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％汇合。一个10厘米的组织培养皿（BD猎鹰）包含约1.5×10 6个细胞。 </li><li>洗涤细胞，用PBS并孵育2毫升胰蛋白酶溶液（0.25％，Invitrogen公司制）1小时，在37℃下</li><li>板用5毫升PBS收集细胞和细胞悬液转移到15毫升猎鹰管。 </li><li>细胞在4°C孵育24小时。 </li><li>准备SKP生长培养基由DMEM-F12，3:1（V / V），40 ng / ml的FGF2（BD Biosciences公司，20 ng / ml的EGF（BD Biosciences公司），B27的无血清补充2％（Invitrogen公司），1微克/毫升两性霉素B（Invitrogen公司）和25微米/毫升庆大霉素（Invitrogen公司）。 </li><li>在1200 rpm离心5分钟沉淀细胞，重悬细胞沉淀直接在4毫升SKP生长介质中。细胞悬液转移到25厘米2组织培养瓶（BD猎鹰）。 </li><li>在37℃孵育烧瓶，，监测文化球体形成每天3至4个星期。 </li></ol> 2。球体培养条件<ol><li>在25cm 2的烧瓶（BD Falcon）中于37℃，5％CO 2中培养细胞。 </li><li>每日大力摇动烧瓶，以避免细胞附着在烧瓶的底部。如果有必要，吸管向上和向下2毫升的无菌吸管分离贴壁细胞和文化转移到一个新的烧瓶。 </li><li>第一球开始建立在3吨内O 4天。 </li><li>让球沉积物的底部的烧瓶中，并改变一半的培养基，每3天。为了保持同样的最终浓度生长因子的增加2X生长因子新鲜配制的SKP生长培养基。 </li><li>保持体积不变最多4毫升。 </li><li>当球〜200毫米，达到相当的规模，他们应该被打破的领域，以更小的尺寸剧烈吹打向上和向下2毫升吸管传代。 </li><li>将培养被分成两个25cm 2的培养瓶（BD Falcon）中和球体文化步骤2.4）中所描述的饲料。 </li><li>所收集的干细胞标记物筛选的16天至21球。 </li></ol> 2.6）和2.7）中描述的程序的传播领域（1）允许营养物和生长因子在SKP介质中，以访问在每个球体之内的所有单元格，（2）扩大SKP球体培养前使用。 通常情况下，球在我们立方米文化lture条件维持的能力增长超过3个月的一段时间，并传代3〜4倍之间。 3。干细胞标记物的免疫细胞化学<ol><li>球体培养收获PBS由16天至21日。培养物在1,000 rpm离心5分钟沉淀。 </li><li>微球在一个小体积的PBS重悬。 </li><li>画两个圆，一个直径约为0.5微米，在显微镜玻片（费舍尔品牌）DAKO笔。 </li><li>球体悬浮液中加入50μl进圈子。 </li><li>通过光镜检查，在每个下拉领域的存在。 </li><li>引擎盖下，让滴干。 </li><li>无论是冻结在-80°C或幻灯片进行免疫荧光染色协议。 </li><li>免疫荧光染色，修复用预冷的甲醇（100％），在-20°C，10分钟的幻灯片。 </li><li>洗涤用PBS幻灯片。 </li><li>座幻灯片胎牛血清PBS/10％/ 0.02％的Triton X100为至少1小时。 </li><li>与初级抗体（ 如抗巢蛋白，Oct4的反，反TG30，抗-Nanog的抗体， 表3），在封闭缓冲液稀释:PBS/10％FBS，在室温下搅拌2小时或过夜在4℃下孵育</li><li>洗3次，用封闭缓冲液，并温育1小时，在室温下与第二抗体。 </li><li>阻断缓冲液和3次，用PBS洗3次。 </li><li>山幻灯片安装介质Vectashield（矢量股份有限公司）。 </li><li>免疫荧光显微镜分析样品的干细胞标记物的表达。 </li></ol> 4。干细胞标记物的表达水平的实时荧光PCR分析<ol><li>约2-3毫升球体培养，从18天到21。 </li><li>颗粒球在5分钟1200转，吸的所有介质。 </li><li>分离RNA用RNeasy MINIKIT（Qiagen公司，瓦伦西亚，CA）领域。 </li><li>评估RNA的纯度，光谱和琼脂糖凝胶。/ LI&gt; <li>合成cDNA（Omniscript逆转录酶（Qiagen公司））使用的细胞总RNA为模板。 </li><li>验证干细胞标记物的干细胞标志物（如表1所示），在一个电源的SYBR Green PCR反应试剂（Applied Biosystems公司），在20微升的浓度为375纳米的每种引物和50 ng的模板的实时PCR用引物反应体积。 GAPDH内对照作为。 </li><li>用于扩增使用的为2.5分钟，然后在95℃下进行40个循环，在95℃初始变性5秒和60℃下持续20秒。 </li><li>分析运行2（ΔΔCT）方法8。 </li></ol> 5。定向分化成平滑肌细胞<ol><li>从21天到26球接种于6孔培养板（BD猎鹰）SKP介质。 </li><li>细胞被允许72小时坚持和长大从SKP媒体领域。 </li><li>平滑肌细胞（校董会）分异离子开始发起的替换介质与SMC分化培养基组成的高葡萄糖Dulbecco氏修改的鹰培养基（Invitrogen公司），含有5％胎牛血清，5 ng / ml的血小板衍生生长因子-BB（Invitrogen公司），和2.5纳克/毫升TGF-B1（Invitrogen公司） 。 </li><li>培养基每3天更换新鲜配制的SMC分化培养基在培养一段时间的3〜4周。 </li><li> SMC标记的筛选3〜4周后进行免疫组化。 </li><li>在PBS中10分钟，用4％多聚甲醛溶液固定细胞。 </li><li>将细胞透性化，用含0.3％的Triton X-100的30分钟。 </li><li>细胞与抗αSMA（MO851，Dako公司，1:100），或者调宁蛋白（Dako公司的M3556，1:100）在室温下1小时和进一步的加工中所描述的3.12）3.15）。 </li></ol>

<ol>
	<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>

我们什么都没有透露。

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。此方法的的从哺乳动物皮肤样品10 SKPS隔离以前报道的方法提供了几个优点。 SKP可以隔绝初级成纤维细胞培养，要么刚建立了从皮肤活检或从初级成纤维细胞培养，已经存在，并可以得到从细胞世界各地的银行。这种新方法提供了可能的成体干细胞研究的含义在皮肤样本不容易获得的各种遗传疾病和罕见疾病的发病机制。最后，这种方法提供了一个窗口的机会，探索成人干细胞生物学和解剖生理和疾病状态时的冲击。总之，这种方法提供了一种新的，快速的方式来隔离皮肤源性前体细胞。

<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（也被称为胚体EB）共享一个类似的生长特性描述以前直接派生SKPS从皮肤样本7 9。 在这里，我们表明，使用方法，我们最近报道7时 ，我们隔离人口CELLS从初级纤维母细胞的多能神经干细胞/前体细胞具有相似的属性，使用神经球培养条件5，10，11在人类和小鼠真皮分离和鉴定。这些SKP来自初级人类成纤维细胞培养自我更新的效力，因为它们可以扩大和传代，至少在期为三个月， 在体外 （请参阅方法部分）。当球体直径在培养后18至21天达到150至200微米大小的，它们被机械地细分成平均直径在50μM，并允许再扩大SKP介质中悬浮培养。这些细分领域被允许成长，直到他们达到200微米前再次传中所描述的方法部分。 SKP微球可传至少三次。本观察结果显示，在该领域内的细胞能够自我更新和扩大神经球培养条件下为o我们和友控股报5，7。 此外，这些SKP来源于成纤维细胞培养细胞表达神经嵴和神经元干细胞的标记物巢蛋白以及胚胎干细胞转录因子Nanog和Oct4和多能性的细胞表面标记物TG30（ 图2），同样地直接来自皮肤的SKP活检5，7，10。免疫组化上SKP领域表示巢在细胞质中的积极信号，10月4日展出打断的核信号和Nanog表现出更为分散的核信号（ 图2）。此外，细胞表面标志物的人多能性胚胎干细胞TG30上的SKP球体制剂（ 图2）给予了积极的胞质膜染色。采用实时荧光定量PCR，我们也证实了存在的mRNA编码巢和Oct4在从SKP领域收集培养18天（ 图3分离RNA制备</stroNG&gt;）。这些结果表明，这些SKP球的细胞内表达的多能干细胞的标记物，如以前报道的是来自于小鼠和人的皮肤活检5,10 SKPS。为了进一步测试的的SKP来自成纤维细胞培养多能，诱导这些细胞分化成平滑肌细胞（ 图4）。诱导培养基中的生长因子PDGF-BB和TGF-β112，3周后，免疫细胞化学证实的的SMC标志物，αSMA，调宁蛋白在来自这些SKPS的平滑肌细胞（ 图4）的表达。 总的来说，这个方法和研究结果表明，从初级成纤维细胞培养分离出的SKP领域有着相似的特性来自皮肤活检的，必须来自人体皮肤的真皮层内的成体干细胞人口居住。 <table border="1"><tbody><tr><td> 名 </td><td> Entrez的ID </td><td> 引物序列 </td><td> 扩增子 </td></tr><tr><td rowspan="2"> GAPDH </td><td rowspan="2"> 2597 </td><td> F-CTC TGC TCC TCC TGT TCG交流</td><td rowspan="2"> 144个基点</td></tr><tr><td> R-TTA AAA GCA GCC CTG GTG交流</td></tr><tr><td rowspan="2">巢</td><td rowspan="2"> 10763 </td><td> F-GCCCTGACCACTCCAGTTTA </td><td rowspan="2"> 200个基点</td></tr><tr><td> R-GGAGTCCTGGATTTCCTTCC的</td></tr><tr><td rowspan="2"> 10月4日</td><td rowspan="2"> 5460 </td><td> F-GAT GGC GTA CTG TGG GCC C </td><td rowspan="2"> 195个基点</td></tr><tr><td> R-TGG GAC TCC TCC GGG TTT TG </td></tr></tbody></table> 表1中。用于实时定量PCR和qPCR引物名单。 <table border="1"><tbody><tr><td> 抗体 </td><td>公司</td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>抗巢</td><td> Chemicon公司</td><td> MAB5326 </td><td> 1/100 </td></tr><tr><td>抗Oct4的</td><td> ABCAM </td><td> ab19857 </td><td> 1/400 </td></tr><tr><td>反TG30 </td><td> Millipore公司</td><td> TG30 </td><td> 1/400 </td></tr><tr><td>抗Nanog的</td><td> ABCAM </td><td> ab21624 </td><td> 1/400 </td></tr><tr><td>抗αSMA </td><td> Dako公司</td><td> MO851 </td><td> 1/100 </td></tr><tr><td>反调宁1 </td><td> Dako公司</td><td> M3556 </td><td> 1/100 </td></tr><tr><td>的Alexa Fluor 555驴抗兔IgG（H + L） </td><td> Invitrogen公司</td><td> A31572 </td><td> 1/800 </td></tr><tr><td>的Alexa Fluor 555驴抗鼠IgG（H + L） </td><td> Invitrogen公司</td><td> A31570 </td><td> 1/800 </td></tr><tr><html><td>的Alexa Fluor 488驴抗鼠IgG（H + L） </td><td> Invitrogen公司</td><td> A21202 </td><td> 1/800 </td></tr><tr><td>的Alexa Fluor 488的驴抗兔IgG（H + L） </td><td> Invitrogen公司</td><td> A21206 </td><td> 1/800 </td></tr></tbody></table> 表3中。抗体来源名单。 <img src="/files/ftp_upload/50185/50185fig1.jpg" alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/50185/50185fig1highres.jpg"/> 图1。概述隔离从初级成纤维细胞培养SKPS。的方法为的SKP球文化开始从初级的真皮成纤维细胞培养，在12代GMO3349C。 0天显示SKP生长培养基中的悬浮液中的成纤维细胞。第4天出现明显的球体形成。第17天显示了一个典型的三维SKP球体。比例尺条:50毫米和100μm的表示。arge.jpg"目标="_blank"&gt;点击这里查看大图。 <img src="/files/ftp_upload/50185/50185fig2.jpg" alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/50185/50185fig2highres.jpg"/> 图2。免疫荧光染色SKP球来自初级成纤维细胞培养，免疫荧光法检测人原代成纤维的文化（GMO3349C）来自SKPS。球体从第16天，在显微镜玻片上沉积，并具有抗巢蛋白，抗TG30，抗Oct4和抗-Nanog的抗体染色。细胞核进行复用DNA DAPI染色（蓝色）。比例尺:20毫米。 <a href="https://www.jove.com/files/ftp_upload/50185/50185fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/50185/50185fig3.jpg" alt="图3" fo:content-width="3.5in" fo:src="/files/ftp_upload/50185/50185fig3highres.jpg"/> <strong&gt;图3。干细胞标记物Nestin和Oct-4的实时荧光PCR分析，分析总RNA孤立的来自SKP领域的来自成纤维细胞GMO8398A在培养18天。 Nestin和Oct-4的相对表达水平相比，亲代成纤维细胞培养所表示。所有的值都为平均值+ / - SD（P &lt;0.05，N = 3）。 <img src="/files/ftp_upload/50185/50185fig4.jpg" alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/50185/50185fig4highres.jpg"/> 图4。平滑肌细胞派生从SKP领域从初级成纤维细胞培养分离 。 SKP-球来自初级成纤维细胞定向分化成平滑肌细胞（校董会）。 （A）相衬成像扼要的步骤，从不同的文化的SKP领域文化向平​​滑肌细胞的分化（上面板）。 （B）平滑肌细胞进行免疫染色来自SKP领域的指示SMC标志，α-斯穆特ħ肌动蛋白（αSMA）和调宁表示。 <a href="https://www.jove.com/files/ftp_upload/50185/50185fig4large.jpg" target="_blank">点击这里查看大图</a> 。

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

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

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 ...

nave-adult-stem-cells-isolation-from-primary-human-fibroblast-cultures

Nanyang Technological University

Research

JoVE Journal

Biology

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

14.1K 次观看.  Technische Universität München. 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。

视频: Naïve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

科研

生物学

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

建立从小学成人成纤维细胞培养鼠害

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

人胰腺癌移植瘤的干细胞的分离

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

快速高密度的人类胚胎干细胞培养成纤维细胞去除

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...