Summary

生成心室样HiPSC衍生的心肌细胞和用于钙处理特征的高质量细胞制剂

Published: January 17, 2020
doi:

Summary

在这里,我们描述和验证一种持续产生强大的人类诱导多能干细胞衍生心肌细胞的方法,并描述其功能。这些技术可能有助于发展对信号通路的机械洞察力,为大规模药物筛查提供平台,并可靠地模拟心脏病。

Abstract

人类诱导多能干细胞衍生心肌细胞(iPSC-CMs)为研究钙(Ca2+)处理和信号通路以及高通量药物筛选和毒性检测的基本科学提供了宝贵的人类来源。在此,我们详细介绍了用于生成高质量 iPSC-CM 的方法,这些方法可以持续再现不同细胞系的分子和功能特征。此外,还介绍了一种方法,通过评估 Ca2+处理特性,可靠地评估其功能特性。低氧 (O2) 条件、 乳酸盐选择和长时间培养产生高纯度和高质量的心室状心肌细胞.与分离的成年大鼠心肌细胞 (ARCMs) 类似,3 个月大的 iPSC-CM 表现出更高的 Ca2+振幅、更快的 Ca2+再摄取率(衰变-tau),以及与第 30 天 iPSC-CM 相比,对 β-肾上腺素刺激的正热性反应。该策略在技术上简单、经济高效且可重现。它为心脏病建模和针对Ca2+处理蛋白的大规模药物筛查提供了一个强大的平台。

Introduction

人类诱导多能干细胞衍生心肌细胞(iPSC-CMs)是一个有吸引力的基于人的平台,用于在体外模拟各种心脏疾病1、2、3、4、5、6、7、8。此外,iPSC-CM可用于预测患者对新药或现有药物的反应,筛选命中化合物,并开发新的个性化药物9,10。然而,尽管取得了显著进展,但在使用iPSC-CM11时,需要考虑一些限制和挑战。因此,改进心脏分化方案、提高iPSC-CMs效率和成熟以及产生特定心肌细胞亚型(心室、心房、心房和节点)的方法已被深入研究,并已导致许多文化策略克服这些障碍12、13、14、15。

尽管这些协议具有鲁棒性,但使用 iPSC-CM 的一个主要问题是,为了获得能够确保相同性能和可重复结果的高质量心肌细胞,需要重复和复杂的程序。重复性不仅在比较具有不同遗传背景的细胞系时,而且在重复同一细胞系的细胞和分子比较时都是至关重要的。细胞变异性(如 iPSC 密度的差异)可能会影响心脏分化,产生低产量和低质量的心肌细胞。这些细胞仍可用于执行不需要纯 VM 总体的实验(例如,执行 Ca2+瞬态测量时)。事实上,在进行电生理分析时,非CM不会跳动,无论是自发的,也不是电刺激的,所以很容易将它们排除在分析之外。然而,由于质量差,iPSC-CM 可以显示改变的电生理特性(例如,不规则的 Ca2+瞬态,低 Ca2+振幅),这些不是由于它们的遗传组成。因此,特别是在使用 iPSC-CM 对心脏病进行建模时,不要将劣质 CM 的结果与疾病表型混淆。在进行电生理学研究之前,需要仔细的筛选和排除过程。

该方法包括优化的协议,以生成高纯度和高质量的心肌细胞,并通过使用钙和收缩性采集和分析系统执行 Ca2+瞬态测量来评估其功能。该技术是一种简单而强大的方法,用于区分高效和低效率的 iPSC-CM 制剂,并提供更具有生理相关性的人类 iPSC-CM 表征。

Protocol

在这项研究中,使用成年大鼠心肌细胞的实验是在西奈山伊坎医学院批准的机构动物护理和使用委员会(IACUC)协议下进行的。成年大鼠心肌细胞被分离出斯普拉格道利鼠的心脏,通过兰根多夫的方法,如前16所述。 1. 媒体的准备 准备 hiPSC 介质。 平衡补充剂和基础介质到室温 (RT)。确保补充已完全解冻。使用 0.22 μm 真空驱动过滤器混合 400 m…

Representative Results

图1A中描述的协议生成了高度纯的心肌细胞,这些细胞在培养中具有随时间而获取心室/成人样表型。通过心房和心室肌苷调节光链2异种的免疫荧光染色评估,该协议产生的大多数细胞在诱导心脏分化后的第30天是MLC2A阳性,而MLC2V在同一时间点以更低的数量表示(图2A,顶部面板)。随着文化时间的增加(第 60 天和第 90 天),?…

Discussion

使用人类 iPSC-CM 作为实验模型的关键步骤是:1) 生成高质量的心肌细胞 (CM),以确保一致的性能和可重现的结果;( 1) 生成高质量的心肌细胞 (CM),2) 允许细胞在培养中成熟至少90天,以充分评估其表型;3) 进行电生理学研究,例如钙(Ca2+)瞬态测量,为人类iPSC-CM提供生理相关功能表征。我们开发了一种基于单层的分化方法,可生产高质量的心室状 iPSC-CM。我们的方法依赖于几个…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究得到了AHA科学家发展赠款17SDG33700093(F.S.) 的支持;西奈山KL2学者奖临床和转化研究职业发展KL2TR001435(F.S.);NIH R00 HL116645 和 AHA 18TPA34170460 (C.K.)。

Materials

Anti-Actin, α-Smooth Muscle antibody, Mouse monoclonal Sigma Aldrich A5228
Alexa Fluor 488 goat anti mouse Invitrogen A11001
Alexa Fluor 555 goat anti rabbit Invitrogen A21428
B27 Supplement Gibco 17504-044
B27(-) insulin Supplement Gibco A18956-01
CHIR-99021 Selleckchem S2924
DAPI nuclear stain ThermoFisher D1306
DMEM/F12 (1:1) (1X) + L- Glutamine + 15mM Hepes Gibco 11330-032
Double Ended Cell lifter, Flat blade and J-Hook Celltreat 229306
Falcon Multiwell Tissue Culture Plate, 6 well Corning 353046
Fluidic inline heater Live Cell Instrument IL-H-10
Fura-2, AM Invitrogen F1221
hESC-qualified matrix Corning 354277 Matrigel Matrix
hPSC media Gibco A33493-01 StemFlex Basal Medium
IWR-1 Sigma Aldrich I0161
Live cell imaging chamber Live Cell Instrument EC-B25
MLC-2A, Monoclonal Mouse Antibody Synaptic Systems 311011
Myocyte calcium and contractility system Ionoptix ISW-400
Myosin Light Chain 2 Antibody, Rabbit Polyclonal (MLC2V) Proteintech 10906-1-AP
Nalgene Rapid Flow Sterile Disposable Filter units with PES Membrane ThermoFisher 124-0045
PBS with Calcium and Magnesium Corning 21-030-CV
PBS without Calcium and Magensium Corning 21-031-CV
Premium Glass Cover Slips Lab Scientific 7807
RPMI medium 1640 (-) D-glucose (1X) Gibco 11879-020
RPMI medium 1640 (1X) Gibco 11875-093
Sodium L-lactate Sigma Aldrich L7022
StemFlex Supplement Gibco A33492-01
Thiazovivin Tocris 3845
Trypsin-EDTA (0.25%) ThermoFisher 25200056
Tyrode's solution Boston Bioproducts BSS-355w Adjust pH at 7.2. Add 1.2mM Calcium Chloride

References

  1. Karakikes, I., et al. Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy. Nature Communications. 6, 6955 (2015).
  2. Moretti, A., et al. Patient-specific induced pluripotent stem-cell models for long-QT syndrome. The New England Journal of Medicine. 363 (15), 1397-1409 (2010).
  3. Davis, R. P., et al. Cardiomyocytes derived from pluripotent stem cells recapitulate electrophysiological characteristics of an overlap syndrome of cardiac sodium channel disease. Circulation. 125 (25), 3079-3091 (2012).
  4. Fatima, A., et al. In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells. Cellular Physiology and Biochemistry: International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology. 28 (4), 579-592 (2011).
  5. Novak, A., et al. Cardiomyocytes generated from CPVTD307H patients are arrhythmogenic in response to beta-adrenergic stimulation. Journal of Cellular and Molecular Medicine. 16 (3), 468-482 (2012).
  6. Lan, F., et al. Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells. Cell Stem Cell. 12 (1), 101-113 (2013).
  7. Sun, N., et al. Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy. Science Translational Medicine. 4 (130), (2012).
  8. Stillitano, F., et al. Modeling susceptibility to drug-induced long QT with a panel of subject-specific induced pluripotent stem cells. eLife. 6, (2017).
  9. Matsa, E., Burridge, P. W., Wu, J. C. Human stem cells for modeling heart disease and for drug discovery. Science Translational Medicine. 6 (239), (2014).
  10. Mordwinkin, N. M., Lee, A. S., Wu, J. C. Patient-specific stem cells and cardiovascular drug discovery. Journal of the American Medical Association. 310 (19), 2039-2040 (2013).
  11. Youssef, A. A., et al. The Promise and Challenge of Induced Pluripotent Stem Cells for Cardiovascular Applications. Journal of the American College of Cardiology: Basic to Translational Science. 1 (6), 510-523 (2016).
  12. Cyganek, L., et al. Deep phenotyping of human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes. Journal of Clinical Investigation: Insight. 3, 12 (2018).
  13. Keung, W., Boheler, K. R., Li, R. A. Developmental cues for the maturation of metabolic, electrophysiological and calcium handling properties of human pluripotent stem cell-derived cardiomyocytes. Stem Cell Research, Therapy. 5 (1), 17 (2014).
  14. Bhattacharya, S., et al. High efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry. Journal of Visualized Experiments: JoVE. (91), e52010 (2014).
  15. Ronaldson-Bouchard, K., et al. Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature. 556 (7700), 239-243 (2018).
  16. Gorski, P. A., et al. Measuring Cardiomyocyte Contractility and Calcium Handling In Vitro. Methods in Molecular Biology. 1816, 93-104 (2018).
  17. Kim, C., et al. Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation. Stem Cells and Development. 19 (6), 783-795 (2010).
  18. Lian, X., et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions. Nature Protocols. 8 (1), 162-175 (2013).
  19. Patterson, A. J., Zhang, L. Hypoxia and fetal heart development. Current Molecular Medicine. 10 (7), 653-666 (2010).
  20. Correia, C., et al. Combining hypoxia and bioreactor hydrodynamics boosts induced pluripotent stem cell differentiation towards cardiomyocytes. Stem Cell Reviews. 10 (6), 786-801 (2014).
  21. Tohyama, S., et al. Glutamine Oxidation Is Indispensable for Survival of Human Pluripotent Stem Cells. Cell Metabolism. 23 (4), 663-674 (2016).
  22. Tohyama, S., et al. Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell. 12 (1), 127-137 (2013).
  23. Tu, C., Chao, B. S., Wu, J. C. Strategies for Improving the Maturity of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Circulation Research. 123 (5), 512-514 (2018).
  24. Lundy, S. D., Zhu, W. Z., Regnier, M., Laflamme, M. A. Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Stem Cells and Development. 22 (14), 1991-2002 (2013).
  25. Hwang, H. S., et al. Comparable calcium handling of human iPSC-derived cardiomyocytes generated by multiple laboratories. Journal of Molecular and Cellular Cardiology. 85, 79-88 (2015).
  26. Scuderi, G. J., Butcher, J. Naturally Engineered Maturation of Cardiomyocytes. Frontiers in Cell and Developmental Biology. 5, 50 (2017).
  27. Jung, G., et al. Time-dependent evolution of functional vs. remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. 30 (4), 1464-1479 (2016).
check_url/60135?article_type=t

Play Video

Cite This Article
Oh, J. G., Dave, J., Kho, C., Stillitano, F. Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization. J. Vis. Exp. (155), e60135, doi:10.3791/60135 (2020).

View Video