We thank Dr. Leonid Gnatovskiy for his carefully and critical reading of the paper. This work was supported in part by grants from the National Institutes of Health (HL109054) and the Samuel and Jean Frankel Cardiovascular Center, University of Michigan (Inaugural Fund) to WZ and from the Leon H Charney Division of Cardiology, New York University School of Medicine to BL.

从鼠标胚胎干细胞衍生1.小鼠胚胎的每次点击成本<ol><li>准备小鼠胚胎成纤维细胞（MEF中）饲养层。 <ol><li>暖MEF培养基（在DMEM 10％胎牛血清）至37℃。 </li><li>准备明胶涂层板。 <ol><li>加入1毫升水的0.1％明胶的成井的6孔板或5毫升到10cm的培养皿中。 </li><li>离开，在37℃的板或盘或室温下至少30分钟。渴望在使用前明胶。 </li></ol></li><li>解冻在37℃水浴中照射的MEF很快，轻轻摇动。 </li><li>轻轻转移细胞到15ml或50ml锥形管中。加入5 ml预热的MEF培养基。漩涡的细胞缓慢和离心，在200×g离心5分钟。 </li><li>重悬的细胞在MEF培养基和计数存活细胞。从10cm的培养皿2毫升每孔的细胞从6孔板和10毫升的细胞：使用MEF培养基的下列卷。 </li><li>板细胞向6孔板或10 cm的培养皿在1-2×10 6每盘/碟。 </li><li>孵育MEF平板/菜在37℃下，在使用前，5％CO 2的至少24小时。 注：放弃MEF板/超过一个星期菜。 </li></ol></li><li>准备鼠标胚胎干细胞<ol><li>文化Isl1-CRE; Rosa26YFP / MEF2C-CRE; Rosa26YFP鼠胚胎干细胞对MEF中，直到90％汇合。利用ES细胞培养基由410毫升DMEM，75毫升FBS，0.1mM的MEM NEAA，2mM的L-谷氨酰胺，0.1mM的丙酮酸，100U青霉素/链霉素和0.1mMβ巯基乙醇。 </li><li>如1.1.2中所述制备明胶包被的6孔板中。 </li><li>从板块吸中，冲洗细胞DPBS。 <ol><li>抽吸DPBS添加0.4毫升0.25％胰蛋白酶进6孔板一个孔。孵育细胞在37℃，5％的CO 2为5分钟。加入1毫升预热的ES培养基以停止胰蛋白酶反应。 </li></ol></li><li>收获细胞和细胞离心机在200 XG和抽吸介质。重悬的细胞在1ml ES细胞培养基，并计数细胞。&lt;/ li&gt; <li>板胚胎干细胞在明胶包被的板在3×10 4个/ cm 2的密度。孵育在37℃，5％CO 2的约2天，当胚胎干细胞达到约90％汇合。媒体每天都在发生变化。 </li><li>当胚胎干细胞是90％汇合，重复步骤1.2.2-1.2.5彻底删除MEF饲养。 </li></ol></li><li>中共诱导分化形成EB <ol><li>制备分化培养基如下：36毫升IMDM，12毫升火腿的F12，2mM的L-谷氨酰胺，0.34毫升BSA（7.5％），加入0.25ml N 2，0.5毫升B27，50微克/毫升的AA，和0.45毫摩尔1-硫代甘油。 </li><li>从细胞中吸中，用清水冲洗DPBS。抽吸DPBS添加0.4毫升0.25％胰蛋白酶进6孔板一个孔。孵育在37℃下5分钟。 </li><li>加入1毫升分化培养基停止反应和移液器上下分散细胞簇为单细胞。 </li><li>在200×g离心5分钟，并弃去培养基离心的胚胎干细胞。 <ol><li>重悬1×10 6 &lt;/ SUP&gt; 1毫升分化培养基的胚胎干细胞。计数细胞，并培养所述细胞在低脱离培养皿以1×10 5细胞/在分化培养基ml的浓度，在37℃下进行第一次电子束的聚集。 </li></ol></li><li> EB形成后两天，从菜传输EBS公司50ml试管。旋转，在200×g离心1分钟。除去培养基和冲洗胚用10毫升DPBS不含Ca 2+和Mg 2+。 </li><li>吸DPBS并加入1 ml 0.25％胰蛋白酶。孵育在37℃下5分钟。添加4.5毫升分化培养基以停止反应。吸管上下解离胚成单个细胞。 </li><li>离心细胞，在200×g离心5分钟。吸中型和悬浮细胞1毫升分化培养基。 </li><li>计数细胞，并稀释为2×10 6个细胞至1×10 5个细胞/在分化培养基毫升。添加VEGF，BMP4和激活素A的培养基中在5毫微克/毫升，0.8纳克/毫升最终浓度，并5毫微克/毫升。丘尔再将细胞在培养皿中用于第二EB形成在37℃。 </li><li>第二个EB形成后的40小时，转移到胚50ml试管。用DPBS冲洗一次，游离的EB与1ml 0.25％胰蛋白酶在37℃下5分钟。吸管上下解离胚成单个细胞。 </li><li>将细胞重悬于Stempro-34培养基与5毫微克/毫升VEGF，10ng / ml的bFGF和12.5毫微克/毫升的FGF10。板中的细胞以2×10 5 / cm 2的在明胶包被的平板上。文化在37℃的孵化器前，中共中央隔离约32小时。 </li></ol></li><li>隔离mCPCs <ol><li>吸中，用清水冲洗DPBS。添加0.5毫升0.25％胰蛋白酶培养板在37℃下5分钟。添加4.5毫升分化培养基以停止反应。吸管上下解离胚成单个细胞。收集的细胞通过离心和重悬细胞的2％FBS / PBS用于FACS纯化。 </li><li>运行在分拣机作为阴性对照未分化的胚胎干细胞。改变电压来调节前进SCAT之三（FSC）和侧向散射（SSC），选择所需的人口。使用前向散射与侧向散射和前向散射的高度与宽度，以排除碎片和双峰。在所选择的子群，根据ESC控制的分布，绘制YFP的栅极上直方图正，以消除细胞的自体荧光。收集YFP +门YFP +的每次点击费用。 </li><li>板中的纯化的每次点击费用（YFP +细胞）在6孔板用分化培养基（1.3.1）。文化额外的3-5天在37℃分化的每次点击费用为心肌和平滑肌细胞。 </li></ol></li></ol>从人类胚胎干细胞衍生2.人类胚胎的每次点击成本<ol><li>准备给料机<ol><li>制备小鼠胚胎成纤维细胞（MEFs）饲养层作为以2×10 4 / cm 2的密度如上所述。 </li></ol></li><li>维持人类胚胎干细胞<ol><li>维持人类ISL1：CRE;对并购pCAG-FLOX-STOP-FLOX-GFP或RFP常规胚胎干细胞EF使用常规的hESC培养基（敲除DMEM / F-12385毫升，敲除的SR 100毫升，2毫摩尔L-谷氨酰胺，0.1mM的NEAA，0.1毫β巯基乙醇，10纳克/毫升碱性FGF）。之前心肌分化，在无饲养条件为至少2次传代转移人类胚胎干细胞。 </li></ol></li><li>准备在无饲养条件下人类胚胎干细胞<ol><li>准备涂层板为人类胚胎干细胞的文化。 <ol><li>解冻基质胶在4℃下过夜。放50ml管在冰上，加入30毫升冷的DMEM / F12培养基到管。 </li><li>加入1毫升基质胶放入冷水中，拌匀。添加1ml冷基质胶的DMEM / 12培养基成良好的6孔板或5毫升到10cm的培养皿中。 </li><li>离开板/菜在4℃过夜或室温下搅拌2小时。抽吸培养基在使用前。 </li></ol></li><li>分裂人类胚胎干细胞到板：吸媒体从MEF板和冲洗人类胚胎干细胞用DPBS。然后加入1 ml分散酶（1毫克/毫升）于6孔板的孔中。孵育细胞在37℃孵化器，直至菌落边缘开始脱落。 </li><li>除去中性蛋白酶的解决方案。用DPBS冲洗两次，然后加入2.5毫升/每孔mTeSR培养基或MEF条件培养基的板上。 </li><li>刮去使用细胞刮刀细胞，并仔细吸管上下打破人类ESC集落成小团块。 </li><li>传送ESC团块并稀释1：3到包被的板。 </li><li>加入2毫升/ mTeSR中等或MEF条件培养基，改变媒体每天。 </li><li>培养人类胚胎干细胞在mTeSR培养基中或在包被的板MEF条件培养基中，至少2次传代。 </li></ol></li><li>诱导人类胚胎干细胞分化CPC <ol><li>当细胞是〜90％汇合时，吸出介质并冲洗用DPBS。然后孵育在0.5毫克/毫升分散酶的人类胚胎干细胞在37℃下20分钟。 </li><li>冲洗DPBS的人类胚胎干细胞的两倍，从板与细胞升降去除细胞，然后悬浮在分化培养基细胞夹（含18％FBS，0.1毫米NEAA，2毫米DMEM / F12L-谷氨酰胺，0.1mM的β巯基乙醇和50微克/毫升抗坏血酸）。 </li><li>转移细胞进入6孔超低附着板的EB形成。 </li><li>第二天，改变分化培养基。 </li><li>更换培养基，每3天，并在悬浮培养中维持的EB。 </li></ol></li><li>隔离HCPCS <ol><li>最迟分化为人类CPC隔离9天收集胚。 </li><li>吸媒体和冲洗胚与DPBS两次。添加0.5毫升0.25％胰蛋白酶至6孔培养板每孔中，在37℃进行5分钟。 </li><li>加入分化培养基的体积相等，使反应停止。吸管上下解离胚成单个细胞。收集细胞通过离心机在200×g离心5分钟，并悬浮细胞在2％FBS / DPBS。过滤单元与40微米的细胞过滤和FACS纯化的RFP + CPC细胞中1.4.3所述。 </li><li>板排序HCPCS到geltin包被的平板。在7分化培养基文化HCPCS-9天分化成心肌细胞和平滑肌细胞。电镀后第7天，培养分化的细胞在DMEM / F12培养基的5％敲除的SR。 </li></ol></li></ol>

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The authors declare no competing financial interests.

This protocol combines a method using growth factors to guide mESCs differentiation and spontaneous differentiation of human ESCs into CPCs. CPC lineage marked with fluorescent reporter is used to efficiently identify and isolate CPCs by FACS. The FACS-purified CPCs retain the capacity to differentiate into cardiomyocytes, smooth muscle, and endothelial cells and have a comparable expression profile to the in vivo cells. Thus, these CPCs can serve as a great resource for cell based heart therapy because of their ability ...

A correction to the author list was made to: <a href="http://www.jove.com/video/52047/derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells">Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells</a>.
The following author is now listed as a co-corresponding author:
Lei Bu

A correction to the author list was made to: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells.

Erratum: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

心脏疾病仍然是世界上领先的原因今天和死亡率在过去二十年（美国心脏协会）大致维持不变。有用于开发新的治疗策略，以有效地防止或逆转心脏衰竭的重要需求。一个有希望的战略是基于细胞治疗造血干细胞生物学1的快速发展。在这点上，多能每次点击费用可能是一个很好的细胞来源的治疗，由于其增殖，但致力于仅心脏谱系分化的能力。因此，有效和可靠的方法来产生和分离的每次点击费用是对心脏细胞疗法的研究非常重要的。 该协议的重点是在早期胚胎发育，以及如何他们这一代人从胚胎干细胞识别胚胎的每次点击费用。不同的每次点击费用已分离出胚胎和成人的心，甚至从骨髓2。在胚胎发育，骨骼morphogeNETIC蛋白（BMP），无翅型MMTV整合位点家族成员（Wnts）和节点信号诱导Mesp1 +多能胚层3的承诺。 Mesp1 +细胞然后分化成胚胎每次点击费用4。这些每次点击费用通常标记HCN4，NK 2同源框5（NKX2-5），ISL LIM同源框1（Isl1），T型箱5（Tbx5中），和肌细胞增​​强因子2C（MEF2C），形成初级和第二心脏领域，并心脏发生5-10时导致心脏的主要部分。既NKX2-5 +和Isl1 + / + MEF2C每次点击费用是能够分化成心肌细胞，平滑肌细胞（SMC）和内皮细胞5-8。因此，这些每次点击费用会引起心脏血管系统以及心脏组织并且是一种理想的细胞源细胞系心脏疗法。因此， 在体外产生的每次点击成本一直是一大研究热点心血管研究。由于胚胎干细胞具有无限扩展能力次代表ICM细胞的囊胚阶段，下面自然胚胎发育胚胎干细胞分化成胚胎的每次点击成本被认为是获得每次点击费用的合理和有效的方法。 一个被广泛应用的方法，从胚胎干细胞获得的每次点击成本是胚胎干细胞聚集成胚11。以提高分化效率的基础上，心脏发育的知识定义的化学和生长因子已被用于12-14。然而，没有明确的中共标记，尤其无细胞表面标志物，其已被广泛接受的领域。为了解决这个问题，胚胎干细胞被设计为纪念Isl1 +或MEF2C +每次点击费用和他们使用的Cre / loxP系统荧光记者衍生物。 Cre重组酶是Isl1 / MEF2C子/增强子的控制下，在敲。改性荧光蛋白RFP或YFP基因由组成型启动子驱动的可通过FLOX终止密码子与Cre重组酶切除被激活（ISL1：CRE; pCAG-FLOX-STOP-FLOX-GFP或RFP / Isl1-CRE; Rosa26YFP / MEF2C-CRE; Rosa26YFP）5,6。一旦胚胎干细胞分化为第二心脏领域的每次点击费用Isl1 / MEF2C子/增强推动华创会激活荧光记者和每次点击费用，可以通过FACS净化富集。简言之，EB凝集法用于启动ESC分化。以提高分化的效率，所述分化细胞用抗坏血酸（AA）和生长因子如BMP4，活化素A和VEGF 13,15。该协议允许用小鼠和人胚胎干细胞强大的和高效率的CPC分化。

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derivation of cardiac progenitor cells from embryonic stem cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

该协议显示了几个胚胎干细胞系的每次点击费用的推导。胚胎干细胞聚集形成EB分化为每次点击费用。胚胎干细胞是经常保持在MEF饲养（ 图1A，F）和馈线分化前被移除。经聚集的胚胎干细胞的成胚在分化培养基（ 图1B，G）中，所述第二形成的EB与BMP4和活化素A处理，以增强在小鼠细胞系（ 图1C）中胚层的分化。胚胎干细胞分化成心肌谱系所确定的荧光蛋白质YF...

Watch this Scientific Journal Video about Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells at JoVE.com

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

从胚胎干细胞衍生心脏祖细胞

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells

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Developmental Biology

8.0K 次观看.  University of Michigan. In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.