作者感谢提供有用的意见，手稿，丹妮拉泰勒和Margaretha神父hwirth提供技术援助，莫妮卡语言编辑法雷尔，乌韦郎，马尔吉特Holzapfel和桑德拉部Eppich妇产科pHPL，伊娃罗德和妮可霍夫曼卡塔琳娜Schallmoser提供的线。 这项工作一直由奥地利研究基金会（FWF，授予N211南到DS），奥地利研究促进署（FFG，授予N200到DS）和成体干细胞研究基金会（AR）的支持。 AR是博士课程的格拉茨医科大学分子医学研究员。

第1部分：设置 1。细胞培养液的制备在开始培养基的配制之前，收集所有的材料和工具，你需要。 解冻2 × 56毫升分装汇集了人类的血小板裂解（pHPL，自制：参考文献1的Jove＃1523），一个100X青霉素/链霉素溶液，10毫升× 5毫升（L -谷氨酰胺两个西格玛）。 解冻的细胞因子和生长因子等分（血管内皮生长因子，碱性成纤维细胞生长因子，表皮生长因子，胰岛素样生长因子，氢化可的松，抗坏血酸，肝素，两性霉素作为“单一quots”提供），补充EGM - 2培养基（龙沙）1瓶（500毫升）调整。 从冰箱里取一个500毫升瓶装（I）α-修改最小的必不可少的媒介（A - MEM）及（ii）内皮细胞培养基（EBM）。 以下所有步骤都是层流组织培养罩在无菌条件下进行。 准备到终浓度为1000 IU / mL的无菌水溶解的粉末不含防腐剂的肝素（例如，Biochrom）。 MSC介质： 使用500毫升的A - MEM，添加56毫升解冻pHPL（亦见进一步的细节参考1）和2 IU /毫升（= 224μL原液）（避免通过介质凝固结块的防腐剂，无肝素在血浆中的纤维蛋白原），达到了10％pHPL的终浓度。此外，添加青霉素（100U/mL）/链霉素（100μg/mL）解决方案的L - Glutamin 2MM（均为Sigma公司）。 过滤介质，通过一个20微米孔径真空过滤机（Millipore公司）。瓶适当的标签（内容，日期）。 ECFC，中等： 使用EBM的瓶（500毫升），添加细胞因子等分pHPL，10国际单位/毫升（原液1120μl），56毫升的不含防腐剂的肝素，青霉素（100U/mL）/链霉素（100克/毫升）溶液和培养基和20μL孔径真空过滤机（微孔）过滤器的L - 2MM Glutamin。瓶适当的标签（内容，日期）。 2。手术器械的消毒钳，一双锋利的手术剪刀和手术刀持有人必须是热消毒无菌一次性矿石。 3。线集合管的制备 500 IU的防腐剂 - 免费肝素添加50毫升聚丙烯管（猎鹰），并调整到终体积为20毫升，用PBS。转移到产房管。 4。知情同意（由您当地的伦理委员会或IRB确认）。 要求家长在交付之前，如果他们想捐一块的电线，并充分告知他们的目的，它将成为。 5。线捐赠分娩后，胎盘和脐带血检查切断一个10厘米的片，并立即传输到你的管与肝素预充式，以避免其余索血管内血液凝固。尽快继续进行细胞分离。 第2部分：细胞分离<ol><li>准备Incidin或70％乙醇清洗表面层流组织培养罩。放置无菌150厘米2细胞培养板，75厘米的细胞培养瓶（两个康宁），PBS，消毒手术器械，细胞刮削器，管座，25毫升stripettes，5毫升注射器钝端针头清洗油烟机的适当配备。 </li><li>预温您准备的α- MEM和EGM - 2细胞培养液至37 ° C水浴中。 </li><li>把电源线从产房到您的实验室配备了细胞培养罩。转移到层流线后洗两次在PBS（转移到一个新的板块），以获得摆脱污染血细胞。 canulating一方5ML配备一个钝端，用PBS液，直到线的另一端​​出来的针，用无菌注射器冲洗干净后脐静脉变得完全透明（偏红表明血液污染）。 </li></ol> ECFCs： <ol start="4"><li>准备一个75厘米的 2瓶（康宁），标签，并填写预热EGM - 2培养基15-20毫升。 </li><li>放入无菌PBS填充一个新的板块的线。 以手术剪脐带切成约5厘米长的片（小品更容易处理，因为脐部血管沿其轴扭曲）。 </li><li>试着找到脐静脉（大血管），您的手术剪刀插入一个刀片和纵向切开静脉。在这一点上可能会有所帮助，如果助理可以容纳两个钳已经切开静脉，当你进一步削减。 </li><li>将线馅饼行政长官在没有新盘PBS切开静脉，脐静脉内（内皮细胞）从一端到另一面拓片细胞刮刀和刮。细胞传送到您准备的烧瓶，在中期清洁刮板的一角。重复此过程，至少10倍。 </li><li>关闭您的容量瓶中，并把它成一个孵化器（37 ° C，5％的CO 2，湿度95％） 。 </li></ol> MSCS： <ol start="4"><li>准备了一个150平方厘米培养板（康宁）和标签。 </li><li>线片（刮后的内皮细胞层）和整线使用无菌剪刀，无菌手术刀切成非常小的碎片。线片的最大尺寸应该是1-2毫米。 </li><li>转移到您预先标记的培养板无菌镊子线片。离开板打开填充介质的前5分钟，附件件塑料表面得到加强。 </li><li>一旦适当的塑料连接线片，轻轻地添加30-35毫升修改纪念预热阿尔法。尝试使用的最低速度，以确保所有的作品则附加。 </li><li>关闭板，并把它孵化器（37 ° C，5％CO 2，湿度95％） 。 </li></ol>第3部分：细胞扩增和免疫表型流式细胞仪检测确认细胞扩增 EFCFs： <ol><li>检查第二天的内皮细胞附着在一个倒置显微镜和交流整个EGM - 2摆脱所有非贴壁细胞和颗粒的介质。 </li><li>展开，直至汇合和交流的一个中期的三分之一每星期两次的细胞。达到汇合时，0.05％Trypsin/0.7 mM的EDTA分离​​细胞和它们转移到新的培养容器。根据你的T75烧瓶内的细胞数量，你应该选择你的新烧瓶的大小和数量，以保证低播种密度为进一步扩大。 </li><li>交易所三分之一每周两次在扩大细胞的培养基。 </li></ol> MSCS： <ol><li>从线的MSCs生长需要更多的时间，这样你就可以等待至少3-4天，直到你在一个倒置显微镜检查梭形细胞连接的组织块周围的外观。当有足够的出细胞生长，件往往要分离，因为他们失去了与塑料的接触。 </li><li>后10-12天取出组织块，从塑料中分离MSCs的0.05％Trypsin/0.7 mM的EDTA，他们转移到新的培养容器。根据你的盘子内对细胞的数量，你应该决定您的新烧瓶的大小和数量进一步扩大，以保证接种密度低。 </li><li>交易所三分之一每周两次在扩大细胞的培养基。 </li></ol>流式细胞仪所需设备： 流式细胞仪（BD FACSCalibur），micronic管（康宁），管座，羊血清（阻断剂），Eppifuge（Eppendorf公司），单克隆的抗体 MSCS：CD45，CD14，CD19，HLA - DR，CD31，CD73，CD90，C​​D105，同型对照 ECFCs：CD45，CD14，CD19，HLA - DR，CD31，CD34，CD90，C​​D105，CD144，CD146，同型对照<ol><li>达到汇合后，用0.05％Trypsin/0.7 mM的EDTA和离心分离的细胞为7分钟，4300克° C。 </li><li>悬浮细胞浓度1 × 10 6细胞/ mL，在修改的PBS和块用10％V / V羊血清20分钟在4 ° C的非特异性抗体结合中</li><li>标签micronic管，并添加适当浓度的荧光标记抗体。 </li><li>每管添加50μL细胞悬液（封锁），孵育30分钟，在4 ° C。 </li><li>洗净加入修改PBS删除非约束性抗体的细胞。 </li><li>您的标记细胞，流式细胞仪进行分析。 </li></ol>第4部分：ECFC菌落生长，固定，染色和层次分析法<ol><li>种子几乎纯粹的收获非常低的密度每平方厘米3或10细胞在细胞培养板电镀ECFCs允许单菌落生长。 </li><li>交易所三分之一的介质，每周两次。 </li><li>后14天（文化底）去除培养基，用PBS洗两次，并修复与冰冷的固定解决方案（2丙酮：甲醇= 3）的殖民地，在冰箱15分钟。 </li><li>放弃固定的解决方案，并离开板打开干10分钟。 </li><li> 10分钟，用蒸馏水补充水分细胞。 </li><li>添加哈里斯苏木素溶液染色12分钟的固定殖民地。 </li><li>删除H matoxylin解决方案，并用自来水冲洗板摆脱其余染色液。 </li><li>采取每个殖民地的照片使用立体显微镜和导入*. jpg或*. TIF格式的文件到您的计算机。 </li><li> ImageJ软件打开照片和动画中的描述分析的准确的细胞数，每个殖民地。 </li><li>打开ImageJ软件，并在下拉菜单中使用“文件\打开”功能导入一个殖民地的形象。 </li><li>继续使用“过程\减去背景”功能</li><li>使用ImageJ菜单中的“选择*椭圆*或刷”功能，以纪念殖民地保证金。 </li><li>清除周围的图像，使用“编辑\清除外”的功能和作物的图片，选择“图像\作物。 </li><li>使用“图像手动调整阈值\调整\门槛”的功能，使所有的细胞都完全红色。 </li><li>通过选择“分析\分析粒子的计数殖民地的细胞数。选择适当的设置（针对不同的细胞类型进行了优化），并选择“确定”。结果框，包括细胞计数和一个新的形象包含的计数细胞相应的轮廓会出现。 <img src="/files/ftp_upload/1525/1525op2.jpg" alt="图1" /></li></ol>

<ol>
	<li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+can+replace+fetal+bovine+serum+for+clinical-scale+expansion+of+functional+mesenchymal+stromal+cells.">Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.</a> Transfusion. 47, 1436-1446 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+system+to+propagate+cord+blood-derived+multipotent+mesenchymal+stromal+cells+for+clinical+application.">Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application.</a> Regen Med. 2, 371-382 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. 113, 6716-6725 (2009).</li><li>Strunk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+and+preclinical+production+of+human+lineage-negative+cells+for+regenerative+stem+cell+therapy.">Phenotypic characterization and preclinical production of human lineage-negative cells for regenerative stem cell therapy.</a> Transfusion. 45, 315-326 (2005).</li></ol>

作者没有财务申报任何利益冲突。

有一个从中获取干细胞或ECFCs包括骨髓，脂肪组织，脐带血，等各种来源从相同的源祖细胞两种类型直接比较的样组织和血管再生或肿瘤进展的贡献过程中的不同类型的细胞参与，这是必要的。 隔离，并随后从同一供体研究ECFCs和干细胞自体对缓解使得这种方法排除在个别实验的捐助变化的优势。 与造血干细胞可能造成的污染降到最?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>alpha modified MEM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-GLutamin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EBM</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Single cytokine quots</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>75 cm2 flasks</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>150 cm2 plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cell scraper</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin/EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Monoclonal antibodies</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>500 mL filter 20 &mu;m pore size </td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Preservative free heparin</td>
<td>&nbsp;</td>
<td>Biochrom</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and animal serum free expansion of human umbilical cord derived mesenchymal stromal cells (mscs) and endothelial colony forming progenitor cells (ecfcs)

脐带是一种具有高增殖潜能，包括间质的基质干细胞（也称为间质干细胞，MSCS）和内皮细胞集落形成祖细胞（ECFCs）的祖细胞的丰富来源。这两种类型的细胞，在维持组织的完整性关键人物，并可能也参与再生过程和肿瘤的形成。 要比较的方式研究它们的生物学和功能，它是重要的，都从同一供体细胞类型。这也可能是来自同一组织的干细胞和ECFCs，有利于再生的目的。 由于细胞疗法，最终找到自己的方式从替补到床边，我们建立了一个新的方法来隔离和进一步扩大不使用动物性蛋白质的祖细胞。在我们的协议中的所有步骤汇集人类血小板裂解液（pHPL）所取代胎牛血清完全避免接触细胞异种蛋白质。 该视频演示了从一个脐带祖细胞的分离和扩张的方法。 所有材料和程序进行说明。

Watch this Scientific Journal Video about Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

这个协议描述不使用动物血清中产生自体对实验移植的目的的间质基质细胞和内皮细胞集落形成细胞的分离和随后的扩大。

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

isolation-animal-serum-free-expansion-human-umbilical-cord-derived

Microsoft Bing

Research

JoVE Journal

Biology

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

15.3K 次观看.  Medical University of Graz, Austria. 这个协议描述不使用动物血清中产生自体对实验移植的目的的间质基质细胞和内皮细胞集落形成细胞的分离和随后的扩大。

视频: Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

分离人脐静脉内皮细胞（HUVEC）

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

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 ...

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

分离和富集大鼠间质干细胞（MSCS）及衍生的干细胞分离单细胞集落

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

人脐动脉平滑肌细胞的分离（HUASMC）

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

免费接驳适应，文化和传代使用完整的基因敲除血清替代馈线中的人类iPS细胞

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

来自人类脐带血内皮细胞集落形成细胞的表型和功能特性

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

米色/布里特细胞的间质血管细胞的分离和分化

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146的分离<sup&gt; +</sup&gt;从大鼠肺组织驻地肺间质基质细胞

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...