从人胚胎干细胞中提取合适的视网膜色素上皮组织的工程移植
1U861, I-Stem, Association Française contre les Myopathies (AFM),Institut National de la Santé et de la Recherche Médicale (INSERM), 2U861, I-Stem, Association Française contre les Myopathies (AFM),Université Evry Val-d’Essonne (UEVE), 3I-Stem, Association Française contre les Myopathies (AFM),Centre pour L’Etude des Cellules Souches (CECS), 4Banque de tissus humain, Hôpital Saint Louis, Assistance Publique – Hôpitaux de Paris (AP-HP), 5Sorbonne Université, INSERM, CNRS, Institut de la Vision, F-75012
Summary
我们描述了一种由人类羊膜顶部培养的人多能干细胞的视网膜色素上皮细胞组成的视网膜组织的方法及其在动物模型中的移植准备。
Abstract
眼睛的几个病理条件影响视网膜色素上皮 (RPE) 的功能和/或生存。这包括一些形式的视网膜色素变性 (RP) 和年龄相关的黄斑变性 (AMD)。细胞治疗是建议治疗这些疾病的最有希望的治疗策略之一, 在人类身上已经有了令人鼓舞的初步结果。然而, 移植物的制备方法对其在体内的功能结果有显著的影响。事实上, 作为细胞悬架移植的视网膜色素上皮细胞的功能比作为视网膜组织移植的细胞要少。在此, 我们描述了一个简单的和可重复的方法来设计视网膜色素组织和它的准备在体内植入。从人类多潜能干细胞中提取的视网膜色素细胞是在生物支持下播种的, 即人羊膜 (火腿)。与人工支架相比, 这种支持有一个基底膜接近刷的膜, 其中内源性视网膜色素细胞附加的优势。然而, 它的操作不容易, 我们制定了一些策略, 以适当的培养和准备移植在体内。
Introduction
RPE 是至关重要的生存和稳态的感光细胞, 它是紧密关联1。一些病理状况改变其功能和/或生存, 包括 RP 和 AMD。
RP 是一组遗传性的基因突变, 影响感光细胞或视网膜色素的功能, 或2,3。据估计, 特别影响视网膜色素上皮细胞的突变占5% 的 RP2。AMD 是另一种情况下, 视网膜色素层被改变, 最终导致中心视力损失。AMD 是由遗传和环境因素复杂的相互作用引起的, 影响老年人4,5,6。根据预测, AMD 将是全球1亿9600万名患者的关注, 到 2020年7。对于这些疾病, 目前还没有有效的治疗方法, 建议的策略之一是移植新的视网膜色素细胞, 以弥补死/非功能性视网膜色素上皮细胞8。
最终产品的配方, 是必不可少的, 以确保最佳的功能效果。RPE 细胞作为细胞悬浮液注入, 尽管是一种简单而直接的分娩方法, 但对其生存、整合和功能9、10、11、12等问题提出了关注。,13. 科学家现在正在开发更复杂的配方, 以提供工程化的视网膜组织9、13、14、15、16。在这种情况下, 我们开发了一种原始的方法来生成体外视网膜色素组织, 可用于移植9。
从人类胚胎干细胞中提取的 RPE 细胞库用于本协议。然而, 不同细胞源 (人诱导的多潜能干细胞、原发性视网膜色素上皮细胞等) 的替代视网膜色素细胞库也适用于该协议。它包括定向分化协议使用细胞因子和/或小分子17,18,19,20,21,22。
要移植, 工程组织应在脚手架上准备。在过去的几年中, 不同的脚手架是基于聚合物或生物起源13,23,24的矩阵开发的。在这里, 使用的生物基质是火腿, 但其他基质, 如裸露的刷膜, 可以实施。此处描述的方法具有使用与 RPE 本地环境更相关的生物支架的优点。
人类 ES 细胞源性视网膜色素上皮细胞培养至少4周, 以充分组织为鹅卵石单层。在该阶段, 获得的上皮细胞功能和极化9。最后, 由于这种组织容易皱纹, 它嵌入在薄层的水凝胶载体, 使其更刚性和弹性, 并在注射过程中保护它。该产品然后存储在4°c, 直到嫁接。
Protocol
本议定书使用的所有人类材料都是按照欧洲联盟的规定使用的。本研究使用的人类 ES 细胞系来源于一个独特的胚胎。捐出胚胎的夫妇完全知情, 并同意匿名捐赠。一个临床级的人类 ES 细胞系来源于这个胚胎, 储存, 合格, 并正确记录的罗丝琳细胞 (英国)。在产妇剖宫产期间, 根据医院的指导原则 (APHP, Hôpital 圣路易斯) 签署了知情同意的胎盘捐献, 火腿是在无菌条件下采购的。 1、…
Representative Results
火腿含有上皮层, 应在视网膜色素细胞播种前除去。用 thermolysin 在震动下对膜进行酶处理。为了不失去膜的极性 (上皮是在一侧), 它是固定在一个支持, 组成可能会有所不同取决于提供者 (图 1A)。在本步骤中检查膜的附着力, 并在必要时添加剪辑。在固定的文化插入时, 要小心地避免在膜上制造孔, 并保持其极性与基底膜朝上 (图 1B</stron…
Discussion
我们描述了一种生物支架上的 RPE 细胞培养方法及其在动物模型中的植入准备。该协议的关键步骤之一是保持火腿的方向一直沿着过程, 直到它纳入到明胶。事实上, 膜的本机上皮被去除, 其基底膜暴露9。视网膜色素上皮细胞必须在基底膜的顶端播种。在制备明胶时, 必须在规定的温度下与所有产品配合使用。事实上, 明胶的性质是刚性在4°c 和液体在体温 (37 °c)9…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者要感谢 Jérôme Larghero 和 valériemarcel Vanneaux (Hôpital 圣路易斯, 巴黎, 法国) 在这里所描述的方法的建立过程中的投入。
这项工作得到了来自情报局的赠款的支持 [GPiPS: ANR-2010-RFCS005;SightREPAIR: ANR-16-CE17-008-02], 基金会倒 la 研究所 Médicale [生物工程项目 DBS20140930777] 和从 LABEX 复活 [ANR-10-LABX-73] 到奥利弗 Goureau 和 Christelle Monville。它得到了 NeurATRIS (Investissements d ‘ 艾文莉) 的支持, 为神经 [ANR-11-INBS-0011] 和 INGESTEM 的 biotherapies, 国家基础设施 (Investissements d ‘ 艾文莉) 工程为多潜能和分化的干细胞 [ANR-11-INBS-000] Christelle Monville。Barek 是由暗淡的 Stempole 和 LABEX 复兴 [ANR-10-LABX-73] 的研究金支持。Biotherapies 是法国反肌病 (AFM)-Téléthon 协会支持的罕见疾病研究所的一部分。
Materials
| Sterile biosafety cabinet | TechGen International | Not applicable | |
| Liquid waste disposal system for aspiration | Vacuubrand | BVC 21 | |
| CO2-controlled +37 °C cell incubator | Thermo Electron Corporation | BVC 21 NT | |
| 200 µL pipette: P200 | Gilson | F144565 | |
| 1 mL pipette: P1000 | Gilson | F144566 | |
| Pipet aid | Drummond | 75001 | |
| +4 °C refrigerator | Liebherr | Not applicable | |
| Vibratome | Leica | VT1000S | |
| Fine scissors | WPI | 501758 | |
| Forceps (x2) | WPI | 555227F | |
| Water bath | Grant subaqua pro | SUB6 | |
| Precision balance | Sartorius | CP225D | |
| Centrifuge | Eppendorff | 5804 | |
| Microscope | Olympus | SC30 | |
| Horizontal Rocking Shaker | IKA-WERKE | IKA MTS 214D | |
| Vortex | VWR | LAB DANCER S40 | |
| Disposable Scalpel | WPI | 500351 | |
| plastic paraffin film | VWR | PM992 | |
| 0.200 µm single use syringe filter | SARTORIUS | 16532 | |
| Syringe without needle 50 mL | Dutscher | 50012 | |
| Bottles 250mL | Dutscher | 28024 | |
| 15 mL sterile Falcon tubes | Dutscher | 352097 | |
| 50 mL sterile Falcon tubes | Dutscher | 352098 | |
| culture insert | Scaffdex | C00001N | |
| 60 mm cell culture disches: B6 | Dutscher | 353004 | |
| 12 well cell culture plate | Corning | 3512 | |
| 6-well culture plates | Corning | 3506 | |
| Razor blades | Ted Pella, Inc | 121-9 | |
| Cyanoacrylate glue | Castorama | 3178040670105 | |
| PBS 1X (500 mL) | Sigma | D8537 | |
| Thermolysine | Roche | 5339880001 | |
| DMEM, high glucose, GlutaMAX | Invitrogen | 61965-026 | |
| KSR CTS (KnockOut SR XenoFree CTS) | Invitrogen | 12618-013 | |
| MEM-NEAA (100X) | Invitrogen | 11140-035 | |
| b-mercaptoethanol (50 mM) | Invitrogen | 31350-010 | |
| Penicillin/Streptomycin | Invitrogen | 15140122 | |
| CO2-independent medium | GIBCO | 18045-054 | |
| Gelatin | MERCK | 104078 | |
| human amniotic membrane | Tissue bank St Louis hospital (Paris, France) | Not applicable |
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Citer Cet Article
Ben M’Barek, K., Habeler, W., Plancheron, A., Jarraya, M., Goureau, O., Monville, C. Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells. J. Vis. Exp. (139), e58216, doi:10.3791/58216 (2018).