Name: na&iuml;ve adult stem cells isolation from primary human fibroblast cultures
Uploaded: 2013-05-03T13:00:00
Description: Watch this Scientific Journal Video about NaÃ¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures Video at JoVE.com

这项工作是由亚历山大·冯·洪堡基金会（5090371），恭Kuhne将过敏研究和教育中心（CK-CARE），和Bayerischen Staatsministerium（KD）的支持。

1。 SKP从主成纤维细胞培养分离<ol><li>无论是从细胞库，或直接从皮肤活检获得保持在培养的成纤维细胞培养在含有成纤维细胞生长培养基DMEM为15％小牛血清，2mM谷氨酰胺，10毫克/毫升青霉素和10毫克/毫升链霉素。 </li></ol>人类成纤维细胞GMO3349C GMO8398A取自卡瑞尔医学研究所（新泽西州Camden），在这项研究中使用。 <ol start="2"><li>文化群体倍增（PPD的）的20〜35使用为SKP培养在80...

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	<li>Jahoda, C. A., Whitehouse, J., Reynolds, A. J., Hole, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+follicle+dermal+cells+differentiate+into+adipogenic+and+osteogenic+lineages.">Hair follicle dermal cells differentiate into adipogenic and osteogenic lineages.</a> Exp. Dermatol. 12, 849 (2003).</li><li>Watt, F. M., Celso, L. o., C, V., Silva-Vargas, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+stem+cells:+an+update.">Epidermal stem cells: an update.</a> Curr. Opin. Genet. Dev. 16, 518 (2006).</li><li>Blanpain, C., Horsley, V., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+stem+cells:+turning+over+new+leaves.">Epithelial stem cells: turning over new leaves.</a> Cell. 128, 445 (2007).</li><li>Hunt, D. P., Jahoda, C., Chandran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+skin-derived+precursors:+from+biology+to+clinical+translation.">Multipotent skin-derived precursors: from biology to clinical translation.</a> Vurr. Opin. Biotechnol. 20, 522 (2009).</li><li>Toma, J. 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=Isolation+of+multipotent+adult+stem+cells+from+the+dermis+of+mammalian+skin.">Isolation of multipotent adult stem cells from the dermis of mammalian skin.</a> Nat. Cell Biol. 3, 778 (2001).</li><li>Fernandes, K. J. L., 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=A+dermal+niche+for+multipotent+adult+skin-derived+precursor+cells.">A dermal niche for multipotent adult skin-derived precursor cells.</a> Nat. Cell Biol. 6, 1082 (2004).</li><li>Wenzel, V., 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=Na&#239;ve+adult+stem+cells+from+patients+with+Hutchinson-Gilford+progeria+syndrome+express+low+levels+of+progerin+in+vivo.">Na&#239;ve adult stem cells from patients with Hutchinson-Gilford progeria syndrome express low levels of progerin in vivo.</a> Bio. Open. 1, (2012).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25, 402 (2001).</li><li>Biernaskie, J. A., McKenzie, I. A., Toma, J. T., Miller, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+skin-derived+precursors+(SKPs)+and+differentiation+and+enrichment+of+their+Schwann+cell+progeny.">Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny.</a> Nature Protocol. 1, 2803 (2006).</li><li>Toma, J. G., McKenzie, I., Bagli, D., Miller, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+multipotent+skin-derived+precursors+from+human+skin.">Isolation and characterization of multipotent skin-derived precursors from human skin.</a> Stem Cells. 23, 727 (2005).</li><li>Fernandes, K. 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=Analysis+of+the+neurogenic+potential+of+multipotent+skin-derived+precursors.">Analysis of the neurogenic potential of multipotent skin-derived precursors.</a> Electrophoresis. 201, 32 (2006).</li><li>Hill, l. K. L., 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=Human+embryonic+stem+cell-derived+vascular+progenitor+cells+capable+of+endothelial+and+smooth+muscle+cell+function.">Human embryonic stem cell-derived vascular progenitor cells capable of endothelial and smooth muscle cell function.</a> Exp. Hematol. 38, 246 (2010).</li></ol>

使用本文所述的方法，的天真皮肤干细胞可以分离出原代皮肤成纤维细胞培养。使用这种方法，我们最近报道的来自患者患有一种罕见的遗传性综合征，哈钦森-Gilford早衰综合症7成纤维细胞培养成体干细胞分离和鉴定。此处显示这些前体细胞表达干细胞标记物是能够自我更新和定向分化成不同的细胞系，包括成纤维细胞和平滑肌细胞（请参阅图4 Wenzel 等人 ，2012）7?...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Invitrogen</td>
			<td>31966-047</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM low glucose</td>
			<td>Invitrogen</td>
			<td>21885-108</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>10270-106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td>Final conc.: 200 mM</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>Final conc.: 10 mg/ml /10 mg /ml</td>
		</tr>
		<tr>
			<td>trypsin solution (0.25%)</td>
			<td>Invitrogen</td>
			<td>25200-056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>F-12 Nutrient Mixture (Ham)</td>
			<td>Invitrogen</td>
			<td>21765-029</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>BD Biosciences</td>
			<td>4114-TC-01M</td>
			<td>Final conc.: 40 ng/ml</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>BD Biosciences</td>
			<td>236-EG-200</td>
			<td>Final conc.: 20 ng/ml</td>
		</tr>
		<tr>
			<td>PDGFBB</td>
			<td>Invitrogen</td>
			<td>PHG0043</td>
			<td>Final conc.: 5 ng/ml</td>
		</tr>
		<tr>
			<td>TGF-b1</td>
			<td>Invitrogen</td>
			<td>PHG9204</td>
			<td>Final conc.: 2.5 ng/ml</td>
		</tr>
		<tr>
			<td>25 cm2 flask</td>
			<td>Omnilab</td>
			<td>FALC353109</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS w/o CaMg</td>
			<td>Invitrogen</td>
			<td>14190-169</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Roth</td>
			<td>8388.2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector Inc.</td>
			<td>H-1200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNeasy Minikit</td>
			<td>Qiagen, Valencia, CA</td>
			<td>74104</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Omniscript Reverse Transcriptase</td>
			<td>Qiagen</td>
			<td>205113</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SsoFast EvaGreen Supermix</td>
			<td>BioRad</td>
			<td>172-5201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Invitrogen</td>
			<td>15290-018</td>
			<td>Final conc.:1 mg/ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 2. Specific reagents and equipment.</td>
		</tr>
	</tbody>
</table>

na&iuml;ve adult stem cells isolation from primary human fibroblast cultures

在过去的十年中，一些成人干细胞种群已确定在人体皮肤1-4。多能成体皮肤前体的隔离首先报道了米勒楼D实验室5，6。这些早期的研究描述了一种多能前体细胞人口从成年哺乳动物真皮5。这些细胞-称为SKPS，皮肤源性前体-从啮齿目动物和人体皮肤进行分离和扩增和分化成神经和中胚层的后代，包括从来没有发现在皮肤的细胞类型，如神经元5。免疫细胞化学对培养SKPS的研究发现，细胞表达波形蛋白和巢蛋白，中间丝蛋白的表达在神经和骨骼肌中的前体，另外，纤连蛋白和多能干细胞的标记物6。到现在为止，已分离出的成体干细胞人口SKPS的新鲜采集的哺乳动物皮肤活检。 假设那几成体干细胞可能会驻留在初级成纤维细胞培养早期人口倍增ve_content“最近，我们已经建立和报道人口的皮肤衍生前体细胞能够保持目前在初级成纤维细胞培养，建立了从皮肤活检7。基于以下的观察：（1）SKPS和初级成纤维细胞培养来自从真皮的，因此，少数SKP细胞可能仍然存在于原代皮肤成纤维细胞培养，（2）已经从冷冻的等分试样初级成纤维细胞生长的培养受到不利的温度，在储存或传输载有少数细胞存活7。这些罕见的细胞能够扩大和可传代数次。推测（1），一小部分细胞具有高增殖能力和抗应力目前在人类成纤维细胞培养7。我们利用这些研究结果，建立初级成纤维细胞培养，可随时从世界各地的银行组织（ 图1）的成人干细胞的快速分离的协议。此方法具有重要的意义，因为它允许隔离前体细胞皮肤样本时无法访问而纤维母细胞可能是从组织银行，从而打开了新的机遇，剖析罕见的遗传性疾病的分子机制，以及在建模疾病菜。

我们表明，细胞选择性地扩展到控制生长条件下产生SKP领域包括EGF和FGF2的人口目前在原发性皮肤成纤维细胞培养（ 图1），为大家报道最近7。 本研究中使用的成纤维细胞培养从PPD的15至25，通常对应于原代成纤维细胞株有细胞库。提交由低温治疗的双重治疗，连同24小时，在这个方法中描述的营养素枯竭的成纤维细胞培养可重复产生的文化，包含的浮动球...

Watch this Scientific Journal Video about NaÃ¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures Video at JoVE.com

NaÃ¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures Video (Video) | JoVE

我们报告一个幼稚多能隔离的皮肤衍生成前体细胞（SKP）的从初级人类成纤维细胞培养的方法。我们表明，这些SKPS来自成纤维细胞培养干细胞有着相似的属性直接来源于人体皮肤活检的。这些细胞表达神经嵴标记，巢蛋白，除了如OCT4和Nanog的多能标记。

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

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Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

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