The authors acknowledge funding agencies, the IMI StemBANCC European community programme, the leducq Foundation (SHAPEHEART) and the Agence Nationale de la Recherche (Genopath)

1. DNA-蛋白质交联<ol><li>从ES细胞和胚胎的心脏组织中产生的固定在15ml管中收获-ES细胞（2×10 6个细胞进行常规的ChIP，2×10 5个细胞的微芯片），胚状体（EB）从E9.5小鼠胚胎解剖（房室管，使用在PBS中的1％甲醛细胞或胚胎组织中透PB2缓冲器流出道和心室）。放置在轨道摇床管在室温下以60rpm的速度恰好10分钟。 </li><li>通过加入甘氨酸至125毫的终浓度停止交联反应，并在60 rpm的速度孵育在上轨道摇床室温下5分钟。 </li></ol> 2.细胞裂解和染色质碎片<ol><li>洗两次悬浮于10交联的细胞毫升的PBS 1x或胚胎组织重新悬浮于1ml PB2。离心机在1000×g离心在4℃下5分钟。弃去上清液。&lt;/ LI&gt; <li>添加蛋白酶抑制剂来缓冲PB1或PB2（2微克/毫升亮肽素，1μg/ ml的抑酶肽，和0.1mM PMSF）中（ 表1）。重悬从步骤2.1细胞沉淀或胚胎组织在1ml缓冲PB1或PB2的分别。吸管上下和重悬的细胞或组织。用1ml注射器用21号针头匀浆的细胞或组织。孵育10分钟4℃（在车轮上）。 </li><li>降速在3000×g离心5分钟，4℃，弃上清。 </li><li>重悬沉淀300μl的在一个干净的试管补充有蛋白酶抑制剂的各自的SB缓冲器（ 表1）的（认证无RNA酶，DNase-，和无热原的），以防止任何DNA降解。孵育在冰上15至30分钟。 </li><li>超声处理的样品在4℃下以剪切使用根据表2中的每个的材料列出的程序超声波仪的染色质。根据下一个应用程序调整超声时间（&lt;EM&gt;即PCR或排序）。剪切DNA大小应为约500 bp的PCR的和300bp的测序。 </li><li>离心机超声处理细胞裂解物10分钟，在6000 xg离心在4℃下，在一个干净的1.5毫升管转移上清液（染色质）弃沉淀。 </li><li>稀释溶液与水的10倍的染色质（上清液缓冲液B），并评估用1.5微升在纳米分光光度计的光密度。获得使用以下公式中微克/微升的蛋白质的浓度:1.55点¯xOD 280 - 0.76点¯xOD 260。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 缓冲 </td><td> 采用 </td><td colspan="4"> 组成 </td><td> 物料 </td></tr><tr><td rowspan="2">一个</td><td rowspan="2">透</td><td colspan="4"> PB1:5毫摩尔PIPES pH值8; 85米米氯化钾; 0.5％NP40 </td><td>所有</td></tr><tr><td colspan="4"> PB2:15mM的HEPES pH 7.6中，15毫摩尔NaCl，4mM的氯化镁60毫米氯化钾，0.5％Triton X-100 </td><td>胚胎组织</td></tr><tr><td rowspan="3">乙</td><td rowspan="3">裂解/超声</td><td colspan="4"> SB1:1％SDS中的10mM EDTA;的50mM的Tris-HCl pH为8 </td><td>退出</td></tr><tr><td colspan="4"> SB2:50mM的HEPES-KOH pH7.9; 140毫米; 1mM的EDTA; 0.1％的脱氧胆; 0.1％SDS中</td><td>胚</td></tr><tr><td colspan="4"> SB3:15毫米的HEPES pH7.6的，15毫米氯化钠; 60毫米氯化钾，1mM的EDTA，0.5毫米EGTA; 1％Triton X-100，0 .1％SDS，0.5％十二烷基肌氨酸</td><td>胚胎组织</td></tr><tr><td> C </td><td>沉淀缓冲</td><td colspan="4"> 150毫摩尔NaCl;的50mM的Tris-HCl pH为7.5; 5mM EDTA的; 0.5％NP40; 1％Triton X-100。 </td><td>所有</td></tr><tr><td> ð </td><td> DNA /蛋白质洗脱</td><td colspan="4"> D1:1％SDS 100毫米碳酸氢钠 D2:50毫摩尔Tris pH值7.6 / 5mM EDTA的，15毫摩尔DTT，2％SDS中</td><td></td></tr><tr><td> Ë </td><td> DNA结合珠的制备</td><td colspan="4"> E:20％PEG 8000; 2.5 M氯化钠;的10mM的Tris-HCl pH值8; 1mM的EDTA </td><td></td></tr></tbody></table> 表1.内部缓冲器 。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 材料 </td><td> 超声方案 </td></tr><tr><td>胚胎干细胞</td><td> 30秒，以15次和30秒关闭</td></tr><tr><td>胚体</td><td> 30秒ON的30个循环和30秒关闭</td></tr><tr><td>胚胎组织</td><td> 30秒对21周期和30秒关闭</td></tr></tbody></table> 表2.超声程序。 3.免疫沉淀和淘<ol><li>洗3次蛋白A结合珠缓冲液C。取100微升珠在干净的1.5 ml管中，加入1毫升缓冲液C（ 表1）。然后将管转移到一个磁铁;等待1分钟，吸出上清液。重复洗涤步骤两次。 </li><li>孵育20微升洗涤蛋白的缀合珠子和1ml缓冲液C在一个干净的1.5毫升管补充有蛋白酶抑制剂产生抗修饰组蛋白或转录因子（ 表3中列出的浓度）抗体。在4℃下设置在旋转器上的样品在40rpm下为至少2小时。 </li><li>洗抗体珠复合3次用1毫升缓冲液C（3.1操作说明）。 </li><li>准备2管，一个包含150染色质和抗体 - 珠子复合物微克和含有150染色质微克和20μl洗涤珠的其它管;加入1ml缓冲液C补充有蛋白酶抑制剂，以每管并在4℃下以40rpm孵育在旋转轮上的样品过夜。 注:染色质的浓度必须至少为300微克免疫沉淀转录因子或用于顺序沉淀。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 标准 </td><td> 浓度（ng /μL） </td><td> 体积（微升） </td><td> 总DNA浓度（ng） </td></tr><tr><td>一个</td><td> 10.0 </td><td> 1 </td><td> 10.0 </td></tr><tr><td>乙</td><td> 5.00 </td><td> 1 </td><td> 5.00 </td></tr><tr><td> C </td><td> 2.50 </td><td> 1 </td><td> 2.50 </td></tr><tr><td> ð </td><td> 1.25 </td><td> 1 </td><td> 1.25 </td></tr><tr><td> Ë </td><td> 0.625 </td><td> 1 </td><td> 0.625 </td></tr><tr><td> F </td><td> 0.3125 </td><td> 1 </td><td> 00.3125 </td></tr><tr><td> G </td><td> 0.156 </td><td> 1 </td><td> 0.156 </td></tr><tr><td> H </td><td> 0.0 </td><td> 1 </td><td> 0.0 </td></tr></tbody></table> 表3.标准的浓度。 4. DNA洗脱，交叉链接和逆转蛋白酶K消化<ol><li>在磁性架设置样品。回收含有未结合的抗体染色质上清。 注:样品可以在该步骤液氮快速冷冻并储存在-80℃。染色质可以与其它抗体其他免疫沉淀实验中使用。用1ml缓冲液C（之前描述的操作）洗涤各样品3次。 </li><li>通过加入150微升缓冲D1或D2的洗脱抗体结合的蛋白质，如果连续的芯片具有要完成（ 表1），以洗涤沉淀材料和在加热块在50℃孵育样品20分钟。 </li><li>取下管加热块，把样品中的磁性架，回收上清液在一个干净的1.5毫升管（认证无RNA酶，DNase-，和无热原的），丢弃该珠子。 </li><li>使用上清液顺序芯片2体积的缓冲液C中加入1到第二抗体的3微克或反向通过加入5M的氯化钠，得到的200mM的终浓度交联。 </li><li>在一个干净的1.5毫升管制备染色质的输入样本（认证无RNA酶，DNase-，和无热原的）拍摄染色质的相同量，在150微升的水最终体积知识产权样品（150微克）的（RNA酶的DNA酶游离水）。添加5M NaCl至200mm的最终浓度。在65℃孵育样品过夜。 </li><li>第二天，从加热块中移除样品中，添加250毫摩尔EDTA，得到12.5毫和蛋白酶K的最终浓度，以获得在芯片材料250微克/ ml的终浓度，并在55℃消化在2小时加热块。 </li></ol><ol><li>珠准备<ol><li>开始，使在室温下将珠粒至少30分钟，涡流珠子非常彻底。珠安定下来，所以当吹打工作速度快。传输1毫升珠干净的1.5 ml管（认证RNA酶，DNase-和无热原的）。 </li><li>坐落于磁铁，等待2 - 3分钟的解决方案，以澄清，弃上清。从磁铁取出，加入1ml 0.5M的EDTA，并通过短暂的震荡混匀。 </li><li>重复步骤5.1.2两次，并在最后丢弃上清液。 </li><li>从磁铁取出，加入1毫升缓冲液E和悬浮慢慢珠。传送整个混合物（在缓冲液E珠）于50ml缓冲大肠杆菌地点振荡器上，并让它在室温下慢慢地搅拌1小时。 </li><li>测试DNA结合珠以下以下，使用3实验条件描述的纯化方案:将50μl珠子/ 50微升的DNA（1体积/ 1体积），100微升的小珠/ DNA的50微升（2体积/ 1体积）和125微升的小珠/ DNA的50微升（2.5体积/ 1体积）。让迁移上的1.5％琼脂糖凝胶纯化的DNA，以检查片段（ 图1A）的大小。 </li></ol></li><li> DNA纯化<ol><li>添加DNA的425微升（2.5倍体积）的磁珠结合每一个DNA样本（约170微升）。通过上下吹打（约10倍）缓慢重悬并在室温下孵育10分钟。 </li><li>上磁体放置5分钟，然后吸出上清液并弃去（在磁铁）。 </li><li>加入600微升新鲜出炉的80％的乙醇在管上的磁铁，等待1分钟，吸上清并丢弃，然后重复前面的步骤一次。 </li><li>举一个快速旋转，5秒（微量），放置在磁铁管取出乙醇的最后一滴。让干在室温下1分钟</li><li>通过加入20μlDNA酶无RNase水洗脱DNA。从磁铁取出，涡旋样品。 </li><li>孵育在室温下3分钟，然后放置试样上磁铁。抽吸洗脱的DNA进新管中（认证无RNA酶，DNase-，和无热原的）。 </li><li>运行上的1.5％琼脂糖凝胶的输入级分的DNA的检查的DNA片段（ 图1B）的大小。 </li></ol></li></ol> 6. DNA定量用荧光检测仪<ol><li>在水中1微克DNA（1kb梯）串联稀释，得到共7稀释液加无DNA的点（聚集在表3稀释）。 </li><li>制备的PCR Green I的染料的溶液中，足以对所有样品（纯化的DNA和标准稀释AH）:稀释10000在1×TE缓冲液的青染料。 </li><li>加入1μl每个DNA样品（纯化的DNA和标准稀释AH），以10微升1个绿色染料混合毛细血管比色皿，以在488纳米波长的荧光读取。对于无DNA分析，使用1微升1×TE缓冲液中。 </li><li>建立使用校准曲线的图形软件和确定的相对浓度的DNA样品以ng /微升的范围内。该DNA然后准备用于PCR或使测序文库。 </li></ol> 7. PCR <ol><li>引物的选择<ol><li>感兴趣的设计引物询问感兴趣的基因组区域。设计引物以扩增用UCSC基因组浏览器的基因的增强子区。增强剂使用H3K4me1或P300芯片起在GEO数据组可用9数据进一步预测。 </li></ol></li><li> PCR <ol><li>在25微升绿色染料混合，2纳克DNA和0.5微升引物混合使用实时PCR热循环45个周期（8秒95℃，8秒60℃，8秒72°C）的PCR反应（20 μM原液）10。 </li></ol></li><li>富集分析<ol><li>计算绝对富集假设核小体的至多1％的免疫沉淀11。 </li><li>求索- [R为丰富，如果相比于0.1纳克的DNA模板时10毫微克IP样品显示更大的富集的基因组。 </li><li>表达结果作为富集通过非富集的区域正常化到输入和调整用非特异性对照样品后增加倍数。 </li><li>考虑输入DNA为100（稀释倍数或DF）稀释。 </li><li>的Ct [输入量x DF] -使用以下等式2-ΔCt×100％，其中-ΔCt=的Ct [IP]正常化输入。 </li><li>使用下面的等式调整控制（ΔCT[IP]-ΔCt[NS]）其中NS为非特异性样品（无抗体的IP，或IgG的IP）。 </li><li>计算出样品的倍富集为2-ΔΔCT。包括与公式计算的所有步骤，其中只有每个PCR样品可输入将便于结果的分析的文件。 </li></ol></li></ol>

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The authors have nothing to disclose.

表观遗传学已成为发育生物学研究的一个重要领域。如何遗传程序在胚胎细胞被激活以允许细胞胚胎谱系中获取的特定身份已经长时间为发育生物学家的一个关键问题。 芯片具有在过去几年内被广泛使用，并结合到DNA测序按照测序分辨率的改善。这已成为以在基因组中广泛依赖性进行调查细胞或特定的胚胎和成年组织的后生景观的强大技术。芯片分析缓冲和超声条件得到极?...

<html>心脏是要形成的第一器官，并成为在胚胎功能。的心脏是从来自第一和第二胚胎心脏字段1出现许多细胞谱系建造。从受精后的囊胚阶段到心脏形，胚胎细胞具有从而使许多细胞命运的决定。基因转录是在时间和空间依赖性管制，是胚胎发育过程中underlies细胞命运决定的关键生物过程。这样的方法是由基因组，包括增强子和心组成的基因的启动子中的绑定调节区特异性转录因子介导的。的DNA是围绕经受修饰例如乙酰化，甲基化，泛素化和/或磷酸化组蛋白包裹。组蛋白修饰导致取决于压抑，活化或蓄势基因转录在其上的组蛋白的赖氨酸残基被修饰2。 jove_content"&gt;染色质免疫沉淀（芯片）已经成立年前3，是目前最广泛使用的技术，以确定任何修饰组蛋白或转录因子4。随着组蛋白或转录因子的免疫目标，结合的DNA可或者通过聚合酶链式反应（PCR）扩增或测序。芯片有技术上克服更具挑战性凝胶阻滞测定法5。然而芯片不意味着直接的转录因子的DNA，凝胶阻滞分析的优点的结合。在另一方面，沉淀结合DNA测序已开通的基因调节的新的全基因组的角度。 胚胎干细胞（ES细胞）的胚状体中的概括（ 即 ，细胞聚集），或在二维培养心脏发育6的早期步骤，并在原则上足够的材料芯片提供。此外，人ES细胞分别表示Ca人细胞模型rdiogenesis虽然他们的心源性潜力取决于它们的表观遗传签名7。在开发的后期阶段，小鼠胚胎组织允许调查测定细胞特性所需的特定后生景观。然而，基因组在一个时间和细胞类型特异性方式8转录。基因转录的表观遗传调控具有局部区域内进行研究。在此，我们描述接着用胚胎干细胞，胚状体和心肌特异性胚胎地区PCR或测序沉淀，顺序芯片的协议。这些协议允许调查心脏基因转录的表观遗传调控。

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			<td height="22" style="height:22px;width:216px;">Formaldehyde&#160;</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:147px;">F8775</td>
			<td style="width:167px;">Cell Fixation&#160;</td>
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		<tr height="22">
			<td height="22" style="height:22px;">Glycine</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td>Cross-link stop</td>
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			<td>Fluka</td>
			<td>10820</td>
			<td>Proteases inhibitor</td>
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			<td height="22" style="height:22px;">Leupeptin hemisulfate</td>
			<td>Sigma</td>
			<td>L2882</td>
			<td>Proteases inhibitor</td>
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		<tr height="22">
			<td height="22" style="height:22px;">PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td>Proteases inhibitor</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Protein A magnetic beads&#160;</td>
			<td>Life technologies</td>
			<td>10001D</td>
			<td>Immunoprecipitation</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">SPRI magnetic beads</td>
			<td>Thermo Scientific</td>
			<td>15002-01</td>
			<td>DNA purification</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Proteinase K</td>
			<td>Life technologies</td>
			<td>25530-015</td>
			<td>Protein digestion&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DNA BR standard&#160;</td>
			<td>Life technologies</td>
			<td>Q32850</td>
			<td>Calibration range&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Syber green</td>
			<td>Molecular Probes</td>
			<td>S-11484</td>
			<td>DNA quantification</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">TE buffer&#160;</td>
			<td>Invitrogen</td>
			<td>P7589</td>
			<td>DNA quantification</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">PBX 1X</td>
			<td>Life technologies</td>
			<td>14190-094</td>
			<td>Washing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DNase RNase free water</td>
			<td>Life technologies</td>
			<td>10977-035</td>
			<td>Dilution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Axygen tube</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td>ChIP purifiction</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Antibody&#160;</td>
			<td>Company</td>
			<td>Reference</td>
			<td style="width:167px;">ChIP concentration</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">H3K27ac</td>
			<td>Abcam</td>
			<td>ab4729</td>
			<td style="width:167px;">3 &#181;g for ESC and EBs, 1 &#181;g for tissues</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">H3K4me1</td>
			<td>Diagenode</td>
			<td>C15410194 (pAb-194-050)</td>
			<td style="width:167px;">3 &#181;g for ESC and EBs, 1 &#181;g for tissues</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">H3K36me3</td>
			<td>Diagenode</td>
			<td>C15410058 (pAb-058-050)</td>
			<td style="width:167px;">3 &#181;g for ESC and EBs, 1 &#181;g for tissues</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">H3K9me2</td>
			<td>Diagenode</td>
			<td>C15410060 (pAb-060-050)</td>
			<td style="width:167px;">3 &#181;g for ESC and EBs, 1 &#181;g for tissues</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">H3K4me3</td>
			<td>Diagenode</td>
			<td>C15410030 (pAb-030-050)</td>
			<td style="width:167px;">3 &#181;g for ESC and EBs, 1 &#181;g for tissues</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">H3K27me3</td>
			<td>Diagenode</td>
			<td>C15410069 (pAb-069-050)</td>
			<td style="width:167px;">3 &#181;g for ESC and EBs, 1 &#181;g for tissues</td>
		</tr>
	</tbody>
</table>

epigenetic regulation of cardiac differentiation of embryonic stem cells and tissues

具体的基因转录是一个重要的生物学过程，在胚胎发育过程中underlies细胞命运决定。生物过程是通过其结合基因组的调节区，包括增强子和心组成的基因的启动子的转录因子介导的。的DNA是围绕经受化学修饰组蛋白包裹。组蛋白的修饰进一步导致压抑，激活或​​泰然自若的基因转录，从而使基因转录微调调控的另一个层次。胚胎干细胞（ES细胞）概括胚状体（ 即 ，细胞聚集体）内，或在2D培养心脏发育的早期步骤。它们提供原则上足够的材料为染色质免疫沉淀（ChIP），一个技术广泛用于鉴定基因的调节区。此外，人ES细胞代表心脏发生的人类细胞的模型。在发育的后期阶段，小鼠胚胎组织允许调查测定细胞特性所需的特定后生景观。在此，我们描述然后进行PCR或使用胚胎干细胞，胚状体和心肌特异性胚胎地区的ChIP测序沉淀，顺序芯片的协议。这些协议允许调查心脏基因转录的表观遗传调控。

图1A首先说明了DNA结合珠和使用不同尺寸（1kb梯）的DNA质量控制的准备。 0NE，珠子2和2.5体积（1至3）加入到样品中纯化高和低分子量大小的DNA片段的一个卷。 图1 B，C，D是从分别从小鼠ES细胞，胚状体或心脏胚胎区域（池25 E9.5心室）萃取整个声振的DNA的DNA凝胶的典型例子。 <p class="jove_content" fo:keep...

Watch this Scientific Journal Video about Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues at JoVE.com

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

基因转录的微调调控underlies胚胎细胞命运决定。在此，我们描述了用于研究干细胞和小鼠胚胎的心脏发育的两个心脏分化的表观遗传调控染色质免疫沉淀测定法。

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

epigenetic-regulation-cardiac-differentiation-embryonic-stem-cells

Research

JoVE Journal

Developmental Biology

8.0K 次观看.  Université Aix-Marseille. 基因转录的微调调控underlies胚胎细胞命运决定。在此，我们描述了用于研究干细胞和小鼠胚胎的心脏发育的两个心脏分化的表观遗传调控染色质免疫沉淀测定法。 

视频: Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

从胚胎干细胞衍生心脏祖细胞

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

科研

发育生物学

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

隔离和脂肪干细胞分化的猪从皮下脂肪组织

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

在微孔聚集尺寸优化人类多能干细胞的基于悬浮心脏分化

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

犬卵巢间充质干细胞的提取与分化

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...