Name: derivation of cardiac progenitor cells from embryonic stem cells
Uploaded: 2015-01-12T13:00:00.000+00:00
Duration: 8 min
Description: Watch this Scientific Journal Video about Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells at JoVE.com

We thank Dr. Leonid Gnatovskiy for his carefully and critical reading of the paper. This work was supported in part by grants from the National Institutes of Health (HL109054) and the Samuel and Jean Frankel Cardiovascular Center, University of Michigan (Inaugural Fund) to WZ and from the Leon H Charney Division of Cardiology, New York University School of Medicine to BL.

从鼠标胚胎干细胞衍生1.小鼠胚胎的每次点击成本<ol><li>准备小鼠胚胎成纤维细胞（MEF中）饲养层。 <ol><li>暖MEF培养基（在DMEM 10％胎牛血清）至37℃。 </li><li>准备明胶涂层板。 <ol><li>加入1毫升水的0.1％明胶的成井的6孔板或5毫升到10cm的培养皿中。 </li><li>离开，在37℃的板或盘或室温下至少30分钟。渴望在使用前明胶。 </li></ol></li><li>解冻在37℃水浴中照射的MEF很快，轻轻摇动。 </li><li>轻轻转移细胞到15ml或50ml锥形管中。加入5 ml预热的MEF培养基。漩涡的细胞缓慢和离心，在200×g离心5分钟。 </li><li>重悬的细胞在MEF培养基和计数存活细胞。从10cm的培养皿2毫升每孔的细胞从6孔板和10毫升的细胞:使用MEF培养基的下列卷。 </li><li>板细胞向6孔板或10 cm的培养皿在1-2×10 6每盘/碟。 </li><li>孵育MEF平板/菜在37℃下，在使用前，5％CO 2的至少24小时。 注:放弃MEF板/超过一个星期菜。 </li></ol></li><li>准备鼠标胚胎干细胞<ol><li>文化Isl1-CRE; Rosa26YFP / MEF2C-CRE; Rosa26YFP鼠胚胎干细胞对MEF中，直到90％汇合。利用ES细胞培养基由410毫升DMEM，75毫升FBS，0.1mM的MEM NEAA，2mM的L-谷氨酰胺，0.1mM的丙酮酸，100U青霉素/链霉素和0.1mMβ巯基乙醇。 </li><li>如1.1.2中所述制备明胶包被的6孔板中。 </li><li>从板块吸中，冲洗细胞DPBS。 <ol><li>抽吸DPBS添加0.4毫升0.25％胰蛋白酶进6孔板一个孔。孵育细胞在37℃，5％的CO 2为5分钟。加入1毫升预热的ES培养基以停止胰蛋白酶反应。 </li></ol></li><li>收获细胞和细胞离心机在200 XG和抽吸介质。重悬的细胞在1ml ES细胞培养基，并计数细胞。&lt;/ li&gt; <li>板胚胎干细胞在明胶包被的板在3×10 4个/ cm 2的密度。孵育在37℃，5％CO 2的约2天，当胚胎干细胞达到约90％汇合。媒体每天都在发生变化。 </li><li>当胚胎干细胞是90％汇合，重复步骤1.2.2-1.2.5彻底删除MEF饲养。 </li></ol></li><li>中共诱导分化形成EB <ol><li>制备分化培养基如下:36毫升IMDM，12毫升火腿的F12，2mM的L-谷氨酰胺，0.34毫升BSA（7.5％），加入0.25ml N 2，0.5毫升B27，50微克/毫升的AA，和0.45毫摩尔1-硫代甘油。 </li><li>从细胞中吸中，用清水冲洗DPBS。抽吸DPBS添加0.4毫升0.25％胰蛋白酶进6孔板一个孔。孵育在37℃下5分钟。 </li><li>加入1毫升分化培养基停止反应和移液器上下分散细胞簇为单细胞。 </li><li>在200×g离心5分钟，并弃去培养基离心的胚胎干细胞。 <ol><li>重悬1×10 6 &lt;/ SUP&gt; 1毫升分化培养基的胚胎干细胞。计数细胞，并培养所述细胞在低脱离培养皿以1×10 5细胞/在分化培养基ml的浓度，在37℃下进行第一次电子束的聚集。 </li></ol></li><li> EB形成后两天，从菜传输EBS公司50ml试管。旋转，在200×g离心1分钟。除去培养基和冲洗胚用10毫升DPBS不含Ca 2+和Mg 2+。 </li><li>吸DPBS并加入1 ml 0.25％胰蛋白酶。孵育在37℃下5分钟。添加4.5毫升分化培养基以停止反应。吸管上下解离胚成单个细胞。 </li><li>离心细胞，在200×g离心5分钟。吸中型和悬浮细胞1毫升分化培养基。 </li><li>计数细胞，并稀释为2×10 6个细胞至1×10 5个细胞/在分化培养基毫升。添加VEGF，BMP4和激活素A的培养基中在5毫微克/毫升，0.8纳克/毫升最终浓度，并5毫微克/毫升。丘尔再将细胞在培养皿中用于第二EB形成在37℃。 </li><li>第二个EB形成后的40小时，转移到胚50ml试管。用DPBS冲洗一次，游离的EB与1ml 0.25％胰蛋白酶在37℃下5分钟。吸管上下解离胚成单个细胞。 </li><li>将细胞重悬于Stempro-34培养基与5毫微克/毫升VEGF，10ng / ml的bFGF和12.5毫微克/毫升的FGF10。板中的细胞以2×10 5 / cm 2的在明胶包被的平板上。文化在37℃的孵化器前，中共中央隔离约32小时。 </li></ol></li><li>隔离mCPCs <ol><li>吸中，用清水冲洗DPBS。添加0.5毫升0.25％胰蛋白酶培养板在37℃下5分钟。添加4.5毫升分化培养基以停止反应。吸管上下解离胚成单个细胞。收集的细胞通过离心和重悬细胞的2％FBS / PBS用于FACS纯化。 </li><li>运行在分拣机作为阴性对照未分化的胚胎干细胞。改变电压来调节前进SCAT之三（FSC）和侧向散射（SSC），选择所需的人口。使用前向散射与侧向散射和前向散射的高度与宽度，以排除碎片和双峰。在所选择的子群，根据ESC控制的分布，绘制YFP的栅极上直方图正，以消除细胞的自体荧光。收集YFP +门YFP +的每次点击费用。 </li><li>板中的纯化的每次点击费用（YFP +细胞）在6孔板用分化培养基（1.3.1）。文化额外的3-5天在37℃分化的每次点击费用为心肌和平滑肌细胞。 </li></ol></li></ol>从人类胚胎干细胞衍生2.人类胚胎的每次点击成本<ol><li>准备给料机<ol><li>制备小鼠胚胎成纤维细胞（MEFs）饲养层作为以2×10 4 / cm 2的密度如上所述。 </li></ol></li><li>维持人类胚胎干细胞<ol><li>维持人类ISL1:CRE;对并购pCAG-FLOX-STOP-FLOX-GFP或RFP常规胚胎干细胞EF使用常规的hESC培养基（敲除DMEM / F-12385毫升，敲除的SR 100毫升，2毫摩尔L-谷氨酰胺，0.1mM的NEAA，0.1毫β巯基乙醇，10纳克/毫升碱性FGF）。之前心肌分化，在无饲养条件为至少2次传代转移人类胚胎干细胞。 </li></ol></li><li>准备在无饲养条件下人类胚胎干细胞<ol><li>准备涂层板为人类胚胎干细胞的文化。 <ol><li>解冻基质胶在4℃下过夜。放50ml管在冰上，加入30毫升冷的DMEM / F12培养基到管。 </li><li>加入1毫升基质胶放入冷水中，拌匀。添加1ml冷基质胶的DMEM / 12培养基成良好的6孔板或5毫升到10cm的培养皿中。 </li><li>离开板/菜在4℃过夜或室温下搅拌2小时。抽吸培养基在使用前。 </li></ol></li><li>分裂人类胚胎干细胞到板:吸媒体从MEF板和冲洗人类胚胎干细胞用DPBS。然后加入1 ml分散酶（1毫克/毫升）于6孔板的孔中。孵育细胞在37℃孵化器，直至菌落边缘开始脱落。 </li><li>除去中性蛋白酶的解决方案。用DPBS冲洗两次，然后加入2.5毫升/每孔mTeSR培养基或MEF条件培养基的板上。 </li><li>刮去使用细胞刮刀细胞，并仔细吸管上下打破人类ESC集落成小团块。 </li><li>传送ESC团块并稀释1:3到包被的板。 </li><li>加入2毫升/ mTeSR中等或MEF条件培养基，改变媒体每天。 </li><li>培养人类胚胎干细胞在mTeSR培养基中或在包被的板MEF条件培养基中，至少2次传代。 </li></ol></li><li>诱导人类胚胎干细胞分化CPC <ol><li>当细胞是〜90％汇合时，吸出介质并冲洗用DPBS。然后孵育在0.5毫克/毫升分散酶的人类胚胎干细胞在37℃下20分钟。 </li><li>冲洗DPBS的人类胚胎干细胞的两倍，从板与细胞升降去除细胞，然后悬浮在分化培养基细胞夹（含18％FBS，0.1毫米NEAA，2毫米DMEM / F12L-谷氨酰胺，0.1mM的β巯基乙醇和50微克/毫升抗坏血酸）。 </li><li>转移细胞进入6孔超低附着板的EB形成。 </li><li>第二天，改变分化培养基。 </li><li>更换培养基，每3天，并在悬浮培养中维持的EB。 </li></ol></li><li>隔离HCPCS <ol><li>最迟分化为人类CPC隔离9天收集胚。 </li><li>吸媒体和冲洗胚与DPBS两次。添加0.5毫升0.25％胰蛋白酶至6孔培养板每孔中，在37℃进行5分钟。 </li><li>加入分化培养基的体积相等，使反应停止。吸管上下解离胚成单个细胞。收集细胞通过离心机在200×g离心5分钟，并悬浮细胞在2％FBS / DPBS。过滤单元与40微米的细胞过滤和FACS纯化的RFP + CPC细胞中1.4.3所述。 </li><li>板排序HCPCS到geltin包被的平板。在7分化培养基文化HCPCS-9天分化成心肌细胞和平滑肌细胞。电镀后第7天，培养分化的细胞在DMEM / F12培养基的5％敲除的SR。 </li></ol></li></ol>

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Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+medium+supporting+cardiomyocyte+differentiation+of+human+embryonic+stem+cells.">Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells.</a> Differentiation: Research in Biological Diversity. 76 (9), 958-970 (2008).</li><li>Lian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+via+temporal+modulation+of+canonical+Wnt+signaling.">Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (27), E1848-1857 (2012).</li><li>Bizy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+light+chain+2-based+selection+of+human+iPSC-derived+early+ventricular+cardiac+myocytes.">Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes.</a> Stem Cell Research. 11 (3), 1335-1347 (2013).</li></ol>

The authors declare no competing financial interests.

This protocol combines a method using growth factors to guide mESCs differentiation and spontaneous differentiation of human ESCs into CPCs. CPC lineage marked with fluorescent reporter is used to efficiently identify and isolate CPCs by FACS. The FACS-purified CPCs retain the capacity to differentiate into cardiomyocytes, smooth muscle, and endothelial cells and have a comparable expression profile to the in vivo cells. Thus, these CPCs can serve as a great resource for cell based heart therapy because of their ability ...

A correction to the author list was made to: <a href="http://jove.unicartagenaproxy.elogim.com/video/52047/derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells">Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells</a>.
The following author is now listed as a co-corresponding author:
Lei Bu

A correction to the author list was made to: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells.

Erratum: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

心脏疾病仍然是世界上领先的原因今天和死亡率在过去二十年（美国心脏协会）大致维持不变。有用于开发新的治疗策略，以有效地防止或逆转心脏衰竭的重要需求。一个有希望的战略是基于细胞治疗造血干细胞生物学1的快速发展。在这点上，多能每次点击费用可能是一个很好的细胞来源的治疗，由于其增殖，但致力于仅心脏谱系分化的能力。因此，有效和可靠的方法来产生和分离的每次点击费用是对心脏细胞疗法的研究非常重要的。 该协议的重点是在早期胚胎发育，以及如何他们这一代人从胚胎干细胞识别胚胎的每次点击费用。不同的每次点击费用已分离出胚胎和成人的心，甚至从骨髓2。在胚胎发育，骨骼morphogeNETIC蛋白（BMP），无翅型MMTV整合位点家族成员（Wnts）和节点信号诱导Mesp1 +多能胚层3的承诺。 Mesp1 +细胞然后分化成胚胎每次点击费用4。这些每次点击费用通常标记HCN4，NK 2同源框5（NKX2-5），ISL LIM同源框1（Isl1），T型箱5（Tbx5中），和肌细胞增​​强因子2C（MEF2C），形成初级和第二心脏领域，并心脏发生5-10时导致心脏的主要部分。既NKX2-5 +和Isl1 + / + MEF2C每次点击费用是能够分化成心肌细胞，平滑肌细胞（SMC）和内皮细胞5-8。因此，这些每次点击费用会引起心脏血管系统以及心脏组织并且是一种理想的细胞源细胞系心脏疗法。因此， 在体外产生的每次点击成本一直是一大研究热点心血管研究。由于胚胎干细胞具有无限扩展能力次代表ICM细胞的囊胚阶段，下面自然胚胎发育胚胎干细胞分化成胚胎的每次点击成本被认为是获得每次点击费用的合理和有效的方法。 一个被广泛应用的方法，从胚胎干细胞获得的每次点击成本是胚胎干细胞聚集成胚11。以提高分化效率的基础上，心脏发育的知识定义的化学和生长因子已被用于12-14。然而，没有明确的中共标记，尤其无细胞表面标志物，其已被广泛接受的领域。为了解决这个问题，胚胎干细胞被设计为纪念Isl1 +或MEF2C +每次点击费用和他们使用的Cre / loxP系统荧光记者衍生物。 Cre重组酶是Isl1 / MEF2C子/增强子的控制下，在敲。改性荧光蛋白RFP或YFP基因由组成型启动子驱动的可通过FLOX终止密码子与Cre重组酶切除被激活（ISL1:CRE; pCAG-FLOX-STOP-FLOX-GFP或RFP / Isl1-CRE; Rosa26YFP / MEF2C-CRE; Rosa26YFP）5,6。一旦胚胎干细胞分化为第二心脏领域的每次点击费用Isl1 / MEF2C子/增强推动华创会激活荧光记者和每次点击费用，可以通过FACS净化富集。简言之，EB凝集法用于启动ESC分化。以提高分化的效率，所述分化细胞用抗坏血酸（AA）和生长因子如BMP4，活化素A和VEGF 13,15。该协议允许用小鼠和人胚胎干细胞强大的和高效率的CPC分化。

<table><tbody><tr><td>FBS</td><td>Thermo scentific</td><td>SH30070.03E</td><td></td></tr><tr><td>Knockout SR</td><td>Life technology</td><td>10828028</td><td></td></tr><tr><td>Knockout DMEM</td><td>Life technology</td><td>10829018</td><td></td></tr><tr><td>DMEM</td><td>Life technology</td><td>11965118</td><td></td></tr><tr><td>NEAA</td><td>Life technology</td><td>11140050</td><td></td></tr><tr><td>GlutaMAX</td><td>Life technology</td><td>35050061</td><td></td></tr><tr><td>N2</td><td>Life technology</td><td>17502048</td><td></td></tr><tr><td>B27</td><td>Life technology</td><td>12587010</td><td></td></tr><tr><td>Ham&amp;rsquo;s F12</td><td>Life technology</td><td>11765062</td><td></td></tr><tr><td>IMDM</td><td>Life technology</td><td>12440061</td><td></td></tr><tr><td>Pen/Strep</td><td>Life technology</td><td>15140122</td><td></td></tr><tr><td>Pyruvate</td><td>Life technology</td><td>11360070</td><td></td></tr><tr><td>Dispase</td><td>Life technology</td><td>17105041</td><td></td></tr><tr><td>Stempro-34</td><td>Life technology</td><td>10639011</td><td></td></tr><tr><td>DMEM/F12</td><td>Life technology</td><td>11330032</td><td></td></tr><tr><td>BSA&amp;nbsp;</td><td>Life technology</td><td>15260037</td><td></td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Life technology</td><td>25200056</td><td></td></tr><tr><td>Ascobic Acid&amp;nbsp;</td><td>Sigma</td><td>A5960</td><td></td></tr><tr><td>1-Thioglycerol</td><td>Sigma</td><td>M1753</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>VEGF</td><td>R&amp;amp;D</td><td>293-VE</td><td></td></tr><tr><td>Bmp4</td><td>R&amp;amp;D</td><td>314-BP</td><td></td></tr><tr><td>ActivinA</td><td>R&amp;amp;D</td><td>338-AC</td><td></td></tr><tr><td>bFgf</td><td>R&amp;amp;D</td><td>233-FB</td><td></td></tr><tr><td>Fgf10</td><td>R&amp;amp;D</td><td>345-FG</td><td></td></tr><tr><td>mTeSR</td><td>Stemcell technologies</td><td>5850</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>354277</td><td></td></tr></tbody></table>

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.

该协议显示了几个胚胎干细胞系的每次点击费用的推导。胚胎干细胞聚集形成EB分化为每次点击费用。胚胎干细胞是经常保持在MEF饲养（ 图1A，F）和馈线分化前被移除。经聚集的胚胎干细胞的成胚在分化培养基（ 图1B，G）中，所述第二形成的EB与BMP4和活化素A处理，以增强在小鼠细胞系（ 图1C）中胚层的分化。胚胎干细胞分化成心肌谱系所确定的荧光蛋白质YF...

Watch this Scientific Journal Video about Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells at JoVE.com

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

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 ...

derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.1K 次观看.  University of Michigan. 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.

视频: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

科研

生物学

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

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

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

从多能干细胞使用BMP拮抗剂Grem2心房心肌细胞分化

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

发育生物学

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

心脏细胞治疗中干细胞 dna 完整性的评估

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

遗传学

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 ...

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 ...

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

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

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

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

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

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

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

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

人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议

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

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

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

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

人类多能干细胞的高效分化为心肌细胞，并鉴定流式细胞仪

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