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 ...

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

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 ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

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 ...

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

胚胎干细胞衍生的内皮细胞治疗肢体缺血

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates 2, 3. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm 4.
Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section 5, 6, 7. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10&mu;m) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.

Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.

使用胚体冰冻切片分析的多能干细胞

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

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 ...

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 ...