从外周单核细胞生成诱导神经干细胞,分化至多巴胺能神经元前体,用于移植研究
Summary
该协议提出了外周血单核细胞的重新编程,以诱导神经干细胞由仙台病毒感染,将iNSCs分化为多巴胺能神经元,将DA前体移植到单方面病变帕金森病小鼠模型,并评价iNSC衍生DA前体用于PD治疗的安全性和有效性。
Abstract
帕金森病 (PD) 是由心内脑素 (VM) 中基拉多巴胺能 (DA) 神经元的退化引起的。细胞替代疗法对PD的治疗大有希望。 最近,诱导神经干细胞(iNSCs)由于肿瘤形成的风险降低和可塑性增加而成为细胞替代治疗的潜在候选者。区域特定的神经元和胶质。iNSCs可以从自体体细胞源(如成纤维细胞、外周血单核细胞(PBMC)和各种其他类型的细胞)重新编程。与其他类型的体细胞相比,PBMC是一个有吸引力的起始细胞类型,因为易于访问和扩展的培养。仙台病毒(SeV),一种RNA非整合性病毒,编码重新编程的因素,包括人类OCT3/4,SOX2,KLF4和c-MYC,具有负感,单链,非分段基因组,不集成到宿主基因组,但仅在受感染细胞的细胞质中复制,为重新编程提供了高效和安全的工具。在本研究中,我们描述了一种协议,其中通过重新编程PBMC获得iNSC,并通过两阶段方法分化成专门的VM DA神经元。然后DA前体被移植到单方面的6-羟基多巴胺(6-OHDA)病变PD小鼠模型中,以评估PD治疗的安全性和有效性。该方法为研究患者特异性DA神经细胞在体外和体内的功能和治疗效果提供了一个平台。
Introduction
帕金森病 (PD) 是一种常见的神经退行性疾病,由心内脑素 (VM) 的底状尼格拉帕压实 (SNpc) 的多巴胺能 (DA) 神经元退化引起,60 岁以上人群的患病率超过 1%1,2.在过去十年中,细胞疗法,旨在取代退行性或受损的细胞,或滋养退化神经元周围的微环境,在PD3的治疗中显示出潜力。同时,重新编程技术也取得了显著进展,为替代疗法提供了有前途的细胞源。人类诱导多能干细胞(iPSCs)和胚胎干细胞(ESCs)已被证明能够分化成DA神经细胞,当移植到大鼠和非人类灵长类动物PD模型中时,这些神经细胞可以存活、成熟,并改善运动功能5 ,6,7,8.iPSC是细胞再编程技术的一个里程碑,在细胞移植方面有着巨大的潜力;然而,人们仍然担心肿瘤从不完全分化的细胞形成的风险。细胞移植的替代细胞来源是通过直接重新编程获得的系世型成人干细胞,如诱导神经干细胞(icCS),可以从不稳定的中间体中提取,绕过多能性阶段9,10,11.
iPSC 和 iNSCs 都可以从自体细胞源(如成纤维细胞、外周血单核细胞 (PBMC)和各种其他类型的细胞12、13、14)重新编程,从而减少移植细胞的免疫原性在很大程度上。此外,与iPSCs相比,iNSCs是固有的,肿瘤形成和系骨的可塑性风险降低,只能分化成神经元和胶质11。在最初的研究中,人类或小鼠iPSC和iNSCs是由从皮肤活检中获得的成纤维细胞产生的,这是一个侵入性程序14,15。在这方面,PBMNCs是一个有吸引力的起始细胞源,因为侵入性较小,并且易于在短时间内获得大量的细胞扩展时间16。初始重新编程研究采用综合输送系统,如慢病毒或抗逆转录病毒载体,这些系统在多种细胞中高效且易于实施17;然而,这些传递系统可能导致突变和残留转基因的重新激活,这提出了临床治疗目的的安全问题12。仙台病毒(SeV)是一种非整合性RNA病毒,具有负感的单链基因组,不集成到宿主基因组中,但只在受感染细胞的细胞质中复制,为重新编程提供了高效和安全的工具 ,19.可组合SeV向量,包含重新编程的因素,包括人类OCT3/4,SOX2,KLF4和c-MYC在其开放阅读帧。此外,通过引入温度敏感突变,可以进一步改善SeV病毒载体,以便在培养温度升高至39°C20时迅速去除。在本文中,我们将介绍一种使用 SeV 系统将 PBMC 重新编程到 iNSC 的协议。
许多研究已经报告从人类ESCs或iPSC衍生的DA神经元使用各种方法6,8,21。然而,在细节上描述DA神经元与iNSCs分化的协议很缺乏。在此协议中,我们将使用两阶段方法描述从 iNSC 高效生成 DA 神经元。DA神经元前体可移植到PD小鼠模型的纹状体中,用于安全性和有效性评估。本文将介绍一个详细的方案,涵盖从仙台病毒生成诱导神经干细胞,将iNSCs分化为DA神经元,建立小鼠PD模型,到将DA前体移植到纹状体的各个阶段PD 模型。使用该协议,人们可以从患者和健康捐赠者生成iNSC,并得出安全、标准化、可扩展和均匀的DA神经元,用于细胞移植目的,或用于在培养皿中建模PD和研究机制潜在的疾病开始和发展。
Protocol
Representative Results
Discussion
在这里,我们提出了一个协议,涵盖了PD模型的iNSC-DA细胞治疗的不同阶段。该协议的关键方面包括:(1) PBMC 的隔离和扩展,以及通过 SeV 感染将 PBMC 重新编程到 iNSC;(2) 将 iNSC 分化为 DA 神经元,(3) 建立单方面 6-OHDA 病变 PD 小鼠模型和行为评估,以及(4)DA前体的细胞移植和行为评估。
在此协议中,第一部分涉及在无血清介质(MNC介质)中收集和扩展PBMC,该介质优先扩展红细胞,不支持淋巴细胞?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
该课题资助项目为:干细胞与翻译国家重点项目(2016YFA0101403)、国家自然科学基金(81661130160、81422014、81561138004)、北京市自然科学基金(5142005)、北京人才基金会(201700021223TD03),北京市”十三五”期间高层次人才资助项目(CIT&TCD20180333),北京医疗系统高层次人才奖(2015-3-063),北京市卫生局基金(PXM 2018_026283_000002)、北京市百千人才基金(2018A03)、北京市医院临床医学发展专项资金支持(ZYLX201706)和皇家学会-牛顿高级奖学金(NA150482)。
Materials
| 15-ml conical tube | Corning | 430052 | |
| 1-Thioglycerol | Sigma-Aldrich | M6145 | Toxic for inhalation and skin contact |
| 24-well plate | Corning | 3337 | |
| 50-ml conical tube | Corning | 430828 | |
| 6-OHDA | Sigma-Aldrich | H4381 | |
| 6-well plate | Corning | 3516 | |
| Accutase | Invitrogen | A11105-01 | Cell dissociation reagent |
| Apomorphine | Sigma-Aldrich | A4393 | |
| Ascorbic acid | Sigma-Aldrich | A92902 | Toxic with skin contact |
| B27 supplement | Invitrogen | 17504044 | |
| BDNF | Peprotech | 450-02 | Brain derived neurotrophic factor |
| Blood collection tubes containing sodium heparin | BD | 367871 | |
| BSA | yisheng | 36106es60 | Fetal bovine serum |
| cAMP | Sigma-Aldrich | D0627 | Dibutyryladenosine cyclic monophosphate |
| CellBanker 2 | ZENOAQ | 100ml | Used as freezing medium for PBMNCs |
| Chemically defined lipid concentrate | Invitrogen | 11905031 | |
| CHIR99021 | Gene Operation | 04-0004 | |
| Coverslip | Fisher | 25*25-2 | |
| DAPI | Sigma-Aldrich | D8417-10mg | |
| DAPT | Sigma-Aldrich | D5942 | |
| Dexamethasone | Sigma-Aldrich | D2915-100MG | |
| DMEM-F12 | Gibco | 11330 | |
| DMEM-F12 | Gibco | 11320 | |
| Donkey serum | Jackson | 017-000-121 | |
| EPO | Peprotech | 100-64-50UG | Human Erythropoietin |
| FGF8b | Peprotech | 100-25 | |
| Ficoll-Paque Premium | GE Healthcare | 17-5442-02 | P=1.077, density gradient medium |
| GDNF | Peprotech | 450-10 | Glial derived neurotrophic factor |
| GlutaMAX | Invitrogen | 21051024 | 100 × Glutamine stock solution |
| Ham's-F12 | Gibco | 11765-054 | |
| HBSS | Invitrogen | 14175079 | Balanced salt solution |
| Human leukemia inhibitory factor | Millpore | LIF1010 | |
| Human recombinant SCF | Peprotech | 300-07-100UG | |
| IGF-1 | Peprotech | 100-11-100UG | Human insulin-like growth factor |
| IL-3 | Peprotech | 200-03-10UG | Human interleukin 3 |
| IMDM | Gibco | 215056-023 | Iscove's modified Dulbecco's medium |
| Insulin | Roche | 12585014 | |
| ITS-X | Invitrogen | 51500-056 | Insulin-transferrin-selenium-X supplement |
| Knockout serum replacement | Gibco | 10828028 | Serum free basal medium |
| Laminin | Roche | 11243217001 | |
| Microsyringe | Hamilton | 7653-01 | |
| N2 supplement | Invitrogen | 17502048 | |
| NEAA | Invitrogen | 11140050 | Non-essential amino acid |
| Neurobasal | Gibco | 10888 | Basic medium |
| PDL | Sigma-Aldrich | P7280 | Poly-D-lysine |
| SAG1 | Enzo | ALX-270-426-M01 | |
| SB431542 | Gene Operation | 04-0010-10mg | Store from light at -20℃ |
| Sendai virus | Life Technologies | MAN0009378 | |
| Sucrose | baiaoshengke | ||
| TGFβⅢ | Peprotech | 100-36E | Transforming growth factor βⅢ |
| Transferrin | R&D Systems | 2914-HT-100G | |
| Triton X 100 | baiaoshengke | Nonionic surfactant | |
| Trypan blue | Gibco | T10282 | |
| Xylazine | Sigma-Aldrich | X1126 |
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