患者外周血单核细胞中人心肌细胞的生成和扩增
1Center for Cardiovascular Research, The Abigail Wexner Research Institute,Nationwide Children’s Hospital, 2The Heart Center,Nationwide Children’s Hospital, 3Department of Physiology and Cell Biology,The Ohio State University College of Medicine, 4Department of Anatomy,The Ohio State University College of Medicine, 5MCDB Graduate Program,The Ohio State University, 6Department of Pediatrics,The Ohio State University College of Medicine
Summary
在这里，我们提出了一种方案，可以从患者外周血单核细胞中稳健地产生和扩增人心肌细胞。
Abstract
通过单次抽血产生患者特异性心肌细胞，引起了人们对心血管疾病精准医学的极大兴趣。与人类诱导多能干细胞（iPSCs）的心脏分化由对胚胎心脏发育至关重要的明确信号通路调节。已经开发了2-D和3-D平台上的许多心脏分化方法，具有不同的效率和心肌细胞产量。这让该领域以外的调查人员感到困惑，因为这些方法的多样性可能难以遵循。在这里，我们提出了一个全面的方案，详细阐述了外周血单核细胞（PBMC）患者特异性心肌细胞的稳健生成和扩增。我们首先描述了使用非整合仙台病毒载体从患者血液样本中获取的高效iPSC重编程方案。然后，我们详细介绍了一种小分子介导的单层分化方法，该方法可以从大多数人类iPSC系中稳健地产生跳动的心肌细胞。此外，还引入了一种使用小分子（CHIR99021）的可扩展心肌细胞扩增方案，该方案可以快速扩增患者来源的心肌细胞，用于工业和临床级应用。最后，描述了这些iPSC-CM的分子鉴定和电生理学表征的详细方案。我们希望该协议对于心血管发育和干细胞生物学知识有限的初学者来说是务实的。
Introduction
人类诱导多能干细胞的发现彻底改变了现代心血管医学1，2。人类iPSCs能够自我更新和产生心脏中的所有细胞类型，包括心肌细胞，内皮细胞，平滑肌细胞和心脏成纤维细胞。患者iPSC衍生的心肌细胞（iPSC-CM）可以作为无限期的资源，用于模拟遗传性心血管疾病（CVD）和测试新药的心脏安全性3。特别是，患者 iPSC-CM 已准备好研究源自心肌细胞缺陷的 CVD 的遗传和分子病因，例如长 QT 综合征4和扩张型心肌病 （DCM）5。结合CRISPR / Cas9介导的基因组编辑，患者iPSC-CM开辟了一条前所未有的途径来了解CVD的复杂遗传基础，包括先天性心脏缺陷（CHD）6，7，8。人类iPSC-CM还显示出在心脏病发作期间作为自体细胞来源补充受损心肌的潜力9。近年来，产生具有明确亚型（心房，心室和淋巴结）的高质量人iPSC-CM对于心脏再生和药物检测至关重要10。
在过去十年中，心脏与人类iPSCs的鉴别已经取得了长足的进步。分化方法已经从基于胚体（EB）的自发分化到化学定义和定向的心脏分化11。对胚胎心脏发育至关重要的关键信号分子，如 Wnt ， BMP ，节点和 FGF 纵以增强心肌细胞与人类 iPSCs 的分化10 ， 12 。重大进展包括Wnt信号传导的顺序调节（激活然后抑制），用于从人iPSCs中稳健地产生心肌细胞13，14。化学定义的心脏分化配方已被探索，以促进大规模生产跳动的心肌细胞15，16，这些细胞有可能升级到工业和临床水平的生产。此外，早期人类iPSC-CM的稳健扩增是通过使用小化学物质（CHIR99021）暴露于构成性Wnt活化来实现的17。最近，亚型特异性心肌细胞是通过在人类iPSCs的心肌细胞谱系承诺期间在特定分化窗口中操纵视黄酸（RA）和Wnt信号通路而产生的18，19，20，21，22。
在该协议中，我们详细介绍了来自患者外周血单核细胞的人CM的稳健生成和增殖的工作程序。我们提出了1）将人类PBMC重新编程为iPSCs的协议，2）从人iPSCs中稳健地产生跳动的心肌细胞，3）早期iPSC-CM的快速扩增，4）人类iPSC-CM的分子表征，以及5）通过膜片钳在单细胞水平上对人类iPSC-CM进行电生理学测量。该协议涵盖了将患者血细胞转化为跳动心肌细胞的详细实验程序。
Protocol
人类受试者的实验方案和知情同意由全国儿童医院的机构审查委员会（IRB）批准。 1. 细胞培养基、溶液和试剂的制备 准备 PBMC 介质 混合20 mL基础PBMC培养基（1x）和0.52 mL补充剂。分别加入20μL SCF和FLT3（储备浓度：100μg/ mL），分别加入4μLIL3，IL6和EPO（储备浓度：100μg/ mL）和200μLLLLLLLLLLLL谷氨酰胺替代品（100x）。将它们彻底混合。使用0.22μm过滤装置在无菌罩中过?…
Representative Results
从 PBMC 进行人类 iPSC 重编程用全血培养基预培养7天后，PBMC变大，可见细胞核和细胞质（图1B），表明它们已准备好进行病毒转染。在用仙台病毒重编程因子转染后，PBMC将再经历一周的表观遗传重编程过程。通常，我们从1 x 10 5 PBMC的转染中获得30-50 个iPSC菌落，重编程效率为0.03%-0.05%。完全重编程的细胞在被引入到完整的E8培养…
Discussion
在 iPSC 重编程期间，培养 PBMC 持续 1 周至关重要，直到它们被透明细胞核和细胞质扩增。由于PBMC不会增殖，因此用于病毒转导的适当细胞数对于成功的iPSC重编程非常重要。应考虑并调整PBMC的细胞数，感染的多重性（MOI）和病毒的滴度，以达到最佳的转导结果。对于心脏分化，初始播种密度对于iPSCs在施用CHIR99021当天达到90%以上的汇合至关重要。一方面，如果iPSCs在心脏分化时汇合较少，CHIR99021将…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了美国心脏协会（AHA）职业发展奖18CDA34110293（M-T.Z.），其他风险投资AVIF和SVRF奖（M-T.Z.），美国国立卫生研究院（NIH / NHLBI）拨款1R01HL124245，1R01HL132520和R01HL096962（I.D.）的支持。赵明涛博士还得到了全国儿童医院阿比盖尔·韦克斯纳研究所（Abigail Wexner Research Institute）的启动资金支持。
Materials
| ABI 7300 Fast Real-Time PCR System | Thermo Fisher Scientific | ||
| Axon Axopatch 200B Microelectrode Amplifier | Molecular Devices | Microelectrode Amplifier | |
| B27 supplement | Thermo Fisher Scientific | 17504044 | |
| B27 supplement minus insulin | Thermo Fisher Scientific | A1895601 | |
| BD Cytofix/Cytoperm Fixation/Permeabilization Kit | BD Biosciences | 554714 | Fixation/Permeabilization solution, Perm/Wash buffer |
| BD Vacutainer CPT tube | BD Biosciences | 362753 | Blood cell separation tube |
| CHIR99021 | Selleck Chemicals | S2924 | |
| CytoTune-iPS 2.0 Sendai Reprogramming Kit | Thermo Fisher Scientific | A16517 | Sendai virus reprogramming kit |
| Digidata 1200B | Axon Instruments | Acquisition board | |
| Direct-zol RNA Miniprep kit | Zymo Research | R2050 | RNA extraction kit |
| DMEM/F12 | Thermo Fisher Scientific | 11330057 | |
| Essential 8 medium | Thermo Fisher Scientific | A1517001 | E8 media for iPSC culture |
| GlutaMAX supplement | Thermo Fisher Scientific | 35050061 | L-glutamine alternative |
| Growth factor reduced Matrigel | Corning | 356231 | Basement membrane matrix |
| iScript cDNA Snythesis Kit | Bio-Rad | 1708891 | cDNA synthesis |
| IWR-1-endo | Selleck Chemicals | S7086 | |
| KnockOut Serum Replacement (KSR) | Thermo Fisher Scientific | 10828028 | |
| pCLAMP 7.0 | Molecular Devices | Electrophysiology data acquisition & analysis software | |
| Recombinant human EPO | Thermo Fisher Scientific | PHC9631 | |
| Recombinant human FLT3 | Thermo Fisher Scientific | PHC9414 | |
| Recombinant human IL3 | Peprotech | 200-03 | |
| Recombinant human IL6 | Thermo Fisher Scientific | PHC0065 | |
| Recombinant human SCF | Peprotech | 300-07 | |
| RPMI 1640 medium | Thermo Fisher Scientific | 11875093 | |
| RPMI 1640 medium, no glucose | Thermo Fisher Scientific | 11879020 | |
| SlowFade Gold Antifade Mountant | Thermo Fisher Scientific | S36936 | Mounting media |
| StemPro-34 SFM | Thermo Fisher Scientific | 10639011 | PBMC culture media |
| TaqMan Fast Advanced Master Mix | Thermo Fisher Scientific | 4444964 | qPCR master mix |
| TrypLE Select Enzyme 10x, no phenol red | Thermo Fisher Scientific | A1217703 | CM dissociation solution |
| UltraPure 0.5 M EDTA | Thermo Fisher Scientific | 15575020 | iPSC dissociation solution |
| Y-27632 2HCl | Selleck Chemicals | S1049 |
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Cite This Article
Ye, S., Wan, X., Su, J., Patel, A., Justis, B., Deschênes, I., Zhao, M. Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells. J. Vis. Exp. (168), e62206, doi:10.3791/62206 (2021).