Name: derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation
Uploaded: 2015-03-18T15:00:00.000+00:00
Duration: 11 min 53 s
Description: Watch this Scientific Journal Video about Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose S

This work was supported in part by the NIH/NHBI (U01 HL099776-5), the NIH Director&#8217;s New Innovator Award (DP2 OD004411-2), the California Institute of Regenerative Medicine (RB3-05129), the American Heart Association (14GRNT18630016) and the Endowed Faculty Scholar Award from the Lucile Packard Foundation for Children and the Child Health Research Institute at Stanford (to SMW). We also acknowledge funding support from the American Heart Association Predoctoral Fellowship 13PRE15770000, and National Science Foundation Graduate Research Fellowship Program DGE-114747 (AS).

注:供应商信息，在这个协议中使用的所有试剂已列于表1和 材料清单。所有解决方案和设备进入与细胞接触必须是无菌的，并且无菌技术应相应使用。除非另有规定执行所有培养孵育在加湿37℃，5％CO 2的培养箱中培养。在这个协议中，所有的分化在6孔板中进行，其中的人iPS细胞是种子。以下分化和纯化，细胞可被解离和再接种供下游使用。 1.准备中<ol><li>为E8介质，使原液的每个介质组分（ 碳酸氢钠 ，L-抗坏血酸-2-磷酸，亚硒酸钠，转铁蛋白，胰岛素，FGF2，TGFB1）和存储溶液在-20℃。加入储备溶液适量至瓶子的DMEM / F12，并过滤灭菌。该解决方案精矿ATION和反应设置为库存组分和E8介质可以在表1中找到。 </li><li>对于细胞外基质溶液中，解冻基底膜基质（10毫克/毫升通常供应）在4℃CO / N。基质胶是常用的多能性干细胞培养，因为人多能干细胞可以使用的α-6-β-1整联9很好地粘附到该材料。一旦解冻，使使用冰冷的枪头和管冰上2.25毫克（250微升）等分并储存在等分-80℃。 <ol><li>当ECMS预涂板，解冻和分装冰的细胞外基质溶液（ECMS）。在冰上200:混合ECM和冰冷的DMEM / F12培养基中的比例为1。保持冷静，以防止过早凝固ECMS。 </li></ol></li><li>对于RPMI / B27无胰岛素中，加10减B27胰岛素毫升到500ml RPMI媒体。 </li><li>对于RPMI / B27（胰岛素）中，加入10 mL B27补充（带insulin）的和5ml青霉素 - 链霉素抗生素到500ml RPMI培养基中。 </li><li>对低葡萄糖培养基中，加入10 mL B27添加剂和5ml的青霉素 - 链霉素抗生素到500ml葡萄糖的RPMI介质。 </li><li>用于冷冻/冷冻保存介质中，一起混合胎牛血清用二甲基亚砜（DMSO）以9:1的比例。冷冻介质可以存储长达1个月，在4℃。 注意:所有媒体应该被过滤并存储在4℃下并且除非另有说明在2周内使用。下面所用的所有试剂/溶液体积是供单个井在一6孔板，除非另有规定。 </li></ol> 2.预涂覆的6孔板的ECMS <ol><li>使ECMS通过混合基底膜基质（ 例如 ，基质胶）用冰冷的DMEM / F12培养基中的比例为1:200，然后应用2毫升新鲜制成ECM的到各孔为一个6孔板中。每个孔在6孔板将获得约90毫克ECMS。 </li><li>孵育ECMS上37℃下包被板1小时。之前立即镀细胞每孔，吸出DMEM / F12溶液。那么板块将准备接收细胞。 </li></ol> 3.解冻冰封人iPS细胞注:紧跟此过程，因为它可能导致最佳的培养人iPS细胞和成hiPSC-CMS的以下的冷冻/解冻循环下游分化。 <ol><li>人iPS细胞解冻一小瓶的6孔板的每个孔中。制备的15毫升锥形管中9ml冷E8介质与ROCK抑制剂（10μM）对每个小瓶。 ROCK抑制剂提高了人iPS细胞的生存以下冷冻保存10。保持在37℃的水浴的小瓶解冻所述细胞直至约5毫米直径的冰晶体在左边。 </li><li>喷用70％乙醇小瓶的外部。将它们转移到无菌层流罩之前，先用纸巾小瓶。 </li><li>转移细胞进入准备锥形管。冲洗吨他小瓶一次用500μlE8培养基并传送包含该解冻细胞相同的锥形管的平台。离心机在室温下以200×g下4分钟。 </li><li>离心后吸出上清液，并轻轻重悬细胞沉淀用2ml E8培养基与ROCK抑制剂（10μM）。转移的悬浮细胞至一个6孔板预先涂覆有ECM的一个阱。 </li><li>培养24小时后更换而不ROCK抑制剂与E8介质的介质。细胞应该坚持这个时候。 </li></ol> 4.传代人iPS细胞的<ol><li>通常情况下，在一个6孔板达到75％-80％汇合后通道人iPS细胞。吸出培养基。用1毫升无菌，RT PBS快速洗井。吸PBS。 <ol><li>加入500微升0.5毫摩尔EDTA或1x细胞剥离溶液（ 如的Accutase），并孵育在室温下1-7分钟。适当的时候EDTA孵化随hiPSC线。期待看到明显的卷曲Ø边缘周围的菌落ř增稠，因为这将表明，EDTA或细胞剥离溶液准备好被除去。 </li></ol></li><li>吸出的EDTA或细胞分离溶液。加入1毫升E8培养基补充有10μM的ROCK抑制剂。置换hiPSC菌落在井通过反复上下吹吸，并与一个P1000移液器喷雾菌落关闭。菌落被分成完成后5-10细胞团块。 </li><li>传送被在1ml E8介质与ROCK抑制剂的悬浮入15ml锥形管中的菌落。 </li><li>破坏大细胞团块成在E8中小团块。与抑制剂添加E8媒体的适当体积的细胞悬浮液稀释细胞。媒体量添加稀释取决于细胞密度，也可以接种井的数量。理想的情况下，人iPS细胞的一个单一的，80％的汇合以及应稀释1:12 <ol><li>添加11毫升E8介质ROCK抑制剂现有1毫升的CE在上述的15毫升锥形管LL溶液中以获得1:12的稀释。 </li></ol></li><li>进一步磨碎的细胞团块成小的细胞碎片（约50-200细胞在每个片段中是最好的）。避免过度捣碎，因为这减少了细胞的存活。 </li><li>吸ECMS来自预包被的板并加2ml悬浮细胞向每个孔中。每一个6孔板的约100,000个细胞是理想的。旨在均匀地分配围绕井的细胞，以避免在井的中心小区的聚类。 </li><li>经过培养24小时，更换E8介质中无ROCK抑制剂。改变培养基每24小时，直到细胞成为80％汇合。然后，将细胞准备通道一次。它通常需要段落之间的3-6天达到80％汇合。 </li></ol> 5.冻人iPS细胞注:紧跟这一程序，因为它可能导致最佳的培养和髋部的下游分化雪旺成hiPSC-CMS以下的冷冻/解冻循环。 <ol><li>标号低温管与所述细胞系的名称，细胞类型，通道号，以及冷冻时间。作为一般准则，用一个小瓶的6孔板的每个孔中。制备的15毫升锥形管中填充用9ml E8介质与ROCK抑制剂（10μM）。制备冷冻培养基用90％FBS和10％DMSO中，并保持该介质在4℃下，直到准备使用。 </li><li>吸出培养基，加入500微升0.5毫摩尔EDTA或1x细胞分离液，孵育在室温下2-7分钟。的EDTA或细胞剥离溶液接触的持续时间从细胞系而异的细胞系。 </li><li>吸EDTA或细胞分离液。加入1毫升E8介质。 </li><li>置换在阱hiPSC菌落通过上下抽吸并用P1000移液器喷雾菌落关闭。菌落不应被破碎成小于100的细胞团块。转移悬浮的细胞，以制备锥形管含有E8。离心机为RT代表在200×g下4分钟。 </li><li>吸出上清液，并添加500μl的冷冻介质中的颗粒。吹打的一至两倍重悬沉淀。细胞存活是通过保持细胞大团块改善。 </li><li>转移悬浮细胞成标低温管。快速移动的小瓶进入冷冻含有异丙醇的容器中，这将允许为逐步冷却。保持容器与细胞在-80℃下放置24小时，然后将细胞转移到液氮中用于长期贮存。 </li></ol>人类的iPSC 6.心脏分化注:加入时，所有的媒体应该至少在RT。 <ol><li>离解用EDTA或1x细胞剥离溶液后，种子就ECMS涂覆的6孔培养板中进行分化大约100,000人类iPSC（相同的步骤，细胞传代过程）。当细胞达到85％汇合，改变介质为RPMI / B27没有与6中胰岛素μMGSK3-β抑制剂CHIR99021（CHIR）并保持48小时。 </li><li> 48小时后，更换RPMI / B27含CHIR培养基无胰岛素中，独自离开24小时（到3天）。 </li><li>在第3天，无胰岛素与5μM的Wnt抑制剂IWR1改变媒体RPMI / B27并保持48小时（直到第5天）。 注:的Wnt抑制还可以使用其它小分子化合物尝试，如在早先的研究11说明。 IWR1被选择比其它小分子Wnt信号的抑制剂，由于在它已被证明可有效地抑制Wnt信号11的上升范围。 </li><li>第5天，改变介质回RPMI / B27无胰岛素中，留下48小时（到7天）。 </li><li>在第7天，更换用R​​PMI / B27培养基（胰岛素）培养基中，并以同样的培养基其后每3天更换培养基。心肌细胞的自律搏动首先应该是可见约8日复一日10。 </li></ol>经过葡萄糖饥饿7.净化人类心肌细胞的<ol><li>在第10天的后分化，改变在6孔板至2ml低葡萄糖培养基的每孔中的培养基，并维持细胞在该培养基中3天（直至第13天）。 </li><li>在第13天，恢复细胞的RPMI / B27培养基（胰岛素）。 </li><li>任选地，replate之前第二轮葡萄糖饥饿，以帮助放松从培养板的非心肌细胞，允许对非心肌细胞中葡萄糖饥饿容易解离的心肌细胞。 <ol><li>在第13天，吸出培养基，用PBS洗一次，并用500微升的细胞解离的酶进行5分钟，在37℃下解离的细胞分化成单细胞。具体地讲，5分钟酶处理后，可以使用一个1000微升吸管反复拉起细胞离解酶和喷涂它靠在cardiomyo手动解离从6孔板心肌CYTE单层。高达30移液重复可能需要解离的心肌细胞为单细胞。 </li><li>后的细胞被离解，并在单细胞形式，收集所有细胞入15ml锥形管中填充用5ml RPMI / B27培养基的胰岛素来稀释出细胞离解酶和离心机在200×g下4分钟。吸并丢弃上清液。 </li><li>重新悬浮细胞用2ml的RPMI / B27培养基和板到一个新ECMS被覆6孔板中。通常情况下，心肌更高的融合有助于细胞存活replating期间。旨在replate每个新的6孔盘以获得最佳的生存2000000细胞replating期间。 </li></ol></li><li>在第14天，改变介质回〜2毫升的低血糖中的第二个缺糖周期。培养的细胞在这种低血糖状态持续3天以上。大多数非心肌细胞会死在这种低血糖培养条件。 </li><li>在17天，改变R的中型〜2毫升PMI / B27介质胰岛素。剩余的细胞将是高度纯化心肌细胞。这些心肌细胞可用于基因表达分析，药物筛选，代谢分析，以及其他各种下游测定。 </li></ol>

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The authors do not declare competing interests.

得到大量的高纯度的hiPSC衍生的心肌细胞是基本心脏研究以及临床和转化应用的关键。心脏分化协议已经在最近几年发生了巨大的改进，从利用心源性生长胚状体为基础的方法转变因子2，以矩阵夹心方法12，最后以小分子调制和单层为基础的方法5。上述协议，这里描述的方案中显示的最高和最可重复的心脏分化效率不同hiPSC细胞系通过使用小分子CHIR和IWR 5,7调制的W...

原代人心肌细胞是因为侵入性心脏活检，难以解离为单细胞的，而且由于差的长期存活的细胞在培养中的要求很难获得。鉴于这种缺乏原代人心肌细胞，针对具体患者的诱导的人多能干细胞衍生的心肌细胞的（hiPSC-CM）的技术已被认为是一个功能强大的替代来源的心肌细胞的基础研究以及临床和翻译的应用，如疾病建模和药物发现1。在分化的多能干细胞分化为心肌细胞的早期努力采用使用胚状体（EB）分化的协议，但这种方法是低效的生产心肌细胞，因为常常少于25％的细胞在使用EB均跳动的心肌细胞2,3。相比较而言，用激活素A和BMP4的细胞因子的单层基分化方案所显示的更高的效率比的EB，但该协议还比较低效率的，需要昂贵的生长因子，并在人多能干细胞系的4有限数量的唯一功能。近日，一个高效，hiPSC单层型心肌细胞分化协议是通过调控Wnt信号/β-catenin信号5开发。这些hiPSC-CM的表达肌钙蛋白T和α辅肌动蛋白，双肌蛋白是心肌6标准的标记。这里的协议描述是这样的小分子为基础，馈线无细胞，单层分化法5,7的适应。我们能够获得后7-10天（ 图1），从人iPS细胞跳动的心肌细胞。然而，下面的一个心肌细胞的分化导致50％的跳动细胞，始终如一的免疫染色显示了非心肌细胞是阴性的心肌特异性标记物，如心脏特异性肌钙蛋白T和α-辅肌动蛋白的群体的存在。来福rther纯化心肌细胞，并消除非心肌细胞，异质分化细胞群经受由具有非常低葡萄糖培养基中多天（ 图2）处理它们对葡萄糖饥饿。这种处理有选择地消除非心肌细胞由于心肌细胞的能力，但不是非心肌细胞，代谢乳酸作为主要能量来源，以便在一个低葡萄糖环境8生存。在此之后的纯化步骤中，在心肌细胞中，以非心肌细胞的比率增加了40％被观察到的，（ 图3，图4），并且这些细胞可以用于下游的基因表达分析，疾病建模和药物筛选试验。

<table><tbody><tr><td>Matrigel (9-12 mg/ml)</td><td>BD Biosciences</td><td>354277</td><td></td></tr><tr><td>RPMI media</td><td>Invitrogen</td><td>11835055</td><td></td></tr><tr><td>Glucose free RPMI media</td><td>Invitrogen</td><td>11879-020</td><td></td></tr><tr><td>B27 Minus Insulin</td><td>Invitrogen</td><td>A1895601</td><td></td></tr><tr><td>B27 Supplement (w/ insulin)</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Pen-strep antibiotic</td><td>Invitrogen</td><td>15140122</td><td></td></tr><tr><td>Fetal bovine serum</td><td>BenchMark</td><td>100-106</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D-2650</td><td></td></tr><tr><td>ROCK inhibitor Y-27632</td><td>EMD Millipore</td><td>688000</td><td></td></tr><tr><td>CHIR99021</td><td>Thermo Fisher</td><td>508306</td><td></td></tr><tr><td>IWR1</td><td>Sigma</td><td>I0161</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575-020</td><td></td></tr><tr><td>Accutase</td><td>Millipore</td><td>SCR005</td><td></td></tr><tr><td>Cell lifter</td><td>Fisher</td><td>08-100-240</td><td></td></tr><tr><td>Cryovial</td><td>Fisher (NUNC tubes)</td><td>375418</td><td></td></tr><tr><td>TrypLE Select Enzyme</td><td>Invitrogen</td><td>12563-011</td><td></td></tr></tbody></table>

derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation

诱导的人多能干细胞衍生的心肌细胞（hiPSC-CMS）已成为一种重要的细胞源，以解决缺乏可用于基础研究和翻译的应用程序主心肌。分化人iPS细胞为心肌细胞，已经开发了各种协议，包括胚状体（EB）为基础的分化和生长因子诱导。然而，这些协议是低效，并在其产生纯化的心肌细胞的能力高度可变。最近，的Wnt /β-catenin信号的小分子为基础的协议利用调制被证明促进心脏分化效率高。与此协议中，分化的细胞大于50％-60％为心肌肌钙蛋白阳性心肌细胞持续观察。为了进一步增加心肌的纯度，分化的细胞进行葡萄糖饥饿基于代谢差异特异性消除非心肌细胞s心肌细胞和非心肌细胞之间。使用此选择策略，我们一致地得到增加心肌非心肌细胞的比率在分化细胞群一大于30％。这些高度纯化心肌要增强人类iPSC的基于体外疾病模型研究和药物筛选测定法的结果的可靠性。

在hiPSC分化的形态变化。 所述hiPSC在无饲养平板上培养生长为平坦的，二维的菌落。当达到约85％汇合，人iPS细胞分别用6微米的CHIR处理分化（ 图1A）。细胞死亡，一个正常的和普遍的现象，显着量的CHIR治疗的24小时后进行观察。后CHIR治疗两天，人iPS细胞持续分化朝向中胚层的命运。相较于细胞的hiPSC克隆，细胞大小，这些天2细胞增多。手机号码也可...

Watch this Scientific Journal Video about Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose S

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

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 ...

derivation-highly-purified-cardiomyocytes-from-human-induced

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

18.6K 次观看.  Stanford University School of Medicine. 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. 

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

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 ...

科研

发育生物学

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 ...

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 ...

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

患者外周血单核细胞中人心肌细胞的生成和扩增

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

用于循环作用电位记录的多井微电极阵列上的人类 iPSC 衍生心肌细胞网络

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

生物工程

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

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

使用人iPSC衍生的心肌细胞，心脏成纤维细胞和内皮细胞制造3D心脏微组织阵列

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

使用成熟的人诱导多能干细胞来源心肌细胞单层进行高通量心脏毒性筛选

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

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 Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

生成心室样HiPSC衍生的心肌细胞和用于钙处理特征的高质量细胞制剂

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...