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
1Department of Biomedical Engineering, School of Medicine, School of Engineering,University of Alabama at Birmingham, 2Division of Cardiovascular Diseases, School of Medicine, School of Engineering,University of Alabama at Birmingham, 3Department of Cardiac Surgery,The Second Xiangya Hospital of Central South University
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 中等准备 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。 对于 RPMI 1640/B27…
Representative Results
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…
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
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.)。
Materials
| Reagent | |||
| Accutase (stem cell detachment solution) | STEMCELL Technologies | #07920 | |
| B27 minus insulin | Fisher Scientific | A1895601 | |
| B27 Supplement | Fisher Scientific | 17-504-044 | |
| CHIR99021 | Stem Cell Technologies | 72054 | |
| DMEM (1x), high glucose, HEPES, no phenol red | Thermofisher | 20163029 | |
| Fetal bovine serum | Atlanta Biologicals | S11150 | |
| Fluo-4 AM (calcium indicator) | Invitrogen/Thermofisher | F14201 | |
| Glucose-free RPMI 1640 | Fisher Scientific | 11879020 | |
| IWR1 | Stem Cell Technologies | 72562 | |
| Matrigel (extracellular matrix ) | Fisher Scientific | CB-40230C | |
| mTeSR (human pluripotent stem cells medium) | STEMCELL Technologies | 85850 | |
| Pen-strep antibiotic | Fisher Scientific | 15-140-122 | |
| Pluronic F-127 (surfactant polyol) | Sigma-Aldrich | P2443 | |
| Rho activator II | Cytoskeleton | CN03 | |
| RPMI1640 | Fisher Scientific | 11875119 | |
| Sodium DL-lactate | Sigma-Aldrich | L4263 | |
| TrypLE (cell-dissociation enzymes) | Fisher Scientific | 12-605-010 | |
| Verapamil | Sigma-Aldrich | V4629 | |
| Y-27632 | STEMCELL Technologies | 72304 | |
| Name | Company | Catalog Number | Comments |
| Equipment and Supplies | |||
| IVIS Lumina III Bioluminescence Instruments | PerkinElmer | CLS136334 | |
| 15 mm Coverslips | Warner | CS-15R15 | |
| Centrifuge | Eppendorf | 5415R | |
| Confocal Microscope | Olympus | IX81 | |
| Cryostat | Thermo Scientific | NX50 | |
| Dual Automatic Temperature Controller | Warner Instruments | TC-344B | |
| Electrophoresis Power Supply | BIO-RAD | 1645050 | |
| Fluoresence Microscope | Olympus | IX83 | |
| High Speed Camera | pco | 1200 s | |
| Laser Scan Head | Olympus | FV-1000 | |
| Low Profile Open Bath Chamber (mounts into above microincubation system) | Warner Instruments | RC-42LP | |
| Microincubation System | Warner Instruments | DH-40iL | |
| Minivent Mouse Ventilator | Harvard Apparatus | 845 | |
| NOD/SCID mice | Jackson Laboratory | 001303 | |
| Precast Protein Gels | BIO-RAD | 4561033 | |
| PVDF Transfer Packs | BIO-RAD | 1704156 | |
| Trans-Blot System | BIO-RAD | Trans-Blot Turbo | |
| Hot bead sterilizer | Fine Science Tools | 18000-45 | |
| Name | Company | Catalog Number | Comments |
| Antibody | |||
| Anti-human Nucleolin (Alexa Fluor 647) | Abcam | ab198580 | |
| Cardiac Troponin T | R&D Systems | MAB1874 | |
| Cardiac Troponin C | Abcam | ab137130 | |
| Cardiac Troponin I | Abcam | ab47003 | |
| Cy5-donkey anti-mouse | Jackson ImmunoResearch Laboratory | 715-175-150 | |
| Cy3-donkey anti-rabbit | Jackson ImmunoResearch Laboratory | 711-165-152 | |
| Fitc-donkey anti-mouse | Jackson ImmunoResearch Laboratory | 715-095-150 | |
| GAPDH | Abcam | ab22555 | |
| Human Cardiac Troponin T | Abcam | ab91605 | |
| Integrin β1 | Abcam | ab24693 | |
| Ki67 | EMD Millipore | ab9260 | |
| N-cadherin | Abcam | ab18203 | |
| Phospho-Myosin Light Chain 2 | Cell Signaling Technology | 3671s | |
| Name | Company | Catalog Number | Comments |
| Software | |||
| Matlab | MathWorks | R2016A | |
| Image J | NIH | 1.52g |
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Citar este artigo
Zhao, M., Tang, Y., Ernst, P. J., Kahn-Krell, A., Fan, C., Pretorius, D., Zhu, H., Lou, X., Zhou, L., Zhang, J., Zhu, W. Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity. J. Vis. Exp. (149), e59452, doi:10.3791/59452 (2019).