Name: 视频: Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth
Uploaded: 2010-05-20T17:00:00
Description: 12.7K 次观看. University of California. 视频生物信息学的自动化处理，分析，理解，并从微观的影片中提取的生物时空数据的数据挖掘。本文的目的是为了演示使用视频生物信息学方法测量人类胚胎干细胞集落增长的方法。

这种方法的发展成为可能由加州烟草相关疾病的研究计划，加州再生医学研究所，美国国家科学基金会IGERT授予视频生物信息学（＃0903667）在加州大学河滨分校（ <a href="http://www.cris.ucr.edu/IGERT/index.php">资金</a> http://www.cris.ucr <a href="http://www.cris.ucr.edu/IGERT/index.php">.edu / IGERT / index.php文件</a> ）。肖恩Forteno萨布丽娜林是从论文奖学金研究生部的支持下，由美国国立卫生研究院的MARC奖学金支持，并Shruthi萨蒂什南比亚是由研究生部奖学金支持。我们感谢为她准备的数字帮助安娜Trtchounian。我们也感谢山姆Alworth和斯内德Jastromb教我们如何使用CL - QUANT软件。

第1部分：实验步骤视频数据包含了丰富的信息。然而，这些信息往往是难以提取和手工完成人类和人员时间可能需要许多小时才能完成。人类的人工分析也受到解释和错误的变异。视频生物信息学涉及计算机软件的使用矿从视频图像中的特定数据。这种分析方法是快速，并能消除发生的错误的分析是通过人类手动。本文的目的是表现出人类胚胎干细胞菌落生长，使用视频生物信息学方法进行量化的方法。 在本文中，时间的推移视频图像采集使用尼康BioStation CT孵化单元，允许随着时间的推移成像等多个领域的细胞（图1）。在这份报告中所描述的方法适用于任何视频显微成立收集到的视频数据。 在我们的实验设计中，我们在48小时12孔组织培养板镀H9的人类胚胎干细胞。在此期间，菌落让其充分重视和蔓延对基质胶。然后，附加的人类胚胎干细胞殖民地的板块转移到BioStation的CT和一个额外的48小时内孵化。虽然在BioStation，殖民地的图像采集在7分钟的时间间隔，后来被用于创建延时视频序列。每个殖民地的影片，然后进行分析，以量化菌落生长，使用视频生物信息学与CL - QUANT软件开发的食谱。分析使用与CL - QUANT软件检查的真实性，手动使用Adobe Photoshop中创建的菜谱。 第2部分：附加的人类胚胎干细胞殖民地的制备<ol><li>生长在基质胶人类胚胎干细胞涂6孔板，一旦达到70％汇合，replate如下分为5个井12井基质胶涂层板6孔板。 </li><li>吸从以及含有人类胚胎干细胞的培养基。 </li><li>冲洗干净2倍，而与PBS1毫升。 </li><li>添加Accutase和孵化1分钟1毫升，在37 ° C和5％的CO 2 。 </li><li>添加入井10-12玻璃珠，轻轻地摇板的殖民地，直到完全脱离井底。 </li><li>抵消Accutase使用1毫升mTeSR中等或MEF条件培养基。 </li><li>在200克离心3分钟15毫升锥形管的细胞悬液。 </li><li>倒出新鲜mTeSR培养基500μL上清，打破颗粒。 </li><li>板块下跌明智的人类胚胎干细胞悬浮液在12孔板，每孔100μL。 </li><li>岩石板来回轻轻地，光镜下观察文化。确保细胞团块均匀分布在整个板块。 </li><li>将进入孵化器的12孔板回。 </li><li>允许人类胚胎干细胞附着和增长为48小时（可以使用更短的时间内）。 </li><li> 48小时后，倒出中期和500ul的PBS洗井，以消除独立的细胞。 </li><li> mTeSR中等1ml到每口井。 </li><li>评论：立即放入孵化器/视频成像显微镜板。 </li><li>开始收集时间推移图像。尽量选择与分立，是不可能成长为其他殖民地的单菌落领域。 </li><li>培养和建立人类胚胎干细胞的其他方法可以用来代替上述协议。 </li></ol>第3部分：生物信息学视频 CL - QUANT软件是用来创建三个“食谱”，按顺序运行时，将决定每个随着时间的推移殖民地微米的像素数或面积。在此应用程序的顺序和内置的三个食谱包括分割，增强和测量。 分析人类胚胎干细胞菌落生长，使用CL - QUANT软件/配方开发： <ol><li>首先创建分割配方 。 </li><li>扫描整个视频，确保视频可用。检查，以确保整个殖民地在视野和其他殖民地不进入这个领域仍然。 </li><li>打开分割向导，然后单击“下一步”按钮。 </li><li>选择正确的图像通道（即阶段，绿色，红色等），并单击“下一步”按钮。 </li><li>挑选出3个选择的“软匹配”，并点击“下一步”。 </li><li>盘旋在图像上通常的外缘中部地区的殖民地的殖民地地区，选择“希望”地区。 <ul><li>此区域应尽可能小，但应该是代表整个殖民地。 </li><li>使用相衬显微镜时，一定要包括的光环围绕软件精确的殖民地选择殖民地。 </li><li>选1或2个地区的利益，可能会呈现出不同的的像素模式内的殖民地，但不要挑太多“希望”，这可能会导致不准确的面膜中的应用。 </li></ul></li><li>点击“下一步”按钮。 </li><li>选择“不想”盘旋在图像上的区域不属于殖民地的一部分，而不是背景的一部分地区。这些模式，你想等文物碎片和死细胞，抑制和包括像素的地区。点击“下一步”。 </li><li>盘旋你想压制的地区，选择“背景”。 <ul><li> “不想要”和“背景”地区在这样的背景下不同，应该是统一的和可能出现在每帧。 </li><li>通常情况下，我们选择的灰色背景，周围的殖民地。 </li></ul></li><li>点击“下一步”按钮。 </li><li>彩色面具应显示在你感兴趣的区域（图2A）。 </li><li>如果掩码不准确覆盖地区的利益，分割阈值范围内（位于右下角的软件显示的右上角）可能会增加或减少。 </li><li>如果有必要进一步屏蔽区域微调分割向导提示。 </li><li>如果你想改变你的面具，选择“更新软匹配的地区”。这允许你改变“要”，“不想”，或“背景”的范畴。 </li><li>如果掩码是令人满意的，选择“申请门槛，并保存我的面具”，并点击“下一步”按钮。 </li><li>面具便会显示出不同的颜色。 </li><li>选择“完成”。 </li><li>配方应显示在右边的软件右上角，它应该被称为“分割配方”。 </li><li>重命名的配方，如果你希望通过右键单击“重命名”。 </li><li>对于抽查中，右键单击和鼠标放在“申请配方”。 </li><li>将显示一个菜单，允许你选择你想的帧的数量，使用时，抽查的配方。抽查面具安置的准确性每10帧运行配方。 </li><li>如果掩码拿起小不需要的地区或碎片的部分，你可以创建一个“增强配方”的面具，以改善装修感兴趣的区域。 </li><li>要创建一个增强配方 ，右击“增强配方”文件夹，然后单击“新建”。应显示一个新的增强配方。 </li><li>从原始字段的视角（FOV），点击“切换增强模块”按钮，在图​​像的右侧。鼠标悬停在工具栏上的图标，以确定​​正确的。 </li><li>选择“标签”下拉菜单。 </li><li>选择“标签4连接”。 </li><li>选择“标签4 connected2”。 </li><li>一个新的任务栏应该显示。 </li><li>抓斗从最初的面具（mask0）分割配方，将其拖动到“输入”框。 </li><li>酒吧现在应该显示“输入掩码＃”。 </li><li>拖动图标“输入掩码＃”面具0图标。 </li><li>在任务栏上的“最小尺寸”查找和调整的数量，直到显示唯一感兴趣的地区。请务必点击“执行”，通过每个接头试验。 </li><li>一旦你与你的最小尺寸调整满意后，单击你以前创建的“增强配方”。 （图2B） </li><li>在屏幕的底部，选择“保存到配方”。 </li><li>该软件现在提示“覆盖所选的配方吗？”，选择“是” </li><li>抽查抽查分割使用上述同样的程序增强配方。 </li><li>如果满意的分割和增强配方，选择右手工具栏上的“创建测量模板“。 </li><li>如果你想重命名模板。 </li><li>在测量模板“窗口，对剪贴画细胞的身影的光标移动。按住Ctrl键并选中该单元格。 </li><li>取消选中“默认参数”和检查“区域参数”下的“形态”。 </li><li>退出“模板”窗口。 </li><li>选择测量配方中创建的图标。 </li><li>重命名的配方。 </li><li>移动到“模板”标签，并在右上角选择。 </li><li>拖放到测量窗口您以前创建的测量模板。 </li><li>点击“是”继续。 </li><li>在测量配方窗口中，选择“频道映射”，然后“全细胞”。 </li><li>选择“面具映射”。 </li><li>然后选择“全细胞”选项增强的面具。 </li><li>选择测量下在主窗口中的配方“选项卡的”良方“。 </li><li>测量配方窗口内选择“保存”图标。 </li><li>然后关闭窗口。 </li><li>现在，关闭并重新打开“视野”。 </li><li>右键单击“配方”图标，选择“锁定配方列表和顺序上运行您的视频数据的食谱。 </li></ol>第4部分：使用Photoshop软件检查配方的准确性为了验证（CL - QUANT softw创建配方都是），相同的数据，可以手动分析与Adobe Photoshop。对于这种分析，每10帧（每70分钟的时间点）进行了分析，超过48小时的殖民地的大小来衡量。 <ol><li>打开在Adobe Photoshop的殖民地图像帧。 </li><li>点击工具栏上的魔术棒工具。 </li><li>各地的殖民地面积，使整场除了殖民地地区覆盖。 </li><li>点击“编辑快速蒙版模式”在工具栏上，单击殖民地，所以选择殖民地。 </li><li>确保各地的殖民地的虚线适合对周围殖民地的边缘，而不是它里面。如果虚线环绕周边，改变上部的工具栏上相应的公差值。 </li><li>转到“窗口”下拉菜单，并点击“直方图”。 </li><li>写下的像素值缓存= 1时。如果缓存是2，点击“！”改变缓存1，然后注意的像素值。 </li><li>为每10帧重复上述过程。如果需要的话，可以用更少的总帧数。 </li><li>然后，可以使用Photoshop和CL - QUANT软件收集的数据绘制在一起。如果CL - QUANT的食谱是真实的，为Photoshop和CL - QUANT分析的曲线应该是非常相似（图3-5）。 </li><li>如果CL - QUANT开发的食谱是诚实的，他们可以可靠地应用到其他的视频数据。 </li><li>在这种分析中使用视频的生物信息学的价值是，一旦分割，增强和测量配方开发和验证，他们将执行的分析，更迅速，更准确地比人类使用Photoshop。 </li></ol>代表性的成果<img src="/files/ftp_upload/1933/1933fig1.jpg" alt="图1" /> 图1：一个人类胚胎干细胞的殖民地，在BioStation CT的增长在48小时内显示在不同的时间帧的电影带。在7分钟的时间间隔帧。 <img src="/files/ftp_upload/1933/1933fig2.jpg" alt="图2" /> 图2：（一）形象与人类胚胎干细胞置于殖民地分割配方口罩。碎片和背景等方面也掩盖表明增强配方需要，以提高评价的准确性。 （二）已执行相同的殖民地的形象，增强后在“A”所示。面具现在选择只有殖民地和噪音已经被淘汰选择。 <img src="/files/ftp_upload/1933/1933fig3.jpg" alt="图3" /> 图3：图表显示增加聚落的大小（以像素为单位）超过48小时，使用CL - QUANT软件和Photoshop。此图的原始数据表明，菌落开始在大小不同的地块。它也表明，这两种方法的测量好的协议。 <img src="/files/ftp_upload/1933/1933fig4.jpg" alt="图4" /> 图4：图表显示正常化后，如图3相同的数据显示超过48小时的群体规模增加百分之。这表明，增长率不管开始殖民地的大小和分析这两项措施提供了类似的结果相似。 <img src="/files/ftp_upload/1933/1933fig5.jpg" alt="图5" /> 图5：图表显示在图4中的规范化数据的手段。该图清楚地显示之间使用生物信息学（CL - QUANT软件）和Photoshop视频，并建立配方是真实的分析的好协议。面积为325帧的Photoshop数据略有下滑是由于不被包含在Photoshop分析的几部影片。

迅速从视频图像中提取数据，视频生物信息学工具是强大的。我们量化人类胚胎干细胞菌落生长的协议演示视频生物信息学的应用到生物问题。这种方法是定量的和有趣的功能，从个别的人类胚胎干细胞的殖民地揭示数据。视频生物信息学的配方，可以发展到其他细胞的过程进行监控，如增殖，迁移，凋亡，细胞附着到基板，到邻近的细胞和细胞附着。配方的准确性进行了验证使用Photoshop使用CL -...

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		<th>Catalogue Number</th>
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<td>mTeSR1 Human Embryonic Stem Cell Maintenance Medium</td>
<td>&nbsp;</td>
<td>Stem Cell Technologies</td>
<td>05850</td>
<td>or any suitable medium for hESC culture.</td>
</tr>
<tr>
<td>BD Matrigel</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>356234</td>
<td>or other suitable substrate.</td>
</tr>
<tr>
<td>DMEM/F12 Basal Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-032</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline without Ca2+ and Mg2+</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Accutase Enzyme Cell Detachment Medium</td>
<td>&nbsp;</td>
<td>eBioscience</td>
<td>00-4555-56</td>
<td>or other suitable detachment enzyme.</td>
</tr>
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<td>3mm Glass beads</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>11-312A</td>
<td>optional.</td>
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<tr>
<td>12-well Tissue Culture Plates</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353043</td>
<td>or any other plate format.</td>
</tr>
<tr>
<td>Nikon BioStation CT/IM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>or other incubator/microscope suitable for collecting video data.</td>
</tr>
<tr>
<td>CL-Quant software (Nikon) and/or Photoshop (Adobe).</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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video bioinformatics analysis of human embryonic stem cell colony growth

由于视频数据是复杂的，包括许多图像，视频素材的挖掘信息是很难做到的，没有计算机软件的援助。视频生物信息学是一个强大的的，从视频图像中提取的时空数据，使用计算机软件来进行约会的挖掘和分析的定量方法。在这篇文章中，我们引入了视频生物信息学方法，通过分析时间推移视频配备了视频成像相机在尼康BioStation的CT孵化器收集量化的人类胚胎干细胞（胚胎干细胞）的生长。在我们的实验中，连接到基质胶的人类胚胎干细胞菌落，48小时在BioStation CT拍摄。要确定这些殖民地的增长速度，配方使用CL - QUANT软件，该软件使用户能够从视频图像的数据中提取各类开发。为了准确地评价菌落生长，创建三个配方。第一分段成殖民地和背景，第二次提高了图像准确定义整个视频序列的殖民地，第三的图像测量的像素数，在随着时间的推移殖民地。这三个配方在BioStation CT收集的数据来分析个别的人类胚胎干细胞菌落生长率超过48小时的视频序列中运行。为了验证CL - QUANT食谱的真实性，同样的数据进行了分析手动使用Adobe Photoshop软件。当所获得的数据使用CL - QUANT食谱和Photoshop进行了比较，结果几乎相同，表明CL - QUANT食谱是真实的。这里所描述的方法可以适用于任何视频数据来衡量人类胚胎干细胞或其他细胞生长在殖民地的生长率。此外，其他视频生物信息学的配方可以如其他细胞迁移，细胞凋亡和细胞粘附过程的未来发展。

Watch this Scientific Journal Video about Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth at JoVE.com

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

视频生物信息学的自动化处理，分析，理解，并从微观的影片中提取的生物时空数据的数据挖掘。本文的目的是为了演示使用视频生物信息学方法测量人类胚胎干细胞集落增长的方法。

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 ...

video-bioinformatics-analysis-human-embryonic-stem-cell-colony

Research

JoVE Journal

Biology

12.7K 次观看.  University of California. 视频生物信息学的自动化处理，分析，理解，并从微观的影片中提取的生物时空数据的数据挖掘。本文的目的是为了演示使用视频生物信息学方法测量人类胚胎干细胞集落增长的方法。

视频: Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

人类胚胎干细胞传代的色调线与胰蛋白酶

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

科研

生物学

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

人类胚胎干细胞（ES细胞）的繁殖

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 ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

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 ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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 ...

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 ...