Name: enhancing the engraftment of human induced pluripotent stem cell-derived cardiomyocytes via a transient inhibition of rho kinase activity
Uploaded: 2019-07-10T12:30:00
Description: Watch this Scientific Journal Video about Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity at JoVE.com

作者感谢Joseph C. Wu博士(斯坦福大学)为Fluc-GFP结构提供了良好的帮助,并感谢刘燕文博士提供了出色的技术援助。这项研究得到国家卫生研究院RO1授予HL95077、HL114120、HL131017、HL138023、UO1HL134764(至J.Z.)和HL121206A1(至L.Z.)和R56资助HL142627(至W.Z.)的资助。16SDG30410018,和阿拉巴马大学伯明翰分校的教师发展补助金(到W.Z.)。

本研究中的所有动物程序均获得伯明翰阿拉巴马大学机构动物护理和使用委员会(IACUC)的批准,并基于国家卫生研究院实验室动物护理和使用指南(NIH出版物No.85-23)。
1. 文化媒体和文化板块的编制
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中等准备
	<ol>
<li>对于hiPSC介质,混合400 mL的人类多能干细胞(hPSC)基底培养基(材料表1)和100 mL的hPSC 5x补充剂;将混合物储存在4°C。</li>
		<li>对于 RPMI 1640/B27...

<ol>
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本研究的关键步骤包括获得纯hiPSC-CM,通过Y-27632预处理提高hiPSC-CM的活性,最后,将精确数量的hiPSC-CM移植到小鼠MI模型中。
这里讨论的关键问题是:首先,我们优化了无葡萄糖纯化方法19,建立了新型高效纯化系统。系统程序包括应用细胞分离酶,在明胶涂层板中重新植入细胞,在重新镀层后在中和培养培养24小时,以及尽量减少移植前细胞的消化时间。其中执行,以达到...

干细胞疗法已经显示出相当大的潜力,作为治疗由MI1造成的心脏损伤。差别化的hiPSC的使用为hiPSC-CMs2提供了取之不尽的源,并为突破性治疗的快速发展打开了大门。然而,治疗转化仍然存在许多限制,包括植入细胞移植率极低的挑战。
分离细胞与胰蛋白酶启动阿诺基斯3,这是加速,只有当这些细胞被注射到恶劣的环境,如缺血性心肌,其中缺氧环境加速走向细胞死亡。在剩余的细胞中,很大一部分从植入部位被冲出到血液中,并扩散到整个周围。其中一个关键的凋亡途径是RhoA/ROCK通路4。根据以前的研究,RhoA/ROCK通路调节行为素细胞骨骼组织5,6,这是负责细胞功能障碍7,8。ROCK抑制剂Y-27632在体细胞分离和传血过程中广泛使用,以增加细胞附着力,减少细胞凋亡9、10、11。在这项研究中,Y-27632用于在移植前治疗hiPSC-CM,以试图提高细胞移植率。
建立了几种旨在提高细胞移植率的方法,如热冲击和基底膜基质涂层12。除了这些方法,基因技术还可以促进心肌细胞增殖13或逆转非心肌细胞进入心肌细胞14。从生物工程的角度来看,心肌细胞被播种在生物材料支架上,以提高移植效率15。不幸的是,这些方法大多复杂且成本高昂。相反,这里提出的方法简单、经济、有效,可作为移植前的基础治疗,以及与其他技术结合使用。

<table>
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase (stem cell detachment solution)</td>
			<td>STEMCELL Technologies</td>
			<td>#07920</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 minus insulin</td>
			<td>Fisher Scientific</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Fisher Scientific</td>
			<td>17-504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stem Cell Technologies</td>
			<td>72054</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (1x), high glucose, HEPES, no phenol red</td>
			<td>Thermofisher</td>
			<td>20163029</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4 AM (calcium indicator)</td>
			<td>Invitrogen/Thermofisher</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose-free RPMI 1640</td>
			<td>Fisher Scientific</td>
			<td>11879020</td>
			<td></td>
		</tr>
		<tr>
			<td>IWR1</td>
			<td>Stem Cell Technologies</td>
			<td>72562</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel (extracellular matrix )</td>
			<td>Fisher Scientific</td>
			<td>CB-40230C</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR (human pluripotent stem cells medium)</td>
			<td>STEMCELL Technologies</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-strep antibiotic</td>
			<td>Fisher Scientific</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127 (surfactant polyol)</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Rho activator II</td>
			<td>Cytoskeleton</td>
			<td>CN03</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Fisher Scientific</td>
			<td>11875119</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium DL-lactate</td>
			<td>Sigma-Aldrich</td>
			<td>L4263</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE (cell-dissociation enzymes)</td>
			<td>Fisher Scientific</td>
			<td>12-605-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Verapamil</td>
			<td>Sigma-Aldrich</td>
			<td>V4629</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment and Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina III Bioluminescence Instruments</td>
			<td>PerkinElmer</td>
			<td>CLS136334</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mm Coverslips</td>
			<td>Warner</td>
			<td>CS-15R15</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Thermo Scientific</td>
			<td>NX50</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual Automatic Temperature Controller</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis Power Supply</td>
			<td>BIO-RAD</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresence Microscope</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Camera</td>
			<td>pco</td>
			<td>1200 s</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scan Head</td>
			<td>Olympus</td>
			<td>FV-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Profile Open Bath Chamber (mounts into above microincubation system)</td>
			<td>Warner Instruments</td>
			<td>RC-42LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Microincubation System</td>
			<td>Warner Instruments</td>
			<td>DH-40iL</td>
			<td></td>
		</tr>
		<tr>
			<td>Minivent Mouse Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>845</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD/SCID mice</td>
			<td>Jackson Laboratory</td>
			<td>001303</td>
			<td></td>
		</tr>
		<tr>
			<td>Precast Protein Gels</td>
			<td>BIO-RAD</td>
			<td>4561033</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Transfer Packs</td>
			<td>BIO-RAD</td>
			<td>1704156</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot System</td>
			<td>BIO-RAD</td>
			<td>Trans-Blot Turbo</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human Nucleolin (Alexa Fluor 647)</td>
			<td>Abcam</td>
			<td>ab198580</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac Troponin T</td>
			<td>R&#38;D Systems</td>
			<td>MAB1874</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac Troponin C</td>
			<td>Abcam</td>
			<td>ab137130</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac Troponin I</td>
			<td>Abcam</td>
			<td>ab47003</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5-donkey anti-mouse</td>
			<td>Jackson ImmunoResearch Laboratory</td>
			<td>715-175-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-donkey anti-rabbit</td>
			<td>Jackson ImmunoResearch Laboratory</td>
			<td>711-165-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Fitc-donkey anti-mouse</td>
			<td>Jackson ImmunoResearch Laboratory</td>
			<td>715-095-150</td>
			<td></td>
		</tr>
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enhancing the engraftment of human induced pluripotent stem cell-derived cardiomyocytes via a transient inhibition of rho kinase activity

提高细胞治疗治疗对心肌再生的一个关键因素是安全有效地提高细胞移植率。Y-27632 是 Rho 相关、含线圈蛋白激酶 (RhoA/ROCK) 的强效抑制剂,用于防止分离引起的细胞凋亡(阿诺基斯)。我们证明,Y-27632在植入前对人类诱导多能干细胞衍生心肌细胞(hiPSC-CMs+RI)进行预处理,导致急性心肌梗死(MI)小鼠模型中的细胞移植率提高。在这里,我们描述了使用Y-27632进行hiPSC-CM分化、纯化和细胞预处理的完整过程,以及由此产生的细胞收缩、钙瞬态测量和移植到小鼠MI模型中。该方法提供了一种简单、安全、有效、低成本的方法,显著提高了细胞移植率。该方法不仅与其他方法相结合,进一步提高细胞移植效率,而且为研究其他心脏疾病的发病机制提供了良好的依据。

本研究中使用的hiPSC-CMs源自人类起源,具有荧光酶报告基因;因此,通过生物发光成像(BLI)17(图1A,B)检测出移植细胞在体内的存活率。对于组织学心脏部分,人类特异性心脏肌钙蛋白T(hcTnT)和人类核抗原(HNA)双阳性细胞被归类为移植的hiPSC-CMs(图1C)。两种结果表明,Y-27632预处理显著改善了细胞移植率。与hiPSC...

Watch this Scientific Journal Video about Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity at JoVE.com

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

在该协议中,我们演示并阐述了如何使用人类诱导多能干细胞进行心肌细胞分化和纯化,并进一步说明如何利用Rho相关蛋白激酶抑制剂提高移植效率小鼠心肌梗死模型的预处理。

通过对Rho激酶活性的暂时抑制,增强人类诱导多能干细胞衍生心肌细胞的诱导

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

我们的数据表明，在将Rho-激酶活性的暂时抑制移植到梗塞动物心脏后，可增强粘附、存活和附着hiPSC衍生的心肌细胞。我们提供一种简单、低成本、高效的方法，以增加移植的hiPSC衍生心肌细胞在受伤动物心脏中的移植。由于该技术可以安全高效地提高急性心肌梗塞和心力衰竭中的细胞移植率，有助于改善心脏功能、血管和凋亡。虽然我们只在hiPSC中测试了我们的协议，但这些方法可以应用于其他类型的细胞，如胚胎干细胞、中生期干细胞等。Y-27632治疗的剂量和持续时间至关重要。可视化演示可以非常详细地演示协议中的所有关键步骤，这对以前从未执行过此技术的人是有益的。在hiPSC-CM分化后的第21天，吸气介质，用无菌PBS清洗细胞一次。每井在37摄氏度下用0.5毫升细胞分离酶孵育细胞5至7分钟。孵育时间后，使用一毫升移液器反复移液，以彻底分离细胞。然后，将一毫升中和溶液添加到每一个井中，将细胞混合物收集在15毫升离心管中，以200倍g离心3分钟。离心后，丢弃上经剂，并在中和溶液中重新暂停细胞。然后，将细胞重新植入明胶涂层的六孔板上，密度为每孔200万个细胞。24至48小时后，用净化介质更换培养介质3至5天。移植前，培养治疗组的细胞在RB+介质中培养12小时，辅以10微摩尔Y-27632。同样，用一微莫维帕米尔对RB+介质中的细胞进行12小时的维拉帕米尔治疗。治疗后，用PBS清洗hiPSC-CMs一次，用每井0.5微升细胞分离酶孵育细胞，最多两分钟。使用单毫升移液器反复移液细胞悬浮液，以彻底分离细胞。接下来，用中和溶液中和细胞，将细胞混合物收集在15毫升离心管中，以200倍g离心3分钟。离心后，每5微升的浓度为0.10万细胞，以制备注射。立即在心肌梗塞诱导后，将五微升控制hiPSC-CMs、Y-27632预处理的hiPSC-CMs、Verapamil治疗的hiPSC-CMs或相同体积的PBS注射到每个站点的小鼠心肌中，一个在梗塞区域，两个在梗塞周围区域。在进行钙瞬态和收缩检测前 12 小时，将 10 微摩尔 Y-27632、100 纳米摩尔 RA、1 微摩尔维拉帕米尔或等量的 PBS 添加到 hiPSC-CM 的培养介质中。接下来，丢弃介质，用 PBS 清洗一次板。将介质更改为苯酚无红色 DMEM，每体积含有 0.02 重量，即表面活性质聚醇、五微摩尔钙指示器以及相应的 Y-27632、RA、verapamil 或等量的 PBS，并在 37 摄氏度下孵育板 30 分钟。孵育后，丢弃上经剂，用无染料DMEM洗涤细胞三次，让介质休息30分钟，使映射染料去酯化。将细胞种子盖玻片放在开放式浴室中，通过自动温度控制器将室插入到平衡至 37 摄氏度的微孵化系统中。使用泰洛德的解决方案进行灌注，并将 RI、RA 或 PBS 不断添加到灌注解决方案中。使用倒置荧光显微镜和激光扫描头，通过 x-t 线扫描记录自发钙瞬态。使用共合显微镜的差分干扰对比度功能记录 hiPSC-CMs 的自发收缩。移植细胞的存活率由荧光素酶活性增加得到证实。增加的人体心脏肌，T和人类核抗原表达表明，Y-27632预处理可以显著提高细胞移植率。Y-27632预处理细胞表现出更大、更明确的棒状细胞骨骼结构。此外，Y-27632预处理减少了 TUNEL 阳性细胞，表明体内移植的 hiPSC-CM 凋亡减少。西方印迹表明，Y-27632预处理增加了蛋白β-1和N-卡德林的表达，并减少了磷-米奥辛光链2的表达。此更改在 Y-27632 退出后 72 小时被规范化。体外小鼠HL-1细胞附着实验表明，Y-27632预处理显著增加了与HL-1相关的hiPSC-CM依从性。Y-27632预处理减少了收缩力、峰值钙瞬态荧光和钙瞬态持续时间。Y-27632预处理通过显著减少cTnI和cTnT的表达来调节心肌细胞收缩。与Y-27632类似，维帕米尔预处理增加了荧光酶活性和hcTnT/HNA双阳性细胞的数量。最重要的步骤是培养治疗组的细胞在Rb+培养12小时，辅以10微摩尔Y-27632。基于这种方法，慢释放Y-27632进一步提高细胞移植率是一个很有前途的策略。该技术为研究人员在体内研究CMshiPSC的生理学和功能铺平了道路。用于心肌细胞分化的小分子CHIR99021是危险的，可能导致呼吸刺激。请不要吸入或吸入任何方式。

Research

JoVE Journal

Biochemistry

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

使用硝酸纤维素结合分析细菌组氨酸激酶自身磷酸化的定量分析

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

科研

生物化学

Assaying Protein Kinase Activity with Radiolabeled ATP

用放射性标记的ATP测定蛋白激酶活性

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

人类外周血神经细胞分离通过评估Rab10磷酸化来询问帕金森氏症相关的LRRK2激酶途径

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

利用多功能生化方法鉴定新型 ck2 激酶基材

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

丝状真菌曲霉菌的单步富集蛋白脱乙酰化酶的酶活性测定

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

使用新激酶蛋白的强生化方法在分子水平上进行表征

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...