Name: pluripotent stem cell derived cardiac cells for myocardial repair
Uploaded: 2017-02-03T15:00:00.000+00:00
Duration: 6 min 37 s
Description: Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

This work was supported by US Public Health Service grants NIH RO1s HL67828, HL95077, HL114120, and UO1 HL100407-project 4 (to JZ), an American Heart Association Scientist Development Grant (16SDG30410018) and a Research Voucher Award from University of Alabama at Birmingham Center for Clinical and Translational Science (to WZ).

所有的实验过程符合阿拉巴马大学伯明翰大学的动物指导进行。 1.区分iPS细胞成hiPSC-CMS <ol><li>外套预冷生长因子减少的凝胶状蛋白混合物6孔板于4℃过夜的孔中。吸使用前胶状蛋白质混合物。在5％CO 2和37℃下在mTeSR1介质补充有10μM的ROCK抑制剂播种人iPS细胞到预涂覆板，和培养的细胞（每孔1×10 5个细胞）。 </li><li>每日刷新介质直到细胞达到90％汇合;然后，生长因子减少的胶状蛋白质混合物添加到培养基（0.5毫克胶状蛋白质每6孔板混合物，每孔2毫升培养基），并培养所述细胞为5％的CO 2和37℃两天。 <ol><li>要更换介质，轻轻地从经真空培养皿与吸出培养基出触摸的细胞，并使用移液管添加新介质。 </li></ol></li><li>通过更换用补充有RPMI1640培养基培养基引发分化生长因子减少的凝胶状蛋白混合物，B27没有胰岛素，和100ng / ml的激活素A;培养的细胞在5％CO 2和37℃下24小时。 </li><li>替换已补充有B27没有胰岛素的RPMI1640培养基，10纳克/毫升的骨形态发生蛋白4（BMP-4），和10ng / ml的碱性成纤维细胞生长因子（bFGF）的介质;培养的细胞在5％CO 2和37℃下96小时。 </li><li>替换用RPMI1640培养基补充有B27培养基并继续培养细胞在5％CO 2和37℃;刷新介质每3天。 </li><li>观察开始分化后，在显微镜下收缩细胞〜3天的簇。收集集群后〜7天观察第一收缩和Hanks平衡盐溶液洗。 <ol><li>分离聚集细胞在板在含100U / ml胶原酶IV 10分钟，在37ºC轻轻摇动Hanks缓冲液培养他们。然后，5分钟加入乙二胺四乙酸（EDTA）0.25％胰蛋白酶。 </li><li>中和用在RPMI / B27培养基10％胎牛血清的酶溶液中，然后悬浮细胞在RPMI / B27培养基。 </li><li>由血球计数细胞的数目。培养在5％CO 2和37℃至少3小时10 cm的培养皿的细胞中，这将允许非心肌细胞粘附于培养皿的表面上。 </li></ol></li><li>收集细胞悬浮液，其中包含hiPSC-CMS，并通过在凝胶状蛋白质混合物涂覆的表面培养它们维持细胞（约1×10 6每10cm培养皿的细胞）。 </li></ol> 2.区分iPS细胞成hiPSC-ECS <ol><li>通过培养第解离hiPSC人口成单个细胞时间与在5％CO 2和37℃5分钟螯合剂。转移细胞到含新鲜mTeSR1培养基15ml离心管中。 </li><li>降速细胞在200×g离心5分钟。重悬在补充有10微米ROCK抑制剂在mTeSR1培养基中的细胞。确定与血球细胞密度。 </li><li>加入250微升20 NIH单位/ ml凝血酶溶液至24孔板的一个孔中。 </li><li>加1×10 6人iPS细胞以250微升12.5毫克/毫升纤维蛋白原溶液。与250微升的含细胞的纤维蛋白原溶液添加到凝血酶加载好。观察混合物固化以形成分钟内含hiPSC纤维蛋白支架。 </li><li>传送固化，含细胞的支架与盖玻璃镊子一个6孔板的孔中，和通过培养补充有没有胰岛素的2％B27在2ml EBM2培养基支架发起分化，激活素A（50毫微克/毫升）和BMP-4在5％的CO（25毫微克/毫升）24小时<sUB&gt; 2℃和37℃。 <ol><li>轻轻吸出经真空旧媒体不接触电池更换培养基。添加补充有B27没有胰岛素新EBM2培养基，血管内皮生长因子（VEGF，50纳克/毫升），促红细胞生成素（EPO，100纳克/毫升），和转化生长因子β1（TGFβ1，10纳克/毫升）中，并培养的 CO 2和37℃下48小时的支架为5％。 </li><li>刷新培养基和培养在5％CO 2和37℃另外48小时的支架;然后，加入200单位胶原酶IV（100单位/毫升）对介质释放从支架的细胞。 </li><li>降速细胞之后发布。用补充有B27，VEGF-A（50毫微克/毫升）和SB-431542（10μM）的2％2毫升EGM2-MV培养基更换培养基;刷新介质每两天。 </li><li>大约10天后（ 即在14〜分化日开始后），游离细胞用100 U / ml胶原酶IV，隔离ħiPSC的内皮细胞从通过流式细胞分化的细胞的种群分析了EC特异性标志蛋白的表达（ 例如 ，CD31，CD144）3。 </li><li>通过已建立的协议3展开隔离hiPSC-ECS。 </li></ol></li></ol> 3.区分iPS细胞成hiPSC，平滑肌细胞<ol><li>种子的人iPS细胞上已经涂覆有凝胶状蛋白质混合物，其板，以及培养中mTeSR1培养基中的细胞在5％CO 2和37℃，用每日介质的变化，直到汇合（〜2天）。 </li><li>通过在RPMI1640培养基和2％B27加上胰岛素与CHIR99021（5μM）和BMP-4（10毫微克/毫升）培养细胞3天开始分化成中胚层谱系的细胞。 </li><li> 为了生产hiPSC，校董会由于主要是合成型: <ol><li>培养细胞VEGF-A（25纳克/毫升），成纤维细胞生长因子β（FGFβ，5毫微克/毫升）的RPMI1640我<html> dium和2％B27减去胰岛素在5％CO 2和37℃下4天。 </li><li>培养细胞中的VEGF-A（25毫微克/毫升），FGFβ（5毫微克/毫升）的RPMI1640培养基，2％B27加上胰岛素在5％CO 2和37 °下进行2天。 </li><li>培养的细胞在血小板衍生的生长因子β（PDGFβ，10纳克/毫升），TGFβ（3纳克/ ml）的RPMI1640培养基，2％B27加上胰岛素在5％CO 2和37 °下进行4天。 </li></ol></li><li> 为了生产hiPSC-校董会与主要收缩型: <ol><li>培养的细胞在RPMI1640 4天的VEGF-A（25毫微克/毫升）和FGFβ（5毫微克/毫升）和2％B27减去胰岛素。 </li><li>培养的细胞在RPMI1640和2％B27加上胰岛素7天PDGFβ（5毫微克/毫升）和TGFβ（2.5纳克/毫升）。 </li></ol></li><li>通过在含有〜6天RPMI1640代谢平台乳酸盐（4毫摩尔）培养细胞纯化的最终hiPSC-SMC的人群。 </li></ol>jove_title"&gt; 4。创建的含IGF的微球<ol><li>热橄榄油在水浴45℃。 </li><li>保温5毫升10％的明胶溶液至50℃。 </li><li>明胶加入橄榄油，搅拌，然后通过加入冰水浴快速冷却到5℃。 </li><li>二十五分钟后，冷却（4℃）丙酮添加到橄榄油以诱导形成微球。 </li><li>保持5温度 ℃下1小时;然后，收集微球，用预冷却的丙酮洗5次以除去橄榄油，并且允许它们风干，在4℃。 </li><li>悬浮的微球在70％乙醇和0.25％戊二醛溶液在4℃过夜以诱导交联，然后中和用100mM甘氨酸的混合物。 </li><li>负载的IGF成微球混合5毫克微球15微升蒸馏水含有5μg的IGF和0.1％牛血清白蛋白30分钟2 O。 </li></ol> 5.创建了损伤部位的修补程序和注射的细胞<ol><li>在1毫升的最低基本培养基（MEM）一起暂停hiPSC衍生的CMS（2元），平滑肌细胞（100万），和ECS（100万）。 </li><li>暂停在1ml的纤维蛋白原溶液（25毫克/毫升）5毫克微球。 </li><li>填充一个针筒与含细胞溶液，用纤维蛋白原/微球体溶液另一注射器，并用1ml的凝血酶溶液的第三注射器（80 NIH单位/ ml，补充有2微升400mM的氯化钙和200mM的ε氨基己酸）。 </li><li>通过左前为2如前所述冠状动脉前降支结扎手术诱发心肌梗死心脏的猪。 注:简单地说，女性约克夏猪（〜13千克，45日龄）的镇静与Telazol /甲苯噻嗪（4.4毫克/千克）的肌内注射，并用异氟烷（0.5-5％）全身麻醉下插管。 A 4 日 interc执行ostal开胸手术，左冠状动脉前降支被暴露。冠状动脉被结扎60分钟闭塞，然后结扎被除去。立即电除颤在室颤的情况下进行的。 </li><li>猪心脏的梗死区的心外膜无菌塑料圈（约2.5厘米）的位置，然后同时注入微球/纤维蛋白原和凝血酶的解决方案成环。 </li><li>允许该混合物凝固（〜30秒），然后通过固化的混合物插入含细胞注射器的针头插入梗死心肌和注入细胞。关闭肌肉层，皮下组织，与胸壁与取决于动物大小3-0或2-0 Monocryl缝线。丁丙诺啡0.01-0.02毫克/公斤肌注，每8小时将用于控制疼痛为手术后的第3天。 </li><li>使用心脏磁共振评估心脏功能之前的外科手术，一周和手术过程2，3，4四周后成像（MRI）。最终MRI研究后，牺牲动物和处理他们的组织学和分子分析2，3，4心。 </li></ol>

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hiPSC-CMS的改善的产量/纯度为了区分人类干细胞到cms传统的协议通常是由低产量和纯度的限制;例如，通过珀分离和心体形成得到的hESC的CM的只是35-66％表达慢肌球蛋白重链或肌钙蛋白6。分化hiPSC-CM群体的纯度可大幅增加通过选择已连接于启动子的报道基因的表达的CM-特异性蛋白7，8，9，但这...

人类诱导多能干细胞（人iPS细胞）是用于再生细胞治疗的最有前途的试剂中，因为它们可以分化成不由病人的免疫系统拒绝的细胞的潜在无限范围和数量。然而，它们的自我复制和分化能力也能导致肿瘤的形成，因此，人iPS细胞需要充分分化成特定的细胞类型，如心肌细胞（CMS），内皮细胞（EC），和平滑肌细胞（SMC ），给药前。之一的细胞给药的最简单和最常用的方法是直接的心肌内注射，但是这是由天然心肌组织嫁接移植细胞的数量是非常低的。许多这种消耗可以归因于缺血组织的细胞毒性的环境;然而，当鼠胚胎干细胞（ESC）是直接注入未受损伤心中的心肌Ó交付被保留为3-5小时1，这表明施用的细胞的相当比例退出给药部位，这可能是因为它们是由过程中产生的高压通过针轨道挤出5百万细胞的唯一一句〜40％心肌收缩。 这里，我们提出新颖的和基本上更有效的方法，用于产生hiPSC衍生的心肌（hiPSC-CMS）2，内皮细胞（hiPSC-ECS）3，和平滑肌细胞（SMC）4。值得注意的是，此hiPSC-SMC协议是第一模仿广泛的通过引导细胞向一个主要合成的或收缩的SMC表型在体平滑肌5观察到的形态和功能特征。我们还提供细胞递送的方法，其通过创建一个含有细胞因子的血纤维蛋白p提高注射细胞的植入率ATCH在注射部位。补丁似乎改善两种细胞保留，通过密封针轨道，以防止细胞从离开心肌，和细胞存活，通过在一段至少三天的释放胰岛素样生长因子（IGF）。

<table><tbody><tr><td>&lt;strong&gt;Protocol Section 1&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>mTeSR1 medium</td><td>Stem cell technologies</td><td>5850</td><td></td></tr><tr><td>Growth-factor-reduced matrigel</td><td>Corning lifescience</td><td>356231</td><td></td></tr><tr><td>Y-27632</td><td>Stem cell technologies</td><td>72304</td><td></td></tr><tr><td>B27 supplement, serum free</td><td>Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>RPMI1640</td><td>Fisher Scientific</td><td>11875-119</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D</td><td>338-AC-010</td><td></td></tr><tr><td>BMP-4</td><td>R&amp;amp;D</td><td>314-BP-010</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D</td><td>232-FA-025</td><td></td></tr><tr><td>Collagenase IV</td><td>Fisher Scientific</td><td>NC0217889</td><td></td></tr><tr><td>Hanks Balanced Salt Solution (Dextrose, KCl, KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, NaHCO&lt;sub&gt;3&lt;/sub&gt;, NaCl, Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; anhydrous)</td><td>Fisher Scientific</td><td>14175079</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Fisher Scientific</td><td>10438018</td><td></td></tr><tr><td>6-well plate</td><td>Corning Lifescience</td><td>356721</td><td></td></tr><tr><td>10 cm dish</td><td>Corning Lifescience</td><td>354732</td><td></td></tr><tr><td>Cell incubator</td><td>Panasonic</td><td>MCO-18AC</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 2&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Versene</td><td>Fisher Scientific</td><td>15040066</td><td></td></tr><tr><td>Fibrinogen</td><td>Sigma-Aldrich</td><td>F8630-5g</td><td></td></tr><tr><td>Thrombin</td><td>Sigma-Aldrich</td><td>T7009-1KU</td><td></td></tr><tr><td>EMB2 medium</td><td>Lonza</td><td>CC-3156</td><td></td></tr><tr><td>VEGF</td><td>ProSpec-Tany</td><td>CYT-241</td><td></td></tr><tr><td>EPO</td><td>Life Technologies</td><td>PHC9431</td><td></td></tr><tr><td>TGF-&amp;#946;</td><td>Peprotech</td><td>100-21C</td><td></td></tr><tr><td>EGM2-MV medium</td><td>Lonza</td><td>CC-4147</td><td></td></tr><tr><td>SB-431542</td><td>Selleckchem</td><td>S1067</td><td></td></tr><tr><td>CD31</td><td>BD Bioscience</td><td>BDB555445</td><td></td></tr><tr><td>CD144</td><td>BD Bioscience</td><td>560411</td><td></td></tr><tr><td>15 ml centrifuge tube</td><td>Fisher Scientific</td><td>12565269</td><td></td></tr><tr><td>Eppendorff Centrifuge</td><td>Eppendorf</td><td>5702R</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 3&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CHIR99021</td><td>Stem cell technologies</td><td>720542</td><td></td></tr><tr><td>PDGF-&amp;#946;</td><td>Prospec</td><td>CYT-501-10ug</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 4&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Olive oil</td><td>Sigma-Aldrich</td><td>O1514</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G9391</td><td></td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>179124</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818100</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>G5882</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G8898</td><td></td></tr><tr><td>IGF</td><td>R&amp;amp;D</td><td>291-G1-01M</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Fisher Scientific</td><td>15561020</td><td></td></tr><tr><td>Heating plate</td><td>Fisher Scientific</td><td>SP88850200</td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific</td><td>15-462-10Q</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 5&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>&amp;#949;-aminocaproic acid</td><td>Sigma-Aldrich</td><td>A0420000</td><td></td></tr><tr><td>MEM medium</td><td>Fisher Scientific</td><td>12561-056</td><td></td></tr><tr><td>Syringe</td><td>Fisher Scientific</td><td>1482748</td><td></td></tr><tr><td>Anesthesia ventilator</td><td>Datex-Ohmeda</td><td>47810</td><td></td></tr><tr><td>Anesthesia ventilator</td><td>Ohio Medical</td><td>V5A</td><td></td></tr><tr><td>Defibrillator</td><td>Physiol Control</td><td>LIFEPAK 15</td><td></td></tr><tr><td>1.5 T MRI</td><td>General Electric</td><td>Signa Horizon LX</td><td></td></tr><tr><td>7 T MRI</td><td>Siemens</td><td>10018532</td><td></td></tr><tr><td>Gadolinium Contrast Medium (Magnevist)</td><td>Berlex</td><td>50419-188-02</td><td></td></tr><tr><td>2-0 silk suture</td><td>Ethilon</td><td>685H</td><td></td></tr><tr><td>3-0 silk suture</td><td>Ethilon</td><td>622H</td><td></td></tr><tr><td>3-0 monofilament suture</td><td>Ethilon</td><td>627H</td><td></td></tr></tbody></table>

pluripotent stem cell derived cardiac cells for myocardial repair

人类诱导多能干细胞（人iPS细胞）必须充分分化为给药前的特定细胞类型，但常规协议分化人iPS细胞为心肌细胞（hiPSC-CMS），内皮细胞（hiPSC-ECS）和平滑肌细胞（SMC）常常由低收率，纯度和/或表型稳定性差的限制。这里，我们提出用于产生hiPSC-CMS，-ECs是基本上比传统方法更有效的新颖的协议，和-SMCs，以及用于细胞注射超过施用部位产生一个含有细胞因子的贴剂组合的方法。贴剂提高了注射细胞的同时保留，通过密封针轨道，以防止细胞从被挤出心肌，和细胞存活，通过在较长时间内释放胰岛素样生长因子（IGF）。在心肌缺血再灌注损伤的猪模型，植入率超过两倍大于当细胞与含细胞因子的贴剂比较的细胞而不补丁，和治疗与细胞和贴片都施用，但不能与单独的细胞，用心脏功能和梗塞大小显著改善有关。

差异化hiPSC-CMS，-ECs和-SMCs表征人iPS细胞的鉴别能力进行了评价2，3，4。流式细胞仪分析肌钙蛋白T（cTnT）的表达表明，最终hiPSC-CM人口的纯度超过90％（ 图1A，1B，面板B1）。几乎所有的细胞中的表达的慢肌球蛋白重链（ 图1B，</st...

Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

我们提出三种新型和更有效的协议，用于人类诱导多能干细胞分化为心肌细胞，内皮细胞和平滑肌细胞，并且通过细胞注射补丁介导的细胞因子的递送组合提高移植细胞的植入的递送方法。

多能干细胞衍生的心肌修复心肌细胞

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

pluripotent-stem-cell-derived-cardiac-cells-for-myocardial-repair

Research

JoVE Journal

Developmental Biology

8.2K 次观看.  University of Alabama at Birmingham. 我们提出三种新型和更有效的协议，用于人类诱导多能干细胞分化为心肌细胞，内皮细胞和平滑肌细胞，并且通过细胞注射补丁介导的细胞因子的递送组合提高移植细胞的植入的递送方法。 

视频: Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

从胚胎干细胞衍生心脏祖细胞

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

科研

发育生物学

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

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

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

通过微接触印刷的定向功能性心肌组织的产生

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

生物工程

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 ...

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

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

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

促纤维化信号增强因子介导的小鼠胚胎成纤维细胞再编程诱导心肌细胞的抑制

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

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

心肌内细胞交货：观察在小鼠心脏

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

医学

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 ...

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. ...

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

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 ...