Name: retroviral infection of murine embryonic stem cell derived embryoid body cells for analysis of hematopoietic differentiation
Uploaded: 2014-10-20T15:00:00.000+00:00
Duration: 11 min 40 s
Description: 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

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.

<table><tbody><tr><td>DMEM</td><td>Sigma/Aldrich</td><td>D5796</td><td></td></tr><tr><td>&amp;#946;-mercaptoethanol</td><td>Sigma/Aldrich</td><td>M3148</td><td></td></tr><tr><td>FBS (ES Screened)</td><td>Hyclone</td><td>SH30070.03E</td><td></td></tr><tr><td>FBS (Defined)</td><td>Hyclone</td><td>SH30070.03</td><td></td></tr><tr><td>Non-essential Amino Acids</td><td>Life Technologies</td><td>11140</td><td></td></tr><tr><td>L-Glutamine</td><td>Life Technologies</td><td>35050</td><td></td></tr><tr><td>Penn/Strep</td><td>Life Technologies</td><td>15070</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life Technologies</td><td>25200</td><td></td></tr><tr><td>LIF ESGRO</td><td>Millipore</td><td>ESG1107</td><td></td></tr><tr><td>ACCUMAX</td><td>Millipore</td><td>SCR006</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life Technologies</td><td>25200</td><td></td></tr><tr><td>Falcon Tissue Culture Plates 6-well</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>Falcon Non-treated Plates 6-well</td><td>Fisher Scientific</td><td>08-772-49</td><td></td></tr><tr><td>Falcon Petri dish 10 cm</td><td>Fisher Scientific</td><td>08-757-100D</td><td></td></tr><tr><td>Falcon Petri dish 15 cm</td><td>Fisher Scientific</td><td>08-757-148</td><td></td></tr><tr><td>Falcon Tube 5 ml</td><td>Fisher Scientific</td><td>14-959-11A</td><td></td></tr><tr><td>Falcon Tube 5 ml with Cell Strainer Cap</td><td>Fisher Scientific</td><td>08-771-23</td><td></td></tr><tr><td>White sterile resevoirs</td><td>U.S.A. Scientific</td><td>111-0700</td><td></td></tr><tr><td>Rat anti-mouse CD41 PE-conjugated</td><td>Biolegend</td><td>133906</td><td></td></tr><tr><td>Rat anti-mouse CD117 APC/CY7-conjugated</td><td>Biolegend</td><td>105826</td><td></td></tr><tr><td>RW4 ESCs</td><td>ATCC</td><td>CRL-12418</td><td></td></tr></tbody></table>

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 ...

retroviral-infection-murine-embryonic-stem-cell-derived-embryoid-body

Research

JoVE Journal

Biology

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. 

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

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 ...

科研

生物学

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

发育生物学

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

免疫与感染

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 ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

干细胞基工程免疫抗HIV感染的人性化小鼠模型

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

医学

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

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

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 ...

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

干细胞衍生病毒Ag特异性T淋巴细胞抑制小鼠HBV复制

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...