Name: epigenetic regulation of cardiac differentiation of embryonic stem cells and tissues
Uploaded: 2016-06-03T14:00:00.000+00:00
Duration: 13 min 3 s
Description: Watch this Scientific Journal Video about Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues at JoVE.com

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。

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

8.1K 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

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

从多能干细胞使用BMP拮抗剂Grem2心房心肌细胞分化

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

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 ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

使用多电极阵列（MEA）的人多能干细胞来源的心肌细胞（hPSC-CM）的电生理分析

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

在体外人骨髓间充质干细胞分化为心肌样细胞功能

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

基因小鼠胚胎干细胞的基础含量为心源性诱导效率量化

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

生物学

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

人类多能干细胞的高效分化为心肌细胞，并鉴定流式细胞仪

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...