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。
1Division of Regenerative Medicine, Center for Molecular Medicine,Jichi Medical University, 2Department of Pediatrics,Jichi Medical University, 3Institute of Global Innovation Research,Tokyo University of Agriculture and Technology, 4Department of Precision Mechanics, Faculty of Science and Engineering,Chuo University
Summary
该方法可用于使用多能干细胞衍生心肌细胞与荧光标记肉瘤蛋白来检查肉瘤缩短。
Abstract
多能干细胞衍生心肌细胞（PSC-CMs）可以同时从胚胎干细胞和诱导多能干细胞（ES/iPS）细胞中产生。这些细胞为心脏病建模提供了有希望的来源。对于心肌病，肉瘤缩短是标准生理评估之一，用于成人心肌细胞检查其疾病表型。然而，现有的方法不适合评估PCS-CM的收缩性，因为这些细胞的肉瘤发育不全，在相对比显微镜下是看不见的。为了解决这个问题，并用PCC-CMs进行肉瘤缩短，使用了荧光标记的肉瘤蛋白和荧光活成像。细 Z 线和 M 线分别位于肉瘤的两端和中心。Z线蛋白 – α-Actinin （ACTN2）、电传素 （TCAP） 和与 ACTIN 相关的 LIM 蛋白 （PDLIM3） 和一种 M 线蛋白–肌素-2 （Myom2） 被标记为荧光蛋白。这些标记的蛋白质可以从内源性等位基因作为敲击或腺相关病毒 （AAV） 表达。在这里，我们介绍了将小鼠和人类多能干细胞分化到心肌细胞、产生 AAV 以及执行和分析活成像的方法。我们还描述了为 PSC-CM 的图案培养物生产聚二甲基硅氧烷 （PDMS） 邮票的方法，这有助于分析带有荧光标记的蛋白质的肉瘤缩短。为了评估肉瘤缩短，在电刺激（0.5-1 Hz）下，以高帧速率（每秒50-100帧）记录了跳动细胞的延时图像。为了分析细胞收缩过程中的肉瘤长度，录制的延时图像受制于 SarcOptiM，这是 ImageJ/Fiji 的插件。我们的战略为研究 PSC-CMs 中的心脏病表型提供了一个简单的平台。
Introduction
心血管疾病是全世界1人死亡的主要原因，心肌病是心脏相关死亡的第三大原因。心肌病是影响心脏肌肉的一组疾病。诱导多能干细胞（iPS）细胞的最新发展以及iPS细胞对心肌细胞（PSC-CMs）的定向分化，为研究作为心肌病体外模型的患者基因组心肌细胞打开了大门。这些细胞可用于了解心脏病的病理生理学，阐明其分子机制，并测试不同的治疗候选者3。因此，产生了大量的兴趣，因此，产生了患者衍生的iPS细胞（例如，肥大性心肌病[HCM]4，5，心律失常右心室心肌病[ARVC]6，扩张性心肌病[DCM]7，和线粒体相关心肌病8，9）。因为心肌病的特征之一是肉瘤功能障碍和紊乱，因此需要一种能统一测量肉瘤功能的有效工具。
肉瘤缩短是评估肉瘤功能和从动物模型和人类衍生的成人心肌细胞收缩性最广泛使用的技术。要执行肉瘤缩短，需要在相对比下可见的发达肉瘤。然而，PSC-CMs培养的体外显示器不发达和混乱的肉瘤，因此，不能用于正确测量肉瘤缩短10。这种难以正确评估PCS-CMs收缩性的问题阻碍了它们作为体外评估心脏功能的平台的使用。为了间接评估PCS-CMs收缩性，原子力显微镜、微柱阵列、牵引力显微镜和阻抗测量被用来测量这些细胞对周围环境的运动效果。更复杂和侵入性较小的视频显微镜记录的实际细胞运动（例如，SI8000从索尼）可以用来替代评估其收缩性，但是，这种方法不能直接测量肉瘤运动或力生成动能14。
为了直接测量PCC-CMs中的肉瘤运动，新的方法，如荧光标记到肉瘤蛋白，正在出现。例如，Lifeact 用于标记丝状活性素 （F-actin） 来测量肉瘤运动15、16。转基因iPS细胞是另一种选择标记肉瘤蛋白（例如，α-actinin[ACTN2]和Myomesin-2[MYOM2]）由荧光蛋白17，18，19。
在本文中，我们描述了如何使用 Myom2-TagRFP（小鼠胚胎干细胞 [ES] 细胞） 和 ACTN2-mCherry（人类 iPS 细胞）执行测量肉瘤缩短的延时成像。我们还表明，有图案的文化有助于肉瘤的排列。此外，我们使用腺相关病毒 （AAV） 描述肉瘤标记的替代方法，该病毒可广泛应用于患者衍生的 iPS 细胞。
Protocol
1. 小鼠多能干细胞的分化 维护鼠标 ES 单元格 维护介质：混合 50 mL 的胎儿牛血清 （FBS）、5 mL 的 L-丙氨酸-L-谷氨酰胺、5 mL 的非必需氨基酸 （NEAA）、5 mL 的 100 mM 皮鲁瓦酸钠和 909 μl 的 55 mM 2-默卡普托乙醇与 450 mL 格拉斯哥最低基本介质 （GMEM）.。补充白血病抑制因子 （LIF）、CHIR-99021 和 PD0325901，最终浓度分别为 1000 U/mL、1 μM 和 3 μM。通过 0.22 μm 过滤器对介质进行消毒。 FB…
Representative Results
使用敲击 PSC-CMs 记者线测量肉瘤缩短。沙科默标签的 PSC-CM 用于测量肉瘤缩短。线条表达 Myom2 – rfp 和 Actn2 – mcherry 从内源性洛西。TagRFP 入Myom2 ，编码 M 蛋白本地化到 M 线，而 mCherry 被敲入ACTN2 ，编码α – 阿克蒂宁，本地化到 Z 线 18 ， 25 。获得延时图像，并用于确定肉瘤缩短，如图1和…
Discussion
PSC-CMs具有巨大的潜力，可以作为体外 平台来模拟心脏病和测试药物的效果。然而，必须首先建立一种准确、统一的方法来评估 PSC-CM 功能。大多数功能测试都与 PSC-CMs 配合使用，例如电生理学、钙瞬态和代谢26，而第一批患者衍生的 PSC-CM 研究之一是关于长 QT 综合征27。然而，收缩性是心肌细胞最重要的功能之一，仍然难以评估。成人心肌细胞，肉瘤缩?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢济池医科大学再生医学系的所有实验室成员提供有益的讨论和技术援助。这项研究得到了日本医学研究开发厅（AMED）的资助：JP18bm0704012 和 JP20bm0804018），日本科学促进会 （JSPS;JP19KK0219），以及日本流通协会（基础研究补助金）给H.U.
Materials
| 1-Thioglycerol | Sigma-Aldrich | M6145-25 | |
| 2-Mercaptoethanol (55mM) | Thermo Fisher Scientific | 21985-023 | |
| 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, | NOF Corp. | LIPIDURE-CM5206 | |
| 2-Propanol | Fujifilm wako | 166-04836 | |
| 35-mm imaging dish with a polymer coverslip (µ-Dish 35 mm, high) | ibidi | 81156 | |
| AAVproR Helper Free System (AAV6) (vectors; pHelper, pRC6, pAAV-CMV-Vector) |
Takara | 6651 | |
| ACTN2-mCherry (AR12, AR21) hiPSCs | N.A. | We inserted IRES-puromycin resistant casette to 3' UTR of TNNT2 locus and mCherry around the stop codon of ACTN2 in 610B1 hiPSC line, following a method describe elsewhere (Anzai, Methods Mol Biol, in press) | |
| B-27 Supplement (50X), serum free | Thermo Fisher Scientific | 17504-044 | |
| B-27 Supplement, minus insulin | Thermo Fisher Scientific | A18956-01 | |
| B27 supplement (50X), minus Vitamin A | Thermo Fisher Scientific | 12587-010 | |
| Benzonase (25 U/µL) | Merck Millipore | 70746 | |
| Blasticidin S Hydrochloride | Fujifilm wako | 029-18701 | |
| BMP-4, Human, Recombinant, | R&D Systems, Inc. | 314-BP-010 | |
| Bovine Serum Albumin | Sigma-Aldrich | A4503-100g | |
| C59, Wnt Antagonist (WntC59) | abcam | ab142216 | |
| CAD drawing software, | Robert McNeel and Associates, WA, USA | Rhinoceros 6.0 | |
| Centrifugal ultrafiltration unit (100k MWCO), Vivaspin-20 | Sartorius | VS2042 | |
| CHIR99021 | Cayman | 13122 | |
| Chromium etchant | Nihon Kagaku Sangyo Co., Ltd., Japan | N14B | |
| Chromium mask coated with AZP1350 | Clean Surface Technology Co., Japan | CBL2506Bu-AZP | |
| Dr. GenTLE Precipitation Carrier (20mg/mL Glycogen, 3 M Sodium Acetate (pH 5.2)) | Takara | 9094 | |
| Dulbecco’s Modified Eagle’s Medium (DMEM) – high glucose | Sigma-Aldrich | D6429-500 | |
| Dulbecco’s Modified Eagle’s Medium (DMEM) – high glucose, without sodium pyruvate | Sigma-Aldrich | D5796 | |
| Ethanol (99.5) | Fujifilm wako | 057-00456 | |
| Fetal Bovine Serum | Moregate | 59301104 | |
| FGF-10, Human, Recombinant, | R&D Systems, Inc. | 345-FG-025 | |
| Fibroblast Growth Factor(basic), human, recombinant | Fujifilm wako | 060-04543 | |
| Gelatin from porcine skin powder | Sigma-Aldrich | G1890-100g | |
| Glasgow Minimum Essential Medium (GMEM) | Sigma-Aldrich | G5154-500 | |
| GLASS BOTTOM culture plates | MatTek | P24G-1.5-13-F/H | |
| Ham’s F-12 | Thermo Fisher Scientific | 11765-062 | |
| Iscove's Modified Dulbecco's Medium (IMDM) | Thermo Fisher Scientific | 12440-061 | |
| L-alanine-L-glutamine (GlutaMAX Supplement, 200mM) | Thermo Fisher Scientific | 35050-061 | |
| L(+)-Ascorbic Acid Sodium Salt | Fujifilm wako | 196-01252 | |
| Laminin-511 E8 fragment (LN511-E8, iMatrix-511) | Nippi | 892012 | |
| Mask aligner | Union Optical Co., Ltd., Japan | PEM-800 | |
| Maskless lithography tool | NanoSystem Solutions, Inc., Japan | D-Light DL-1000 | |
| MEM Non-Essential Amino Acids Solution (100X) | Thermo Fisher Scientific | 11140-050 | |
| Millex-HV Syringe Filter Unit, 0.45 µm, PVDF (0.45-µm filter) | Merck Millipore | SLHVR33RS | |
| Myom2-RFP (SMM18) | N.A. | Developed in our previous paper (Chanthra, Sci Rep, 2020) | |
| N-2 Supplement (100X) | Thermo Fisher Scientific | 17502-048 | |
| ORCA-Flash4.0 V3 digital CMOS camera | Hamamatsu | C13440-20CU | |
| PD0325901 | Stemgent | 04-0006-10 | |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140-122 | |
| Petri dish | Sansei medical co. Ltd | 01-004 | |
| Phenol/Chloroform/Isoamyl alcohol (25:24:1) | Nippon Gene | 311-90151 | |
| Polydimethylsiloxane (PDMS) elastomer | Dow Corning Corp., MI, USA | SILPOT 184 | |
| polyethylenimine MAX (MW. 40,000) | Polyscience | 24765-1 | |
| Positive photoresist developer | Tokyo Ohka Kogyo Co., Ltd., Japan | NMD-3 | |
| PowerUp SYBR Green Master Mix | Thermo Fisher Scientific | A25742 | |
| Proteinase K | Takara | 9034 | |
| Puromycin Dihydrochloride | Fujifilm wako | 166-23153 | |
| Recombinant Human/Mouse/Rat Activin A Protein | R&D Systems, Inc. | 338-AC-050 | |
| Recombinant trypsin-like protease (rTrypsin; TrypLE express) | Thermo Fisher Scientific | 12604-039 | |
| RPMI1640 Medium | Thermo Fisher Scientific | 11875-119 | |
| Silicon wafer | Matsuzaki Seisakusyo Co., Ltd., Japan | N.A. | |
| Sodium Pyruvate (100 mM) | Thermo Fisher Scientific | 11360-070 | |
| Spin-coater | Mikasa Co., Ltd., Japan | MS-A100 | |
| Spininng confocal microscopy | Oxford Instruments | Andor Dragonfly Spinning Disk System | |
| StemSure LIF, Mouse, recombinant, Solution (10^6U) | Fujifilm wako | 195-16053 | |
| SU-8 3010 | Kayaku Advanced Materials, Inc., MA, USA | SU-8 3010 | |
| SU-8 developer | Kayaku Advanced Materials, Inc., MA, USA | SU-8 developer | |
| Tris-EDTA | Nippon Gene | 314-90021 | |
| Vascular Endothelial Growth Factor-A165(VEGF), Human, recombinant | Fujifilm wako | 226-01781 |
Referências
- Mensah, G. A., Roth, G. A., Fuster, V. The Global Burden of Cardiovascular Diseases and Risk Factors. Journal of the American College of Cardiology. 74 (20), 2529-2532 (2019).
- Wexler, R. K., Elton, T., Pleister, A., Feldman, D. Cardiomyopathy: an overview. American Family Physician. 79 (9), 778-784 (2009).
- Parrotta, E. I., Lucchino, V., Scaramuzzino, L., Scalise, S., Cuda, G. Modeling Cardiac Disease Mechanisms Using Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Progress, Promises and Challenges. International Journal of Molecular Sciences. , 30 (2020).
- Carvajal-Vergara, X., et al. Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome. Nature. 465 (7299), 808-812 (2010).
- 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).
- Kim, C., et al. Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs. Nature. 494 (7435), 105-110 (2013).
- Gramlich, M., et al. Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy. EMBO Molecular Medicine. 7 (5), 562-576 (2015).
- Wang, G., et al. Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nature Medicine. 20 (6), 616-623 (2014).
- Li, S., et al. Mitochondrial Dysfunctions Contribute to Hypertrophic Cardiomyopathy in Patient iPSC-Derived Cardiomyocytes with MT-RNR2 Mutation. Stem Cell Reports. 10 (3), 808-821 (2018).
- Cho, G. -. S., et al. Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy. Cell Reports. 18 (2), 571-582 (2017).
- Yang, X., et al. Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells. Journal of Molecular and Cellular Cardiology. 72, 296-304 (2014).
- Ribeiro, A. J. S., et al. Multi-Imaging Method to Assay the Contractile Mechanical Output of Micropatterned Human iPSC-Derived Cardiac Myocytes. Circulation Research. 120 (10), 1572-1583 (2017).
- Sewanan, L. R., Campbell, S. G. Modelling sarcomeric cardiomyopathies with human cardiomyocytes derived from induced pluripotent stem cells. The Journal of Physiology. 598 (14), 2909-2922 (2020).
- Kopljar, I., et al. Chronic drug-induced effects on contractile motion properties and cardiac biomarkers in human induced pluripotent stem cell-derived cardiomyocytes. British Journal of Pharmacology. 174 (21), 3766-3779 (2017).
- Riedl, J., et al. Lifeact: a versatile marker to visualize F-actin. Nature Methods. 5 (7), 605-607 (2008).
- Ribeiro, A. J. S., et al. Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness. Proceedings of the National Academy of Sciences. 112 (41), 12705-12710 (2015).
- Roberts, B., et al. Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs. Stem Cell Reports. 12 (5), 1145-1158 (2019).
- Chanthra, N., et al. A Novel Fluorescent Reporter System Identifies Laminin-511/521 as Potent Regulators of Cardiomyocyte Maturation. Scientific Reports. 10 (1), 4249 (2020).
- Ribeiro, M. C., et al. A cardiomyocyte show of force: A fluorescent alpha-actinin reporter line sheds light on human cardiomyocyte contractility versus substrate stiffness. Journal of Molecular and Cellular Cardiology. 141, 54-64 (2020).
- Yamanaka, S., Zahanich, I., Wersto, R. P., Boheler, K. R. Enhanced Proliferation of Monolayer Cultures of Embryonic Stem (ES) Cell-Derived Cardiomyocytes Following Acute Loss of Retinoblastoma. PLoS ONE. 3 (12), 3896 (2008).
- Miyazaki, T., Isobe, T., Nakatsuji, N., Suemori, H. Efficient Adhesion Culture of Human Pluripotent Stem Cells Using Laminin Fragments in an Uncoated Manner. Scientific Reports. 7 (1), 41165 (2017).
- Prasad, K. -. M. R., Xu, Y., Yang, Z., Acton, S. T., French, B. A. Robust cardiomyocyte-specific gene expression following systemic injection of AAV: in vivo gene delivery follows a Poisson distribution. Gene Therapy. 18 (1), 43-52 (2011).
- Arakawa, H., et al. Protein evolution by hypermutation and selection in the B cell line DT40. Nucleic Acids Research. 36 (1), e1 (2007).
- Konno, A., Hirai, H. Efficient whole brain transduction by systemic infusion of minimally purified AAV-PHP.eB. Journal of Neuroscience Methods. 346, 108914 (2020).
- Anzai, T., et al., Yoshida, Y., et al. Generation of Efficient Knock-in Mouse and Human Pluripotent Stem Cells Using CRISPR-Cas9. Methods of Molecular Biology. , (2020).
- Ahmed, R. E., Anzai, T., Chanthra, N., Uosaki, H. A Brief Review of Current Maturation Methods for Human Induced Pluripotent Stem Cells-Derived Cardiomyocytes. Frontiers in Cell and Developmental Biology. 8, 178 (2020).
- 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).
- Flores, L. R., Keeling, M. C., Zhang, X., Sliogeryte, K., Gavara, N. Lifeact-GFP alters F-actin organization, cellular morphology and biophysical behaviour. Scientific Reports. 9 (1), 3241 (2019).
- Sparrow, A. J., et al. Measurement of Myofilament-Localized Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy Mutations. Circulation Research. 124 (8), 1228-1239 (2019).
Citar este artigo
Ahmed, R. E., Chanthra, N., Anzai, T., Koiwai, K., Murakami, T., Suzuki, H., Hanazono, Y., Uosaki, H. Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.. J. Vis. Exp. (169), e62129, doi:10.3791/62129 (2021).