组织工程血管在小鼠模型的生成和嫁接
Summary
在这里，我们提出了一个协议，以产生组织工程血管移植物中有功能的移植入小鼠双播部分诱导多能干细胞（PiPSC） – 来源的平滑肌细胞和PiPSC – 在脱细胞血管支架的生物反应器来源的内皮细胞。
Abstract
The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.
Introduction
血管管道的建设是由心血管疾病导致受损和损伤血管修复手术的基本策略。迄今为止，在外科手术中使用的接枝物质包括生物相容的合成聚合物（聚四氟乙烯[聚四氟乙烯]，膨胀的聚四氟乙烯[膨体聚四氟乙烯;奥亚特克斯]或聚对苯二甲酸乙酯[涤纶]），同种异体移植物，自体组织（心包或隐静脉）和异种移植物1。虽然人工移植物（ 例如，戈尔特斯和涤纶）是最常用的，这些材料可能会导致大量的短期和长期的并发症，包括狭窄，钙沉积，血栓栓塞和感染。尽管患者目前生物移植减少血栓栓塞事件，他们还是遇到限制，如二次移植失败，缩短耐久性由于钙化退化2。因此，尽管在手术吨显著改善被echniques多年来，研究人员和临床医生仍然背负着需要用于识别提供了理想的导管用于血管疾病。最近，血管组织工程的研究领域已产生了在其中细胞掺入可生物降解的支架，以创建缩影成功移植1的官能容器仿生环境的目的的概念。根本的是，血管构建体的成功取决于三个主要组分;细胞组成的支架， 即，内皮细胞内层和平滑肌细胞层，含有合适的细胞外基质，以提供相当的机械性能与天然血管的支架，并且所需要用于启动/调节分子/细胞信号修理。
长期移植通畅和新组织的持续发展在很大程度上取决于支架的有效细胞接种，日ereby渲染的至关重要的细胞类型决定。一些报告展示了利用成熟的内皮细胞，并从各种来源的平滑肌细胞的发育小口径管道3-6。尽管有希望，缺乏足够的自体血管，以获得成熟的内皮细胞和平滑肌细胞保持一个相当大的负担。最近，从各种来源的干细胞已被开发用于血管组织工程的应用程序。确实，多种干细胞类型，包括胚胎干细胞7，诱导多能干细胞（iPS细胞）8,9，PiPSC 10,11，骨髓来源的单核细胞12，间质干细胞13，内皮祖细胞和成血管壁衍生干细胞抗原-1（SCA-1）+干/祖细胞14,15都被证明是能够分化成任何官能内皮或平滑肌细胞响应于确定的培养基和培养条件。此外，干细胞的无限自我更新能力使得它们不同于成熟的内皮细胞和平滑肌细胞可以经历生长停滞和衰老之前仅划分为次数有限数量的更好的候选。
支架材料的选择，以产生成功的组织工程血管接枝取决于几个因素，例如生物相容性，生物力学性能，和生物降解的速率。从根本上说，用料打造的支架移植物应该是可生物降解，不会安装不必要的接受者的免疫反应。此外，它必须包括合适的孔隙度和微结构用于细胞附着和随后的存活。迄今为止，用于在血管组织工程支架中最常见的材料包括聚乙醇酸，聚乳酸和聚ε己内酯16的聚合物。最近，脱细胞的生物材料具有也已应用取得了一些成功。几个实验室证明，脱细胞播种人，犬或猪血管自体细胞提供了一个生物学的移植物的抵制凝血和内膜增生17-19。在血管组织工程等战略包括细胞外基质蛋白为基础的血管移植物如种子细胞的纤维蛋白胶13，并产生细胞片无支架支持20，21。
当前协议证明人类PiPSC分化为功能性内皮细胞和平滑肌细胞中，生成的组织工程血管的生物反应器，包括一个脱细胞血管支架怀有官能PiPSC源性血管细胞的，和接枝到重症联合免疫缺陷病（SCID ）小鼠。 PiPSC是一个最佳的细胞类型以用于容器的移植物的组织工程，因为这些细胞不形成在小鼠中的肿瘤或提高的伦理和同种异体的免疫应答。此外，我们已经表明，该策略用于产生PIPS内皮细胞和PIPS平滑肌细胞是有效的和可再现的10,11。此后，我们设计用于PiPSC源性血管细胞密切模仿天然容器内存在的基质蛋白播种去细胞的容器中，从而提高了移植存活和功效。此外，前PiPSC播种血管的脱细胞防止安装在由免疫细胞类型的炎症反应，如巨噬细胞的产生。更重要的是，该协议不只是代表了一种方法，以产生希望的血管导管翻译成人类，而且还提供了学习和理解，通过小鼠模型支配的血管组织再生的分子机制的有价值的手段。
Protocol
Representative Results
Discussion
当前协议指示声音，快速，简单，高效和可重复性，其中功能性的组织工程血管可使用PiPSC从人成纤维细胞产生的策略。这种技术代表了用于再生医学，组织工程和潜在的患者特异性细胞治疗在不久的将来的宝贵工具。关键步骤，以确保协议的功效包括PiPSC的制备中，制备无菌的，且完全脱细胞主动脉移植物，PiPSC在支架成功播种和分化和主动脉移植物保持无菌在血管的生物反应器。
<p class="jove…Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by The British Heart Foundation and The Oak Foundation.
Materials
| Human Fibroblasts CCL-153 | ATCC | CCL-153 | Prenatal human embryonic fibroblasts |
| ATCC F-12K Medium (Kaighn's Modification of Ham's F-12 Medium) | ATCC | 30-2004 | |
| Fetal Bovine Serum | ATCC | 30-2020 | |
| Knockout DMEM medium optimized for embryonic stem cells | Life technologies (Gibco) | 12660-012 | |
| Knockout Serum Replacement | Life technologies (Invitrogen) | 10828-028 | |
| Human Basic FGF-2 | Miltenyi Biotech | 130-093-837 | |
| alpha-MEM medium | Life technologies (Invitrogen) | 32571093 | |
| Human PDGF | R&D System | 120-HD-001 | |
| Gelatin Solution 2% | Sigma | G1393 | |
| Plasmid 20866: pCAG2LMKOSimO (SOX2, OCT4, KLF4, C-MYC) | Addgene | 20866 | |
| PvuI Restriction Enzyme | New England Biolabs | RO150S | |
| SureClean Plus | Bioline | BIO-37047 | |
| Nucelofection Kit (NHDF Kit) | LONZA | VPD-1001 | |
| Neomycin | SIGMA | G418 | Selection of |
| KL 1500 LCD, Illumination for Stereo Microscopy | SCHOTT | KL 1500 LCD | Cold light illumination for stereo microscopy |
| Nikon Zoom Steromicroscope SMZ800 | Nikon | SMZ800 | |
| Heparin sodium salt | Sigma | H3393 | |
| 10% SDS Stock Solution Molecular Biology Reagent | Severn Biotech | CAS 151-21-3 | |
| Dulbecco's Phosphate Buffered Saline | Sigma | D8537 | |
| Matrigel (10mg/ml) | BD | A6661 | |
| Shaker IKA Vibrax with Shaking platform VX 7 | Jepson Bolton's, Janke&Kunkel | S32-102 | |
| Masterflex L/S Digital Pump Drive | Cole-Parmer | WZ-07523-80 | |
| Masterflex L/S 6-channel, 6-roller cartridge pump head | Cole-Parmer | EW-07519-15 | |
| Masterflex L/S large cartridges for pump head | Cole-Parmer | EW-07519-75 | |
| Masterflex platinum-cured silicone pump tubing, L/S 14, 25 ft | Cole-Parmer | WZ-96410-14 | Tubing goes through the peristaltic pump |
| 0.5mm ID, 0.8 mm OD Silicone Tubing | SILEX | N/A | Tubings connect incubation chamber, media reservoir and compliance chamber |
| Fitting Reducer 0.5 to 1.6, natural Polypropyline | Ibidi | 10829 | Adapter connect above two types of tubings |
| 1/32" Tubing, ID 0.01" (250µm) Material: PEEK | LabSmith | T-132-010P | Tubing through the incubation chamber wall which connects the graft with outside tubing |
| One-Piece Fittings | LabSmith | T-132-100 | Fix the above tubings through the incubation chamber wall |
| Nylon tubes (OD 0.9mm, ID 0.75mm) | Smiths Medical | N/A | Tubings insert into two ends of the aorta graft |
| NOD.CB17-Prkdcscid/NcrCrl mouse | Charles River | ||
| Surgical sutures, 8-0 silk | ETHICON | W819 | |
| Hypnorm | Vetapharm | Vm21757/4000 | Neuroleptanalgesic for use in mice |
| Hypnovel (Midazolam) | Roche | 59467-70-8 | Induction of anaesthesia |
| Dissecting microscope | Carl Zeiss | Stemi 2000 | |
| Nylon Tubing | Portex LTD | 800/200/100/200 | 0.65 mm in diameter and 1 mm in length; to make artery cuff |
| Electrocoagulator | Martin | SN 54.131 | Ligation of artery branches on aorta |
| Bipolar micro hemostat forceps | Martin | 80-91-12-04 | Fixation of vessel ends |
| Vessel Dilator | S&T | JFX-7 | |
| Vessel Dilator | S&T | JFL-3dZ | |
| Vessel Dilator | S&T | D-5aZ | |
| Mini applier | AESCULAP | FE572K | |
| Micro hemostats clips | AESCULAP | FE720K | |
| Surgical sutures, 6-0 VICRYL | ETHICON | V489 |
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