1。解冻胚胎干细胞预实验设置<ol><li>前一天，解冻胚胎干细胞，板照射的小鼠胚胎成纤维细胞（MEF），CF1的应变，MEF培养液中的明胶涂层的6孔组织培养板（D - MEM培养基辅以滋养层10％热灭活胎牛血清和1％非必需氨基酸）。孵育过夜，在37 ° C和5％的CO 2。 </li><li>的胚胎干细胞从液氮中删除之前，一定要在所有必要的设备和试剂，并准备使用，以确保有效和成功的解冻。 </li></ol>解冻胚胎干细胞<ol start="3"><li>从液氮存储罐使用金属镊子取出的胚胎干细胞的低温小瓶。 </li><li>卷为3-5秒，戴手套的手之间的小瓶删除霜冻。 </li><li>在小瓶标签上记录的信息。 </li><li>使用金​​属镊子，浸入37℃水浴的小瓶。瓶轻轻摇动，并观察解冻的进展往往小瓶光看到冰晶的大小（但很快！）。不要淹没在水浴小瓶上限，因为这可能污染的细胞。 </li><li>当仍然只是一个很小的冰晶，淹没整个小瓶中的乙醇消毒。 </li><li>在无菌的生物安全柜，低温瓶中的内容直接传送到15 mL锥形管的底部。 </li><li>慢慢胚胎干细胞培养液中添加4毫升（D-MEM/F-12培养基与血清替代20％的淘汰赛，1％非必需氨基酸，1％L -谷氨酰胺，0.2％β-巯基乙醇和4纳克/补​​充毫升碱性成纤维细胞生长因子）管。轻轻摇动，新媒体添加到管，以不断混合细胞。 </li><li> 5分钟后，细胞离心200 × G. </li></ol>电镀的胚胎干细胞<ol start="11"><li>虽然胚胎干细胞在离心机，准备通过适当的胚胎干细胞的信息标签MEF馈线板：细胞株，传代次数，及解冻日期。 </li><li>当有左旋转时间约2分钟，吸MEF培养液中加入1.0毫升的PBS井。 </li><li>带颗粒的胚​​胎干细胞的生物安全柜，并认真吸取上清液。小心不吸细胞沉淀，但消除尽可能上清，这个解决方案包含从冻结介质DMSO。 </li><li>重悬沉淀，轻轻胚胎干细胞培养液中加入2.5毫升用5 mL的玻璃吸管，移液3-4次。 </li><li>以及从MEF馈线的PBS吸，慢慢加入6孔板准备好所有的胚胎干细胞悬液2.5毫升。 </li><li>放置到37℃孵化器板，小心地将板着回来，方方，均匀地分布在整个以及细胞。不要旋流板的圆形图案，以避免集中在中心的殖民地。 </li><li>允许细胞在37 ° C和5％的CO 2一夜之间重视。 </li><li>解冻后的一天，取出培养基（与任何浮动细胞），新鲜胚胎干细胞培养液中加入2.5毫升的良好。 </li><li>可选：移除的介质和细胞可以被转移到一个单独的准备MEF的接驳板。这一步往往会产生新的殖民地，从原来的解冻细胞的同时，可培养的。 </li><li>孵育37 ° C和5％的CO 2一夜之间板块。 </li></ol> 2。冷冻胚胎干细胞<ol><li>虽然胚胎干细胞在离心机，标签内的生物安全柜的低温，小瓶，包括细胞系，传代次数，和冻结日期。典型的冷冻胚胎干细胞的密度为每低温瓶细胞（从6孔板）。 </li><li>当胚胎干细胞，离心，生物安全柜带来的管。从管吸上清，小心不要去打扰松散包装的细胞沉淀。 </li><li>重悬细胞沉淀与胚胎干细胞培养液中适量：0.5毫升胚胎干细胞每低温瓶中的培养基。移液时，胚胎干细胞复苏时更好地冻结在比较大的殖民地件很温柔。 </li><li>慢慢地，一边轻轻摇晃试管，2X胚胎干细胞冷冻培养基（60％定义胎牛血清，20％的胚胎干细胞培养液中，20％二甲基亚砜）添加适量（0.5毫升，每离心管）的细胞。补充说，一旦冻结介质吸管解决方案非常轻轻一到两倍混合</li><li>很快，但轻轻地，加1毫升细胞悬液，以每低温小瓶。 </li><li>异丙醇冷冻集装箱和地方转移到小瓶一个-80 ° C冰箱过夜。细胞将冻结在1℃每分钟异丙醇冷冻集装箱。 </li><li>翌日，迅速转移冷冻瓶液氮储存罐，使用金属镊子。 </li></ol>代表性的成果人类胚胎干细胞解冻后的第一天，可能会出现小菌落透明和可能难以在显微镜下看到。由于刚解冻的胚胎干细胞往往增殖缓慢，他们可能需要几天的时间，显示为建立殖民地（图1）。 图1。细胞的复苏和增长 。胚胎干细胞从液氮解冻成像 1，7日和11日解冻后。

<ol>
	<li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Technique. , (2005).</li><li>Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. <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), 1145-1147 (1998).</li></ol>

附带的视频演示解冻和冷冻胚胎干细胞的方法。应在液态氮中冷冻细胞解冻后尽快洗澡，以获得最佳的复苏。记住，要非常小心，移液解冻细胞时，：尽量减少处理的细胞和吸管轻轻。一小瓶胚胎干细胞可以被镀到任何一个6孔板，或4孔板井，并立即放置在孵化器。由于在四个独立的井培养减少失去整个文化污染的可能性，并可以更容易地找到胚胎干细胞克隆较小的表面积，4孔板时，建议第一次解冻胚胎干细胞。 解冻后的胚胎干细胞的第一天，菌落很小，可能是透明的。每天补充的胚胎干细胞培养液，殖民地，即使在显微镜下明显，因为它可能需要几天在文化为胚胎干细胞出现为建立殖民地。重要的是使用新鲜镀的MEF饲养层细胞解冻时，殖民地常常不能达到一个合适的大小，传代后，直到10月12日解冻。 解冻时的胚胎干细胞的低传小瓶，这是很好的做法，扩大和冻结额外的小瓶，保持低传代细胞，为未来的文化和实验的股票。定期冻结所有“额外”的健康胚胎干细胞保持冻结的股票，这也是一个好主意。作为高品质的，无差别的，积极除以胚胎干细胞克隆的最成功的解冻和随后的文化被冻结。胚胎干细胞恢复冻结，更有效地处理较大的细胞聚集轻轻。最后的总大小应该是相似的（或稍大）后例行通过镀聚合的大小。

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		<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>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965-092</td>
<td>For MEF mediumjavascript:void(0);</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Certified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>Heat-inactivated 
For MEF medium</td>
</tr>
<tr>
<td>D-MEM/F-12</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-057</td>
<td>&nbsp;</td>
<tr>
<td>Knockout Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>Pre-screen to ensure support of hES cells</td>
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<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>03-0002</td>
<td>Use at [4 ng/mL] final</td>
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<tr>
<td>Non-essential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
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<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030-081</td>
<td>200 mM (100X) 
Use at [1X] final</td>
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<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-250</td>
<td>Without Ca2+ or Mg2+</td>
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<tr>
<td>Collagenase IV</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>Make a fresh solution at 1 mg/mL in D-MEM/F-12</td>
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<td>Gelatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1890</td>
<td>Type A, Porcine</td>
</tr>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2560</td>
<td>For freezing medium</td>
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<td>Fetal Bovine Serum (FBS), Defined</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH300.70.01</td>
<td>For freezing medium</td>
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<td>6-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>140675</td>
<td>For general culture</td>
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<tr>
<td>4-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>For thawing</td>
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<tr>
<td>5 mL glass pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-27E</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>15 mL conical tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>Polypropylene</td>
</tr>
<tr>
<td>Cryogenic vials</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>5000-1020</td>
<td>1.5 mL capacity</td>
</tr>
<tr>
<td>Freezing container</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>5100-0001</td>
<td>&#x201C;Mr. Frosty&#x201D;</td>
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freezing and thawing human embryonic stem cells

自詹姆斯汤姆森<em&gt;等</em&gt;在1998年开发出一种技术，隔离，并在文化中成长的胚胎，冷冻细胞以供日后使用，从冻结的股票解冻和扩大细胞已成为进行常规胚胎干细胞培养中的重要程序。由于胚胎干细胞冷冻和解冻的压力非常敏感，必须特别小心。在这里，我们表现出适当的技术迅速解冻胚胎干细胞从液氮股，电镀小鼠胚胎馈线细胞，并慢慢地冻结他们长期储存。

Watch this Scientific Journal Video about Freezing and Thawing Human Embryonic Stem Cells at JoVE.com

Freezing and Thawing Human Embryonic Stem Cells

自从詹姆斯汤姆森等人在1998年开发出一种技术，隔离，并在文化中成长的胚胎，已成为供以后使用，从冻结的股票解冻和扩大细胞的冷冻细胞进行常规胚胎干细胞培养中的重要程序。由于胚胎干细胞的冷冻和解冻的压力非常敏感，必须特别小心。在这里，我们表现出适当的技术迅速解冻胚胎干细胞从液氮股，对小鼠胚胎馈线细胞电镀，慢慢冻结他们长期储存。

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 ...

freezing-and-thawing-human-embryonic-stem-cells

Research

JoVE Journal

Biology

45.6K 次观看.  Stemgent. 自从詹姆斯汤姆森等人在1998年开发出一种技术，隔离，并在文化中成长的胚胎，已成为供以后使用，从冻结的股票解冻和扩大细胞的冷冻细胞进行常规胚胎干细胞培养中的重要程序。由于胚胎干细胞的冷冻和解冻的压力非常敏感，必须特别小心。在这里，我们表现出适当的技术迅速解冻胚胎干细胞从液氮股，对小鼠胚胎馈线细胞电镀，慢慢冻结他们长期储存?...

视频: Freezing and Thawing Human Embryonic Stem Cells

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

冻结人类胚胎干细胞

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

科研

生物学

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

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 ...

Freezing, Thawing, and Packaging Cells for Transport

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks. 

Cultured mammalian cells are used extensively in cell biology studies. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.

冷冻，解冻，和运输包装细胞

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the ...

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 ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

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 ...

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 ...