Name: differentiation of newborn mouse skin derived stem cells into germ-like cells in vitro
Uploaded: 2013-07-16T13:00:00
Description: Watch this Scientific Journal Video about Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro at JoVE.com

PWD understøttes af en canadisk Institutes of Health Research Fellowship (CIHR) og en CIHR tilskud til Gerald M. Kidder på Western University. Forfatteren vil også gerne anerkende Julang Li på University of Guelph om hjælp med udviklingen af ​​protokollen, der præsenteres her.

1.. Medier Forberedelse <ol><li> Forbered medier 2-3 hr forud for brug og sted med en løsnet hætte i cellekultur inkubator hvor forsøget vil finde sted. Medier, der anvendes i den nuværende protokol: <ol><li> Forbered stamcelle medium bestående af DMEM/F12 suppleret med 1 x B27, 40 ng / ml bFGF og 20 ng / ml EGF. </li><li> Forbered kim celledifferentiering medium bestående af M199 suppleret med 0,05 IU FSH 0,03 IU LH, 3 mg / ml BSA, 5 ul / ml ITS, 0,23 mM natriumpyruvat, 1 mg / ml fetuin, og 1 ng / ml EGF. En ba...

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	<li>van den Hurk, R., Zhao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+mammalian+oocytes+and+their+growth,+differentiation+and+maturation+within+ovarian+follicles.">Formation of mammalian oocytes and their growth, differentiation and maturation within ovarian follicles.</a> Theriogenology. 63, 1717-1751 (2005).</li><li>Molyneaux, K. A., Stallock, J., Schaible, K., Wylie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+analysis+of+living+mouse+germ+cell+migration.">Time-lapse analysis of living mouse germ cell migration.</a> Dev. Biol. 240, 488-498 (2001).</li><li>Lawson, K. A., Hage, W. 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H., McLaren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primordial+germ+cells+in+the+mouse+embryo+during+gastrulation.">Primordial germ cells in the mouse embryo during gastrulation.</a> Development. 110, 521-528 (1990).</li><li>Hubner, K., Fuhrmann, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+oocytes+from+mouse+embryonic+stem+cells.">Derivation of oocytes from mouse embryonic stem cells.</a> Science. 300, 1251-1256 (2003).</li><li>Toyooka, Y., Tsunekawa, N., Akasu, R., Noce, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+can+form+germ+cells+in+vitro.">Embryonic stem cells can form germ cells in vitro.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 11457-11462 (2003).</li><li>Wang, L., Cao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-like+Cells+Induced+from+Mouse+Spermatogonial+Stem+Cells.">Oocyte-like Cells Induced from Mouse Spermatogonial Stem Cells.</a> Cell Biosci. 2, 27 (2012).</li><li>Dyce, P. W., Wen, L., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+germline+potential+of+stem+cells+derived+from+fetal+porcine+skin.">In vitro germline potential of stem cells derived from fetal porcine skin.</a> Nat. Cell Biol. 8, 384-390 (2006).</li><li>Danner, S., Kajahn, J., Geismann, C., Klink, E., Kruse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+oocyte-like+cells+from+a+clonal+pancreatic+stem+cell+line.">Derivation of oocyte-like cells from a clonal pancreatic stem cell line.</a> Mol. Hum. Reprod. 13, 11-20 (2007).</li><li>Cheng, X., Chen, S., Yu, X., Zheng, P., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP15+gene+is+activated+during+human+amniotic+fluid+stem+cell+differentiation+into+oocyte-like+cells.">BMP15 gene is activated during human amniotic fluid stem cell differentiation into oocyte-like cells.</a> DNA Cell Biol. 31, 1198-1204 (2012).</li><li>Song, S. H., Kumar, B. 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Til dato funktionelle OLCs ikke er blevet udviklet ved hjælp af en helt in vitro-assay. For nylig en undersøgelse af Hayashi et al. Var i stand til at producere afkom fra PGC-lignende celler dannet in vitro og overført in vivo til videreudvikling 14.. Startende med ES-celler eller induceret pluripotente celler, de var i stand til at danne PGC lignende celler, som, når inddrevne efter in vivo transplantation kunne blive modnet, befrugtet, og bruges til at genere...

I den tidlige embryogenese sker gametogenese gennem en række faser, herunder oprindelige kønsceller (PGC) dannelse, migration og endelig kolonisering af de gonadale kamme. I løbet af denne tid PGC&#39;er undergå proliferation og differentiering i stadig mere modne kønsceller 1.. Den omstændighed, at PGC&#39;er migrere fra bunden af allantois i embryo hindgut og endelig langs den dorsale væg sidst koloniserede gonadale kamme gør dem ekstremt vanskeligt at studere 2. Trods fremskridt på området, har undersøgelser forsøger at forstå PGC dannelse og differentiering været hæmmet af deres begrænsede antal, placering og vandrende art 3,4. I de seneste år embryonale stamceller har vist sig at have potentiale til at danne kimceller in vitro 5,6. Ligeledes har flere somatiske stamceller også vist sig at have potentiale til at danne kønsceller Follgrund in vitro dyrkning 7-11. For nylig stamceller isoleret fra nyfødte musehud blev differentieret in vitro i kim-lignende celler og nyuddannede oocytter 12. Under differentiering den delmængde af stamcellerne udtrykker Oct4 steg og strukturer ligner cumulus-oocyt-komplekser blev dannet. Ægget-lignende celler (OLCs) kan plukkes fra kulturer, og i forhold til naturlige oocytter. Brug af denne dyrkningsmetode OLCs måler 40-45 um opnås som udtrykker lignende markører til oocytter såsom Gdf9b, VASA og DAZL. Til dato de resulterende OLCs forbliver ude af stand til at modne eller blive befrugtet 12.. Selvom OLCs forbliver i stand til at fungere den protokol, der bruges til at danne disse OLCs stadig kan have nytte som et assay til at studere dannelsen og udviklingen af tidligere stadier kønscellerne i en lukket in vitro-system. Den originale publikation diskuterer dannelsen af ​​OLCs fra, såmatic stamceller udnyttet føtal svinehud som en stamcelle kilde 8.. Udvidelse på denne undersøgelse af PGC-lignende celler dannet tidligt i induceret differentiering viste sig at være forstadier til OLCs og ekspres sådanne PGC markører som OCT4, VASA, STELLA, C-KIT og DAZL 13.. Disse data understøtter potentialet for at udnytte dette system til at studere kimcelle dannelse og differentiering in vitro. Protokollen anvendes til dannelse OLCs fra nyfødt mus hud vil blive demonstreret i denne video artiklen. Denne protokol giver en in vitro model til at studere kim celle udvikling, der kan give et middel til at belyse faktorer vigtige for deres proliferation og differentiering.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>EGF</td>
			<td>Invitrogen</td>
			<td>PHG0311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>bFGF</td>
			<td>Invitrogen</td>
			<td>PHG0266</td>
			<td></td>
		 </tr>
		 <tr>
			<td>ITS</td>
			<td>Invitrogen</td>
			<td>41400-045</td>
			<td></td>
		 </tr>
		 <tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A-6003</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium Pyruvate</td>
			<td>Invitrogen</td>
			<td>11360-070</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetuin</td>
			<td>Sigma</td>
			<td>F3385</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FSH</td>
			<td>Sioux Biochemicals</td>
			<td>915</td>
			<td></td>
		 </tr>
		 <tr>
			<td>LH</td>
			<td>Sioux Biochemicals</td>
			<td>925</td>
			<td></td>
		 </tr>
		 <tr>
			<td>M199</td>
			<td>Invitrogen</td>
			<td>31100-035</td>
			<td></td>
		 </tr>
		 <tr>
			<td>24 well plates</td>
			<td>Nunc</td>
			<td>144530</td>
			<td></td>
		 </tr>
		 <tr>
			<td>10 cm dishes</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td></td>
		 </tr>
		 <tr>
			<td>15 ml tubes</td>
			<td>BD</td>
			<td>352095</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

differentiation of newborn mouse skin derived stem cells into germ-like cells in vitro

Studere kønsceller dannelse og differentiering har traditionelt været meget vanskeligt på grund af lave celletal og deres placering dybt inde fostre. Tilgængeligheden af en &quot;lukket&quot; in vitro-baseret system kan vise sig uvurderlig for vores forståelse af gametogenese. Dannelsen af ​​oocyt-lignende celler (OLCs) fra somatiske stamceller, isoleret fra nyfødte musehud, er påvist, og kan visualiseres i denne video protokol. De resulterende OLCs udtrykker forskellige markører i overensstemmelse med oocyter såsom Oct4, Vasa, Bmp15 og Scp3. Men de forbliver i stand til at undergå modning eller befrugtning på grund af en manglende færdiggørelse meiose. Denne protokol vil tilvejebringe et system, der er nyttig til at studere den tidlige fase dannelse og differentiering af kimceller i mere modne kønsceller. Under tidlig differentiering antallet af celler, der udtrykker Oct4 (potentielle kim-lignende celler) når ~ 5%, men i øjeblikket fsninger af OLCs fortsat relativt ineffektive. Protokollen er forholdsvis ligetil selvom særlig forsigtighed bør tages for at sikre, startende cellepopulation er sundt og på et tidligt passage.

Oprindeligt cellerne tillægger dyrkningsskålen bunden og spredt ud. Ved 42-72 hr i differentiering små suspenderede OCT4 positive celler dannes og formere (figur 1A). Kort efter visualisering af disse celler flertallet vil forsvinde og de vedhæftede celler vil danne tætte koloni aggregater på kulturen bunden. Efter et par dage disse aggregater vil løsne sig fra den kultur overflade. I nogle af disse aggregater en stor celle kan observeres (figur 1b og 1c). Ved f?...

Watch this Scientific Journal Video about Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro at JoVE.com

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

I de seneste år dannelsen af ​​kønsceller brug af in vitro-dyrkningsmetoder er blevet demonstreret ved hjælp af flere forskellige typer af somatiske stamceller. Denne artikel vil visuelt præsentere differentieringen af ​​nyfødte mus afledte stamceller til tidlige oocyt-lignende celler.

Differentiering af Nyfødte musehud afledt stamceller i Germ-lignende celler<em&gt; In vitro</em

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Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

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