1.建议开始之前<ol><li>不要使用被污染的产品分成合同与支原体，因为他们不会分化成正常窦房结细胞。启动协议之前，测试支原体污染。用PCR试剂盒进行快速，灵敏地检测支原体做到这一点，并按照manufacturer`s协议。 </li><li>对于每个培养皿（步骤2.3.4），涂层1 10cm 2的细胞培养皿用无菌7毫升0.1％明胶从冷水鱼皮1小时，在37℃。除去明胶，并让在无菌条件下将培养皿干燥在无菌台上。 </li><li>在你开始分化协议，你需要一个双稳定转染的小鼠胚胎干细胞系克隆包含以下特点:一）构TBX3使用哺乳动物表达载体过表达。 ⅱ）MYH6启动子5的控制下的G418抗性基因。 </li><li>未分化细胞的物业服务公司应共同培育ð与标准条件如所述1下照射饲养细胞。 </li></ol> 2.分化过程<ol><li>两天之前分化 - 从饲养层细胞中取出产品分成合同。 <ol><li>吸出介质，用10毫升的磷酸盐缓冲盐水PBS中洗涤细胞，添加7毫升胶原酶IV溶液，并在37℃下孵育细胞10分钟。放置在50ml管中的无菌40μm的过滤器。 </li><li>仔细冲洗PSC殖民地（避免去除饲养层细胞）吹打胶原酶溶液向上和向下的5倍。 </li><li>细胞悬液转移到一个40微米的过滤器，冲洗过滤器三次用8ml PBS中。转身过滤器，并将其放置在培养皿倒挂。吹打10毫升PBS中至过滤器的底部取出的PSC细胞集落。 </li><li>细胞悬液转移到15毫升管离心5分钟，在300×g下。 </li><li>取出PBS，悬浮细胞1毫升的Accutase和我ncubate在37℃下5分钟。 </li><li>加入10毫升的PBS，吹打它向上和向下的5倍，离心5分钟，在300×g下混合的细胞溶液。 </li><li>除去PBS，悬浮细胞在10ml培养基中并测定细胞数。 </li><li>种子15000细胞/ cm 2上的75cm 2的过滤器烧瓶中，并加以培养2天，在37℃，5％CO 2。 2天后将烧瓶应50-70％汇合。 </li></ol></li><li> 0天 - 开始分化<ol><li>除去培养基并用10ml的PBS洗涤细胞。 </li><li>吸出PBS，加2ml的Accutase孵育将细胞在37℃下5分钟。放置在50ml管中的无菌40μm的过滤器。 </li><li>加入10毫升的PBS，将细胞悬浮液转移到40微米的过滤器，通过加入10ml PBS中，离心冲洗过滤器流过5分钟，在300×g下。 </li><li>悬浮细胞在10ml的分化培养基，并确定细胞数。 </li><li>淡化吨他的细胞悬液与分化培养基中至20000个细胞/ ml的终浓度。 </li><li>吸管20ml水和5ml挂在一个二次培养皿，以避免干燥出加氢脱硫的压降（HD）的溶液中。 注:对于每个24以及含板iSABs（见2.8.6.4/2.8.7）启动16个培养皿。 </li><li>吸取最多至50ml细胞悬浮液中的托盘。 </li><li>转身培养皿的盖子。将180日立数据系统每片含20微升（400个细胞/ HD）细胞悬液到使用12道移液器的盖子。 </li><li>小心地将周围的盖子，将它放到培养皿中。 注:扭转盖子的速度是很重要的。如果盖子被打开围绕太慢或太快，将细胞悬浮液的表面张力不够高，保留悬滴在盖。在试图产生日立数据系统首次练习扭转盖子用中的20微升滴。 </li><li>在3培养细胞2天7°C，5％CO 2，让他们形成EB。 注:将一个培养皿盛满水的一叠5培养皿的顶部。否则，HD在上培养皿就会枯竭。 </li></ol></li><li>第2天<ol><li>小心地将周围的二次培养皿的盖子，并从2培养皿（360 EBS）衍生的胚转移至50ml管。 </li><li>等待10分钟，让胚定居在试管底部（不要离心EBS）。 </li><li>吸出尽可能多的介质成为可能，暂停在10ml分化培养基中的胚，悬浮液转移到10cm的培养皿中。 </li></ol></li><li>一天2-6悬浮培养<ol><li>培养在悬浮液4天的EB中，在37℃，5％CO 2，2天后改变介质。 注意:即使在陪替氏培养皿不涂（而不是一个细胞培养皿）的胚往往附加到表面。控制培养皿每天附着胚，并在必要时分离的胚从表面通过温和移液。可选:在悬浮培养期间不断摇晃培养皿。小心振动筛的速度。 EBS的既不应积累在中间，也没有浮到的培养皿边界。 </li></ol></li><li> 6天<ol><li>从一个培养皿转移EBS的一个明胶涂10厘米2细胞培养皿。 </li></ol></li><li> 7-12日<ol><li>细胞应该开始后8-12天打。检查每天在显微镜下跳动的细胞灶。细胞开始形成的层。 </li><li>改变媒体每天。 注意:在改变培养基后，将细胞需要3-4小时，以恢复和重新开始跳动。 </li></ol></li><li> 12-15日选择窦淋巴结细胞。 <ol><li>所述细胞已经开始跳动（约12-15天）后三天，吸新鲜分化培养基含有400微克/毫升G418的细胞的培养基和吸管10毫升</li></ol></li><lI&gt;的选择的开始后的第4天解离的细胞层。 注意:这是可能的细胞层已经从表面脱离。 <ol><li>小心取出介质不吸细胞层。 </li><li>加入10毫升的PBS，并小心地取出PBS不吸细胞层。 </li><li>加入10毫升胶原酶IV溶液，将细胞转移到50毫升管中并孵育所述细胞在37℃下5分钟。 </li><li>大力通过移液器上下吹吸10次混合的悬浮液，并培育的细胞在37℃下5分钟。应该发生的小簇暂停。如果仍有离开层的庞大零件，然后重复步骤2.8.4两次以上。 </li><li>加入20毫升的PBS和离心机在300×g下10分钟。 </li><li>小心吸出PBS。 注:如果iSABs要生成继续步骤2.8.7。如果ISAB衍生窦结单细胞是要产生继续步骤2.9。 </li><li>悬浮细胞于12ml分化培养基中含有400微克/毫升G418和种子它们上6个孔的明胶包被的24孔（2毫升/孔）。 </li></ol></li><li>代单结电池。 <ol><li>暂停从步骤2.8.6在5毫升的Accutase细胞孵育在37℃下5分钟。 </li><li>大力通过移液器上下吹吸10次混合的悬浮液，并培育的细胞在37℃下5分钟。 </li><li>加入20毫升的PBS，离心5分钟，在300×g下。 </li><li>暂停含有400微克/毫升G418的细胞于12ml分化培养基和种子它们上6个孔一个明胶包被的24孔（2毫升/孔）。 </li></ol></li><li>第2天解离后，吸出培养基，用PBS洗涤细胞三次。加入1毫升的分化培养基/孔。 </li><li> 4-8天解离后，改中，每2 次一天。上解离使用iSABs或ISAB衍生窦后第10天的节点的单细胞可用于进一步的分析。 </li></ol>

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作者什么都没有透露。

的能力产生干细胞来源的心脏起搏细胞可允许适当心律重建中的"生物起搏器"的感觉。同样，在体外药物测试将受益于他们的可用性。的PSCs可以引起哺乳动物体包括与起搏器电池特性8,9,10,11,12,13心肌的任何细胞类型。然而，通常为"胚体"内的细胞群体是高度异质的，这不可避免地导致了可靠的选择和分离的策略的要求 - 这尤其适用于心脏结细胞的非常罕见的细胞类型。基于外源性和内源性...

术语"病态窦房结综合征"总结了多种疾病导致的心脏起搏器系统的恶化。它包括病理，对症窦性心动过缓，窦房传导阻滞，窦性停搏以及心动过速，心动过缓综合征。由此，一个"病态窦房结综合征"常伴一般心脏疾病如缺血性心脏疾病，心肌病或心肌炎。目前，治疗方法是基于电起搏器的植入。然而，这伴随着一个数字，如感染和电池故障的风险。总体而言，并发症的发生率还是非常高的患者已经植入人工心脏起搏器。此外，相对于内源性心脏起搏器，这些设备不响应​​激素刺激。 未来的另一种可能依赖于哪些产品分成合同可能成为的"生物起搏器"的可用性作为合适的细胞来源，这也将是用于体外药物测试非常有价值的。然而，一个主要的问题出在胚体（EBS）中非常罕见的外观窦结细胞-这通常不超过约0.5％1。 此前，它表明"向前编程"朝特定心肌亚型是通过不同的早期心血管转录因子如中胚层特异性-后1（MesP1）和NK2转录因子相关的，轨迹5（的Nkx2.5）2的过表达是可行的， 3。对于正常尺寸和窦房结（SAN）的功能，在T-box转录因子TBX3是至关重要的，这已被证明以引发起搏器基因程序，并控制在SAN 4的分化。而这种增强的功能性起搏细胞的外观，内容依然没有整个cardiomyocytic细胞群中超过〜40％。 因此，一个额外的MYH6，促进基于抗生素选择步骤5中我们进行了介绍。这最终导致尚未未观察到心肌细胞聚集体（"诱导窦房机构;"iSABs"），其在体外表现出高度增加的跳动的频率（&gt; 400 BPM），首次近似那些鼠心脏的和可比较的体外培养窦结细胞从鼠心脏6隔离。在异丙肾上腺素管理甚至殴打的550 BPM频率来实现。值得注意的是，iSABs包括超过80％的功能节点细胞从粗放生理得到印证分析7。最近，一些方法来使用直接重新编程19产生窦淋巴结细胞，表面标记14或药物治疗与小分子16,17进行了描述。然而，这些方法导致起搏细胞的这种高纯度和跳动频率接近鼠他艺术在iSABs观察。 此外，在已失去了他们的自发活动的跳动种植成年小鼠脑室片的体外模型中，iSABs能够集成到切片组织，因此剩余的自发主动和有力起搏心脏切片收缩7。这些iSABs的生成一个详细的协议，本文介绍。

<table><tbody><tr><td>Iscove&#039;s liquid medium with stable glutamine</td><td>Biochrom AG</td><td>FG 0465</td><td></td></tr><tr><td>DMEM liquid medium without Na-pyruvate, with stable glutamine</td><td>Biochrom AG</td><td>FG 0435</td><td></td></tr><tr><td>CLS type IV, CLS IV</td><td>Biochrom AG</td><td>C4-22</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Biochrom AG</td><td>K 0293</td><td></td></tr><tr><td>FBS Superior</td><td>Biochrom AG</td><td>S 0615</td><td></td></tr><tr><td>Sodium pyruvate (100 mM)</td><td>Biochrom AG</td><td>L 0473</td><td></td></tr><tr><td>G 418-BC liquid (ready-to-use solution), sterile</td><td>Biochrom AG</td><td>A 2912</td><td></td></tr><tr><td>Reagent reservoir PP f.multichannel pip. 60ml,sterile</td><td>Brand</td><td>703409</td><td></td></tr><tr><td>Accutase</td><td>eBioscience,Inc.</td><td>00-4555-56</td><td></td></tr><tr><td>Eppendorf Xplorer/Xplorer plus, electronic pipette</td><td>Eppendorf</td><td>4861000155</td><td></td></tr><tr><td>Falcon Cell Strainer 40 &amp;micro;m</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp;</td><td>GE Healthcare</td><td>P11-010</td><td></td></tr><tr><td>Petri Dishes</td><td>Greiner BioOne</td><td>663102</td><td></td></tr><tr><td>DPBS without Ca and Mg</td><td>PAN-Biotech</td><td>P04-36500</td><td></td></tr><tr><td>Fetal bovine serum</td><td>PAN-Biotech</td><td>P30-3302</td><td></td></tr><tr><td>Leukemia inhibitory factor</td><td>Phoenix Europe GmbH</td><td>LIF-250</td><td></td></tr><tr><td>Quadratic petri dishes</td><td>Roth</td><td>PX67.1</td><td></td></tr><tr><td>Gelatin from cold water fish skin</td><td>SigmaAldrich</td><td>G7765</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>SigmaAldrich</td><td>M3148&amp;nbsp;</td><td></td></tr><tr><td>1-Thioglycerol</td><td>SigmaAldrich</td><td>M6145</td><td></td></tr><tr><td>Tissue culture dishes</td><td>TPP</td><td>93100</td><td></td></tr><tr><td>Tissue culture flask</td><td>TPP</td><td>90076</td><td></td></tr><tr><td>Tissue culture test plates (24 well)</td><td>TPP</td><td>92424</td><td></td></tr></tbody></table>

generation of murine cardiac pacemaker cell aggregates based on es-cell-programming in combination with myh6-promoter-selection

"病窦综合征"的治疗是根据人工心脏起搏器。这些熊的危害，如电池故障和感染。此外，它们缺乏激素响应性和总体过程是成本密集型的​​。从生成的PSCs"生物起搏器"有可能成为一种替代方法，还起搏细胞的胚状体的典型含量（EBS）是非常低的。所描述的协议将通过窦房结诱导TBX3与MYH6，促进基于抗生素选择"前进编程"鼠产品分成合同。这就产生心肌聚集一致&gt; 80％生理功能起搏细胞。这些"诱导窦房结-体"（"iSABs"）被自发收缩在对应于节点的细胞从小鼠心脏中分离但未得频率（400-500 BPM），并能踱步鼠心肌体外 。使用所描述的协议的高纯度窦可以生成节点单个细胞，其例如可以用于在体外药物测试。此外，根据该协议所产生的iSABs可能成为朝向心脏组织工程的关键步骤。

所描述的协议允许代iSABs与大约450 BPM的产品分成合同（在电影中所示），一个跳动频率附近是对小鼠心脏跳动频率。 iSABs的解离（步骤2.8.8）后所观察到的单个细胞显示窦房结（主轴和蜘蛛细胞）的细胞的典型形状如图1，这些细胞是已知的高表达的蛋白质是该功能所必需像超极化激活环核苷酸门控阳离子通道4（HCN4），连接蛋白（Cx45的），Connexin30.2（Cx30.2）和MYH6（ - C图2A）</str...

Watch this Scientific Journal Video about Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection at JoVE.com

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

本协议描述了如何制作从小鼠多能干细胞（PSC）功能性鼻窦淋巴组织。 T-BOX3（TBX3）表达以及心肌肌球蛋白重链（MYH6）启动抗生素的选择导致了高纯度的起搏细胞聚集。这些"诱导窦房结-机构"（"iSABs"）含有80％以上的起搏细胞，显示高度增加跳动的速度，并能步伐心肌体外 。

基于ES-Cell的编程组合MYH6启动子，选择小鼠心脏起搏器细胞聚集一代

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

generation-murine-cardiac-pacemaker-cell-aggregates-based-on-es-cell

Research

JoVE Journal

Developmental Biology

9.6K 次观看.  University of Rostock. 本协议描述了如何制作从小鼠多能干细胞（PSC）功能性鼻窦淋巴组织。 T-BOX3（TBX3）表达以及心肌肌球蛋白重链（MYH6）启动抗生素的选择导致了高纯度的起搏细胞聚集。这些"诱导窦房结-机构"（"iSABs"）含有80％以上的起搏细胞，显示高度增加跳动的速度，并能步伐心肌体外 。 

视频: Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

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

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

诱发心肌细胞的使用多顺反子构建表达GATA4，MEF2C和Tbx5中的最优比率改善发电

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

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 ...

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

通过微接触印刷的定向功能性心肌组织的产生

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

生物工程

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

促纤维化信号增强因子介导的小鼠胚胎成纤维细胞再编程诱导心肌细胞的抑制

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

小鼠心脏场特定心脏祖细胞的体外生成

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

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 ...

生物学

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 ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Myospheres的产生从人类胚胎干细胞通过表观遗传修饰

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...