设备<ul><li>组织文化的孵化器，37℃，5％的CO 2 </li><li>离心分离</li><li>设置的micropipettors（100，100μL） </li><li>水浴37℃ </li><li>水浴90℃水蒸汽源</li><li>相衬显微镜</li></ul>第一步细胞治疗colcemid 在此过程中，我们使用人类胚胎干细胞线H9的培养灭活的DMEM/F12（Invitrogen公司）与丝裂霉素C（Sigma公司）和保持的小鼠胚胎成纤维细胞（MEF）上，20％的基因敲除血清替代（KSR，GIBCO）和8ng/mL的补充成纤维细胞生长因子（FGF - 2，R＆D）。 为了获得大量的中期利差，这是建议使用高的有丝分裂指数，其中有许多分裂的细胞培养。 <ol><li>以从孵化器的培养瓶中，在层流，添加终浓度在0.1微克/毫升Colcemid。 </li><li>返回烧瓶孵化器，并等待3个小时，所以抑制剂能导致有丝分裂中期，当染色体浓缩，可以很容易地在显微镜下可视的逮捕。 </li></ol>修复细胞<ol start="3"><li>取出介质和添加tripsin / EDTA的0.05％，足以覆盖细胞表面。允许细胞孵育在室温下5分钟，然后加入含血清培养基或刚刚加入血清细胞酶失活。 注：重要的是要确保实现单细胞悬液，以取得良好的中期利差。 </li><li>细胞转移到15 mL离心管中，离心5分钟，在122 克 。 </li><li>弃去上清液，留下约200毫升，并重新悬浮颗粒轻轻攻管，如视频所示。确保细胞分离的，所以你不能可视化解决方案中的任何团块。 </li><li>接下来，您将需要添加5毫升低渗溶液（氯化钾75MM）预热至37 ° C，慢慢的，在下面的描述管壁。首先加3毫升，逆变管到水平位置，只是低渗溶液混合细胞，然后加入剩余的2毫升，并注意保暖在37 ° 15分钟。 注意：此时低渗溶液会导致细胞体积增加，并帮助理清染色体。孵化的时间是很重要的，获得良好的染色体利差。如果时间过长，细胞膜破裂过早的染色体丢失。如果太短，可能很难获得染色体的利差，因为可能不会破坏细胞膜。 </li><li>加入3滴固定液（甲醇/冰醋酸3:1），轻轻地反转管细胞和离心5分钟没有打破122克混合固定液。 注：应该是新鲜的固定液。 注：重要的是使用离心没有打破，以避免细胞消耗，防止材料的流失。 </li><li>弃上清液，留下约200毫升，并轻轻重悬轻敲管底部的沉淀。 注：同样，确保细胞分离的，所以你不能可视化解决方案中的任何团块。 </li><li>添加固定液第3毫升，慢慢管壁，当你旋转管。反转管到水平位置，只是细胞混合，然后添加2毫升的固定液管壁细胞下降。股票在4 ° C过夜。 注：固定液固定细胞可以储存几个月4 ° C。除了保护细胞在其肿胀的状态，固定液去除脂类和蛋白质变性。这些事件使细胞膜脆弱，因此，容易使染色体蔓延。 </li></ol>清洁玻片在开始之前使染色体利差确保您的幻灯片妥善清洁或你可能会失去一些核，也中期分裂。这一步也很重要，如果你想尝试任何杂交技术 9 。 <ol><li>包含6M盐酸在室温下至少3个烧杯中放置幻灯片。 </li><li>过了这段时间，在运行10分钟自来水冲洗幻灯片。 </li><li>幻灯片和蒸馏水冲洗，让他们在室温下干燥。 </li><li>现在的幻灯片，可以存入96％的乙醇，干燥无绒布，使用前。 </li></ol>注：对于原位杂交方法，幻灯片不能存放超过2天到96％的乙醇第二个步骤洗涤细胞<ol start="10"><li>第二天，122 克离心5分钟不间断的细胞。 </li><li> ðiscard上清液，留下约200毫升。轻轻弹管，再加入5毫升的新鲜固定液（甲醇/冰醋酸3:1），管壁慢慢旋转管重悬沉淀。重复此清洗步骤2次以上的共有3个洗。 注：应该是新鲜的固定液。 </li></ol>准备幻灯片<ol start="12"><li>在过去的离心步骤，保留足够数量的解决方案，均匀颗粒细胞和​​细胞获得足够的密度，在幻灯片中（见图1）。 </li><li> 20-30μL细胞悬液加入到一个干净的干滑动移动非常接近平行于表面的吸管。由于固定液的蒸发，滑动面颗粒感。 </li><li>随即，揭露幻灯片，面对30秒成蒸汽，热水（90 °），导致细胞炸毁。 注：在这一刻，从固定液挥发的甲醇，醋酸浓度增加造成的，与水一起刺激染色体蔓延。 </li><li>看在相差显微镜检查的浓度以及细胞分布好（见图1）。 </li><li>现在的幻灯片，准备用来检查染色体G -带（吉姆萨），鱼，天空或比较基因组杂交细胞遗传学方法。 </li></ol>重要的提示如何选择一个适当的中期加以分析选择圆形和孤立的中期（图2d），您可能会避免考虑虚假的非整倍体染色体由两个或两个以上的产生的收益利差混合在一起，或推出的染色体产生染色体的损失。因此，应避免选择靠近原子核的中期，因为有些染色体可能隐藏（图2A和2B）。此外，看一轮中期，确保任何染色体分离是从中期（图2c）。 饲养层细胞和胚胎干细胞染色体核型 G带和SKY分析，饲养层细胞在胚胎干细胞文化的存在不干预的结果，因为饲养层细胞没有下一个分裂的状态（丝裂霉素C或照射灭活）并不会中期人口的一部分。然而，在间期核FISH技术的应用，它是首选分开饲养层细胞收获胰蛋白酶的前殖民地手动FISH分析的人类胚胎干细胞的人口。 <img border="0" src="/files/ftp_upload/1512/1512fig1tmb.jpg" alt="图1" /> 图1：如何知道在幻灯片的合适的细胞密度。 10倍的目标相显微镜的图像。 （一）细胞密度过高。箭头指向太靠近原子核的中期利差。 （二）具有良好的细胞密度的幻灯片。圆圈显示，从原子核或其他中期隔离的中期利差。请<a href="https://www.jove.com/files/ftp_upload/1512/1512fig1.jpg">点击这里</a>看到图1的放大版本。 <img border="0" src="/files/ftp_upload/1512/1512fig2tmb.jpg" alt="图2" /> 图2：如何选择一个适当的中期加以分析。染色体是染色用DAPI荧光显微镜获得的图像在100X目标。 （一）（b）避免靠近原子核的中期（箭头所指）（C）和中期分裂不是圆的，目前的染色体分离（圈）。 （四）全面中期是一个很好的中期加以分析。请<a href="https://www.jove.com/files/ftp_upload/1512/1512fig2.jpg">点击这里</a>看大图图2。

<ol>
	<li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1145 (1998).</li><li>Draper, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+gain+of+chromosomes+17q+and+12+in+cultured+human+embryonic+stem+cells.">Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.</a> Nat Biotechnol. 22 (1), 53-53 (2004).</li><li>Maitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+alterations+in+cultured+human+embryonic+stem+cells.">Genomic alterations in cultured human embryonic stem cells.</a> Nat Genet. 37 (10), 1099-1099 (2005).</li><li>Mitalipova, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+the+genetic+integrity+of+human+embryonic+stem+cells.">Preserving the genetic integrity of human embryonic stem cells.</a> Nat Biotechnol. 23 (1), 19-19 (2005).</li><li>Buzzard, J. J., Gough, N. M., Crook, J. M., Colman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotype+of+human+ES+cells+during+extended+culture.">Karyotype of human ES cells during extended culture.</a> Nat Biotechnol. 22 (4), 381-381 (2004).</li><li>Caisander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+integrity+maintained+in+five+human+embryonic+stem+cell+lines+after+prolonged+in+vitro+culture.">Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture.</a> Chromosome Res. 14 (2), 131-131 (2006).</li><li>Imreh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+conditions+favoring+selection+of+chromosomal+abnormalities+in+human+ES+cells.">In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells.</a> J Cell Biochem. 99 (2), 508-508 (2006).</li><li>Robin-Wesselschmidt, J. L., Loring, J. F., Robin-Wesselschmidt, J. L., Robin-Wesselschmidt, S. c. h. w. a. r. t. z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Stem Cell Manual. , 59-59 (2007).</li><li>Rooney, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cytogenetics. , (2001).</li><li>Heslop-Harrison, P., Schwarzacher, T., Heslop-Harrison, P., Schwarzacher, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical in situ hybridization. , 51-51 (2000).</li></ol>

The authors have nothing to disclose.

染色体利差的准备是成功的胚胎干细胞的遗传状态的由常规技术的G -带，和更先进的技术，如鱼，天空和CGH分析的关键一步。 此过程可应用于不同colcemid孵化的时期，这对细胞周期的长短取决于多种细胞类型。对于胚胎干细胞的殖民地，我们等待了3个小时，而胚体（EB）的时间可长达6个小时。 在细胞聚集（如胚体），则需要很好地分解，为了获得一个单...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>15 ml centrifuge tube</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>91015</td>
<td>&nbsp;</td>
<tr>
<td>Colcemid Karyo MAX</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15212-012</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Isofar</td>
<td>721</td>
<td>&nbsp;</td>
<tr>
<td>Glacial acetic acid</td>
<td>&nbsp;</td>
<td>Isofar</td>
<td>100</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>&nbsp;</td>
<td>Isofar</td>
<td>208</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Chloride (KCl)</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>104.931.000</td>
<td>&nbsp;</td>
<tr>
<td>Slide</td>
<td>&nbsp;</td>
<td>Bioslide</td>
<td>7105-1</td>
<td>&nbsp;</td>
<tr>
<td>Tripsin</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>T4799</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

chromosomal spread preparation of human embryonic stem cells for karyotyping

虽然人类胚胎干细胞（胚胎干细胞）已被证明是目前稳定的二倍体核型1，已经有很多研究报告，根据培养条件下，他们变得容易获得全部或部分染色体此外，如染色体异常。事实上，在长期培养，染色体核型改变时使用酶或化学分解2,3,4，而手动传代的殖民地清扫，保持一个稳定的染色体核型5。此外，作为饲养层细胞的清除等环境的变化也似乎妥协的胚胎干细胞3,6的遗传完整性。一旦染色体改变可能会影响细胞生理学，人类胚胎干细胞在体外的遗传完整性的特性是至关重要的考虑人类胚胎干细胞在胚胎发育的研究和药物试验的一个必不可少的工具。此外，为今后的治疗目的，染色体的变化是一个真正的关注，因为它通常是相关联的癌变。 在这里，我们展示一个简单而有用的方法来获得高品质的染色体利差染色体G显带，鱼，天空或比较基因组杂交技术7,8随后分析。我们建议常规检查每隔5代的染色体状态，以监测易位和非整倍体的外观 Priscila布里托和拉斐拉Sartore贡献同样的文件。

Watch this Scientific Journal Video about Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping at JoVE.com

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

核型分析是一种简单而有用的技术，广泛用于检测遗传变异。在这里，我们描述了一个一步一步监测文化在保持这些细胞的染色体状态的人类胚胎干细胞的染色体蔓延的准备协议。

为核型分析人类胚胎干细胞的染色体蔓延制备

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

chromosomal-spread-preparation-human-embryonic-stem-cells-for

Research

JoVE Journal

Biology

48.5K 次观看.  Federal University of Rio De Janeiro-UFRJ. 核型分析是一种简单而有用的技术，广泛用于检测遗传变异。在这里，我们描述了一个一步一步监测文化在保持这些细胞的染色体状态的人类胚胎干细胞的染色体蔓延的准备协议。

视频: Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

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

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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

在人类胚胎干细胞的线粒体复杂的活动初探

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

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 ...

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 ...

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.

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.