用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道
Summary
来自诱导多能干细胞(iPSC-EP)的内皮祖子有可能彻底改变心血管疾病的治疗,并能够创建更忠实的心血管疾病模型。本文介绍了iPSC-EP在三维(3D)胶原蛋白微环境中的封装,并对这些细胞的血管生成电位进行了定量分析。
Abstract
诱导多能干细胞(iPSCs)是一种患者特异性增殖细胞源,可分化成任何体细胞类型。双能内皮祖细胞(EP),可以分化成组装成熟、功能性血管所必需的细胞类型,从胚胎和诱导多能干细胞中提取。然而,这些细胞在三维环境中尚未得到严格评估,其血管生成潜力的定量测量仍然难以实现。在这里,首先概述了通过荧光激活细胞分类生成和分离 iPSC-EP,然后介绍了 iPSC-EP 在胶原蛋白水凝胶中的封装和培养。这种细胞外基质(ECM)模拟微环境鼓励强健的血管反应;血管网络形成后一个星期的文化。使用开源软件量化此血管反应的计算管道的创建进行了划定。此管道专为保留毛细管丛的 3D 体系结构而设计,以以最少的用户输入可靠地识别分支数、分支点和总网络长度。
Introduction
人类脐带静脉内皮细胞(HUVECs)和其他原发内皮细胞类型已经使用了20年,以模型血管发芽和在体外发育1。这种血管平台有望阐明心血管疾病的分子和组织级机制,并可能为原始血管网络2、3的发展提供生理见解。尽管血管建模领域取得了显著进展,但能够定量建模和评估生理血管发育的”金本位”测定仍然遥遥无期。大多数已公布的协议没有充分重述血管利基,以鼓励成熟、功能性血管的形成,或者没有方法在三维上定量比较评估细胞类型的血管生成电位(3D)。
许多目前的血管模型是有限的,他们的能力,模仿生理血管利基。最常用的体外平台之一是胶状蛋白混合物的管形成测定。简单地说,HUVECs被播种为单细胞,在薄薄的凝胶层上,由从肉瘤细胞外基质(ECM)中采集的蛋白质组成;在一至两天内,HUVECs自组装成原始管4。然而,这个过程发生在双维(2D)和内皮细胞(ECs)用于这个测定不形成封闭的,空心流明,从而限制了这些研究的生理意义。最近,EC和支持细胞(例如,中生干细胞(MSCs)和围细胞)在模拟原生ECM纤维结构的3D微环境中共同培养,如胶原蛋白或纤维蛋白水凝胶5。为了模拟这种微环境下的血管发育,通常采用涂有EC的聚合物珠子。加入其他细胞分泌的外源生长因子和/或生长因子,这些细胞间质嵌入在水凝胶中,可以诱导ECs,涂覆聚合物珠子,发芽并形成单流明;然后可以计算芽和容器的数量和直径。然而,这些芽是奇异的,不形成一个封闭的,连接的网络,如在生理条件下看到,因此更让人想起肿瘤血管模型。微流体装置也被用来模拟血管利基,并促进在EC载水凝胶7,8的血管的形成。通常,血管生成生长因子梯度应用于循环细胞培养基,以诱导EC迁移和发芽。构成发达容器流明的ECs对通过微流体装置的流体流动引起的剪切应力敏感;因此,这些微流体装置捕获静态模型中不可访问的关键生理参数。但是,这些设备需要昂贵的微制造能力。
最重要的是,所有三种血管模型(2D、3D、微流体)绝大多数使用初级EC以及主要支持细胞类型。原发性细胞不能发展成有效的心血管治疗,因为细胞在植入后会产生免疫反应;此外,HUVEC 和类似的原发性细胞类型不是患者特异性的,并且不会捕获遗传倾向或预先存在的健康状况(如糖尿病)患者发生的血管异常。诱导多能干细胞(iPSCs)在过去十年中已经作为一种患者特异性增殖细胞源出现,可以分化成人体中的所有体细胞9。特别是,已经发布了协议,概述了iPSC衍生的内皮祖子(iPSC-EP)10、11的生成和隔离。iPSC-EP 是双能的,因此可以进一步分化为内皮细胞和平滑肌细胞/细胞,成熟、功能性血管的构建基块。只有一项研究令人信服地详细说明了iPSC-EP在3D微环境中的初级毛细管丛的发育12;尽管本研究对于了解iPSC-EP总成和天然和合成水凝胶的分化至关重要,但它没有定量地比较所产生血管的网络拓扑。另一项最近的研究使用聚合物珠模型比较了HUVECs和iPSC衍生的EC5的发芽。因此,显然需要进一步阐明调节iPSC-EP血管生成的物理和化学信号机制,并确定这些细胞是否适合缺血治疗和心血管疾病建 模。
在过去十年中,开发了不同的开源计算管道和骨架化算法,以量化和比较血管网络长度和连接性。例如,Charwat等人开发了一种基于Photoshop的管道,从纤维蛋白基质13、14中脂肪衍生干细胞和生长EC的共同培养中提取出一个过滤的、双光化的血管网络图像。也许最广泛使用的拓扑比较工具是AngioTool,一个由国家癌症研究所15在线发布的程序;尽管该计划被广泛采用和充分记录保真度,该计划仅限于分析两维的容器状结构和其他程序,包括AngioSys和Wimasis,共享相同的维数限制16。开发了强大的软件套件,如 Imaris、Lucis 和变形,以分析工程微血管的网络拓扑;但是,对于大多数学术实验室来说,这些套件成本高昂,并且限制了对源代码的访问,这可能会妨碍最终用户根据特定应用程序自定义算法的能力。3D切片机,一个开源磁共振成像/计算机断层扫描包,包含一个血管建模工具包,可以有效地分析3D血管网络的拓扑17;但是,分析依赖于用户手动放置网络的端点,这在分析大型数据集时可能会变得单调乏味,并且可能受用户潜意识偏见的影响。在本手稿中,详细介绍了一个可量化 3D 血管网络的计算管道。为了克服上述限制,此开源计算管道利用 ImageJ 预处理获取的共聚焦图像,将 3D 卷加载到骨架分析器中。骨架分析仪采用平行中轴变薄算法,最初由Kerschnitzki等人开发,用于分析骨细胞网络18的长度和连通性;该算法可有效地描述工程微血管的长度和连通性。
总之,该协议概述了在3D微环境中微血管网络的创建,并提供了一个开源和用户偏差自由计算管道,以随时比较iPSC-EP的血管电位。
Protocol
Representative Results
Discussion
该协议涉及三种细胞培养基基中细胞的长期培养:E8、LaSR巴塞尔和EGM-2。因此,应非常小心,适当地消毒所有材料。此外,在实验室的层流罩中工作时,应始终佩戴实验室外套和乙醇清洁手套。建议经常检测支原体污染;如果在 iPSC 培养期间观察到大量碎屑或观察到分化效率突然下降,则支原体污染是可能的原因。使用该协议生成的 iPSC-EP 往往会沉积大量的 ECM,从而延长分离时间,并导致单个细胞悬浮液分离?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国心脏协会(授予J.Z.的15SDG25740035号)、美国国家卫生研究院国家生物医学成像和生物工程研究所(NIBIB)的支持(授予C.C.的赠款号EB007507),以及再生康复研究与培训联盟(AR3T,授予编号1 P2C HD086843-01,授予J.Z.)。我们要感谢Jeanne Stachowiak教授(德克萨斯大学奥斯汀分校)就共聚焦显微镜提供的技术建议。我们还感谢与塞缪尔·米赫利奇(德克萨斯大学奥斯汀分校)、艾丽西亚·艾伦博士(德克萨斯大学奥斯汀分校)、朱莉·雷特莱夫斯基博士(适应性生物技术)和莱昂·贝兰博士(范德比尔特大学)的讨论,以表达他们对三维网络的计算分析。最后,我们感谢鲍小平博士(加州大学伯克利分校)关于将iPSC区分为iPSC-EP的建议。
Materials
| µ-Slide Angiogenesis | Ibidi | N/A | A flat, glass bottom tissue-culture plate with side walls enables facile confocal imaging |
| 96 well, round bottom, ultra low attachment microplate, sterile | Corning | 7007 | Prevents the binding of cell-laden collagen hydrogels to the cell culture dish |
| Accutase | STEMCELL Technologies | 7920 | Gentle cell detachment solution; does not degrade extracellular epitopes vital for FACS |
| Advanced DMEM/F12 | Thermo Scientific | 12634010 | The base media for iPSC-EP differentiation. |
| Barnstead GenPure xCAD Plus | Thermo Fisher Scientific | 50136165 | Water purification system; others can be readily substituted |
| Bovine Serum Albumin solution,7.5% in DPBS, sterile-filtered, BioXtra, suitable for cell culture | Fisher Scientific | A8412 | To preserve cell viability when FACs sorting |
| CD34-PE, human (clone: AC136) | Miltenyi Biotec | 130-098-140 | Antibody used for FACs isolation of iPSC-EPs |
| CHIR99021 | LC Laboratories | C-6556 | Induces the formation of mesoderm from pluripotent stem cells |
| Collagen I Rat Tail High Protein 100 mg | VWR | 354249 | Main component of the 3D microenvironment |
| Conical centrifuge tubes (15/50 mL) | Fisher Scientific | 14-959-49D/A | Used to store and mix relatively large volumes of reagents and cell culture media |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher Scientific | D1306 | To counterstain and visualize cell nuclei |
| DMEM/F12 | Thermo Fisher Scientific | 11320-082 | For dilution of Matrigel and thawing of pluripotent stem cells |
| Dulbecco's phosphate-buffered saline (DPBS) | ThermoFisher | 14190-250 | To wash monolayer cultures |
| EDTA | Sigma-Aldrich | E8008 | For passaging of pluripotent stem cell colonies and to prevent cell aggregation when FACs sorting |
| Endothelial Cell Growth Medium 2 | PromoCell | C-22011 | Promotes endothelial cell viability and proliferation |
| Essential 8 Medium | Thermo Fisher Scientific | A1517001 | For maintenance of pluripotent stem cells |
| Glycine,BioUltra, for molecular biology, >=99.0% (NT) | Sigma-Aldrich | 50046 | Neutralizes remaining detergent |
| L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate,>=95% | Sigma-Aldrich | A8960 | Component of iPSC-EP differentiation medium |
| MATLAB | MathWorks | 1.8.0_152 | Multi-paradigm numerical computing environment (free available at most academic institutions) |
| Matrigel Matrix GFR PhenolRF Mouse 10 mL (gelatinous protein mixture) | Fisher Scientific | 356231 | Diluted in DMEM/F12 to coat plates for iPSC-EP differentiation |
| Medium-199 10X | Thermo Fisher Scientific | 1825015 | Used to balance final hydrogel osmolarity and pH |
| Microcentrifuge tubes (1.7 mL) | VWR | 87003-294 | Stores small volumes of reagents |
| Phosphate-buffered saline (PBS) | Sigma-Aldrich | P3813 | The main ingredient of the immunostaining solutions |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140122 | Antibiotic used after sorting to remove possible contamination from FACS instrument |
| Recombinant Human VEGF 165 Protein | R&D Systems | 293-VE | Mitogen that stimulates endothelial cell proliferation and tubulogenesis |
| Rhodamine phalloidin | Themo Fisher Scientific | R415 | To identify F-actin deposition and therfore outline the borders of the vascular networks |
| Triton X-100 (nonionic surfactant) | Sigma-Aldrich | X-100 | Detergent used to gently permeabilize cells |
| Tween-20 (emulsifying reagent) | Fisher Scientific | BP337 | Increases the binding specificity of the added antibodies |
| VE-Cadherin (F-8) | Santa Cruz Biotechnology | sc-9989 | To identify 3D endothelial lumen in collagen hydrogels |
| Vitronectin | ThermoFisher | A14700 | For maintenance of pluripotent stem cells |
| Y-27632 | Selleck Chemicals | S1049 | Preserves pluripotent stem cell and iPSC-EP viability when dissociated and re-seeded |
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