这项研究得到了 (部分) 的支持, 由美国国立卫生研究院 NIH 的内部研究计划。特别感谢 Shyamal Peddada 的统计分析。我们还要感谢汉弗瑞, 加里鸟, 和雪荒尾照片, 以审查这份手稿, 和佛朗哥 DeMayo 继续支持和建议。

所有操作都是作为 NIEHS 击出鼠标核心设施的正常操作的一部分进行的。所有的老鼠都保持在12:12 小时的光: 黑暗的循环, 并喂养 NIH-31 和水的饮食ad 随意.所有的实验都是按照国家环境健康科学研究所的动物护理和使用委员会进行的。
1. 动物制剂
<ol><li>培育捐赠小鼠, B6 (cg)-Tyr < c-2J >/J 雌性小鼠在 8-10 周自然 B6 (Cg)-Tyr < c-2J >/j 雄性小鼠在8和26周的年龄。第二天早上 (E0.5) 目视检查插头。</li>
	<li>养殖受体小鼠, 瑞士韦氏雌性小鼠在6和9周的年龄是自然繁殖输精管切除术瑞士韦氏雄性小鼠。第二天早上 (E0.5) 目视检查插头。</li>
</ol>2. ES 细胞制剂
<ol><li>对于 129-derieved AB2.2 ES 细胞, DMEM 的培养细胞补充15% 的血清, 1% 谷氨酰胺, 1% 佩恩/链球菌, 0.1% β巯基乙醇。使用推荐的协议, 每 es 细胞供应商的说明中培养所有其他 es 细胞。</li>
</ol>3. 胚胎采集
<ol><li>
动物解剖
	<ol>
<li>准备60毫米的菜肴, 大约1每5小鼠加上3额外, 与10毫升爆炸收集媒体 (立方米) (10% DMEM) 和一个4个好菜与 0.5 mL 每井。平衡在37°c, 5% CO2。</li>
		<li>弄死 B6 (Cg)-Tyr < c-2J >/J 母鼠 E3.5 使用 CO2窒息, 流速为10%。</li>
		<li>euthanizing 后, 将小鼠置于仰卧位, 用70% 乙醇彻底湿润腹部。</li>
		<li>通过皮肤的第一切口, 在腹部的中点, 使开口尽可能宽。接下来通过腹壁打开, 再次使切口尽可能宽。 注意: 在两个步骤中做到这一点有助于减少或消除毛皮污染。</li>
		<li>从卵巢开始, 用钝钳抱住卵巢的脂肪垫, 小心地移除卵巢、输卵管和子宫, 格外小心, 使脂肪组织保持最小。将子宫作为单个单位或单独的角移除, 以下过程不发生任何变化。</li>
		<li>取出后, 将子宫放置在含有爆炸收集介质的60毫米盘中。</li>
	</ol>
</li>
	<li>
胚胎切除
	<ol>
<li>准备一个10毫升的注射器, 装满了立方米, 并配合25克针。</li>
		<li>一次子宫, 将其从所持皿中取出, 将其涂抹在组织上以去除多余的血液, 并在低放大率解剖显微镜下放置在60毫米的盘子中。</li>
		<li>当仔细地和轻轻地举在远端末端 (在输卵管附近) 与解剖钳的子宫, 插入针在子宫的中心在平行一架飞机尽可能。插入针只是在钳的尖端。</li>
		<li>对镊子施加一些压力, 将子宫放到针上, 然后通过子宫角排出大约1毫升的立方米。 注意: 在这一步必须小心, 因为太多的压力, 注射器会导致一个无法控制的媒体流, 经常离开盘子。</li>
		<li>冲洗后的子宫, 多达5整个子宫每60毫米菜, 把菜放回孵化器, 直到收集。</li>
	</ol>
</li>
	<li>
胚胎采集
	<ol>
<li>使用手拉玻璃吸管和嘴吸管装置, 收集在解剖范围内的囊胚, 设置在25X 和35X 之间。(图 1)。 注: 口吸管装置由 30-40 厘米的挠性油管组成, 在5毫米的 id 下插入1、000µL 吸管尖端, 首先指向一端, 并在其上贴上玻璃吸管。添加注射器过滤器到另一端, 附有 1 cc 注射器桶, 没有柱塞, 作为一个口片。</li>
		<li>从孵化器中取出菜肴, 使用网格模式, 检查盘子中的每一部分是否有胚胎。胚胎被单独拾起, 并放置在第二道菜的一滴。</li>
		<li>从所有的菜肴中收集后, 再在一滴新鲜的立方米中再洗一次, 然后放在4井的盘子里等待注射。</li>
	</ol>
</li>
</ol>4. 胚胎注射
<ol><li>根据确认的注射针分别取 ES 细胞。<ol>
<li>通过加入 5-7 毫升的注射培养基 (DMEM, 10% 血清和20毫米 HEPES), 将注射盘准备成一个玻璃底部注射皿。注射是在室温下进行的。</li>
		<li>使用空气或油 microinjector, 将真空应用于注射针, 以吸引 ES 细胞。注意尽量在细胞间保持尽可能少的空间。计算 ES 细胞在这里, 或在注射步骤。</li>
		<li>选择与普通细胞相比, 大小适中的 ES 细胞, 表面光滑, 无明显的缺陷, 如空泡。 注意: ES 细胞的形态质量有很大差异, 所使用的标准将需要与之进行调整。</li>
	</ol>
</li>
	<li>注射大约 15 ES 细胞的胚胎 (图 1)。<ol>
<li>将持有吸管尖端连接到第二个空气 microinjector, 并将胚胎定位在支架的开口附近。应用真空将胚胎固定在支架上。</li>
		<li>通过将保持吸管向上或向下移动来调整 z 轴, 这样胚胎才会接触到滑块, 然后细聚焦在透明的上。接下来, 调整注射针的 Z 轴, 使尖端附近的 ES 细胞处于焦点。</li>
		<li>现在调整胚胎, 通过轻轻推从顶部或底部注射针, 旋转它把 ICM 在理想的位置。</li>
		<li>使用一个坚定但控制的推力插入注射针通过透明的, 或者 ICM 或滋养层。对注射针的 microinjector 施加轻微的压力, 以帮助防止囊胚崩溃。</li>
		<li>一旦针穿透囊胚, 施加 microinjector 的压力, 将 ES 细胞转移到胚胎的囊胚。短暂停顿, 因为针的斜角开始离开胚胎, 使胚胎中的压力恢复正常, 同时仍保留细胞。 注: 参考组 es 细胞正常注射, 遵循传统 es 细胞注射协议9, 注意不要接触 ICM。ES 细胞也被注入通过 icm, 直接推针通过 icm (TICM) (图 2, 补充电影)。</li>
	</ol>
</li>
</ol>5. eEmbryos 的转让
<ol><li>在 E2.5, 使用非手术胚胎转移装置, 根据制造程序, 将胚胎移植到瑞士韦伯斯特小鼠体内。这种非手术程序不需要麻醉或特殊术后护理。在胚胎移植后, 通过分娩和随后的断奶, 单独喂养小鼠。</li>
</ol>

<ol>
	<li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Genome+Engineering+Using+CRISPR/Cas+Systems.">Multiplex Genome Engineering Using CRISPR/Cas Systems.</a> Science. 339 (6), 819-823 (2013).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6), 823-826 (2013).</li><li>Friedel, R., Seisenberger, C., Kaloff, C., Wurst, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EUCOMM+the+European+Conditional+Mouse+Mutagenesis+Program.">EUCOMM the European Conditional Mouse Mutagenesis Program.</a> Brief Funct Genomic Proteomic. 6 (3), 180-185 (2007).</li><li>Austin, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+knockout+mouse+project.">The knockout mouse project.</a> Nat Genet. 36 (9), 921-924 (2004).</li><li>Coleman, J. L., Brennan, K., Ngo, T., Balaji, P., Graham, R. M., Smith, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Knockout+and+Reporter+Mouse+Line+Generation+and+Breeding+Colony+Establishment+Using+EUCOMM+Conditional-Ready+Embryonic+Stem+Cells:+A+Case+Study.">Rapid Knockout and Reporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMM Conditional-Ready Embryonic Stem Cells: A Case Study.</a> Front Endocrinol (Lausanne). 30, 105 (2015).</li><li>Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., Roder, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+completely+cell+culture-derived+mice+from+early-passage+embryonic+stem+cells.">Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.</a> Proc Natl Acad Sci U S A. 90 (18), 8424-8428 (1993).</li><li>Eggan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+and+female+mice+derived+from+the+same+embryonic+stem+cell+clone+by+tetraploid+embryo+complementation.">Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation.</a> Nat Biotechnol. 20 (5), 455-459 (2002).</li><li>Eggan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+vigor,+fetal+overgrowth,+and+viability+of+mice+derived+by+nuclear+cloning+and+tetraploid+embryo+complementation.">Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.</a> Proc Natl Acad Sci U S A. 98 (11), 6209-6214 (2001).</li><li>Eakin, G. S., Hadjantonakis, A. K., Papaioannou, V. E., Behringer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+potential+and+behavior+of+tetraploid+cells+in+the+mouse+embryo.">Developmental potential and behavior of tetraploid cells in the mouse embryo.</a> Dev Biol. 288 (1), 150-159 (2005).</li><li>Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+production+of+mice+from+embryonic+stem+cells+injected+into+four-+or+eight-cell+embryos+by+piezo+micromanipulation.">Efficient production of mice from embryonic stem cells injected into four- or eight-cell embryos by piezo micromanipulation.</a> Stem Cells. 26 (7), 1883-1890 (2008).</li><li>Poueymirou, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F0+generation+mice+fully+derived+from+gene-targeted+embryonic+stem+cells+allowing+immediate+phenotypic+analyses.">F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses.</a> Nat Biotechnol. 25 (1), 91-99 (2007).</li><li>Behringer, R., Gertsenstein, M., Nagy, K. V., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse embryo. , (2014).</li><li>Pease, S., Saunders, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advanced Protocols for Animal Transgenesis. , (2011).</li><li>Young, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mechanical+stimulation+on+the+reprogramming+of+somatic+cells+into+human-induced+pluripotent+stem+cells.">Effects of mechanical stimulation on the reprogramming of somatic cells into human-induced pluripotent stem cells.</a> Stem Cell Res Ther. 8 (1), 139 (2017).</li></ol>

作者没有相互竞争的利益来披露。

长期以来, 人们一直认为 ICM 的任何干扰都可能导致死亡率, 事实上, 目前的胚泡微注射协议仍然警告此12,13。我们在这里显示的是, 通过 ICM 的显微注射不损害胚胎, 并增加合体的产量。
这种效果的确切机制尚未确定。然而, ICM 的机械干扰, 以及伴随的 hypoblast, 应将 ES 细胞的发病率增加到新形成的 ICM 后, 显微注射。最近在 iPSCs 领域的...

近25年来, 基因靶向小鼠的产生一直是一个缓慢的过程, 经常受到胚胎干细胞微注射阶段的瓶颈的阻碍, 生殖传送合体的产生。最近的进展, 包括 CRISPR/Cas91、2针对 ES 细胞和高吞吐量 es 细胞生成联盟 (如 KOMP), 改善了基因改良 ES 细胞的生成/可用性。然而, 生殖合体的产生仍然是从这些 ES 细胞中创建基因修饰小鼠的瓶颈3,4。高吞吐量 es 细胞生成项目受到 es 细胞质量高变异性的困扰, 这对于成功创建生殖合体至关重要。此外, 一些常用 ES 细胞系以高体检著称, 生殖主管合体5难以生产。
已经开发了许多方法来创建具有更高的嵌合体的老鼠, 或完全 ES 细胞衍生的老鼠6,7,8,9,10。虽然这些系统都有自己的优点, 但也有其缺点。生成的二、四倍体杂交合体, 同时生成 100% ES 细胞派生的小鼠, 是低效的, 通常限于 outbred线5, 6.新的注入桑椹胚的方法可以产生高百分比合体, 接近 100%, 但通常需要大量额外设备 (激光器或 piezos) 和训练 10, 11.在这些技术已经到位的实验室里, 可能没有必要采用这种新的方法。
本研究的目的是寻找一种利用常用技术和现成设备来提高嵌合体生成率的方法, 增加突变等位基因生殖传播的几率, 建立后续的小鼠群。

<table>
	<tbody>
		<tr>
			<td>ES Cell injection needle</td>
			<td>Humagen</td>
			<td>MSC-20-0</td>
			<td></td>
		</tr>
		<tr>
			<td>NSET device</td>
			<td>Paratechs</td>
			<td>60010</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco (life technologies)</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco (life technologies)</td>
			<td>10439-024</td>
			<td>lots should be individually tested for ES Cell compatibility.</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>b-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro manipulator</td>
			<td>Leica</td>
			<td>Micro manipulator</td>
			<td></td>
		</tr>
		<tr>
			<td>micro injector</td>
			<td>Eppendorf AG</td>
			<td>Cell Tram Air 5176</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM IRB</td>
			<td></td>
		</tr>
		<tr>
			<td>4 well dish</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm dish</td>
			<td>Sarstedt</td>
			<td>83.3901</td>
			<td></td>
		</tr>
		<tr>
			<td>B6(Cg)-Tyr&#60;c-2J&#62;/J</td>
			<td>Jackson Labs, Bar Harbor, Maine, USA</td>
			<td>000058</td>
			<td></td>
		</tr>
		<tr>
			<td>Swiss Webster mice</td>
			<td>Taconic, USA</td>
			<td>Tac:SW</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection Dish</td>
			<td>MatTek Corp.</td>
			<td>P50G-0-30-F</td>
			<td></td>
		</tr>
	</tbody>
</table>

trans-inner cell mass injection of embryonic stem cells leads to higher chimerism rates

为了通过 ES 细胞方法提高转基因小鼠的效率, 我们提出了一种对目前囊胚注射协议的适应。在这里, 我们报告, 胚胎的简单旋转, 并通过跨内细胞质量 (TICM) 的注射增加了嵌合体小鼠的百分比从31% 到 50%, 没有额外的设备或进一步的专门训练。在9月内注射了26种不同的近交无性系克隆和35株总无性系。传统注射技术与 TICM 的妊娠率、胚胎恢复率均无显著性差异。因此, 如果没有任何重大改变的注射过程和简单的定位胚泡和注射通过 ICM, 释放 ES 细胞进入囊胚腔可以有可能提高嵌合生产的数量和随后生殖传输。

以细胞系为随机效应, 采用混合效应线性模型分析软件中的数据 (如 SAS (9.2 版))。每种分析都控制着移植胚胎的数量和细胞系。第二种分析还控制了存活的幼崽数量。
在这项研究中, 每种 ES 细胞系和克隆均采用传统的和 TICM 的技术, 在每个胚胎后交替使用。注射后, 胚胎被隔离在盘子上, 并分组的转移, 一般在12和15胚胎每接受女?...

Watch this Scientific Journal Video about Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates at JoVE.com

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

在这里, 我们提出了一个协议, 以增加嵌合体生产不使用新设备。一个简单的方向改变的胚胎注射可以增加胚胎的数量, 并有可能缩短时间的生殖传播。

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

trans-inner-cell-mass-injection-embryonic-stem-cells-leads-to-higher

Research

JoVE Journal

Biology

10.1K 次观看.  NIEHS/NIH. 在这里, 我们提出了一个协议, 以增加嵌合体生产不使用新设备。一个简单的方向改变的胚胎注射可以增加胚胎的数量, 并有可能缩短时间的生殖传播。

视频: Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

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 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 ...

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 ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

使用LysoTracker检测及鉴别胚胎干细胞在胚胎细胞程序性死亡

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...