celler ved kanten av det nevrale plate, som grenser mellom det neurale og ikke-nevrale ektoderm. Under neurulation, utvikler den nervevev fra et neural-plate for å danne nevrale folder, som konvergerer ved dorsal midtlinjen som resulterer i det nevrale rør 13,14. Neural crest cellene skilles ut fra taket plate av nevralrøret, på motsatt side av ryggstreng, og gjennomgår en epitelial til mesenchymale overgang før den vandrer bort for å gi opphav til en mangfoldig befolkning av differensierte celler. Skjebnen av toppen celler er definert delvis av den anatomiske plassering av taket platen langs kroppens akse for embryoet. Neural crest celle derivater omfatter linjer karakteristisk for både mesoderm (glatte muskelceller, osteoblaster, adipocytter, chondrocytes) og ektoderm celler (melanocytter, Schwann calen, nevroner) 14. Nevrale kløft stamceller oppregulere transkripsjonsfaktor SOX10 og kan isoleres ved fluorescens-aktivert cellesortering med antistoffer mot p75 og HNK1. Neural crest cellene skjebnebestemt til å bli melanocytter passere gjennom en melanoblast scenen og oppregulere KIT og MITF (mikroftalmi-assosiert transkripsjonsfaktor) 6,21 MITF er en mester regulator av melanocyte utvikling og er en transkripsjonsfaktor ansvarlig for å kontrollere mye av melanocytter utvikling 22- 24. Menneske melanoblasts migrere til basal laget av epidermis hvor de bor enten i håret kul eller omgitt av keratinocytter i epidermis (forming pigmentering enheter) å tjene som forløpere for de modne, pigmenterte melanocytter. Differensiering og modning av melanoblasts inn i pigmenterte melanocytter opptrer samtidig med kolonisering av hår pære og ekspresjon av melaninproduksjonen pathway (TYRP1, TYR, og OCA2PMEL) 25,26. Isolere menneskelige melanocytter og melanoblasts fra pasienter er dyrt, vanskelig og begrensende i kvantitet. Denne protokollen gjør det mulig for forskere å skille hPSCs (indusert eller embryonale) i melanocytter eller melanocyte forløpere i en veldefinert, rask, reproduserbar, skalerbar og billig metode uten cellesortering. Protokollen ble brukt tidligere for å identifisere sykdomsspesifikke feil når differensiere iPSCs fra pasienter med pigmentforstyrrelser.

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	<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.

Denne protokollen tilveiebringer en fremgangsmåte for å utlede fullt pigmenterte, modne melanocytter fra hPSCs i en in vitro-mater-fri, kostnadseffektive, og reproduserbar måte. I motsetning til det som tidligere er etablert Fang et al., Protokoll for hPSC-avledede melanocytter, ikke beskrevet protokollen ikke krever kondisjonert medium og reduserer tidskrav. Fang et al. Protokollen benyttes kondisjonert medium fra en WNT3A produserende murine cellelinje og ...

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 Utledning av Melanocytter fra Menneskelig Pluripotent stamceller

Dette arbeidet beskriver en in vitro differensiering protokollen til å produsere pigmenterte, modne melanocytter fra menneskelige pluripotente stamceller via en neural crest og melanoblast mellomstadium ved hjelp av en mater-fri, 25 dagers protokollen.

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

Avledning av Cardiac stamceller fra 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 Utledning av retinal pigment epitelet 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

Avledning av Highly Purified kardiomyocytter fra menneskeskapte Pluripotent stamceller ved hjelp av små Molecule-modulert Differensiering og Følgende 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

Tredimensjonal Kvantifisering av dendrittutløperne fra Pyramideformet Nevroner Avledet fra menneskeskapte Pluripotent 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 Induced Pluripotent stamceller fra menneske perifere T-celler Bruke Sendai Virus i Feeder-free 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

Definert og Scalable Generation of hepatocytter lignende celler fra menneskelige Pluripotent 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

Differensiering av kondrocytter fra perifert blod-avledede humane inducerte 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

Integreringsfri avledning av humane inducerte pluripotente stamceller ved bruk 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 differensiering av Human Pluripotent 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 generasjon av Somite derivater fra menneske indusert stamceller Pluripotent

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