这项工作是由一个赠款的援助从心脏和中风支持加拿大（G-14-0006265）的基础，从健康研究（137352和143066）和普·比克尔基金会授予加拿大学院运营赠款（1013821 ）到SSN。

主设计与制作注意:使用软光刻技术进行器件制造。为二甲基硅氧烷（PDMS）成型制成两层SU-8主机。 <ol><li>使用设计和绘图软件设计设备（ 图1A ，左）。分别绘制主体的每一层。在20,000 dpi的两个光掩模上打印设备设计，对应于主板15的两层。将设备图案设置为透明，周围区域为黑色;掩模将用作通过光刻将器件图案光学转移到SU-8上的模板（如其他15所述）。 </li><li>将约4mL的SU-8 50分配到4英寸硅晶片的中心。使用旋涂机将SU-8 50均匀地涂在晶片上，以每分钟2000转（RPM）旋转30秒。在95°C的电热板上烘烤晶片10分钟。将晶片暴露于紫外线（UV）GHT以200mJ / cm 2以下。 </li><li>在2500 RPM 30秒倒约4毫升的SU-8 2050到晶片和旋转的中心。烤上95℃热板上15分钟。冷却至室温（RT）。 </li><li>重复步骤1.3两次。 </li><li>覆盖与所述第一层的透明度的光掩模的涂覆的晶片。暴露晶片于UV光，在270毫焦/厘米2，以使第一层。烤上95℃热板上15分钟。 </li><li>通过重复步骤1.3两次使SU-8 2050的第二层。 </li><li>对准使用掩模对准器在第一层上的第二层掩模的功能。然后，暴露于UV光，在240毫焦/平方厘米。烘烤在95℃下15分钟。 注:曝光时间由所需要的总UV剂量和在使用前的天测得的UV光输出强度来确定。 </li><li>通过在丙二醇单甲醚乙酸酯（PGMEA）淹没它，并以200RPM在定轨振荡器中振荡30分钟开发晶片。 </li><li>地点主到培养皿并小心倒入PDMS混合物（碱以10:1的比例与交联剂:按重量计1）在晶片的中心。覆盖所有的功能，避免气泡。 </li><li>热在在70℃下2小时的烘箱中。使用锋利的刀片切割围绕PDMS biowire模板的（ 图1A，右图）的边缘。剥离从主的PDMS。修剪与刀片装置。把PDMS biowire模板在灭菌袋中，蒸汽高压釜中于121℃进行20分钟。 </li><li>在生物安全柜（BSC），将biowire模板PDMS到培养皿中。用镊子保持，并通过在信道（ 图1B-I）的两端在槽安置它安装了一块在PDMS微孔的通道的中心无菌手术丝缝线（6-0）的。 </li></ol> 2.电刺激商会创作<ol><li>使一对矩形聚碳酸酯片（长×宽×厚度:2厘米×0.85厘米X0.35厘米），并利用它们作为电刺激室的框架（ 图2）。 </li><li>钻两个宽3毫米的孔相距2厘米沿着各聚碳酸酯框架件的中心线上。 </li><li>切为1.5厘米长炭棒两片。通过关于从端3毫米碳棒钻一个1mm宽的孔。线穿过碳棒孔铂丝和通过包装它的碳棒周围收紧导线。夹子连接到铂线，用于与电刺激器后连接的另一端。 </li><li>插入碳棒在聚碳酸酯框架件。放置与炭棒组装帧分成6孔板的孔中。 </li><li>倾2mL的PDMS的入井。浸没聚碳酸酯帧的底部PDMS同时保持PDMS表面水平接近但不到达炭棒的底部。热在在70℃下2小时的烘箱中。 </li><li>就拿电刺激室出来的六-Well板，并把它在灭菌袋中用于蒸汽消毒在121℃下20分钟。 </li></ol> 3. HPSC-CM的酶解<ol><li>诱导HPSC分化成使用建立的协议4心肌细胞。 注:简单地说，通过产生建立胚状体的心肌细胞（EB）协议在通过顺序补充骨形态发生蛋白4，碱性成纤维细胞生长因子，活化素A，血管内皮生长因子，和同系物可夫1 4干亲介质。从分化的20-34天使用的EB做出biowires。 </li><li>收集从低安装板将EB与P1,000移液管尖端和转移到锥形管中。通过离心在125×g下和RT 5分钟沉淀。与弃上清，重悬沉淀预温，37含1％脱氧核糖核酸酶I℃的I型胶原酶（DNA酶I）。孵育在37℃下2小时消化。 </li><li>添加预热的，37℃的5毫升洗涤介质和离心机在125×g下和室温下5分钟。弃去上清液。重悬用2mL的胰蛋白酶/乙二胺四乙酸（EDTA）溶液中的沉淀，并在37℃下孵育5分钟。 </li><li>加入1毫升含3％光阑培养基（V / V）DNA酶I </li><li>附加一个20 G（0.9毫米）针5毫升注射器，并通过它传递EB悬浮液3-5次。 </li><li>在280×g下加入7毫升洗涤介质和离心机5分钟。弃去上清液。重悬的Iscove氏改良的Dulbecco氏培养基（IMDM）的丸粒并储存在冰上。确定与血球细胞数。取0.5×10 6细胞和沉淀通过离心，在280×g离心5分钟。除去上清液。 </li><li>在冰上在无菌管中混合它们之前预冷所有凝胶组分。混合以下的胶原凝胶的组分（最终浓度）:2.1 mg / mL的鼠尾I型胶原蛋白，24.9 mM葡萄糖，23.8毫将NaHCO 3，14.3毫摩尔的NaOH，10mM的HEPES，和在1×M199培养基10％的细胞外基质。添加胶原蛋白和最后的细胞外基质。吹打向上和向下混合。 </li><li>重悬0.5×10 6细胞与胶原凝胶的3.5μL拌匀。 </li><li>使用P10移液管尖，以4微升的细胞悬浮液加入到0.5厘米长的信道（ 图1B-II）。调整用无菌镊子如果需要缝合的位置。 </li><li>覆盖培养皿用无菌磷酸盐缓冲盐水（PBS）的底部，确保不要触碰凝胶。孵育在37℃下30分钟。 </li><li>用培养基的12毫升（ 例如干Pro或介质推荐的电池制造商），足以覆盖细胞（ 图1B-III）更换PBS。培养在37℃下1周的孵化器。改变介质隔日。 </li></ol> 4.电刺激Biowire栽培<ol><li>在所述BSC，加入2毫升的PBS到6孔板，将容纳电刺激室的每个孔中。使用无菌镊子蒸压电刺激装置轻轻地放入井中。从井中丢弃PBS。 </li><li>覆盖用5ml预温热培养基的炭棒。使用无菌镊子装入biowires在电刺激室和它们定向垂直于碳棒（ 图2）。 </li><li>放置在6孔板的顶部上的盖子，但留下足够的铂丝在到达板的外侧与电刺激器相连接。置于细胞培养箱六孔板中于37℃和铂丝连接到电刺激器。 </li><li>双相脉冲的重复，1毫秒脉冲持续时间，3 V / cm，并且每秒1个脉冲（PPS）:使用具有以下设置的电刺激器刺激biowires。提高PPS每24个小时为下列值:1.83，2.66，3.49，40.82，5.15，和6更改隔日媒体。 </li><li>在10倍放大倍率观察在显微镜下响应于电刺激的心肌收缩性。 注:刺激后一周，该biowires变得成熟（参见结果部分）。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

该手稿描述了改进hPSC-CMs成熟的设计平台的生物线的设置和实现。该装置可以在标准的微细加工设备中制造，并且生物线可以用普通的细胞培养技术和电刺激器生产。 据我们所知，没有报道类似于生物线的方法。该策略表明，改善的成熟性质取决于电刺激方案，如通过较高刺激速率上升方案（6Hz对3Hz）的生物线中获得的较大成熟水平所证明的。生物线中的心肌细胞与培养20天?...

细胞治疗是最有前途和研究战略，以实现心肌修复/再生一个。已经通过心脏组织工程和生物材料1，2的共递送辅助。大多数现有的细胞来源进行了研究，在动物模型为他们的损伤，患病或年老心3潜在的有利影响。尤其显著已经努力用人类多能干细胞（HPSC）衍生的心肌细胞（HPSC-CM），用于心脏组织工程可能无限的自体细胞来源。 HPSC-CMS可以使用多种已建立的方案4，5，6来制造。然而，所获得的细胞相比，成年心室肌7显示胚胎样表型，具有一定范围的未成熟的特性， </sup&gt; 8。这可能是一个障碍HPSC-CMS应用在药物研发和成人心脏疾病模型9的发展成人心脏组织的模型。 为了克服表型不成熟的这种限制，新方法已被积极地研究促进心肌细胞成熟。早期的研究通过循环机械10或电刺激11显露在新生大鼠心肌细胞的有效促成熟性质。凝胶压实和环状机械刺激还显示出改善HPSC-CM熟化12， 图13，与电生理和钙的处理性能的增强最小的某些方面。因此，所谓的"生物丝"（biowire）的平台系统是由同时提供结构线索和电场stimulatio设计n增强hPSC-CMs的成熟14 。该系统使用微制造平台来创建适合于电场刺激的对准的心脏组织。这可以用于改善hPSC-CM的结构和电生理成熟度。在这里，我们描述制作这样的生物线的细节。

<table border="0" cellpadding="0" cellspacing="0" width="582">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">L-Ascorbic acid</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">A-4544</td>
			<td style="width:167px;">hPSC-CM culture media componet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">AutoCAD</td>
			<td style="width:80px;">Autodesk, Inc</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Software to design device</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Carbon rods, &#216; 3 mm</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Electrical stimulator chamber component</td>
		</tr>
		<tr height="206">
			<td height="206" style="height:206px;width:216px;">Collagen, type 1, rat tail</td>
			<td style="width:80px;">BD Biosciences</td>
			<td style="width:119px;">354249</td>
			<td style="width:167px;">Collagen gel: 2.1 mg/ml of rat tail collagen type I in 24.9 mM glucose, 23.8 mM NaHCO3, 14.3 mM NaOH, 10 mM HEPES, in 1xM199 media with 10 % of growth factor-reduced Matrigel.</td>
		</tr>
		<tr height="164">
			<td height="164" style="height:164px;width:216px;">Collagenase type I&#160;</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">C0130</td>
			<td style="width:167px;">0.2% collagenase type I (w/v) and 20% FBS (v/v) in PBS with Ca2+ and Mg2+. Sterilize with 0.22 &#956;m filter and make 12 ml aliquots. Store at -20 &#176;C.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:216px;">Deoxyribonuclease I (DNase I)</td>
			<td style="width:80px;">EMD Millipore</td>
			<td style="width:119px;">260913-25MU</td>
			<td style="width:167px;">Make 1 mg/ml DNase I stock solution in water. Filter sterile and store 0.5 ml aliquots at &#8722;20 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Drill &#38; drill bits (&#216; 1mm and 2 mm)</td>
			<td style="width:80px;">Dremel</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Drill holes in polycarbonate frames</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Electrical stimulator</td>
			<td style="width:80px;">Grass</td>
			<td style="width:119px;">s88x</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Fetal bovine serum (FBS)</td>
			<td style="width:80px;">WISENT Inc.</td>
			<td style="width:119px;">080-450</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">D-(+)-Glucose&#160;</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">G5767</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">L-Glutamine</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25030081</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">H2O</td>
			<td style="width:80px;">MilliQ</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">18.2 M&#937;&#183;cm at 25 &#176;C, ultrapure, to make all solutions</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">HEPES</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">H4034</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Hot plate</td>
			<td style="width:80px;">Torrey Pines</td>
			<td style="width:119px;">HS40</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Iscove&#39;s Modified Dulbecco&#39;s Medium(IMDM)</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12440053</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Mask aligner</td>
			<td style="width:80px;">EVG&#160;</td>
			<td style="width:119px;">EVG 620</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Matrigel, growth factor reduced&#160;</td>
			<td style="width:80px;">Corning</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Medium 199 (M199)</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11150059</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Monothioglycerol (MTG)</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">M-6145</td>
			<td style="width:167px;">hPSC-CM culture media componet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Orbital shaker</td>
			<td style="width:80px;">VWR</td>
			<td style="width:119px;">89032-088</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Penicillin/Streptomycin (P/S)</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15070063</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Phosphate-buffered saline (PBS) with Ca2+ and Mg2+&#160;</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14040133</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Plate (6-well)</td>
			<td style="width:80px;">Corning</td>
			<td style="width:119px;">353046</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Plate (6-well), low attachment</td>
			<td style="width:80px;">Corning</td>
			<td style="width:119px;">3471</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Platinum wires, 0.2 mm</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Electrical stimulator chamber component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:80px;">Dow Corning</td>
			<td style="width:119px;">Sylgard 184</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Propylene glycol monomethyl ether acetate (PGMEA)</td>
			<td style="width:80px;">Doe &#38; Ingalls Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">To develop the wafer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Pouch, peel-open</td>
			<td style="width:80px;">Convertors</td>
			<td style="width:119px;">92308</td>
			<td style="width:167px;">For steam sterilization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Silicon wafer, 4-inch</td>
			<td style="width:80px;">UniversityWafer Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">Sodium bicarbonate (NaHCO3)</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sodium hydroxide</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">S8045</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Sprin coater</td>
			<td style="width:80px;">Specialty Coating Systems</td>
			<td style="width:119px;">G3P-8</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="280">
			<td height="280" style="height:280px;width:216px;">StemPro-34 culture medium</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10639011</td>
			<td style="width:167px;">hPSC-CM culture medium. To make 50 ml, add 1.3 ml supplement, 500 &#956;l of 100&#215; L-Glutamine, 250 &#956;l of 30 mg/ml transferrin, 500 &#956;l of 5 mg/ml ascorbic acid, 150 &#956;l of 26 &#956;l /2 ml monothioglycerol (MTG), and 500 &#956;l (1 %) penicillin/streptomycin.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Stop media</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">&#160;Wash medium:FBS (1:1)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">SU-8 50&#160;</td>
			<td style="width:80px;">MicroChem Corp.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">photoresist, master component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">SU-8 2050&#160;</td>
			<td style="width:80px;">MicroChem Corp.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">photoresist, master component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Transferrin</td>
			<td style="width:80px;">Roche</td>
			<td style="width:119px;">10-652-202</td>
			<td style="width:167px;">hPSC-CM culture media componet</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Trypsin/EDTA, 0.25%</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25200056</td>
			<td style="width:167px;">hPSC-CM culture media componet</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Wash medium</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">IMDM containing 1% Penicillin/Streptomycin</td>
		</tr>
	</tbody>
</table>

maturation of human stem cell-derived cardiomyocytes in biowires using electrical stimulation

人类多能干细胞衍生的心肌细胞（HPSC-CMS）是一个有前途的细胞来源，并从而鼓励他们在心脏研究潜力的应用，包括药物发现，疾病建模，组织工程和再生医学的研究。然而，由现有的协议产生的细胞显示与天然成年心室肌相比的范围不成熟。许多已作出努力以成熟HPSC-CMS，只有迄今达到中等成熟。因此，工程化的系统，称为biowire，已通过提供物理和电线索导致HPSC-CMS 在体外更加成熟的状态设计的。该系统使用微制造平台种子HPSC-CM的I型胶原沿着一个刚性模板缝合组装成对准的心脏组织（biowire），将其进行电场刺激具有逐渐增加的频率胶凝。相比未刺激控制，刺激biowired心肌细胞表现出的结构和电生理学成熟的增强程度。这样的变化是取决于刺激速率。这份手稿详细描述了biowires的设计和创作。

理性在biowires使用的缝合线的是，以作为在一个轴对准并模仿心肌纤维的形状的3D结构的形成的模板。我们发现，之后在biowire培养7天的细胞改造围绕缝合（ 图3A）的凝胶。沿着缝合线的轴线组装细胞形成对准心脏组织（ 图3）。后7天的预培养物，在biowires进行7天电场刺激的，并与心肌细胞的结构和功能的成熟兼容进一步表现出的性质。 <p class="...

Watch this Scientific Journal Video about Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation at JoVE.com

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

心脏biowire平台是使用三维细胞培养与电刺激结合成熟人胚胎和诱导的多能干细胞衍生的心肌细胞（HPSC-CM）的体外方法。这份手稿介绍了心脏biowire平台的详细设置。

使用电刺激人干细胞衍生的心肌细胞的成熟中Biowires

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

maturation-human-stem-cell-derived-cardiomyocytes-biowires-using

Research

JoVE Journal

Developmental Biology

9.0K 次观看.  University Health Network. 心脏biowire平台是使用三维细胞培养与电刺激结合成熟人胚胎和诱导的多能干细胞衍生的心肌细胞（HPSC-CM）的体外方法。这份手稿介绍了心脏biowire平台的详细设置。 

视频: Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

从人类诱导多能干细胞使用小分子调制分化和随后的葡萄糖饥饿高度纯化心肌推导

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

科研

发育生物学

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

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 ...

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 ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

多能干细胞衍生的心肌修复心肌细胞

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

产妇基因产品的官能化操作使用<em&gt;体外</em斑马鱼&gt;卵母细胞成熟

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

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 ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

人诱导多潜能干细胞衍生肝细胞的药物发现方法

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

诱导多潜能干细胞源性肝细胞的家族性高胆固醇血症人肝嵌合小鼠模型

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...