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?...

<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 at JoVE.com

Na&amp;#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

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

14.0K 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: Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

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 ...

科研

生物学

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 ...

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 ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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 ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

从人类Lipoaspirates脂肪来源干细胞的手动隔离

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...