添多伊尔博士和斯坦福大学在体内与生物发光成像援助的成像中心。同时也感谢银黄，博士分享她的技术，干细胞共同注射矩阵解。最后，感谢史蒂夫认为，博士协助兽医照顾动物。

<ol><li> 建设双融合报告基因 <ol><li>为了进行人类胚胎干细胞的生物发光成像，你首先需要获得稳定表达如像泛素或EF1a构推动者驱动的萤火虫荧光素酶的荧光素酶记者基因的细胞。 </li><li>本协议的重点是对报告基因的应用，所以这里不提供详细的程序。然而，我们实验室的总体战略是使用双重融合构建载体，包含萤火虫荧光素酶（fluc）和增强型绿色荧光蛋白（EGFP）间隔内pCDNA 3.1 +分隔。 </li><li>简而言之，我们的双融合基因原位于下游pCDNA 3.1巨细胞病毒启动子（+），所以用NdeI和NotI消化，平端结扎成多个克隆网站的自我失活（单3.3 kbp的片段被切除）慢病毒载体，一个Ubiquitin启动的控制之下。 </li></ol></li><li> 慢病毒转导 <ol><li>人类胚胎干细胞可转通过后3-5天，在感染复数（MOI），10（约107 106个细胞培养的病毒滴度）。 </li><li>解冻病毒的股票，可以直接添加到新鲜的胚胎干细胞媒体。 </li><li>刷新媒体12日和24小时后。 </li><li> 48 h后，转染效率可以通过使用荧光显微镜定性评估。随后，流式细胞仪可用于隔离受感染的细胞。 </li></ol></li><li> 培养人类胚胎干细胞，并介绍到影像设置 <ol><li>文化在您的荧光素酶的积极人类胚胎干细胞的馈线自由的条件。我们通常遵循的标准WiCell协议，并使用6孔板，生长因子减少基质胶涂层，无论是使用空调的媒体或商业，馈线自由的媒体。 </li><li>为了检测荧光素酶阳性细胞，你会需要有记者探头的D -荧光素已经准备好。要做到在1-1.5 ml的终浓度为45毫克/毫升的准备，等分荧光素。保持在-20 ° C的分装时不使用避免暴露在光线下和在没有存储时用纸巾覆盖。 </li><li>为了形象化的荧光素酶阳性细胞中，我们使用IVIS 50 IVIS 200成像系统（精诺真公司，阿拉米达，CA）。后者系统包括一个集成的isoflourane器具临时麻醉的小动物和诱导室。 </li></ol></li><li> 在人类胚胎干细胞的体外生物发光成像<ol><li>体外生物发光成像，重要的是要保持无菌条件下。因此，成像系统应该是无菌的，最好是在细胞培养室。 </li><li>成像前，取出细胞媒体，并添加刚够PBS覆盖细胞。例如，加入1ml PBS将每孔6以及含有胚胎干细胞文化的板。 </li><li>的D -荧光素，以PBS的比例应该是1:100，所以每6孔板以及添加10μL解冻ð -荧光素。 </li><li>等待一分钟，然后取一个形象，使用1秒的曝光时间。如果信号弱，增加曝​​光时间间隔，然后再试一次。 </li><li>发光信号反映了细胞的数量，因此，关联与不同的手机号码信号，可以进行定量分析。 </li></ol></li><li> 制备小鼠和注射细胞在活体成像 <ol><li>记者基因显像提供移植后几天，几周，甚至几​​个月，为追踪细胞的高通量，价格低廉，敏感的方法。这一点尤其重要，因为移植的干细胞通常形成畸胎瘤，是由宿主的免疫系统拒绝，或者干脆死。 </li><li>成像在体内畸胎瘤形成的最简单的方法是注入到SCID小鼠背上的萤火虫荧光素酶报告基因的人类胚胎干细胞，表达和收购IVIS系统的发光图像。 </li><li>当你准备好移植的细胞，用胶原酶dispase或放松的细胞，多洗几次，并暂停在1:1混合生长因子减少，基质胶和DMEM。通常情况下，我们首先冷冻的DMEM混合细胞，然后添加在基质胶。对于每个皮下注射站点，我们注入50-100 UL量在基质胶/ DMEM培养液的混合物悬浮200000细胞。可调节的细胞数，取决于您的应用程序。请记住在注射之前，一切都保持在冰上。 </li><li>一旦细胞准备，麻醉与异氟醚流打开它放置在感应中的鼠标。 1-5分钟后，鼠标是睡着了，可以上手术台连续异氟醚。 </li><li>由于毛皮自然自动发出荧光，并可能掩盖了发光的形象，我们将需要删除的头发从鼠标的背面。要做到这一点最简单的方法是使用一个电动剃须刀，但你也可以使用除毛凝胶。用酒精擦拭消毒皮肤之后</li><li>下一步，画出一个注射器在你的细胞悬液。 23日至27日表针的工作最好，因为它们不会阻塞与细胞。如果过大（&lt;23压力表）针针道可能会离开细胞，通过它可以泄漏出一个大洞。 </li><li>鼠标的背部皮肤下注入细胞。由于皮肤是相当宽松的，用你的拇指和食指捏，伸出你要注入的区域。只是在皮肤下注射，注意不要穿刺太深。此外，尽量保持针头从注射部位滑动，而令人沮丧的柱塞：这将防止创建一个皮肤细胞孔，通过它可以泄漏，如果您尝试再次注入。 </li><li>注入细胞后，等待了几个小时，让鼠标唤醒和运行之前重新麻醉生物发光成像左右。这样做可以避免异氟醚的毒性。 </li></ol></li><li> 移植细胞在体内的生物发光成像<ol><li>对于整个动物生物发光成像技术，我们做的D -荧光素腹腔注射375毫克/公斤体重。称量动物在注射之前，计算用量。 </li><li>在腹膜注入的D -荧光素，就在中线。小心不太深了，或者你可以损伤内脏。如果鼠标开始唤醒，将它早在淘汰赛中，并等待一两分钟。对小鼠，最大的生物发光，通常发生在注射后15-40分钟。 </li><li>记住放置在成像盒磨砂黑纸，以帮助吸收由胚胎干细胞不发出任何光线。鼠标（背面朝上）放置在与异氟醚的成像室，黑纸的顶部。开始与一个10秒的曝光时间。如果信号饱和，尽量减少暴露时间;如果太弱​​，增加曝​​光。 </li><li>一旦信号已经曝光时间优化你的鼠标，开始考虑图像的每一分钟，直到信号达到最大值。最好的做法是通过使用图像处理软件，选择“地区利益率（ROI）”，包括注射部位。通过监控每个ROI的信号强度，你可以很容易地确定信号开始下降时，表明已达到最大的信号强度。作为您的最终数据，应使用最大信号。 </li><li>当您与您的图像满意，除去异氟醚的鼠标，并允许它唤醒在笼子里。通常情况下，鼠标应该在15分钟内醒来。 </li><li>生物发光记者基因显像可以重复，只要每天，每周，每月，作为人类胚胎干细胞在动物生存。信号从一个发展中的畸胎瘤会成倍增加，随着时间的推移，通常在数周的顺序，作为人类胚胎干细胞开始增殖。 </li><li>生物发光图像所需的时间当然是收购后，可能会牺牲和动物组织切片组织学用于。 </li></ol></li></ol>

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相比其他方式，如PET和MRI，生物发光有限的空间分辨率和降低组织的渗透，由于相对薄弱的发射光子的能量（2-3 EV）;，由于这些原因，迄今尚未适用于大型动物。然而，生物发光的优点是低成本，高通量，和非侵入性，使其具有很高的理想，在体内的干细胞在小动物的跟踪。非生物发光记者的基因，如PET和荧光结构，可用于荧光素酶的结合，创造一个融合记者基因，包含个别记者基因的不同的域组成。例如，本...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BD Matrigel&#x2122; Basement Membrane Matrix</td>
<td>Growth factor reduced (optional: phenol-red free)</td>
<td>BD Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>mTeSR1 Maintenance Medium for Human Embryonic Stem Cells</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-Luciferin Firefly, potassium salt</td>
<td>&nbsp;</td>
<td>Biosynth AG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Dissolve 30 mg Collagenase Type IV in 30 mL DMEM-F12 media. Sterile filter and store at 4 degrees (Celsius).</td>
</tr>
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<td>Baked Pasteur pipets</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>6-well tissue culture-treated plates</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>92006</td>
<td>&nbsp;</td>		</tbody>
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in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells

人类胚胎干细胞（胚胎干细胞）的发现大大增加了在再生医学感兴趣的医学科学家可用的工具。然而，直接注射胚胎干细胞，并从人类胚胎干细胞分化的细胞进入生物体，迄今已阻碍了显着的细胞死亡，畸胎瘤的形成，与宿主的免疫排斥反应。了解在体内的人类胚胎干细胞移植后行为，需要新的成像技术的纵向监测人类胚胎干细胞的本地化，增殖和活力。分子成像已发出超过体内细胞的增殖天，星期，甚至几个月的跟踪调查一个高通量，价格低廉，而敏感的手段。这种进步已经大大提高了人类胚胎干细胞植入，增殖和生活科目的畸胎瘤形成的时空动力学的理解。 分子成像中的一个重大进步，已经从分子和细胞生物学的无创性的记者基因检测在体内的多模态成像平台的延伸。这些记者的基因，控制工程的推动者和促进剂，利用宿主细胞转录机制的优势下，引入使用各种向量和非向量方法的细胞。一旦在细胞中，记者的基因可以被转录组成或只在特定的生物或细胞的条件，对启动子的类型而定。成具有生物活性的蛋白质，记者基因的转录和翻译是那么敏感的，非侵入性的仪器使用探针信号产生的D -荧光素等（例如，CCD摄像头）检测。 为了避免兴奋的光需要跟踪干细胞在体内荧光成像，生物发光报告基因成像系统的要求只是一个外生管理探针诱导光发射。萤火虫荧光素酶，从萤火虫Photinus pyralis的，编码一种酶，它催化D -荧光素的光学活性的代谢产物，oxyluciferin。光学活性，然后可以与外部的CCD摄像头监视。稳定转染细胞进行的记者在他们的染色体DNA的构造，将通过记者构造子细胞的DNA，使人类胚胎干细胞在体内的生存和扩散的纵向监测。此外，记者基因产物的表达，因为信号产生所需，将创建唯一可行的父母和女儿细胞生物发光信号;凋亡​​或死亡的细胞不会。 在这个视频，跟踪干细胞的增殖和畸胎瘤的形成与生物发光成像所需的具体材料和方法进行说明。

Watch this Scientific Journal Video about In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells at JoVE.com

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

分子成像技术在干细胞疗法的兴趣与日俱增，是理想的监测干细胞移植后的行为。荧光素酶基因已启用非侵入性，重复评估细胞的存活，位置，并在体内扩散。本视频将演示如何跟踪生活在一个小鼠胚胎干细胞增殖。

在体外和体内生物发光记者人类胚胎干细胞的基因显像

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

in-vitro-vivo-bioluminescence-reporter-gene-imaging-human-embryonic

Research

JoVE Journal

Biology

21.7K 次观看.  Stanford University School of Medicine. 分子成像技术在干细胞疗法的兴趣与日俱增，是理想的监测干细胞移植后的行为。荧光素酶基因已启用非侵入性，重复评估细胞的存活，位置，并在体内扩散。本视频将演示如何跟踪生活在一个小鼠胚胎干细胞增殖。

视频: In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

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 ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

在人类胚胎干细胞体外培养的标签用于磁共振成像

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

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 ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...