转录组水平上人类原发性肠系膜动脉内皮细胞的分离和分析
Summary
该方案描述了从人肠系膜动脉中分离、培养和分析内皮细胞。此外，还提供了一种制备用于空间转录组学的人类动脉的方法。蛋白质组学，转录组学和功能测定可以在分离的细胞上进行。该方案可以重新用于任何中型或大型动脉。
Abstract
内皮细胞（EC）通过其对环境线索的动态反应，对血管和全身功能至关重要。阐明EC的转录组和表观基因组对于了解它们在发育，健康和疾病中的作用至关重要，但分离的原代细胞的可用性有限。最近的技术已经实现了EC转录组和表观基因组的高通量分析，从而鉴定了以前未知的EC细胞亚群和发育轨迹。虽然EC培养物是探索EC功能和功能障碍的有用工具，但培养条件和多个传代可以引入改变天然EC性质的外部变量，包括形态学，表观遗传状态和基因表达程序。为了克服这一局限性，本文展示了一种从供体肠系膜动脉中分离出人类原发性EC的方法，旨在捕获其天然状态。内膜层中的ECs通过使用特定的酶在机械和生化上解离。所得细胞可直接用于本体RNA或单细胞RNA测序或接种用于培养。此外，描述了用于制备用于空间转录组学的人类动脉组织的工作流程，特别是用于市售平台，尽管该方法也适用于其他空间转录组分析技术。该方法可以应用于从健康或疾病状态的各种供体收集的不同血管，以深入了解EC转录和表观遗传调节，这是内皮细胞生物学的一个关键方面。
Introduction
内皮细胞（EC）衬里血管腔内，是血管张力和组织灌注的关键调节因子。EC在对细胞外环境做出反应并适应血流动力学和组成变化的能力方面非常出色。这些动态反应通过细胞内信号传导事件网络介导，包括具有时空分辨率的转录和转录后调制。这些反应的失调与许多病理学有关，包括但不限于心血管疾病，糖尿病和癌症1，2。
很大一部分研究利用细胞系或动物模型来询问EC转录组。前者是一个有用的工具，因为相对易于使用和便宜。然而，连续培养可以给EC引入表型改变，例如成纤维细胞特征和缺乏极化，使它们与 体内 状态3断开连接。原代细胞，例如人脐静脉EC（HUVEC）自20世纪80年代以来一直是一种流行的选择，但来源于成人中不存在的发育性血管床，因此不太可能完全代表成熟的EC。动物，特别是小鼠模型，更好地代表了EC的生理或病理生理环境，并允许由于遗传扰动而对转录组进行询问。鼠EC可以从各种组织中分离，包括主动脉，肺和脂肪组织，使用基于酶的程序4，5，6，7。然而，分离的细胞不能用于多次传代，除非转化6 并且通常数量有限，这需要从多个动物5，8，9中汇集。
在转录组学水平上探索血管结构的新技术的出现，特别是单细胞分辨率，通过揭示ECs 5，10，11，12，13，14的新功能和特性，实现了内皮生物学的新时代。由Tabula Muris研究人员建立的丰富资源收集了100，000个细胞的单细胞转录组谱，包括来自20个不同小鼠器官的EC15的EC，揭示了具有组织间和组织内差异的独特转录组特征5，13。然而，小鼠和人类在基因组，表观基因组和转录组之间存在明显差异，特别是在非编码区域16，17，18中。上述缺点强调了使用人类样本分析EC的重要性，以便获得EC在健康和疾病中的原生状态的忠实特征。
大多数EC分离方法依赖于通过均质化，精细切割和组织切碎的物理解离，然后与蛋白水解酶一起孵育不同时间。酶和条件在组织类型之间也有很大差异，从胰蛋白酶到胶原酶，单独使用或组合使用19，20，21。通常包括进一步的基于抗体的富集或纯化，以提高EC的纯度。通常，针对EC膜标志物的抗体，例如CD144和CD31与磁珠偶联并加入细胞悬浮液22，23中。这种策略通常可以适用于从多个人和小鼠组织中分离EC，包括该方案中引入的技术。
在它们的天然状态下，EC与多种细胞类型相互作用，并且可能存在于血管壁龛中，其中细胞接近性对功能至关重要。虽然单细胞和单核RNA测序（scRNA和snRNA-seq）研究对于最近在描述EC异质性方面取得的突破至关重要，但解离过程破坏了组织环境和细胞 – 细胞接触，这对于理解EC生物学也很重要。空间转录组分析于2012年开发，并于2020年被评为年度方法24，已用于分析全球基因表达，同时保留各种组织中的空间特征，包括脑25，肿瘤26和脂肪组织27。这些技术可以靶向，使用特异性于附着在亲和试剂或荧光标签上的特定RNA序列的专用探针，从而以亚细胞分辨率28，29，30，31检测选择基因。它们也可以是无靶向的32，33，通常使用空间条形码的寡核苷酸来捕获RNA，它们一起转化为cDNA用于随后的seq文库制备，因此具有以无偏倚方式推断整个组织基因表达的优点。然而，目前还没有使用市售技术在单一蜂窝水平上实现空间分辨率。这可以通过与scRNA-seq数据的数据集成在一定程度上克服，最终允许在复杂组织环境中绘制单细胞转录组，同时保留其原始空间信息34。
在本文中，描述了使用人肠系膜上动脉（一种用于研究血管舒张，血管重塑，氧化应激和炎症的外周动脉）来分析EC转录组的工作流程35，36，37。描述了两种技术：1）从血管的内膜中分离和富集EC，结合机械解离和酶消化，适用于单细胞转录组测序或随后 的体外 培养;2）准备用于空间转录组分析的动脉切片（图1）。这两种技术可以独立进行，也可以互补地用于分析EC及其周围细胞。此外，该工作流程可以适应任何中型或大型动脉。
Protocol
人体组织研究是从希望之城的南加州胰岛细胞资源中心获得的去识别标本上进行的。使用死后人体组织的研究同意是从捐赠者的近亲那里获得的，并且这项研究的伦理批准由希望之城的机构审查委员会（IRB No. 01046）授予。 1.物理解离（估计时间：1-2小时） 将新鲜动脉放在10厘米的培养皿上，并用无菌的Dulbecco磷酸盐缓冲盐水（D-PBS）清洗。 用无菌?…
Representative Results
这里描述了使用机械和酶解离或冷冻保存的组合来分析肠系膜动脉的EC，用于各种下游测定（图1）。可以使用以下步骤在肠系膜动脉中分析EC：A）从内膜的机械解离与胶原酶消化到培养细胞相结合;B）产生用于scRNA-seq的单细胞悬浮液;或C）动脉的横截面可以嵌入OCT中进行冷冻切片以分析空间转录组（图1 和 图2）。 <p class="jove_con…
Discussion
所介绍的工作流程详细介绍了一组技术，以单细胞和空间分辨率从单条人类动脉中分析EC。协议中有几个关键步骤和限制因素。转录组分析的一个关键是组织的新鲜度和RNA的完整性。在加工之前尽可能多地将组织保持在冰上以尽量减少RNA降解非常重要。通常，在死亡后8-14小时之间处理死后组织。然而，建议在从供体中提取后尽快开始分离或冷冻保存。特别是对于空间转录组映射，组织应保持在干?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH拨款R01HL108735，R01HL145170，R01HL106089（Z.B.C.）的支持;DP1DK126138 和 DP1HD087990（转 S.Z.）;艾拉·菲茨杰拉德基金会的赠款和瓦内克家庭项目（向Z.B.C.）;以及人类细胞图谱种子网络赠款（授予Z.B.C.和S.Z.）。本出版物中报告的研究包括在希望之城的综合基因组学核心中进行的工作，该工作由美国国立卫生研究院国家癌症研究所支持，奖励编号为P30CA033572。作者要感谢希望之城胰岛移植团队的Ismail Al-Abdullah博士和Meirigeng Qi博士隔离人体组织，City of Hope的Dongqiang Yuan博士协助进行scRNA-seq分析，以及约翰霍普金斯大学医学院心血管病理学部门的Marc Halushka博士对血管组织学的宝贵见解。
Materials
| 1.5 mL micro-centrifuge tube | USA Scientific | 1615-5500 | |
| 10 cm dish | Genesee Scientific | 25-202 | |
| 23G needles | BD | 305145 | |
| 2-methylbutane | Thermo Fisher | AC327270010 | |
| 40 µm strainer | Fisher | 14100150 | |
| 4200 TapeStation System | Agilent Technologies | G2991BA | |
| 5 mL tube | Thermo Fisher | 14282300 | |
| 6-well plate | Greiner Bio-One | 07-000-208 | |
| Attachment factor | Cell Applications | 123-500 | Attachment reagent in the protocol |
| Black wax | Any commercial black wax can be used | ||
| Bovine serum albumin heat shock treated | Fisher | BP1600-100 | |
| CaCl2 | Fisher | BP510 | |
| Centrifuge | Eppendorf | ||
| Chloroform | Fisher | C607 | |
| Collagenase D | Roche | 11088866001 | |
| Cryostat | Leica | ||
| Cryostat brushes | |||
| D-Glucose | Fisher | D16-1 | |
| Dimethyl sulfoxide | Fisher | MT25950CQC | |
| Dispase II | Roche | 4942078001 | Bacteria-derived protease in the protocol |
| Disposable Safety Scalpels | Myco Instrumentation | 6008TR-10 | |
| D-PBS | Thermo Fisher | 14080055 | |
| Ethanol | Fisher | BP2818-4 | |
| Fetal bovine serum | Fisher | 10437028 | |
| Hemocytometer | Fisher | 267110 | |
| HEPES | Sigma Aldrich | H3375-100g | |
| High sensitivity D1000 sample buffer | Agilent Technologies | 5067-5603 | |
| High sensitivity D1000 screen tape | Agilent Technologies | 5067-5584 | |
| Incubator | Kept at 37 °C 5% CO2 | ||
| Isopropanol | Fisher | BP26324 | |
| KCl | Fisher | P217-3 | |
| Liquid nitrogen | |||
| Medium 199 | Sigma Aldrich | M2520-10X | |
| Metal cannister | |||
| Microscope | Leica | To assess cell morphology | |
| Microvascular endothelial culture medium | Cell Applications | 111-500 | |
| NaCl | Fisher | S271-1 | |
| New Brunswick Innova 44/44R Orbital shaker | Eppendorf | ||
| Optimal Cutting Temperature compound | Fisher | 4585 | |
| Plastic cryomolds | Fisher | 22363553 | |
| RNA screen tape | Agilent Technologies | 5067-5576 | |
| RNA screen Tape sample buffer | Agilent Technologies | 5067-5577 | |
| RNase ZAP | Thermo Fisher | AM9780 | |
| RNase-free water | Takara | RR036B | RNase-free water (2) in kit |
| Sterile 12" long forceps | F.S.T | 91100-16 | |
| Sterile fine forceps | F.S.T | 11050-10 | |
| Sterile fine scissors | F.S.T | 14061-11 | |
| Superfrost PLUS Gold Slides | Fisher | 1518848 | |
| TRIzol reagent | Fisher | 15596018 | |
| Trypan Blue | Corning | MT25900CI | |
| TrypLE Express Enzyme (1X) phenol red | Thermo Fisher | 12605010 | Cell-dissociation enzyme in the protocol |
| Visium Accessory Kit | 10X Genomics | PN-1000215 | |
| Visium Gateway Package, 2rxns | 10X Genomics | PN-1000316 | |
| Visium Spatial Gene Expression Slide & Reagent Kit, 4 rxns | 10X Genomics | PN-1000184 |
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Citar este artículo
Malhi, N. K., Luo, Y., Tang, X., Sriram, K., Calandrelli, R., Zhong, S., Chen, Z. B. Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level. J. Vis. Exp. (181), e63307, doi:10.3791/63307 (2022).