The authors wish to thank Shonna Johnston, Claire Cryer, Fiona Rossi and Will Ramsay at the University of Edinburgh and Alison Logar and Megan Blanchard at the University of Pittsburgh for their expert assistance with flow cytometry. We also wish to thank Anne Saunderson and Lindsay Mock for their help with obtaining human tissues. Human adult and fetal heart tissue samples were procured with full ethics permission of the NHS Scotland Tayside Committee on Medical Research Ethics and the NHS Lothian Research Ethics Committee (REC08/S1101/1) respectively. This work was supported by grants from the Medical Research Council (BP), British Heart Foundation (BP), Commonwealth of Pennsylvania (BP), Children's Hospital of Pittsburgh (BP), National Institute of Health R01AR49684 (JH) and R21HL083057 (BP), and the Henry J. Mankin Endowed Chair at University of Pittsburgh (JH). JEB was supported by a British Heart Foundation Centre of Research Excellence doctoral training award (RE/08/001/23904). WC was supported in part by an American Heart Association predoctoral fellowship (11PRE7490001).

1.人力心脏样品处理<ol><li>确保所有的液体，容器，仪器，和专用业务区域是无菌的。 </li><li>放置心脏组织样品含有20％胎牛血清（FBS），并在冰上1％青霉素 - 链霉素（P / S）冷冻Dulbecco改进的Eagle培养基（DMEM）的组成，在存储介质（由组织库或外科团队购买）交通3。 </li><li>从存储介质中删除心脏样品并用补充有2％FBS和1％P / S的磷酸盐缓冲盐水（PBS）组成的洗涤介质洗涤。当处理样品，用细尖镊子把握大血管或心包和减少心肌的破碎。 </li><li>淹没在洗涤介质中的样品在培养皿和描述3取出心包，心内膜及大血管用消毒剪刀虹膜和细尖镊子。 </li><li>切剩下的myocardium成约1mm 3小块使用单一侧刀片或一对锋利的虹膜剪的。注意:如果样品立即处理最高细胞产量将获得。如果延迟是不可避免的，在新鲜的存储介质的存储处理后的心脏组织的（&gt;5毫升每1cm 3组织）于4℃多达72小时是可能的，但是推测可能与在细胞中产量相关联的下降。 </li></ol> 2.细胞的组织和隔离消化<ol><li>新鲜弥补消解液含有1.5-2.4毫升每胶原酶I，II胶原酶，和胶原酶IV（均在DMEM 0.5毫克/毫升），并以温暖的37℃水浴。 注:音量和胶原酶的浓度是适宜的约1cm 3个样本。 </li><li>通过100微米滤网过滤悬浮组织碎片，用PBS洗两次。转移组织块到无菌30毫升容器一个密封的盖子。 注:短而宽的盆，而不是高的细管给出更好的结果。可替代地，把组织片段在无菌50ml管中，如果在30ml容器不可用水平放置。 </li><li>添加全部消化解决方案（4.5毫升总数）并放置一个密封的塑料袋内的容器设置为120转一个37℃摇动的水浴。另外，与封蜡膜和地方的容器中加热轨道摇床设置为120 RPM。 </li><li> 15分钟后，取出和更换再15分钟前三次颠倒了锅。除去和淬火用5ml的DMEM补充有20％FBS和1％P / S的胶原酶。 </li><li>用10毫升血清吸管，打破了任何组织团块轻轻吹打十到二十次磨碎的摘要。通过100微米过滤悬浮液顺序地，70μm和最后40微米的细胞滤网以除去未消化的物质，将获得单细胞悬浮液。 注意:不细胞滤网之间冲洗，以避免被酶消化过程debrisgenerated细胞。 </li><li>离心机在200×g离心4分钟的细胞悬浮液，小心地倒出上清液并用4ml洗涤介质的稀释前重新悬浮在1ml红细胞裂解缓冲液，在室温下2分钟的沉淀。 </li><li>重复离心步骤和重新悬浮在1ml洗涤培养基的沉淀。拌匀取10微升细胞悬浮液为细胞计数。 </li><li>混合10微升细胞悬浮液与10微升台盼蓝染色，并放置10微升该混合物的对细胞计数标准血细胞计数器。算上明亮，圆形细胞存在于四角方块（每个细分成十六小方块）的数量除以4，让每角方的平均值。乘以2的平均值以帐户为污渍稀释因子，然后通过10 4，得到每1ml样品中的细胞的总数。 </li></ol><ol><li>加入50μl的小鼠血清对细胞悬浮液，并在4℃下孵育20分钟以阻断非特异性抗体结合。 </li><li>离心机在200×g离心4分钟小鼠血清的细胞悬浮液，取出上清液，并在洗涤介质中以每100微升1×10 6个细胞的浓度重悬。 </li><li>等分每个细胞悬浮液的同种型和未染色对照的50微升成两个聚苯乙烯圆底流式细胞管然后将剩余体积为第三管为多色染色。 </li><li> CD34-PE，CD45-APC-Cy7的，CD56-PE-Cy7的，CD144-PerCP-经Cy5.5，和CD146 - AF647抗体添加（总共1:100）的单细胞悬浮液用于多色染色。对于同种型对照，加入PE-，APC-Cy7-，PE-Cy7-，PerCP-Cy5.5-和AF647共轭合同种型抗体的等效容积。轻轻移液管混合与细胞抗体，并在4℃下孵育所有试管20分在黑暗中。 </li><li>通过在五个聚苯乙烯圆底流式细胞仪管加入正珠一滴100微升洗涤介质的准备补偿控制珠。添加的CD34-PE，CD45-APC-Cy7的，CD56-PE-Cy7的，CD144-PerCP-经Cy5.5，和CD146 - AF647抗体（所有1:100）分别到每个5管子。孵育所有试管于4℃在黑暗中20分钟。 </li><li>在温育，制备各用500μl内皮生长培养基-2在4℃（EGM-2）培养基和地点的细胞收集管中。 </li><li>以下抗体孵育，添加5毫升洗涤介质洗涤的细胞和小珠以除去未结合的抗体。离心机在200 XG 4分钟都管。重复洗涤和离心步骤。小心倒出上清，轻轻吸管重新悬浮细胞时。 </li><li>在洗涤介质中以每250微升约1×10 6个细胞的浓度重悬。重悬在100μl的洗涤微米珠edium。 </li><li>交通建设在暗处的冰所有细胞和珠悬浮液的细胞分选仪。负珠1滴添加到正珠补偿控制管，并使用这些设置荧光补偿。 </li><li>运行，根据制造商的说明来建立背景荧光和电压设置为以下未染色的对照细胞:FSC 150V; SSC 290V; V450 / 50 350V; V710 / 50 620V; B530 / 30 400V; B710 / 50 530V; YG780 / 60 460; R670 / 30 480V; R780 / 60 460V。根据制造商的说明运行同种型对照以建立与非特异性结合背景荧光阈值。运行多色染色样品和收集周细胞和外膜细胞群进入收集管（ 图1）。 注:最佳可行的细胞产量是周细胞的总活细胞解离的大约3％和外膜细胞的4％。 </li></ol> 4.细胞培养<ol><li>根据FACS分选的结果，选择合适的尺寸培养板中。加入100微升无菌0.2％的明胶溶液的每生长面积的平方厘米和手动搅动涂覆整个孔中。在4℃下进行10分钟孵育板和完全除去明胶溶液。保持收集的细胞在冰上而制备培养板。 注:当在30,000到40,000细胞/ cm 2的明胶包被培养表面的密度接种新鲜整理周细胞外膜和细胞生长良好。 </li><li>离心新鲜采集的细胞以200×g离心4分钟，并在EGM-2.Seed适量在明胶包被的平板的细胞轻轻重悬浮细胞沉淀。 注意:每孔和待添加的EGM-2的量被接种的细胞的实际数量取决于板的选择。例如加入0.5ml和1ml EGM-2则分别需要每孔为48和24孔板中。 </li><li>交换EGM-2为血管周围细胞生长培养基（的DMEM补充有20％FBS和1％P / S）为周细胞和外膜细胞一次细胞已（后至少72小时）定居并粘附到板上。从此改变介质每72小时。进行初次和所有后续的孵育在5％CO 2，并在37℃。 </li><li>使用0.05％胰蛋白酶的EDTA当周细胞和外膜细胞达到80〜90％汇合解离的细胞。用在PBS中的20％的FBS，离心机在200×g离心4分钟淬火，以1重悬浮在血管周围细胞的生长培养基中，然后通过将细胞:3的比例到未涂覆聚苯乙烯培养板中。从通道2开始，传代细胞以1:5的比例（或在约7000名细胞/ cm 2），以未涂覆的聚苯乙烯培养板或烧瓶中。 </li></ol>

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The authors have no conflict to disclose.

越来越多的证据支持成人心脏损伤后有限的再生能力。负责受伤的心如再生反应本土前体细胞的识别和鉴定是相关的机制和信号通路双方的理解和制定办法来治疗利用这些细胞的关键。 先前协议已经从人骨骼肌25中所述的血管周围的前体细胞亚群的分离。然而，这些技术对心脏组织的直接应用经常导致非常差的细胞产量。因此，我们以丰富血管周围的前体细胞，即周?...

该心脏一直被认为是一个有丝分裂后的器官。然而，最近的研究已经证明有限心肌周转的成年人的心脏1的存在。原生干/祖细胞，心肌细胞分化的潜能也被认定为成人啮齿动物和人的心，其中的Sca-1 +，C-KIT +，cardiosphere形成心肌内，以及最近的血管周围前体细胞2,3。这些细胞代表了旨在通过细胞移植或原位增殖的刺激增强心脏修复/再生疗法具有吸引力的候选者。 间充质干细胞/基质细胞（MSC）已经从几乎每个人组织4,5的MSC的治疗应用的临床试验已进行了多种病理条件下分离如心血管修补6，移植物抗宿主病7 </sup&gt;和肝硬化8。有益的作用已被归因于MSCs向的能力:家里炎症9的部位;分化成不同细胞类型10;分泌促修复分子11;和调节宿主的免疫反应12。干细胞的分离历来依靠其优惠的坚持塑料基板。然而，所得到的细胞群是典型地显着异质13。通过使用荧光激活细胞分选（FACS）用的关键血管周围细胞标记物的结合，我们已经能够分离和纯化多能的MSC样前体群体（CD146 + / CD31 - / CD34 - / CD45 - / CD56 - ）从多种人体组织包括成人骨骼肌和白色脂肪14。 在各种非心脏组织的血管周围细胞群已被显示出具有干/祖细胞特性的第二正在研究在心血管设置临床使用。周细胞，最知名的血管周围细胞亚群之一，是一个异质人口玩几个病理生理作用，包括在新船15的发展，血压16和血管完整性17,18的维护管理。由于在多种组织所示，周细胞的特定子集本身表达MSC抗原和FACS纯化后14维持在原代培养的MSC样表型。此外，这些细胞稳定地维持培养于他们的长期的表型，并表现出多谱系分化潜能，类似的MSCs 19,20。这些结果表明，周细胞是难以捉摸的MSC 14的起源地之一。周细胞的治疗潜力已经证明在心肌疤痕的减小和增强移植后的心脏功能到缺血性损伤心21。最近，我们成功地从人类心肌纯化周细胞和无骨骼肌肉发生3展示了他们MSC样表型和多能（脂肪形成，软骨和骨）。此外，心肌当周细胞与其他器官纯化的同行相比表现差心肌潜力和血管生成能力。 多能血管周围干/祖细胞，外膜细胞的第二群体，已经从人隐静脉分离阳性CD34的表达22的基础上的。静脉外膜细胞已被证明具有克隆的潜力，中胚层分化能力和体外促血管生成潜力。这些细胞注入小鼠的缺血性损伤的心移植导致间质纤维化的减少，增加血管生成和心肌血流量，减少心室稀通货膨胀，而增加心脏射血分数23。有趣的是，脂肪外膜细胞已被证明失去CD34的表达和上调响应于血管生成素II治疗中培养CD146表达，提示通过一个周细胞表型与刺激24。内的心脏，然而，外膜细胞群体尚未前瞻性通过FACS和/或良好表征纯化。利用在以下各节中描述的细胞分离方法，我们目前正在表征心肌外膜细胞和调查其对再生的应用潜力。 这里我们描述了分离和从人胎儿或成年心肌纯化血管周围干/祖细胞的两个亚群的方法。本前瞻性细胞分离方法将使研究人员能够从人的心脏活检的比较研究和furthe获得等基因血管周围干/祖细胞亚群- [R探讨其在各种心脏病理状态的治疗潜力。

<table><tbody><tr><td>AbC Anti-mouse Bead Kit</td><td>Molecular Probes</td><td>A-10344</td><td></td></tr><tr><td>Collagenase I</td><td>Gibco</td><td>17100-017</td><td>Reconstitute powder as required and filter sterilise</td></tr><tr><td>Collagenase II</td><td>Gibco</td><td>17101-015</td><td></td></tr><tr><td>Collagenase IV</td><td>Gibco</td><td>17104-019</td><td></td></tr><tr><td>anti-human CD34-PE</td><td>BD Pharmingen</td><td>555822</td><td>Keep sterile</td></tr><tr><td>anti-human CD45-APC-Cy7</td><td>BD Pharmingen</td><td>557833</td><td>Keep sterile</td></tr><tr><td>anti-human CD56-PE-Cy7</td><td>BD Pharmingen</td><td>557747</td><td>Keep sterile</td></tr><tr><td>anti-human CD144-PerCP-Cy5.5</td><td>BD Pharmingen</td><td>561566</td><td>Keep sterile</td></tr><tr><td>anti-human CD146-AF647</td><td>AbD Serotec</td><td>MCA2141A647</td><td>Keep sterile</td></tr><tr><td>EGM2-BulletKit</td><td>Lonza</td><td>CC-3162</td><td>For collection of cells and culture until adhered</td></tr><tr><td>DMEM, high glucose, GlutaMAX without sodium pyruvate</td><td>ThermoFischer Scientific</td><td>10566-016</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ThermoFischer Scientific</td><td>10500-064</td><td>Freeze in aliquots and keep sterile</td></tr><tr><td>Gelatin</td><td>Sigma Aldrich</td><td>G1393</td><td>Dilute with sterile water</td></tr><tr><td>IgG1k-PE</td><td>BD Pharmingen</td><td>559320</td><td>Keep sterile</td></tr><tr><td>IgG1k-APC-Cy7</td><td>BD Pharmingen</td><td>557873</td><td>Keep sterile</td></tr><tr><td>IgG1k-PE-Cy7</td><td>BD Pharmingen</td><td>557872</td><td>Keep sterile</td></tr><tr><td>IgG1k-PerCP-Cy5.5</td><td>BD Pharmingen</td><td>561566</td><td>Keep sterile</td></tr><tr><td>IgG1k-647</td><td>AbD Serotec</td><td>MCA1209A647</td><td>Keep sterile</td></tr><tr><td>Mouse serum</td><td>Sigma Aldrich</td><td>M5905</td><td>Keep sterile</td></tr><tr><td>Paraffin Film - Parafilm M</td><td>Sigma Aldrich</td><td>P7793</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15979-063</td><td>Freeze in aliquots and keep sterile</td></tr><tr><td>Phosphate buffered saline pH 7.4</td><td>ThermoFischer Scientific</td><td>10010-023</td><td>Keep sterile</td></tr><tr><td>Red Blood Cell Lysing Buffer Hybri-Max</td><td>Sigma Aldrich</td><td>R7757</td><td>Keep sterile</td></tr><tr><td>Trypan Blue Solution</td><td>Sigma Aldrich</td><td>T8154</td><td></td></tr><tr><td>Trypsin-EDTA 0.5%(10x)</td><td>Invitrogen</td><td>15400-054</td><td></td></tr><tr><td>&amp;nbsp;FACSARIA FUSION</td><td>BD Pharmingen</td><td></td><td>Fluorescence Activated Cell Sorter</td></tr></tbody></table>

isolation of perivascular multipotent precursor cell populations from human cardiac tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

单细胞从碎片和双峰正向和侧向散射分布的基础上区别。活细胞被他们未能采取了DAPI染料标识。选通策略被选择这个活，全心脏细胞的解离（ 图1）的同种型对照标记的基础上。从活细胞，CD45 +细胞被第一选通出来，接着CD56 +细胞。 CD144 +内皮细胞然后从CD56取出-级分。从这个最后的人口，CD146 + / CD34 -被选择/ C...

Watch this Scientific Journal Video about Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue at JoVE.com

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

人体心脏组织窝藏多能血管周围的前体细胞群可能适合于心肌再生。这里描述的技术允许与天然血管， 即 CD146 + CD34相关联的两个多能基质干细胞群的同时分离和纯化-周细胞和CD34 + CD146 -外膜细胞，从人的心肌。

从人类心脏组织血管周围多能前体细胞群的分离

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

isolation-perivascular-multipotent-precursor-cell-populations-from

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

7.3K 次观看.  University of Edinburgh. 人体心脏组织窝藏多能血管周围的前体细胞群可能适合于心肌再生。这里描述的技术允许与天然血管， 即 CD146 + CD34相关联的两个多能基质干细胞群的同时分离和纯化-周细胞和CD34 + CD146 -外膜细胞，从人的心肌。 

视频: Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

验尸报告组织心肌细胞核分离

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

科研

医学

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

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

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

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

在体外人骨髓间充质干细胞分化为心肌样细胞功能

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

发育生物学

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

人多潜能干细胞第一心场样心脏祖细胞和心室样细胞的生成

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

低血清条件下心脏祖细胞内皮分化的诱导作用

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

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 ...

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. ...

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 ...

生物工程

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

从小鼠心脏中分离主黑素细胞样细胞

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

生物学

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

鼠心围细胞的分离与纯化

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...