人成体和胎心标本中原发性心外膜细胞的分离培养
Summary
心外膜在心脏的发育和修复中起着至关重要的作用, 它为心肌壁提供细胞和生长因子。在这里, 我们描述了一种培养人原发性心外膜细胞的方法, 能够对它们的发育和成人特征进行研究和比较。
Abstract
心外膜是一种覆盖心肌的上皮细胞层, 在心脏发育过程中, 以及在缺血性损伤后心脏的修复反应中具有重要作用。当激活时, 心外膜细胞经历了称为上皮间质转移的过程, 为再生心肌提供细胞。此外, 心外膜通过分泌基本的分泌因子而做出贡献。为了充分了解心外膜的再生潜能, 需要一个人类细胞模型。在这里, 我们概述了一个新的细胞培养模型, 从人类成年和胎儿心脏组织中提取原发性心外膜衍生细胞 (EPDCs)。为了分离 EPDCs, 心外膜从心脏标本的外部进行解剖, 并处理成单个细胞悬浮。其次, EPDCs 被镀和培养在 EPDC 培养基含有5激酶抑制剂 SB431542 保持其上皮表型。TGFβ刺激诱发急诊。这种方法首次能够在控制的环境中对人心外膜急诊的过程进行研究, 并有助于在 EPDCs 的 secretome 中获得更多的洞察力, 从而有助于心脏再生。此外, 这种统一的方法可以直接比较成人和胎儿的心外膜行为。
Introduction
心外膜, 一个单细胞上皮层, 信封心脏, 是至关重要的心脏发育和修复 (在史密特et 。1). 发育, 心外膜产生于 proepicardial 器官, 一个小结构位于发展的心脏基地。在发展天 E9.5 在老鼠和4星期以后构想在人, 细胞开始从这个花椰菜结构迁移并且包括开发的心肌2。一旦形成单个上皮细胞层, 心外膜细胞的一部分就会进行上皮间质的转移 (急诊室)。在急诊室, 细胞失去了上皮特征, 如细胞粘连, 并获得一个间充质表型, 使他们有能力迁移到发展中的心肌。经形成的心外膜衍生细胞 (EPDCs) 可分化为多种心肌细胞类型, 包括成纤维细胞、平滑肌细胞和潜在心肌细胞和内皮干细胞3, 虽然后两个细胞的分化人口仍需进行辩论 (在史密特等) 中进行审查。4). 此外, 心外膜提供了指导分泌信号的心肌调节其生长和血管化5,6,7,8。多项研究表明, 心外膜形成受损导致心肌发育缺陷9,10, 血管11, 传导系统12, 强调至关重要的心外膜对心脏形成的贡献。
虽然在成人心脏心外膜是存在作为休眠层数, 它在缺血13以后变得激活。心外膜重新激活损伤后概括为心脏发育, 包括增殖和急诊14所描述的几个过程, 尽管效率较低。有趣的是, 虽然确切的机制还没有完全理解, 但通过治疗可以改善心外膜对修复的贡献,例如, 胸腺素β415或修改 VEGF-mRNA16, 从而改善心脏心肌梗死后功能。因此, 心外膜被认为是一个有趣的细胞来源, 以加强内源性修复受伤的心脏。
心脏发育的机制经常概括在损伤期间, 虽然以一种较不有效的方式。为了寻找心外膜激活剂, 我们可以确定和比较胎儿和成人心外膜的全部容量, 这是至关重要的。此外, 从治疗的角度来看, 重要的是, 除了动物实验外, 我们还要对人类心外膜的反应提供知识。在这里, 我们描述了一种方法, 以分离和培养人成体和胎儿心外膜衍生细胞 (EPDCs) 在上皮细胞样的形态学和诱发急诊。本模型旨在探讨和比较成人和胎儿心外膜细胞的行为。
该协议的主要优点是使用了人的心外膜材料, 尚未深入研究。重要的是, 所描述的隔离和细胞培养协议提供了一个统一的方法来获得胎儿和成人鹅卵石 EPDCs, 使这两个细胞来源的直接比较。此外, 由于心外膜是根据其位置隔离的, 因此确保单元格实际上是 epicardially 派生的17。
虽然以前已经建立了人类的 EPDC 隔离方法, 但它们大多依赖于将心脏或心外膜组织镀到细胞培养皿18,19的生长协议。这种方法可以专门为部分丧失其上皮表型的细胞进行移植, 更容易接受自发的急诊急救。在当前的协议中, 心外膜首先被处理成一个单一的细胞溶液, 允许隔离的 EPDCs 保持其上皮状态。因此, 该方法提供了一个固体的体外模型来研究心外膜急诊。
Protocol
Representative Results
Discussion
在这里, 我们描述了一个详细的协议, 以隔离和培养从人类成年和胎儿心脏的原发性心外膜细胞。这些单元格的广泛描述以前已发布17。我们已经表明, 两种细胞类型可以保持作为上皮鹅卵石样细胞, 当培养与 ALK5 激酶抑制剂 SB431542。在发育和损伤后反应过程中, 急诊急救是心外膜激活的体内的一个组成部分。通过添加 TGFβ, 可以对急诊医师进行研究。重要的是, 我们以前观察…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究得到了荷兰科学研究组织 (NWO) (VENI 016.146.079) 的支持, 还有一个 LUMC 研究金与 AMS, LUMC Bontius Stichting (MJG)。
Materials
| Dulbecco’s modified Eagle’s medium + GlutaMAX | Gibco | 21885-025 | |
| Medium 199 | Gibco | 31150-022 | |
| Fetal Bovine Serum | Gibco | 10270-106 | |
| Trypsin 0.25% | Invitrogen | 25200-056 | |
| Penicillin G sodium salt | Roth | HP48 | |
| Streptomycin sulphate | Roth | HP66 | |
| Trypsin 1:250 from bovine pancreas | Serva | 37289 | |
| EDTA | Sigma | E4884 | |
| Gelatin from porcine skin | Sigma-Aldrich | G1890 | |
| Culture plates 6 well | Greiner bio-one | 657160 | |
| Culture plates 12 well | Corning | 3512 | |
| Culture plates 24 well | Greiner bio-one | 662160 | |
| SB 431542 | Tocris | 1614 | |
| Dimethyl Sulfoxide (DMSO) | Merck | 102931 | |
| 100-1000µL Filtered Pipet Tips | Corning | 4809 | |
| 10-ml pipet | Greiner bio-one | 607180 | |
| 5-ml pipet | Greiner bio-one | 606180 | |
| Cell culture dish 100/20 mm | Greiner bio-one | 664160 | |
| PBS | Gibco | 10010056 | Or home-made and sterilized |
| Eppendorf tubes 1.5 mL | Eppendorf | 0030120086 | |
| 15-ml centrifuge tubes | Greiner bio-one | 188271 | |
| 50-ml centrifuge tubes | Greiner bio-one | 227261 | |
| 10 mL Syringe | Becton Dickinson | 305959 | |
| Needles 19 Gauge | Becton Dickinson | 301700 | |
| Needles 21 Gauge | Becton Dickinson | 304432 | |
| EASYstrainer Cell Sieves, 100 µm | Greiner bio-one | 542000 | |
| TGFβ3 | R&D systems | 243-B3 | |
| Monoclonal Anti-Actin, α-Smooth Muscle | Sigma | A2547 | |
| Anti-Mouse Alexa Fluor 555 | Invitrogen | A31570 | |
| Alexa Fluor 488 Phalloidin | Invitrogen | A12379 | |
| Equipment | |||
| Name | Company | Catalog Number | Comments |
| Pipet P1,000 | Gilson | F123602 | |
| Pipet controller | Integra | 155 015 | |
| Stereomicroscope | Leica | M80 | |
| Inverted Light Microscope | Olympus | CK2 | |
| Centrifuge | Eppendorf | 5702 | |
| Waterbath | GFL | 1083 |
Referências
- Smits, A. M., Dronkers, E., Goumans, M. J. The epicardium as a source of multipotent adult cardiac progenitor cells: Their origin, role and fate. Pharmacological research. 127, 129-140 (2017).
- Risebro, C. A., Vieira, J. M., Klotz, L., Riley, P. R. Characterisation of the human embryonic and foetal epicardium during heart development. Development. 142 (21), 3630-3636 (2015).
- Zhou, B., Ma, Q., et al. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature. 454 (7200), 109-113 (2008).
- Smits, A., Riley, P. Epicardium-Derived Heart Repair. Journal of Developmental Biology. 2 (2), 84-100 (2014).
- Chen, T. H. P., Chang, T. C., et al. Epicardial Induction of Fetal Cardiomyocyte Proliferation via a Retinoic Acid-Inducible Trophic Factor. Biologia do Desenvolvimento. 250 (1), 198-207 (2002).
- Pennisi, D. J., Ballard, V. L. T., Mikawa, T. Epicardium is required for the full rate of myocyte proliferation and levels of expression of myocyte mitogenic factors FGF2 and its receptor, FGFR-1, but not for transmural myocardial patterning in the embryonic chick heart. Developmental Dynamics. 228 (2), 161-172 (2003).
- Lavine, K. J., Yu, K., et al. Endocardial and epicardial derived FGF signals regulate myocardial proliferation and differentiation in vivo. Developmental Cell. 8 (1), 85-95 (2005).
- Stuckmann, I., Evans, S., Lassar, A. B. Erythropoietin and retinoic acid, secreted from the epicardium, are required for cardiac myocyte proliferation. Developmental biology. 255 (2), 334-349 (2003).
- Männer, J., Schlueter, J., Brand, T. Experimental analyses of the function of the proepicardium using a new microsurgical procedure to induce loss-of-proepicardial-function in chick embryos. Developmental Dynamics. 233 (4), 1454-1463 (2005).
- Weeke-Klimp, A., Bax, N. A. M., et al. Epicardium-derived cells enhance proliferation, cellular maturation and alignment of cardiomyocytes. Journal of Molecular and Cellular Cardiology. 49 (4), 606-616 (2010).
- Eralp, I., Lie-Venema, H., et al. Coronary Artery and Orifice Development Is Associated With Proper Timing of Epicardial Outgrowth and Correlated Fas Ligand Associated Apoptosis Patterns. Circulation Research. 96 (5), (2005).
- Kelder, T. P., Duim, S. N., et al. The epicardium as modulator of the cardiac autonomic response during early development. Journal of Molecular and Cellular Cardiology. 89, 251-259 (2015).
- van Wijk, B., Gunst, Q. D., Moorman, A. F. M., van den Hoff, M. J. B. Cardiac regeneration from activated epicardium. PloS one. 7 (9), e44692 (2012).
- Zhou, B., Honor, L. B., et al. Adult mouse epicardium modulates myocardial injury by secreting paracrine factors. The Journal of clinical investigation. 121 (5), 1894-1904 (2011).
- Smart, N., Bollini, S., et al. De novo cardiomyocytes from within the activated adult heart after injury. Nature. 474 (7353), 640-644 (2011).
- Zangi, L., Lui, K. O., et al. Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Nature Biotechnology. 31 (10), 898-907 (2013).
- Moerkamp, A. T., Lodder, K., et al. Human fetal and adult epicardial-derived cells: a novel model to study their activation. Stem Cell Research & Therapy. 7 (1), 1-12 (2016).
- Clunie-O’Connor, C., Smits, A. M., et al. The Derivation of Primary Human Epicardium-Derived Cells. Current Protocols in Stem Cell Biology. 35, 2C.5.1-2C.5.12 (2015).
- Van Tuyn, J., Atsma, D. E., et al. Epicardial Cells of Human Adults Can Undergo an Epithelial-to- Mesenchymal Transition and Obtain Characteristics of Smooth Muscle Cells In Vitro. Stem Cells. 25 (2), 271-278 (2007).
- Bax, N. A. M., van Oorschot, A. A. M., et al. In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGFβ-signaling and WT1. Basic research in cardiology. 106 (5), 829-847 (2011).
- Chechi, K., Richard, D. Thermogenic potential and physiological relevance of human epicardial adipose tissue. International Journal of Obesity Supplements. 5 (Suppl 1), S28-S34 (2015).
