定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成
Summary
多潜能干细胞的专业视网膜细胞的产生是干细胞治疗视网膜疾病的一个转折点。本文介绍了一种有效生成视网膜 organoids 和视网膜色素上皮的简单方法, 用于基础、平移和临床研究。
Abstract
多潜能干细胞的专业细胞的生产为再生医学的发展提供了有力的工具。使用人类诱导的多能干细胞 (iPSCs) 对神经退行性疾病研究特别有吸引力, 其中包括视网膜白斑, iPSC 视网膜细胞模型标志着一个重要的步骤, 以了解和消除失明。在本文中, 我们描述了一个简单和可扩展的协议, 以生成, 成熟, cryopreserve 视网膜 organoids。在媒介变化的基础上, 该方法的主要优点是避免了在 iPSCs 的引导分化过程中通常需要的多次和耗时的步骤。模拟视网膜发育的早期阶段由被定义的媒介的连续的变动在黏附人的 iPSC 文化, 这个协议允许同时产生自已形成的 neuroretinal 结构和视网膜色素上皮 (RPE) 细胞在4周内重现性和高效的方式。这些结构包含视网膜祖细胞 (rpc) 可以很容易地分离, 以进一步成熟的漂浮培养条件, 使 rpc 分化为七视网膜细胞类型存在于成人视网膜。此外, 我们还描述了快速保存视网膜 organoids 和视网膜色素上皮细胞长期贮存的方法。结合起来, 这里描述的方法将是有用的生产和银行人类 iPSC 的视网膜细胞或组织的基础和临床研究。
Introduction
视网膜是中枢神经系统的一个组成部分, 在外伤或疾病后自发再生的能力有限。因此, 退行性病变导致明确的视网膜细胞丢失, 如年龄相关黄斑变性 (AMD), 视网膜色素变性 (RP), 青光眼和糖尿病视网膜病变, 通常导致不可逆转的失明。抢救视网膜退化是一个主要的挑战, 以干细胞为基础的治疗, 旨在取代受损或丢失的细胞是最有希望的方法1,2,3。多潜能干细胞作为人类胚胎干细胞 (ESCs) 细胞或人类诱导的多潜能干细胞 (iPSCs) 有能力在培养中无限期扩展, 它们有可能产生任何细胞类型。我们对视网膜发育的认识和改善人 iPSC 分化的体外协议的进展, 导致了视网膜 organoids7,8,9的产生, 10,11,12。所有的主要视网膜细胞, 包括视网膜神经节细胞 (RGCs), 感光细胞, 视网膜色素上皮 (RPE), 已经成功地分化为人类 ESCs 和 iPSCs4,5, 6. 以 Eiraku等为基础, 建立了 SFEB (胚状体样骨料) 的无血清培养方法。13、在定义的胞外基质成分7、10、14中, 可从 ESC 或 iPSC 衍生的胚体样骨料中获得视网膜 organoids 的自形成。但是这些协议是错综复杂的, 需要大量的步骤并不总是与大规模生产细胞的治疗方法或药物筛选。因此, 人类视网膜细胞生成方法的选择是至关重要的, 该方法需要健壮、可伸缩性和高效性。
在这里, 根据我们以前的出版物15, 我们描述了一个简单和高效的视网膜细胞的每一个步骤, 通过视网膜 organoid 自形成从黏附人 iPSCs 培养在无饲养和异的条件。从人类 iPSCs 的常规文化出发, 该协议只需要一个简单的连续培养基改变, 以允许在4周内生成 iPS 源性 RPE (hiRPE) 细胞和 neuroretinal 结构。人工隔离后, hiRPE 可以扩展, 视网膜结构可以培养为漂浮 organoids, 在那里视网膜祖细胞能够按照与体内人一致的顺序依次分化为所有视网膜细胞类型。retinogenesis。最后, 对于研究进展或临床翻译, 我们描述了一种冷冻方法, 允许长期储存的整个视网膜 organoids 和 hiRPE 细胞不影响其表型特征和功能。
Protocol
Representative Results
Discussion
该协议描述了如何在无异和无饲养条件下, 从人类多能干细胞中产生视网膜 RGCs 和感光细胞的视网膜色素 organoids。与良好的生产实践 (GMP) 过程兼容, 这里提出的方法允许大量生产 iPSC 视网膜细胞作为 RPE 细胞, RGCs, 和光传感器, 以发展干细胞为基础的治疗和药物未来治疗视网膜退行性疾病的发现方法。整个视网膜 organoids 或 hiRPE 细胞的超低温保存也为建立中间细胞库提供了一个重要的优势, 这是今?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢 Goureau 小组成员在这里所描述的方法的建立过程中的投入, 以及 g. Gagliardi 和 m. 拉·加里对他们的批判性阅读。这项工作得到了来自情报局 (GPiPS: ANR-2010-RFCS005 的赠款的支持;SightREPAIR: ANR-16-CE17-008-02), 视网膜法国协会和技术转让公司 SATT Lutech。它还在 Investissements d ‘ 艾文莉计划 (ANR-11-IDEX-0004-02) 的 LABEX LIFESENSES (ANR-10-LABX-65) 的框架内执行。
Materials
| Vitronectin (VTN-N) Recombinant Human Protein, Truncated | ThermoFisher Scientific | A14700 | Coating |
| CTS Vitronectin (VTN-N) Recombinant Human Protein, Truncated | ThermoFisher Scientific | A27940 | Coating |
| Essential 8 Medium | ThermoFisher Scientific | A1517001 | medium |
| Essential 6 Medium | ThermoFisher Scientific | A1516401 | medium |
| CTS (Cell Therapy Systems) N-2 Supplement | ThermoFisher Scientific | A1370701 | supplement CTS |
| N-2 Supplement (100X) | ThermoFisher Scientific | 17502048 | supplement |
| B-27 Supplement (50X), serum free | ThermoFisher Scientific | 17504044 | supplement |
| CTS B-27 Supplement, XenoFree | ThermoFisher Scientific | A1486701 | supplement CTS |
| DMEM/F-12 | ThermoFisher Scientific | 11320074 | medium |
| MEM Non-Essential Amino Acids Solution (100X) | ThermoFisher Scientific | 11140035 | supplement |
| Penicillin-Streptomycin (10,000 U/mL) | ThermoFisher Scientific | 15140122 | antibiotic |
| CellStart CTS | ThermoFisher Scientific | A1014201 | Matrix CTS |
| Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix | ThermoFisher Scientific | A1569601 | Matrix |
| Gentle Cell Dissociation Reagent | Stemcell Technologies | 7174 | dissociation solution |
| Cryostem Freezing Media | clinisciences | 05-710-1D | Cryopreservation medium |
| Fibroblast growth factor 2 (FGF2) | Preprotech | 100-18B | FGF2 |
| Fibroblast growth factor 2 (FGF2) animal free | Preprotech | AF-100-18B | FGF2 Xeno free |
| AGANI needle 23G | Terumo | AN*2332R1 | Needle |
| Flask 25 cm² Tissue Culture Treated | Falcon | 353109 | T-25 cm² |
| 24 well plate Tissue Culture Treated | Costar | 3526 | 24-well plate |
| 6 well plate Tissue Culture Treated | Costar | 3516 | 6-well plate |
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