目前支持人类胚胎干细胞研究在Teitell实验室由加州再生医学研究所（CIRM）种子批RS1 - 00313。我们感谢卡拉克勒博士（加州大学洛杉矶分校）和克勒实验室成员在这些问题和进一步的研究提供意见和见解。

高分辨率清除母语凝胶Electophoresis线粒体复合物（hrCNE）活性测定 - 凝胶单层ˠ照射或丝裂霉素C处理过的小鼠胚胎成纤维细胞（MEFs）然后切换MEF空调介质中，使用标准的条件下5天的基质胶上的增长，对维持人类胚胎干细胞（胚胎干细胞）。为了去除污染MEFs，人类胚胎干细胞被重新镀上基质胶和MEF空调额外3天的中期增长。 Oct - 4和SSEA - 4的多能性标志物免疫荧光显微镜是用来确认的人类胚胎干细胞分化的缺乏。 <ol><li>细胞与温暖的1X PBS，pH值7.4，胰蛋白酶（1毫升1X胰蛋白酶在6孔板，每孔）5分钟洗净。 </li><li>添加新鲜的胰蛋白酶抑制剂等体积（1mg/ml的1X PBS，pH值7.4）。在200克收获细胞和离心5分钟，室温。 </li><li>丢弃在0.8上清液和悬浮颗粒细胞 - 1.7毫升冰冷的线粒体隔离缓冲区（83MM蔗糖; 6.6mM咪唑/盐酸，pH值7.0）（根据细胞沉淀的大小）新鲜配制的1蛋白酶抑制剂。在冰上孵育10分钟。 </li><li> Dounce细胞与均质机（〜40 - 50杆）。 dounced细胞台盼蓝染色等份，以同质化的使用光学显微镜的完整性检查。 </li><li>在20000克离心10分钟，4 ° C至获得原油线粒体还包含细胞核和细胞碎片较大的颗粒。倒出并丢弃上清液，并权衡沉淀原油线粒体颗粒。颗粒可以保存于-80 ° C以下液氮的瞬间凝固，如果这是一个停止点。 注：可以用于分离和收集一个纯粹的线粒体膜分数差速离心或其他基于梯度程序。见文献。有关详细信息，4。这种替代的方法将产生每毫克线粒体蛋白复杂的活动，而不是每毫克体重细胞或细胞数量的线粒体复杂的活动。线粒体蛋白的浓度，将需要净化，使这个比较后确定。此外，这种方法可能需要不同的隔离缓冲。溶解纯线粒体膜分数，请注意以下步骤6。 </li><li>冰冰冷的增溶缓冲液（氯化钠50MM，50MM咪唑/盐酸pH值7.0，为1mM EDTA，2MM 6 - 氨基己酸）涡旋溶解原油线粒体颗粒。 6 - 氨基己酸，可以被替换为1个蛋白酶抑制剂。使用颗粒重量的20毫克每增溶缓冲液35μl。加入20μL的10％W / V每20毫克的颗粒重量和组合由轻弹管毛地黄皂苷。在冰上孵育5-10分钟。十二烷基- BD -麦芽糖或Triton X - 100可替代毛地黄皂苷溶解的蛋白质。洗涤剂和数量的选择可能会影响supercomplexes线粒体复合物多聚体形式的形成。欲了解更多信息，请参阅参考资料1。 注：所需数量的洗涤剂溶解一个纯粹的线粒体膜分数对应的洗涤剂/蛋白质2 - 6G /克不等的重量比。溶解线粒体膜的最佳比例是2.5克/克为十二烷基- BD -麦芽糖，3G / G为Triton X - 100，和6g /毛地黄皂苷克。 </li><li> 10万克离心15分钟在4 ° C溶解的沉淀收集上清液。 </li><li>样缓冲液加入到最后的5％W / V甘油和0.01％W / V丽春红（使用50％W / V甘油，0.1％W / V丽春红加载缓冲库存解决方案）。 </li><li>装载卷包含原油线粒体颗粒每通道20 - 40毫克凝胶井。使用3.5％的浓缩胶，丙烯酰胺梯度凝胶电泳（4 -13％）。 注：凝胶的制备及电泳条件： 凝胶法制备，使用如下的丙烯酰胺/双丙烯酰胺（AA / bisAA）3组合：丙烯酰胺和双丙烯酰胺1.5克（49.5％T，3％，其中T是AA和bisAA和C总浓度是48克总单体交联剂（bisAA）的百分比）。 胶缓冲液（3）：1.5M 6 - 氨基己酸; 75MM咪唑，pH值7.0 </li></ol><table style="padding-left: 60px;" border="0"><tbody><tr><td>梯度凝胶： </td><td> 4％ </td><td> 13％ </td><td>浓缩胶： </td><td> 3.5％ </td></tr><tr><td> AA / bisAA 3混合</td><td> 2毫升</td><td> 6.5毫升</td><td></td><td> 0.6毫升</td></tr><tr><td>凝胶缓冲3“ </td><td> 8.3毫升</td><td> 8.3毫升</td><td></td><td> 2.7毫升</td></tr><tr><td>甘油</td><td></td><td> 5克</td><td></td><td></td></tr><tr><td> H 2 O </td><td> 14.7毫升</td><td> 5毫升</td><td></td><td> 4.7毫升</td></tr><tr><td>总成交量</td><td> 25毫升</td><td> 25毫升</td><td></td><td> 8毫升</td></tr><tr><td> 10％的APS </td><td> 139μL </td><td> 125μL </td><td></td><td> 67μL </td></tr><tr><td> TEMED </td><td> 14μL </td><td> 12.5μL </td><td></td><td> 6.7μL </td></tr></tbody></table>阳极缓冲：25MM咪唑/盐酸，pH值7.0 阴极缓冲液：50MM，7.5MM咪唑甘氨酸/盐酸，0.02％W / V十二烷基- BD -麦芽糖，0.05％，pH值7.0 W / V脱氧胆酸 在寒冷的房间（4-7℃）倒入和运行凝胶。所使用的初始电压是100V。当样品进入浓缩胶，引发电压与电流限制到500V至15mA（0.15&#39;14“14厘米凝胶）。电泳后3-4小时，当红色的丽春红染料的急剧行的方法凝胶前停止。另外，凝胶可以用恒压运行在70 - 100V过夜。 <ol><li>凝胶活性测定。每次检测的孵化时间取决于蛋白质增溶的效率和材料的数量，每孔装。我，IV和V配合物通常会产生更强的信号比复合物II和III。 </li></ol>复杂的I（CI） 孵育CI缓冲区（5MM的Tris -盐酸，pH值7.4 2.5mg/ml NBT;为0.1mg/ml NADH）的凝胶片在室温下了好几个小时。 CI为缓冲区：每5MM的Tris - HCl，pH值7.4百毫升添加： 0.01克NADH 0.25克NBT 修复凝胶在50％甲醇和10％醋酸为15-45分钟。固定凝胶是保存在10％醋酸。 复合物Ⅱ（CII） 孵育与CII的缓冲区（20MM琥珀酸钠5MM的Tris - HCl pH值7.4; 0.2MM吩嗪methasulfate; 2.5mg/ml NBT）凝胶片几个小时，在室温下。 CII的缓冲区：每5MM的Tris - HCl，pH值7.4100毫升添加： 200μL1M琥珀酸钠 80μl吩嗪methasulfate（250MM的PMS DMSO溶解） 0.25克NBT 修复复杂所述的凝胶综合三（CIII） 孵育CIII缓冲区（50MM磷酸钠，pH值7.2; 0.05％3,3&#39; - 二氨基tetrahydrochloride（DAB））的凝胶片在室温下了好几个小时。 CIII缓冲液：每50毫米的钠磷酸盐，pH值7.2100毫升： 50毫克二氨基tetrahydrochloride 修复复杂所述的凝胶综合四（CIV） 孵育CIV缓冲区（50MM磷酸钠，pH值7.2; 0.05％3,3&#39; - 二氨基tetrahydrochloride（DAB），50μM马心细胞色素c）的凝胶片几个小时，在室温下。 CIV缓冲液：每50毫米的钠磷酸盐，pH值7.2百毫升： 50毫克二氨基tetrahydrochloride 0.1克马心细胞色素c 修复复杂所述的凝胶综合五（简历） 孵育凝胶片CV缓冲区（35MM三基地，270甘氨酸，pH值8.3在25 ° C，14mm的硫酸镁 ，0.2％W / V PB（NO 3）2，8mm的三磷酸腺苷）在室温下了好几个小时。 CV缓冲液：每100毫升的解决方案中添加： <table border="0"><tbody><tr><td>三基</td><td> 0.424克</td></tr><tr><td>甘氨酸</td><td> 2.026克</td></tr><tr><td> 硫酸镁 </td><td> 0.345克</td></tr><tr><td> PB（NO 3）2 </td><td> 0.29克</td></tr></tbody></table>无需调整pH值（应该是8.3） - 不使用酸或碱。过滤和添加<table border="0"><tbody><tr><td>三磷酸腺苷</td><td> 0.441克</td></tr></tbody></table>修复凝胶在50％甲醇30分钟，并转移到水。

<ol>
	<li>Wittig, I., Braun, H. -. P., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> Nature Protocols. 1, 418-428 (2006).</li><li>Wittig, I., Karas, M., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Resolution+Clear+Native+Electrophoresis+for+In-gel+Functional+Assays+and+Fluorescence+Studies+of+Membrane+Protein+Complexes.++6.">High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes. 6.</a> Molecular &amp; Cellular Proteomics. 6, 1215-1225 (2007).</li><li>Wittig, I., Carrozzo, R., Santorelli, F. M., Sch&#747;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assays+in+high-resolution+clear+native+gels+to+quantify+mitochondrial+complexes+in+human+biopsies+and+cell+lines.">Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines.</a> Electrophoresis. 28, 3811-3820 (2007).</li><li>Castle, J. D., Coligan, J. E., Dunn, B. M., Speicher, D. W., Wingfield, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Organelles+from+Mammalian+Cells.">Purification of Organelles from Mammalian Cells.</a> Current Protocols in Protein Science. 4.2, 4.2.1-4.2.57 (2007).</li></ol>

在这段视频中，我们已经描述了从人类胚胎干细胞，他们高分辨率的清晰本地凝胶电泳（hrCNE）分离，以及随后的分析凝胶的催化活性检测线粒体蛋白质复合物的提取。 hrCNE解析线粒体膜蛋白复合物，并在一项决议，与蓝色本地凝胶电泳凝胶活性测定优越。可以采用这种技术不仅量化线粒体呼吸复合物IV也分析，无论是在人类胚胎干细胞和其衍生的后代的任何线粒体膜蛋白复合物的亚基组成。

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<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>0.5M EDTA</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP120-1</td>
<td>adjust pH to 8.0 with NaOH </td>
</tr>
<tr>
<td>1M NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3014-500MG</td>
<td>&nbsp;</td>
<tr>
<td>1M Sodium succinate</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>S413-500</td>
<td>&nbsp;</td>
<tr>
<td>0.1M Sodium phosphate buffer pH 7.2</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP332-1 and BP329-1</td>
<td>Mix 68.4 ml of 1M Na2HPO4 and 31.6 ml of 1M NaH2PO4 to prepare 0.1M sodium phosphate buffer with pH 7.2 at 25 C </td>
</tr>
<tr>
<td>1M Tricine, pH 7.0 (adjusted with NaOH)</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>9010</td>
<td>store at 4 C </td>
</tr>
<tr>
<td>1M imidazole/HCl, pH 7.0</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-2399</td>
<td>store at 4 C</td>
</tr>
<tr>
<td>1M Tris-HCl, pH 7.4</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP153-1</td>
<td>&nbsp;</td>
<tr>
<td>20% (wt/vol) dodecyl-b-D-maltoside (DDM)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>44205-500MG</td>
<td>Dissolved in water. Store 1 ml aliquots at &#x2013;20 C.</td>
</tr>
<tr>
<td>10% (wt/vol) digitonin (>50% purity, used without recrystallization)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>37008-1G</td>
<td>Dissolved in water. Store 0.1&#x2013;1 ml aliquots at &#x2013;20 C. Should be warmed up to 95 C before use.</td>
</tr>
<tr>
<td>2M 6-aminohexanoic acid</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>07260-100G</td>
<td>store at 4 C</td>
</tr>
<tr>
<td>10x Ponceau S/glycerol stock solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-3504 and G6279-1L</td>
<td>0.1% Ponceau S, 50% glycerol, (wt/vol) in water.</td>
</tr>
<tr>
<td>10% w/v sodium deoxycholate</td>
<td>&nbsp;</td>
<td>Fluka </td>
<td>30970-25G</td>
<td>Dissolved in water. Must be protected from light. Adjust pH to 7.0 with HCl, filter and store at room temperature.</td>
</tr>
<tr>
<td>250 mM phenazine methasulfate (PMS)</td>
<td>&nbsp;</td>
<td>TCI America</td>
<td>P0083</td>
<td>Dissolved in DMSO and aliquoted. Store at &#x2013;20 C.</td>
</tr>
<tr>
<td>Nitro-blue tetrazolium (NBT)</td>
<td>&nbsp;</td>
<td>Amresco </td>
<td>0329-1G</td>
<td>&nbsp;</td>
<tr>
<td>3,3&#x2019;-diaminobenzidine tetrahydrochloride (DAB)</td>
<td>&nbsp;</td>
<td>TCI America </td>
<td>D0078</td>
<td>&nbsp;</td>
<tr>
<td>Pb(NO3)2</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>L62-100</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP213-1</td>
<td>&nbsp;</td>
<tr>
<td>Tris base </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP152-10</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>G45-212</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2019;-triphosphate disodium salt (ATP)</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>A2383-5G</td>
<td>Grade I, minimum 99%</td>
</tr>
<tr>
<td>beta-nicotineamide adenine dinucleotide (NADH) reduced form </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>N-8129</td>
<td>&nbsp;</td>
<tr>
<td>Cytochrome c From horse heart</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>C2506-250MG</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>BP220-212</td>
<td>&nbsp;</td>
<tr>
<td>100x protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P8340</td>
<td>Store at &#x2013; 20 C.</td>
</tr>
<tr>
<td>Dimethyl sulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>D128-500</td>
<td>&nbsp;</td>
<tr>
<td>Trypan blue Stain 0.4%</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15250-061</td>
<td>&nbsp;</td>
<tr>
<td>10x Trypsin (0.5%)</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15400-054</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin Inhibitor </td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>17075-029</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

probing for mitochondrial complex activity in human embryonic stem cells

线粒体是细胞质的细胞器，有一个细胞代谢和动态平衡的主要作用，细胞信号转导网络调控，和程序性细胞死亡。线粒体产生ATP，调节细胞质的氧化还原状态和Ca2 +的平衡，分解代谢脂肪酸，合成血红素，核苷酸，类固醇激素，氨基酸，并帮助组装蛋白的铁硫簇。线粒体也有产热的重要作用。线粒体基因组的突变引起人类疾病的几种类型。 mtDNA突变的积累与衰老相关，怀疑是在癌症发展的重要作用。由于他们的作用极为重要，在所有类型的细胞，线粒体的功能，还必须干细胞的关键。干细胞活力，增殖和分化能力的认识已经取得了重要进展。但干细胞的功能活动，尤其是他们的能量状态，尚未被详细研究。几乎没有什么是已知的人类胚胎干细胞（胚胎干细胞），和他们的区别的易制毒化学后代线粒体属性。线粒体在人类胚胎干细胞的功能和发展潜力作用来认识和评价的方法之一是直接测定线粒体呼吸复合物的活性。在这里，我们描述了高分辨率的清晰的原生凝胶电泳凝胶作为活动的可视化分析五年的人类胚胎干细胞呼吸链复合物的方法和后续。

Watch this Scientific Journal Video about Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells at JoVE.com

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

在这段视频中，我们将向您可以分析人类胚胎干细胞的线粒体呼吸链复合物如何使用凝胶活性测定。

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 ...

probing-for-mitochondrial-complex-activity-human-embryonic-stem

Research

JoVE Journal

Biology

14.2K 次观看.  University of California, Los Angeles. 在这段视频中，我们将向您可以分析人类胚胎干细胞的线粒体呼吸链复合物如何使用凝胶活性测定。

视频: Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

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 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 ...

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 ...

Freezing and Thawing Human Embryonic Stem Cells

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

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

人类胚胎干细胞冷冻和解冻

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

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

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.