This work was supported by a Pilot and Feasibility Grants from the Indiana University School of Medicine Center (IUSM) for Excellence in Molecular Hematology (NIDDK, 5P30DK090948). Additional funding was provided by a Biomedical Enhancement Grant from IUSM. We would like to thank Dr. Karen Cowden Dahl for commenting on the manuscript.

1，胚状体（EB）形成<ol><li>明胶调整已维持在小鼠胚胎成纤维细胞（MEF中）胚胎干细胞。通路胚胎干细胞在6孔组织培养板3次涂覆有0.1％明胶去除的MEF。 <ol><li>生长的细胞在ESC维持培养基与LIF，以保持未分化（ 表1）的细胞。确保电池不会超过80％汇合。细胞分化（由MEF除去后10个或更少的通道）使用低传代。 </li></ol></li><li>关于前胚胎干细胞可用于EB分化培养一天，通道的细胞，使它们将约为50-70％汇合的第二天。继续维持ESC培养基中培养细胞，保留细胞的多能性。 </li><li>在分化，吸ESC维持培养基和当天洗用1ml磷酸盐缓冲盐水（PBS：137 mM氯化钠，2.7 mM的氯化钾，和11.9 mM磷酸盐缓冲液，pH 7.4）中每一个6孔板的。 <ol><li>加入200微升的0.25％tryps在/ EDTA至各孔中，在37℃下进行3分钟。 </li><li>灭活胰蛋白酶/ EDTA与800微升分化培养基中。吸取上下在2 ml血清吸管用P200尖分手的细胞成单细胞悬液。确保P200尖端完全吻合的末尾，以便媒体不上去的吸管和小费接口之间。 </li><li>吸取上下约5-10倍。小心不要移液过程中产生的气泡。 </li></ol></li><li>细胞转移到15毫升锥形管。调出量10毫升的PBS中。离心机在315×g离心5分钟，在室温下进行。 </li><li>取出上清液。用5毫升的PBS洗两次，以除去含LIF剩余的介质。离心机在每次洗涤步骤1.4。 <ol><li>重悬在2ml的分化培养基（ 表2）的最终的细胞沉淀。 </li></ol></li><li>计数细胞，血球或细胞计数和板10cm的培养皿6000-10000个/ ml（非组织培养处理）。 <ol><li>确定确切的细胞/ ml凭经验每个ESC线。使用通常用于细菌工作或低粘附板的无菌皮氏培养皿。 注：为了获得最佳的差异化效果，胚体应形成保持悬浮而不附着在平板领域。测试几个品牌平板找到那些用最少的坚持。 </li><li>孵育分化的胚胎干细胞在37℃组织培养箱5％的CO 2。 </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0"><colgroup><col /><col span="2" /><col /><col /><col /></colgroup><tbody><tr><td> 试剂 </td><td> 股票 </td><td> 终浓度 </td><td> 成交量 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> DMEM </td><td> 1个</td><td></td><td> 410毫升</td><td> Š<html> IGMA /奥尔德里奇</td><td> D5796 </td></tr><tr><td> FBS（ES屏蔽） </td><td> 100％ </td><td> 15％ </td><td> 75毫升</td><td> Hyclone公司</td><td> SH30070.03E </td></tr><tr><td>非必需氨基酸</td><td> 100X </td><td> 1个</td><td> 5毫升</td><td> Life Technologies公司</td><td> 11140 </td></tr><tr><td> L-谷氨酰胺</td><td> 100X </td><td> 1个</td><td> 5毫升</td><td> Life Technologies公司</td><td> 35050 </td></tr><tr><td> Penncillin /链霉素</td><td> 100X </td><td> 1个</td><td> 5毫升</td><td> Life Technologies公司</td><td> 15070 </td></tr><tr><td> β-巯基乙醇</td><td> 14.3 M </td><td> 114微米</td><td> 4微升</td><td>西格玛/ Aldrich公司</td><td> M3148 </td></tr><tr><td>白血病抑制因子（LIF） </td><td> 10 7个/毫升</td><td> 1000个/毫升</td><td> 50微升</td><td>密理博</td><td> ESG1107 </td></tr></tbody></table>ontent“&gt; 表1：ESC维护媒体。 <table border="0" cellpadding="0" cellspacing="0"><colgroup><col /><col span="2" /><col /><col span="2" /></colgroup><tbody><tr><td> 试剂 </td><td> 股票 </td><td> 终浓度 </td><td> 成交量 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> DMEM </td><td> 1个</td><td></td><td> 410毫升</td><td>西格玛/ Aldrich公司</td><td> D5796 </td></tr><tr><td> FBS（用户筛选最佳的分化） </td><td> 100％ </td><td> 15％ </td><td> 75毫升</td><td> Hyclone公司</td><td> SH30070.03E </td></tr><tr><td>非必需氨基酸</td><td> 100X </td><td> 1个</td><td> 5毫升</td><td> Life Technologies公司</td><td> 11140 </td></tr><tr><td> L-谷氨酰胺</td><td> 100X </td><td> 1个</td><td> 5毫升</td><td>生活TECHNOLO吉斯</td><td> 35050 </td></tr><tr><td> Penncillin /链霉素</td><td> 100X </td><td> 1个</td><td> 5毫升</td><td> Life Technologies公司</td><td> 15070 </td></tr><tr><td> β-巯基乙醇</td><td> 14.3 M </td><td> 114微米</td><td> 4微升</td><td>西格玛/ Aldrich公司</td><td> M3148 </td></tr></tbody></table> 表2：差异化媒体。 2，准备EB细胞病毒感染<ol><li>在发展的所需阶段（第2天第3天之间），从10cm培养皿的胚转移到15毫升锥形管中。用5ml PBS洗涤板和洗涤添加到锥形管中。 </li><li>沉淀在胚胎发育第315 XG在室温下搅拌5分钟。用5毫升的PBS洗EB细胞。 </li><li>加入1毫升解冻细胞分离液（含蛋白酶和胶原溶解酶），置于37℃水浴管（或孵化器）30分钟与OCCasional搅拌（自弹或轻涡旋）。 </li><li>加入1 ml分化培养基中。吸取上下以2毫升移液管与P200的枪头。再次将管子在37℃水浴30〜60分钟。 </li><li>降速的细胞在315×g离心5分钟，并用PBS洗涤细胞。 </li></ol> 3，病毒感染<ol><li>重悬在1.5ml分化培养基中的细胞。细胞悬液转移到6孔非经组织培养物处理板。 </li><li>准备逆转录病毒通过提前的标准技术。添加5〜100μl病毒上清液，以根据病毒滴度的细胞。不添加聚凝胺，以提高感染，因为这抑制了EBS的后续改革。 </li><li>通过在2,120 XG离心，在室温下90分钟Spinoculate细胞与病毒。 </li></ol> 4，区分病毒性感染的EB细胞悬滴<ol><li>加入2mL分化培养基对我每口井nfected EB细胞悬液。 </li><li>移液管将3.5毫升感染EB细胞悬液放入无菌试剂水库multipipettors。使用multipipettor到吸管15-20行8 20微升滴到15厘米培养皿的反转盖子。 </li><li>加入10毫升无菌PBS或水的底部的培养皿的一半。然后反转的下降和地点到培养皿的盖子。确保滴挂在锅盖用PBS或以下的流水倒扣，以保持室内加湿。 </li><li>将菜在37℃组织培养箱有5.0％的CO 2。 </li><li>后第2天，收集滴通过反转盖子和洗涤用4.5ml分化培养基中形成的悬挂的EB。 <ol><li>传输介质和EBS到一个新的10厘米非组织培养培养皿。用3毫升分化培养基并转移至10cm平板的洗盖一次。 </li></ol></li><li>收获细胞共8后流式细胞仪分析流程天的培养，从细胞的初始传送到分化培养基（-LiF）开始。 </li></ol>在流式细胞仪分析5。准备细胞<ol><li>细胞转移到15毫升锥形管，用10毫升吸管。用5ml PBS洗涤该板，并转移到同一个锥形管。 </li><li>颗粒细胞在315×g的。洗粒料用10ml PBS中。 </li><li>加入1 ml的解冻细胞分离溶液，然后将试管在37℃水浴中30分钟，偶尔搅拌。用于检测某些细胞表面抗原，可以使用胰蛋白酶代替。 </li><li>加入1 ml分化培养基和吸管上下2毫升移液管。使用P200尖吸管的一端，以帮助分手EB消化成单细胞悬液。 <ol><li>再次将管在37℃水浴中60分钟，以使整合膜蛋白，以再循环到细胞表面。 </li></ol></li><li>沉淀细胞，在315×g离心5分钟。 </li><li>洗细胞无线网络在PBS（PBS / BSA）中制备次0.1％的牛血清白蛋白级分V（BSA）孵育的细胞的特异性荧光标记的抗体。 </li></ol>对造血祖细胞6流式细胞仪分析<ol><li>将106 EB的细胞在5ml的12×75毫米圆底管。用于调节流动血细胞计数补偿设定，根据制造商的说明使用补偿珠。 </li><li>颗粒细胞在315×g的。倒掉上清，重悬沉淀在管残余的PBS / BSA溶液。 </li><li>用于检测ESC高性能计算机，孵育细胞与荧光标记的抗体：抗小鼠CD41-PE（R-藻红蛋白）和抗小鼠CD117（CKIT）-APC / CY7（别藻蓝蛋白花青7）。 <ol><li>稀释抗体1：在PBS / BSA 100。加入100μl的稀释抗体至每个样品。确定正确的稀释抗体对每个供应商，克隆和地段或抗体。确定最佳稀释单独标记的细胞。 </LI&gt; </ol></li><li>在冰上孵育的样品在黑暗中30分钟。 </li><li>加入2毫升的PBS / BSA和颗粒细胞在315×g离心5分钟。 </li><li>吸出上清液，在300微升PBS或其他缓冲等渗重悬沉淀。 </li><li>通过细胞过滤器盖（0.35微米）过滤悬浮细胞，除去大细胞聚集。 </li><li>在流式细胞仪分析细胞。 </li></ol>

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作者们没有竞争的经济利益披露。

如上所讨论的，可以使用多西环素系统产生ESC克隆诱导地表达感兴趣的基因，然而，产生这些线是耗时且劳动密集型的。另外，在本协议是为了表达从ESC准备单细胞悬液衍生胚目的基因的方法。然后，这些被感染的细胞改造成胚体由悬滴检查后续的分化。在这个例子中（ 图3），它示出了mirn23a簇的表达增强了造血发育时，在分化的第3天表达。在这一点上已发生新生胚层组织的?...

Murine embryonic stem cells (ESCs) are pluripotent, remaining undifferentiated and self-renewing in the presence of the cytokine Leukemia Inhibitory Factor (LIF)1. Upon withdrawal of LIF they will spontaneously differentiate into 3-dimensional (3D) structures called embryoid bodies (EBs)2. The 3D architecture allows for the development of the three germ layers ectoderm, endoderm, and mesoderm, which then later give rise to mature tissue types3. ESCs are an exceptional model for elucidating the molecular mechanisms of cellular differentiation, particularly the investigation of the development of early hematopoietic progenitor cells (HPCs)4. 
Gene expression in ESCs can be manipulated by several techniques that allow for the determination of a role for individual molecules in development. One of the most common techniques is to use homologous recombination to generate ESC lines, which lack a gene of interest5,6. There are also a number of techniques that have been used to overexpress genes. The first technique used to modify gene expression in ESCs was to infect them with recombinant retroviruses7,8. The gene of interest however is often silenced as the ESCs differentiate into progenitor and mature cell types. Use of lentiviruses has been successful in limiting the silencing of virally expressed genes9. Other viral vectors used for overexpression include adenovirus and adeno-associated virus10. In addition standard transfection techniques to stably introduce expression plasmids are widely used for ESC transgene expression11. One difficulty with these systems is that often expression of a specific gene has different effects depending on its temporal expression. For example, the Smad1 protein affects the development of hematopoietic cells differently during different stages of EB development12,13. 
This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest. The most common system for inducibly expressing transgenes in ESCs uses the tetracycline resistance operon from E. Coli (Escherichia coli). Several different tetracycline systems have been developed. One of the more popular strategies was developed by Kyba and colleagues14. They generated an ESC line (Ainv15), which has the reverse tetracycline transactivator gene inserted into the constitutively active ROSA26 locus. A tetracycline response element (TRE), a downstream LoxP site (locus of X-over P1 from bacteriophage P1), and a promoterless neomycin cassette were introduced into the HPRT (Hypoxanthine-guanine phosphoribosyltransferase) locus on the X chromosome. Using a CRE recombination approach a gene of interest along with a eukaryotic promoter to drive the expression of the neomycin resistance gene can be inserted into the LoxP site. Correctly targeted ESCs are isolated by G418 selection. These targeted clones then must be tested for doxycycline (or tetracycline) inducible expression of the transgene. This approach has successfully generated ESC lines that inducibly express HoxB4, Stat5, SCL, and Smad714-17. However, generation of these inducible cell lines is time consuming. Described here is a method to disaggregate ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infect the cell suspensions at different days of development, and then reform the EBs by hanging drop. Downstream differentiation is later evaluated by flow cytometry. In this article an example of how miRNA expression in EB-derived cells effects hematopoietic differentiation is shown. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

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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.

在这些研究中胶凝RW4胚胎干细胞（从129X1 / SVJ小鼠品系派生）被使用。胚中分离在分化的3天。为了达到最佳效果EBS的应是球形和非贴壁（ 图1A，B）。 spinoculation与病毒共表达与感兴趣的基因的荧光标记的GFP后，胚被悬滴法改性。胚成功从2.0，2.5，和3.0天胚制备细胞悬液改革。但是，胚不能从4.0天的EB制备细胞悬浮液改性。受感染的和未感染的细胞之间的EB的嵌合体观察通过荧光显微术?...

Watch this Scientific Journal Video about Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation at JoVE.com

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

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 ...

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8.4K 次观看.  Harper Cancer Research Institute. 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. 