Name: ultrasound-guided induced pluripotent stem cell-derived cardiomyocyte implantation in myocardial infarcted mice
Uploaded: 2022-03-30T15:00:00
Description: Watch this Scientific Journal Video about Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice at JoVE.com

这项工作得到了国家自然科学基金重大研究计划（编号：91539111-JY）、湖南省科技重点项目（编号：2020SK53420至JY）和湖南省科技创新计划（2021RC2106-CF）的支持。

本研究所有动物实验均经中南大学湘雅二医院伦理委员会审核通过。有关本协议中使用的所有材料和设备的详细信息，请参阅 材料表 。细胞注射，成像和安乐死的时间表如下：t0-诱导梗死，t1周-成像和植入细胞，t2周-成像和植入细胞，t4周-最终成像，安乐死和组织收集。
1. hiPSC培养、心肌细胞分化和细胞纯化
<ol><li>将DMEM / F12和基底膜基质储备溶液...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remuscularization+of+the+failing+heart.">Remuscularization of the failing heart.</a> The Journal of Physiology. 595 (12), 3685-3690 (2017).</li><li>Hu, 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=Transplantation+of+hypoxia-preconditioned+mesenchymal+stem+cells+improves+infarcted+heart+function+via+enhanced+survival+of+implanted+cells+and+angiogenesis.">Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis.</a> The Journal of Thoracic and Cardiovascular Surgery. 135 (4), 799-808 (2008).</li><li>Feng, Y., et al. <a target="_blank" 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本研究的关键步骤包括hiPSC培养，心肌细胞分化，hiPSC-CM纯化和hiPSC-CM移植到小鼠心肌梗死部位。关键是使用心脏超声经皮引导治疗到梗死边缘的梗死部位，在那里将hiPSC-CMs注射到该区域。
随着培养时间的延长，hiPSC-CM表型在形态（较大的细胞大小）、结构（肌肉、原纤维密度、排列、微观细胞簇）和生理（钙处理和β-肾上腺素能反应）上随时间变化。本研究在hiPSC分化为心肌?...

当急性心肌梗死发生时，梗死区域的心肌细胞因缺血和缺氧而迅速死亡。细胞死亡和破裂后释放几种炎症因子，而炎性细胞浸润梗死部位引起炎症1。值得注意的是，没有收缩性和导电性的成纤维细胞和胶原蛋白取代梗死部位的心肌细胞形成疤痕组织。由于成年哺乳动物心肌细胞的再生能力有限，大面积梗死后形成的活组织通常不足以维持足够的心输出量2。心肌梗死引起心力衰竭，在心力衰竭严重的情况下，患者只能依靠心脏移植或心室辅助装置来维持正常的心脏功能3，4。
心肌梗死后，理想的治疗策略是用新形成的心肌细胞替换死亡的心肌细胞，与健康组织形成机电耦合。然而，治疗方案通常采用心肌挽救而不是替代。目前，基于干细胞和祖细胞的疗法是MI5后促进心肌修复的最有希望的策略之一。然而，这些细胞的移植有几个问题，主要是成体干细胞无法分化成心肌细胞并且它们的寿命短6。
与使用胚胎干（ES）细胞相关的伦理问题可以通过iPSCs来规避，iPSCs是一种有希望的细胞来源。此外，iPSCs具有很强的自我更新能力，可以分化为心肌细胞7。研究表明，移植到MI位点的hiPSC-CMs可以存活并与宿主细胞形成间隙连接8，9。然而，由于这些移植的细胞位于缺血和炎症的微环境中，它们的存活率极低10，11。
已经建立了几种提高移植细胞存活率的方法，例如移植细胞的缺氧和热休克预处理12，13，基因修饰14，15以及同时移植细胞和毛细血管16。不幸的是，大多数方法都受到复杂性和高成本的限制。因此，本研究提出了一种可重复、方便、侵入性相对较强且有效的hiPSC-CM递送方法。
超声引导下心肌细胞注射只能用高分辨率小型兽医超声机和显微注射器进行，无论部位如何。在超声引导下，剑突下的细胞从心包直接输送到小鼠心肌是一种安全的方案，可以避免肝和肺损伤。该方法可以与其他技术同时使用，以显着提高移植细胞的存活率。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac troponin T</td>
			<td>Abcam</td>
			<td>ab8295</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Mouse IgG H&#38;L (Alexa Fluor 488)</td>
			<td>Abcam</td>
			<td>ab150105</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Mouse IgG H&#38;L (Alexa Fluor 555)</td>
			<td>Abcam</td>
			<td>ab150110</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Rabbit IgG H&#38;L (Alexa Fluor 488)</td>
			<td>Abcam</td>
			<td>ab150073</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Rabbit IgG H&#38;L (Alexa Fluor 555)</td>
			<td>Abcam</td>
			<td>ab150062</td>
			<td></td>
		</tr>
		<tr>
			<td>Human cardiac troponin T</td>
			<td>Abcam</td>
			<td>ab91605</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolectin B4</td>
			<td>Vector</td>
			<td>FL-1201</td>
			<td></td>
		</tr>
		<tr>
			<td>Sarcomeric alpha actinin</td>
			<td>Abcam</td>
			<td>ab9465</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat germ agglutinin</td>
			<td>Thermo Fisher Scientific</td>
			<td>W11261</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Thermo Fisher Scientific</td>
			<td>00-4555-56</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement(minus insulin)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement(serum free)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17&#8211;504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bouin&#39;s solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>SDHT10132</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Selleck</td>
			<td>CT99021</td>
			<td></td>
		</tr>
		<tr>
			<td>cyclosporin A</td>
			<td>Medchemexpress</td>
			<td>HY-B0579</td>
			<td></td>
		</tr>
		<tr>
			<td>DIRECT RED</td>
			<td>Sigma-Aldrich</td>
			<td>365548-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy Blood &#38; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td></td>
		</tr>
		<tr>
			<td>FAST GREEN FCF</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;F7252-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose-free RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>11879020</td>
			<td></td>
		</tr>
		<tr>
			<td>IWR1</td>
			<td>Selleck</td>
			<td>S7086</td>
			<td></td>
		</tr>
		<tr>
			<td>lactic acid</td>
			<td>Sigma-Aldrich</td>
			<td>L6661</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>BD356234</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>Stem Cell Technologies</td>
			<td>72562</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. Compound</td>
			<td>SAKURA</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerUP SYBR Green MasterMix kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A25742</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875119</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdif Cardiomyocyte Freezing Medium/STEMdiff</td>
			<td>Stem Cell Technologies</td>
			<td>5030</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Cardiomyocyte Support Medium</td>
			<td>Stem Cell Technologies</td>
			<td>5027</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>ultrasound coupling agent</td>
			<td>CARENT</td>
			<td>22396269389</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Selleck</td>
			<td>S6390</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment and Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems</td>
			<td>Thermo Fisher Scientific</td>
			<td>7500 Real-Time PCR</td>
			<td></td>
		</tr>
		<tr>
			<td>cryostat</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>fluoresence microscope</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>fine anatomical scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>fine dissecting forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro syringe</td>
			<td>Hamilton</td>
			<td>7633</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal anesthesia machine</td>
			<td>MATRX</td>
			<td>VMR</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-high resolution small animal ultrasound imaging system</td>
			<td>VisualSonics</td>
			<td>Vevo 2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Statistical Product and Service Solutions</td>
			<td>IBM</td>
			<td>21</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>NIH</td>
			<td>1.48</td>
			<td></td>
		</tr>
		<tr>
			<td>Human mitochondrial DNA primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>the forward primer sequence</td>
			<td>CCGCTACCATAATCATCGCTAT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>the reverse primer sequence</td>
			<td>TGCTAATACAATGCCAGTCAGG</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

ultrasound-guided induced pluripotent stem cell-derived cardiomyocyte implantation in myocardial infarcted mice

心肌梗死（MI）后细胞治疗的主要目的是有效提高细胞移植率，人诱导多能干细胞来源的心肌细胞（hiPSC-CMs）是缺血性损伤后心脏修复的有希望的细胞来源。然而，低移植率是移植后有效心脏组织再生的重大障碍。该协议表明，多次hiPSC-CM超声引导经皮注射到MI区域可有效提高细胞移植率。该研究还描述了整个hiPSC-CM培养过程，预处理和超声引导的经皮递送方法。此外，使用人类线粒体DNA有助于检测其他小鼠器官中hiPSC-CMs的缺失。最后，本文描述了小鼠细胞递送后4周梗死边界区心脏功能、血管生成、细胞大小和细胞凋亡的变化。可以得出结论，超声心动图引导下经皮注射左心室心肌是一种可行、侵入性相对较强、令人满意、可重复且有效的细胞治疗。

用于评估每组小鼠左心室功能的超声心动图显示，MD组的MI损伤有效逆转（图2A）。与MI组相比，SD组的射血分数（EF）增加（从30%增加到35%; 图2B）和分数缩短（FS）（从18%增加到22%; 图2C）在MI之后。然而，更值得注意的是，在MD组小鼠中多次注射hiPSC-CMs经皮增加了EF（从30%增加到42%; 图2B）和满量程（从18%降至28...

Watch this Scientific Journal Video about Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice at JoVE.com

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

在小鼠心肌梗死部位周围进行超声引导的细胞递送是一种安全、有效、方便的细胞移植方式。

超声引导诱导多能干细胞来源心肌细胞植入心肌梗死小鼠

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

这是第一份报告，证明超声心动图引导的经皮心肌内注射心肌细胞可显着增强人iPS来源的心肌细胞的修复特性。该方法是可行的、令人满意的、可重复的、侵入性较小的和有效的程序，用于递送人iPS来源的心肌细胞治疗。我们认为，经皮心肌内给药是缺血性心脏病、心肌梗塞和心力衰竭等疾病的局部给药安全有效的方法。本项目由杨金福教授和彭军教授指导。演示该程序的将是我实验室的医学博士Xun Wu，Kele Qin和Kun Xiang以及湖南省心血管研究重点实验室的博士Di Wang。首先，从 hiPSC-CM 培养物的 60 天中真空吸出培养物上清液。用PBS洗涤细胞三次。然后使用37摄氏度的细胞分离酶混合物将hiPSC-CMs解离成单个细胞三到五分钟。将细胞收集在含有5毫升RPMI加B27培养基的15毫升管中并混合。计算单元格数并计算单元格总数。在室温下以300G离心细胞悬液两分钟。弃去上清液并将沉淀重悬于心肌细胞支持培养基中至每微升第4个细胞的浓度为6×10。将试管置于37摄氏度直至注射步骤。小鼠充分麻醉后，用胶带固定四肢和尾巴。然后，用 2/0 丝线和胶带固定鼠标门牙。用三轮交替的甜菜碱和酒精对正中颈部和左胸部进行消毒。使用精细解剖剪刀和精细解剖钳做一个颈部正中切口。切掉几块颈椎前部肌肉，以稳定并充分暴露气管。将 20 号导管穿刺针作为气管插管插入口腔。将气管插管连接到呼吸机并检查胸腔运动，以确保双肺通风良好。调整呼吸参数。接下来，将左后肢重新固定到右下侧，并松开左上肢周围的胶带。用细夹剪和镊子，在左胸的三到四个肋间间隙进行开胸术。使用镊子，剥离心包并定位左前降冠状动脉。使用 6/0 丝线结扎动脉近端。为确保建立急性心肌梗死模型，请等到结扎部位的动脉远端由红色变为白色。逐层关闭胸部，并用4/0线缝合皮肤。对于假动物组，执行除结扎以外的所有外科手术。在心肌梗死后的前两周，将小鼠置于与5%异氟烷连接的麻醉盒中进行气管插管。在给予hiPSC-CMs前三天，以每天每公斤10毫克的剂量将环孢素A注射到腹膜腔内。用脱毛膏去除小鼠胸部和上腹部的毛发。供应吸入异氟烷，直到心率保持在每分钟 400 至 500 次。在检测控制台上用胶带固定鼠标四肢和尾巴，并将平台温度设置为 37 摄氏度。将超声波果冻涂抹在上腹部。使用高分辨率兽医超声探头，获得小鼠肝脏图像。使用5微升微型注射器，将约3 x 10吸入第5个hiPSC-CM。握住注射器并在左旁突插入三毫米针头。在超声引导下，沿着横膈膜的上边缘。观察呼吸节律和范围。在呼气圈末端进入心包，避免损害肝脏和肺部。然后通过B型超声心动图获取小鼠心脏的胸骨旁长轴图像。在超声引导下，将1 x 10至第5个hiPSC-CMs细胞注射到梗死部位的三个边缘区域中的每一个。取下超声探头并冲洗掉果冻。使小鼠脱离麻醉并将其放回笼子中。移植后连续注射环孢素A一个月。左心室超声心动图显示，多剂量组心肌梗死损伤逆转。hiPSC-CMs的移植也导致射血分数增加和分数缩短。使用天狼星红和快绿染色测量心脏参数显示，移植后梗死大小和胶原体积分数减少，而左心室前壁厚度增加。绿色非特异性α肌动蛋白标记，蓝色DAPI标记，红色标记人特异性肌钙蛋白T显示梗死和边缘梗死区域存在移植细胞。与单剂量相比，多剂量组移植细胞的数量和百分比更高。心肌梗死后28天，多剂量组显示表达内皮细胞标志物I-B4的细胞数量增加。心肌细胞上的小麦胚芽凝集素和肌瘤α肌动蛋白染色显示最小纤维直径减小。多剂量组中隧道阳性心肌细胞核数量较少表明细胞凋亡减少。PCR分析仅在心脏中检测到人类线粒体DNA，显示特异性移植。插入针头时，请确保它与横膈膜的上缘和呼气圈的末端一起进入心包腔，以避免对肝脏或肺部造成伤害。

Research

JoVE Journal

Medicine

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

植入小鼠触发剪应力致动脉粥样硬化的颈袖

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

科研

医学

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

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 ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

超声引导下经皮内注射小鼠

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Substernal Thyroid Biopsy Using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration

胸骨后甲状腺活检使用支气管内超声引导下经支气管针吸

Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1. There is a wide variation in the published incidence rates due to the ...

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

利用外束辐射对小型动物模型的多能干细胞衍生畸胎瘤进行靶向选择性治疗

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

研究小鼠心肌梗死的低温损伤模型

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

使用荧光标记肉瘤蛋白缩短多能干细胞衍生心肌细胞的肉瘤。

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...

Endoscopic Ultrasound-Guided Biliary Drainage: Endoscopic Ultrasound-Guided Hepaticogastrostomy in Malignant Biliary Obstruction

内镜超声引导下胆道引流：内镜超声引导下肝胃造瘘术治疗恶性胆道梗阻

Patients with unresectable malignant biliary obstruction often require biliary drainage to decompress the biliary system. Endoscopic Retrograde ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

小鼠心肌梗死和心肌缺血再灌注损伤的诱导

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

微电极阵列和膜片钳记录在人诱导多能干细胞来源心肌细胞上的技术应用

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...