我们感谢，仁泷泽伸之Onai和Markus曼茨宝贵意见。这项工作是由瑞士国家科学基金会，瑞士癌症联盟的Fondazione Ticinese每找实习SUL Cancro。

1。从骨髓细胞悬液的制备<ol><li>安乐死鼠标放置在一个不锈钢锅和喷70％的乙醇，将其腹部和背部的动物。收集后腿和胫骨股骨，脊柱。 </li><li>从脊柱准确地清除所有的软组织残留，用锋利的尖剪刀，从腿的骨头用纱布。在50毫升猎鹰管店骨头与30毫升的RPMI培养基含10％热灭活胎牛血清（FBS），血清，50μM的β-巯基乙醇，100μM的的MEM非必需氨基酸的补充，1毫米的MEM丙酮酸钠，2毫米L-谷氨酰胺，100微克/ ml链霉素和100 U / mL青霉素。 </li><li>覆盖无菌过滤器，先前消毒迫击炮的底部。 </li><li>迫击炮转移到骨骼和他们以组装摩擦表面覆盖层的过滤器。 </li><li>准备一个50毫升的隼管与猎鹰过滤器提供。 </li><li>粉碎的骨头一杵，直至获得均匀的细胞悬液的帮助。 </li><li>转移到管悬挂。 </li><li>添加到砂浆的5毫升新鲜的RPMI完全培养基洗和收获剩余的细胞。 </li><li>重复最后两个点，直到出现明确的同质化骨。 </li><li>转让和筛选，在一个新的管细胞悬液，以消除剩余的组织碎片。 </li><li>在5分钟1500转离心管和吸小心取出上清液。确保不扰乱细胞沉淀。 </li></ol> 2。表型分析<ol><li>悬浮和个别小鼠的细胞在15毫升和2％FBS的PBS洗。 </li><li>在5分钟1500转离心管和吸小心取出上清液。确保不打扰沉淀。 </li><li>暂停在5毫升PBS和2％FBS和计数细胞沉淀。 </li><li>转移1×10 6细胞/ 96井圆底板。 </li><li>离心5分钟1500转板。 </li><li>准备了针对以下的表面标志物的单克隆抗体的组合。稀释他们表示在PBS和2％FBS染色细胞。 <ul type="disc"><li> CD4，CD8细胞，CD3 +，CD45R，CD19 +，GR1，Ter119，NK1.1结合的PE-Cy5的1:200 </li><li> C-套件结合APC的1:100 </li><li> SCA1结合生物素1:50 </li><li> CD34的共轭FITC标记1:50 </li><li> FcγR结合，以体育为1:100 </li><li> IL-7R共轭的PE-Cy7 1:100 </li></ul></li><li>弃上清，每孔加入50μL的单克隆抗体组合。吹打成单个细胞的分离。 </li><li>细胞在4°C孵育20分钟，在黑暗中</li><li> 150μL的PBS和2％FBS洗涤细胞两次。 </li><li>离心5分钟1500转板和丢弃上清液反相板。 </li><li>在PBS稀释链亲和2％FBS，表示染色细胞。 <ul type="disc"><li>链霉亲和共轭门来的APC-Cy7 1:300 </li></ul></li><li>加入50μL的解决方案，以每口井。吹打成单细胞悬液分离。 </li><li>在4°C孵育10分钟，在黑暗中的细胞</li><li> 150μL的PBS和2％FBS洗涤细胞两次。 </li><li>板和5分钟1500转离心，弃上清。 </li><li>重悬于100μLPBS和2％FBS的细胞。 </li><li>购入流式细胞样本。 </li></ol> 3。 C-kit +细胞的磁富集<ol><li>准备一个缓冲液PBS，pH值7.2，0.5％的牛血清白蛋白（BSA），2毫米EDTA。 </li><li>悬浮颗粒骨骨髓细胞来自至少10 1毫升缓冲含c-kit的抗体结合APC的解决方案中的小鼠稀释1:50。 </li><li>细胞在4°C孵育20分钟，在黑暗中</li><li> 40毫升缓冲液洗两次细胞。 </li><li> 5分钟1500转离心管，小心取出ţ他上清愿望。确保不打扰沉淀。 </li><li>重悬细胞沉淀，并添加每个安乐死鼠标50μL的反装甲运兵车微珠直接沉淀。 </li><li>拌匀，并在4°C孵育20分钟，在黑暗中</li><li>清洗细胞，加入40毫升缓冲溶液中，离心5分钟1500转。完全吸出上清液。 </li><li>重悬细胞沉淀在每三个老鼠安乐死的缓冲溶液4毫升多个。 </li><li>地方磁珠在磁场一个合适的磁珠分离LS列。使用1列，每三只老鼠安乐死。 </li><li>准备用4毫升缓冲溶液冲洗列。 </li><li>细胞悬液涂于列。 </li><li>收集未标记的细胞，通过与4毫升缓冲液洗柱。履行加入缓冲溶液4毫升三次洗涤步骤。添加新列时，水库清空缓冲区。 </li><li>删除列的分隔和PLAC北京时间15毫升猎鹰管上每个人。 </li><li>吸取4 mL的缓冲溶液到每列。立即冲洗掉柱塞推入列磁标记细胞。 </li><li>在5分钟1500转离心管和吸小心取出上清液。 </li><li>每1毫升缓冲溶液中和一个15毫升的猎鹰管组颗粒悬浮沉淀。 </li></ol> 4。造血干细胞和传承的承诺祖排序<ol><li>准备了针对以下的表面标志物的单克隆抗体的组合。稀释他们表示，在500μL的PBS和2％FBS染色细胞。 <ul type="disc"><li> CD4，CD8细胞，CD3 +，CD45R，CD19 +，GR1，Ter119，NK1.1结合的PE-Cy5的1:200 </li><li> C-套件结合APC的1:100 </li><li> SCA-1结合到生物素1:50 </li><li> CD34的共轭FITC标记1:50 </li><li> FcγR结合，以体育为1:100 </li></ul></li><li>离心管的上一页s节在5分钟1500转，并小心地取出吸上清。 </li><li>添加单克隆抗体混合颗粒。吹打成单细胞悬液分离。 </li><li>细胞在4°C孵育20分钟，在黑暗中</li><li>洗两次细胞的PBS和2％FBS 14mL。 </li><li>在5分钟1500转离心管，弃上清。 </li><li>稀释链霉表明，在500μLPBS和2％FBS染色细胞。 <ul type="disc"><li>链亲和素结合的APC-Cy7 1:300 </li></ul></li><li>加入该管的解决方案。吹打成单细胞悬液分离。 </li><li>在4°C孵育10分钟，在黑暗中的细胞</li><li>洗两次细胞14毫升PBS和2％FBS。 </li><li>在5分钟1500转离心管，弃上清。 </li><li>重悬于500μLPBS和2％FBS的细胞。 </li><li>传输和过滤在一个新的5 mL管细胞悬液，以ELIminate细胞的集合体，可以干扰与排序程序。 </li><li> 7-AAD的5μL（使用稀释1:100）加入细胞悬液，以7-AAD的+死细胞排除排序人口。 </li><li>与BD FACSAria排序根据以下表型的细胞: <ul type="disc"><li>李嘉诚细胞:林- C-KIT + SCA-1 + </li><li> LT-造血干细胞:CD34的李嘉诚- </li><li> ST-造血干细胞:CD34的李嘉诚+ </li><li>绿化总纲图:林- C-KIT + SCA-1 低 FcγRCD34的高+ </li><li> MEPS:林- C-KIT + SCA-1 低 FcγR 低 CD34的- </li><li>中医:林- C-KIT + SCA-1 低 FcγR 低 CD34的+ </li></ul></li></ol> 5。代表结果 图1显示，例如电子浇注过程得分淋巴共同祖（CLPs）林- C-KIT / LO /白细胞介素（IL）-7R +细胞;林- / C套件+ / SCA-1 +（LKS）和林- / C-KIT + / SCA 劳 -1（C-KIT +）细胞; C-套装+门，共同髓系祖细胞（中医）确定为FcγR 劳 CD34 +细胞，粒细胞/单核细胞祖细胞FcγR 您好 CD34 +细胞和巨核细胞/（绿化总纲图） FcγR 劳 CD34的红细胞祖细胞（MEPS） -细胞;从李嘉诚门，LT-HSC和ST-HSC的被确定为CD34的-和CD34 +细胞分别。可以丰富的骨髓细胞悬液，C-kit +细胞与磁珠（ 图2）; LT-HSC和ST-造血干细胞，然后可以根据CD34的表达，以及其他祖排序根据以上所述的表型。 造血干细胞可以是全日空<html>在现场共聚焦显微镜成像lyzed显示在图4中 ，其中CD34的-细胞染色与核苷酸结合的复合奎纳克林11以及核红（DRAQ5）。在造血干细胞，但没有绿化总纲图，奎纳克林阳性囊泡可见细胞质中的12个月 。此外，排序的造血干细胞可用于功能的实验，图5表明，这表明在胞内Ca 2 + LT-HSC的嘌呤P2受体激活后，接触的增加，腺苷三磷酸（ATP）和离子霉素12。 <img alt="图1" src="/files/ftp_upload/3736/3736fig1.jpg" /> 图1。代表点积林骨髓细胞分析。电子门控过程识别和取得的造血祖细胞和造血干细胞从骨髓细胞悬液，流式细胞仪染色，在协议文本描述。 pload/3736/3736fig2.jpg"/&gt; 图2:C-KIT +骨髓细胞的选择性富集的计划。 <img alt="图3" src="/files/ftp_upload/3736/3736fig3.jpg" /> 图3门李嘉诚电子CD34的细胞周期分析-文67抗体和DAPI染色与鸡传染性法氏囊病的健康对照组和小鼠造血干细胞。固定细胞裂解/修复和权限与FITC标记的Ki-67的和DAPI染色，在1微克/毫升的缓冲区透。骑自行车细胞几乎没有，如果在所有李嘉诚CD34的检测-造血干细胞车厢的12名健康对照。 <img alt="图4" src="/files/ftp_upload/3736/3736fig4.jpg" /> 图4实时成像流式细胞仪排序CD34的共聚焦显微镜-造血干细胞和核苷酸约束力的化合物的奎纳克林及核红（DRAQ5）染色的绿化总纲图。小象限显示奎纳克林POSitive囊泡检测造血干细胞的细胞质中，但没有绿化总纲图。 <img alt="图5" src="/files/ftp_upload/3736/3736fig5.jpg" /> 图5。胞浆Ca 2 +在海拔排序加载的Fura-2与ATP和ionomycin刺激造血干细胞。从单细胞的痕迹。细胞外ATP除了ATP的胞内Ca 2 +后显着增加的响应。

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没有利益冲突的声明。

这里介绍的方法能够快速，准确的分析（ 图1）个别小鼠造血。在分析各种实验设置，包括小鼠模型炎症，自身免疫性疾病，免疫缺陷，退行性疾病，代谢性疾病和癌症，允许解决的病理条件下对造血功能的影响。 图3显示了电子门李嘉诚分析细胞周期活动 CD34 -从健康小鼠的细胞和小鼠炎症性肠病（IBD）的13 Ki-67的人与生物圈计划和DAPI染色。后者显示，在细胞周期的S / G2 / M期细胞显着增加。流式细胞仪分析表明T细胞介导的炎症影响HSC单车活动12。 磁标签和富集骨髓细胞与细胞表达C-套件组合排序流cytometrŸ允许高度纯化造血谱系祖细胞和造血干细胞的分离。获得的细胞群可以使用的检测数量，来研究细胞生物学（ 图4），信号转导（ 图5），发育调控基因表达， 在体外以及体内不同的细胞亚群的功能潜力。事实上，磁丰富的C-kit +细胞成功嫁接的非辐照的主机被用来研究在稳态和炎症14造血干细胞单车活动的动态变化。这里介绍的过程，在我们的手中，可以更加高效，快速，比平均产量200,000李嘉诚小鼠细胞冲洗骨骨髓细胞的恢复。极少数的静态，可以汇集多个小鼠骨髓中分离造血干细胞的实验方法，需要更多的细胞构成的限制。此限制，以避免在初步许多情况下，更丰富的李嘉诚细胞进行研究，可以设想更有针对性和准时分析在CD34 -静态造血干细胞。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> RPMI 1640培养</td><td> Gibco公司</td><td> 42401 </td></tr><tr><td>的MEM NEAA 100X </td><td> Gibco公司</td><td> 11140 </td></tr><tr><td>丙酮酸钠</td><td> Gibco公司</td><td> 11360 </td></tr><tr><td> PenStrep </td><td> Gibco公司</td><td> 15070 </td></tr><tr><td> PBS </td><td> Gibco公司</td><td> 20012 </td></tr><tr><td>胎牛血清</td><td> Gibco公司</td><td> 16000 </td></tr><tr><td>细胞过滤40微米</td><td>屋宇署猎鹰</td><td> 352340 </td></tr><tr><td> 7-AAD的染色溶液</td><td>屋宇署Pharmingen公司</td><td> 559925 </td></tr><tr><td>裂解/修复缓冲区</td><td>屋宇署Pharmingen公司</td><td> 558049 </td></tr><tr><td>烫发缓冲区III </td><td>屋宇署Pharmingen公司</td><td> 558050 </td> &lt;/ TR&gt; <tr><td> Ki-67的</td><td>屋宇署Pharmingen公司</td><td> 556026 </td></tr><tr><td>的DAPI </td><td> Invitrogen公司</td><td> D21490 </td></tr><tr><td> CD4（GK1.5） </td><td> eBioscience </td><td> 150041 </td></tr><tr><td> CD8（53-6.7） </td><td> eBioscience </td><td> 150081 </td></tr><tr><td> CD3（145-2C11） </td><td> eBioscience </td><td> 150031 </td></tr><tr><td> CD45R（，RA3-6B2） </td><td> eBioscience </td><td> 150452 </td></tr><tr><td> CD19的（6D5） </td><td> eBioscience </td><td> 150193 </td></tr><tr><td> GR1（的RB6-8C5） </td><td> eBioscience </td><td> 155931 </td></tr><tr><td> tre119（的TER-119） </td><td> eBioscience </td><td> 155921 </td></tr><tr><td> NK-1.1（PK136） </td><td> eBioscience </td><td> 455941 </td></tr><tr><td> C-包（2B8） </td><td> eBioscience </td><td> 171171 </td></tr><tr><td> （SCA-1的D7） </td><td> eBiosciENCE </td><td> 135981 </td></tr><tr><td> CD34（RAM34） </td><td> eBioscience </td><td> 110341 </td></tr><tr><td> FcγR（2.4G2） </td><td> eBioscience </td><td> 553145 </td></tr><tr><td>反装甲运兵车微珠</td><td>美天旎生物技术</td><td> 130-090-855 </td></tr><tr><td>的LS列</td><td>美天旎生物技术</td><td> 130-042-401 </td></tr></tbody></table>

phenotypic analysis and isolation of murine hematopoietic stem cells and lineage-committed progenitors

骨髓造血干细胞和更成熟的血细胞谱系祖居住在成年生物体和分化的主要站点。造血干细胞构成的多能干细胞能够产生所有的血细胞谱系的生命时间1分钟的细胞群。在骨髓中的造血干细胞稳态的分子解剖，具有造血，肿瘤学和再生医学的重要意义。我们描述了荧光抗体和流式细胞仪，以取得造血祖细胞亚群及个别小鼠造血干细胞的分布（ 图1）在电子门控过程的标签协议。此外，我们描述了一个方法，广泛丰富造血祖细胞，以及长期（LT）和短期（ST）的重组细胞表达c-kit的磁富集的造血干细胞从汇集的骨髓细胞悬液。由此产生的细胞制剂可用于选定的子集，并在体外进行排序在体内的功能性研究（ 图2）。 2,3都小梁成骨细胞和肝窦内皮细胞4构成支持骨髓中的造血干细胞的功能龛。几个机制中的成骨细胞的利基，包括N-cadherin的成骨细胞3子集和Tie2的表达及其配体血管生成素1 5造血干细胞的受体酪氨酸激酶的相互作用同意确定造血平静。在骨髓中的"休眠"是至关重要的，以保护造血干细胞复制和最终用尽后，过多的骑自行车活动6。外源性刺激作用于细胞的先天免疫系统，如Toll样受体配体7和α-干扰素6也将沿袭承诺祖细胞诱导的造血干细胞的增殖和分化。最近，林内的人口处于休眠状态的小鼠造血干细胞- C-KIT + SCA-1 + CD 150 + CD48 - CD34 -人口已8。细胞排序从此处所述的造血祖细胞富集的细胞悬液，CD34的表达，可以隔离两个静态的自我更新的LT-HSCs和ST-造血干细胞9。一个类似的过程基于血统阳性细胞的枯竭和LT-HSC的排序与CD48和Flk2抗体的先前已描述10。在本报告中，我们提供了一个协议表型特征和体外造血祖细胞，它可以用于监测在不同的生理和病理条件下的造血细胞周期分析。此外，我们描述了流式细胞仪分选造血干细胞的过程，它可以用来定义调节他们的自我更新，在细胞生物学和信号转导检测，以及用于移植的扩张和分化的因素和机制。

Watch this Scientific Journal Video about Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors at JoVE.com

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

描述的方法来分析流式细胞仪检测骨髓造血祖细胞的分布以及有效地隔离高度纯化造血干细胞（HSCs）。基本上是基于C-kit +细胞和排序，以净化造血干细胞和分子生物学研究细胞的磁富集分离过程。

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

phenotypic-analysis-isolation-murine-hematopoietic-stem-cells-lineage

Nanyang Technological University

Research

JoVE Journal

Biology

18.6K 次观看.  Bellinzona (Switzerland). 描述的方法来分析流式细胞仪检测骨髓造血祖细胞的分布以及有效地隔离高度纯化造血干细胞（HSCs）。基本上是基于C-kit +细胞和排序，以净化造血干细胞和分子生物学研究细胞的磁富集分离过程。

视频: Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

分离和造血干细胞移植（造血干细胞）

科研

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

小鼠卵黄囊和AGM早期造血干细胞的分离

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

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, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

分离，培养和移植肌卫星细胞

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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 ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

小鼠胚胎的逆转录病毒感染干细胞衍生胚体细胞造血分化的分析

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...