人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成
1iBB – Institute for Bioengineering and Biosciences and Department of Bioengineering, Instituto Superior Técnico,Universidade de Lisboa, 2Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina,Universidade de Lisboa, 3The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus,Universidade de Lisboa, 4PBS Biotech, Inc, Camarillo, CA, USA
Summary
该协议描述了一个动态培养系统，用于利用一种一种用途的生物反应器，产生控制大小的人类多能干细胞，进一步刺激在化学定义和免饲料条件下小脑器官的分化。
Abstract
小脑在维持平衡和运动协调中起着至关重要的作用，不同小脑神经元的功能缺陷可以触发小脑功能障碍。目前关于疾病相关神经元表型的知识大部分基于死后组织，这使得对疾病进展和发育的理解变得困难。动物模型和不朽的细胞系也被用作神经退行性疾病的模型。然而，他们不能完全概括人类疾病。人类诱导多能干细胞（iPSCs）在疾病建模方面潜力巨大，为再生方法提供了宝贵的来源。近年来，来自患者衍生的 iPSC 的脑器官的生成改善了神经退行性疾病建模的前景。然而，在三D培养系统中，缺乏产生大量器官和高产成熟神经元的协议。所提出的协议是使用可扩展的一次使用生物反应器在化学定义条件下可重复和可扩展生成人类 iPSC 衍生器官的新方法，其中有机体获得小脑特性。生成的器官的特点是在mRNA和蛋白质水平上表达特定的标记。对特定蛋白质组的分析可以检测不同的小脑细胞群，其定位对于组织结构的评价非常重要。器官冷冻和进一步免疫污染器官切片用于评估特定小脑细胞群的存在及其空间组织。
Introduction
人类多能干细胞（PSCs）的出现是再生医学和疾病建模的极佳工具，因为这些细胞可以分化成人体1、2的多数细胞系。自从他们发现以来，PSC分化使用不同的方法被报告为不同的疾病建模，包括神经退行性疾病3，4，5，6。,4,5,6
最近，有报道说，3D培养物来自类似于人类大脑结构的PSC;这些被称为大脑器官3，7，8。,83,这些结构的生成来自健康和患者特定的PSC提供了一个宝贵的机会来模拟人类发展和神经发育障碍。然而，用于生成这些组织良好的脑结构的方法很难应用于其大规模生产。为了产生足够大的结构，以重述组织形态，而不在器官内坏死，协议依赖于在静态条件下的初始神经承诺，然后封装在水凝胶和随后的培养在动态系统3。然而，这种办法可能限制器官生产的潜在扩大。尽管已经努力将PSC分化到中枢神经系统的特定区域，包括皮质、神经、中脑,和脊髓神经元9、10、11、12，,10但动态条件下的特定大脑区域的生成仍然是一个挑战。11,12特别是，3D结构中成熟的小脑神经元的生成尚未描述。Muguruma等人开创了培养条件的产生，重新概括了早期小脑发育13，最近报告了一个方案，允许人类胚胎干细胞产生一个极化结构，让人联想到头三个月小脑7。然而，在报告研究中，小脑神经元的成熟需要器官分离，小脑祖细胞的排序，并在,单层培养系统7、14、15、16,14中与馈养细胞15共同培养。因此，在定义的条件下，用于疾病建模所需的小脑器官的可重复生成仍然是与培养和馈送源变异相关的挑战。
该协议提供了使用一用垂直轮生物反应器（参见规格材料表），即使用一种使用垂直轮生物反应器将人类PSC有效分化成小脑神经元Table of Materials的最佳培养条件，以下简称生物反应器。生物反应器配有大型垂直叶轮，与U形底部结合，在容器内提供更均匀的剪切分布，允许温和、均匀的混合和颗粒悬浮，降低搅拌速度17。有了这个系统，可以获得形状和大小控制的细胞聚合体，这对更均匀、更高效的分化非常重要。此外，更多的 iPSC 衍生器官可以以不太费力的方式生成。
器官的主要特征是通常由干细胞形成的3D多细胞结构，是不同细胞类型的自组织，形成特定形状，如人类形态18、19、20,19,中所看到的形状。因此，有机形态是分化过程中需要评价的重要标准。使用一组特定抗体对器官进行冷冻和进一步免疫渗透，使分子标记在空间上可视化，以分析细胞增殖、分化、细胞种群特性和凋亡。有了这个协议，通过免疫控制器官冷冻，在分化的第7天观察到了一个初始有效的神经承诺。在分化过程中，观察到几个具有小脑特征的细胞群。在这个动态系统中35天后，小脑神经皮皮沿着一个轴线组织，有一个增殖的祖细胞和基本定位的后层神经元。在成熟过程中，从第35~90天的分化，可以看到不同类型的小脑神经元，包括Purkinje细胞（卡尔宾丁+），颗粒细胞（PAX6+/MAP2+），高尔基细胞（神经粒蛋白+），单极刷细胞（TBR2+），和深小脑核投影+神经元（TBR1+）。+此外，在培养的90天后，在生成的小脑器官中观察到不重要的细胞死亡量。
在这个系统中，人类 iPSC 衍生的器官成熟成不同的小脑神经元，存活长达 3 个月，无需分离和喂食共培养，为疾病建模提供了人类小脑神经元的来源。
Protocol
1. 在单层文化中传传和维护人类 iPSC 板材的制备 在4°C下 解冻基底膜基质（见材料表）库存，制备60μL等分。将等分在-20°C下冷冻。 要覆盖6孔板的孔，在冰上解冻地下室膜基质的一个等分。解冻后，将 DMEM-F12 的 60 μL 添加到 6 mL。通过上下移液轻轻重新暂停。 将 1 mL 稀释的地下膜基质溶液添加到 6 孔板的每个孔中，并在 RT 中孵育至少 1 小时，然后以 4°C…
Representative Results
该协议是通过使用0.1 L生物反应器促进细胞聚合（图1A）启动的。对 iPSC 进行单细胞接种，在 60 mL 的培养中播种 250，000 个细胞/mL，搅拌速度为 27 rpm。这被定义为第 0 天。24小时后，细胞有效形成球形状聚集体（第1天，图1B），形态学一直保持良好，直到第5天，大小逐渐增加，表明随着时间的推移，在聚合形态和大小方面具有高度的同质性。（<strong cla…
Discussion
需要大量的细胞数量以及定义的培养条件来生成特定的细胞类型，用于药物筛选和再生医学应用，这一直在推动可扩展培养系统的发展。近年来，几组报告了可扩展的神经祖代和功能神经元32、33、34等34，为神经退行性疾病新模型的发展提供了重大进展。32,然而，胚胎发育的一些关键事件的回顾仍然缺乏，长期维持在悬浮过程?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了葡萄牙国家电信协会（FCT）的支持（UIDB/04565/2020通过Lisboa区域项目2020，项目N.007317， PD/BD/105773/2014 至 T.P.S 和 PD/BD/128376/2017 到 D.E.S.N.），项目由 FEDER （POR Lisboa 2020 – 项目区域行动区域葡萄牙 2 和 FCT 通过授予 PAC – 精确 LISBOA – 01 – 0145 – Feder – 016394 和 CEREBEX 生成小脑有机体为 Ataxia 研究授予 LISBOA -01-0145-FEDER-029298。还根据赠款协议第739572号——再生和精密医学发现中心H2020-2020-01-2016-2017年，从欧洲联盟的Horizon 2020研究和创新方案获得资金。
Materials
| 3MM paper | WHA3030861 | Merck | |
| Accutase | A6964 – 500mL | Sigma | cell detachment medium |
| Anti-BARHL1 Antibody | HPA004809 | Atlas Antibodies | |
| Anti-Calbindin D-28k Antibody | CB28 | Millipore | |
| Anti-MAP2 Antibody | M4403 | Sigma | |
| Anti-N-Cadherin Antibody | 610921 | BD Transduction | |
| Anti-NESTIN Antibody | MAB1259-SP | R&D | |
| Anti-OLIG2 Antibody | MABN50 | Millipore | |
| Anti-PAX6 Antibody | PRB-278P | Covance | |
| Anti-SOX2 Antibody | MAB2018 | R&D | |
| Anti-TBR1 Antibody | AB2261 | Millipore | |
| Anti-TBR2 Antibody | ab183991 | Abcam | |
| Anti-TUJ1 Antibody | 801213 | Biolegend | |
| Apo-transferrin | T1147 | Sigma | |
| BrainPhys Neuronal Medium N2-A & SM1 Kit | 5793 – 500mL | Stem cell tecnhnologies | |
| Chemically defined lipid concentrate | 11905031 | ThermoFisher | |
| Coverslips 24x60mm | 631-1575 | VWR | |
| Crystallization-purified BSA | 5470 | Sigma | |
| DAPI | 10236276001 | Sigma | |
| Dibutyryl cAMP | SC- 201567B -500mg | Frilabo | |
| DMEM-F12 | 32500-035 | ThermoFisher | |
| Fetal bovine serum | A3840001 | ThermoFisher | |
| Gelatin from bovine skin | G9391 | Sigma | |
| Glass Copling Jar | E94 | ThermoFisher | |
| Glutamax I | 10566-016 | ThermoFisher | |
| Glycine | MB014001 | NZYtech | |
| Ham’s F12 | 21765029 | ThermoFisher | |
| Human Episomal iPSC Line | A18945 | ThermoFisher | iPSC6.2 |
| IMDM | 12440046 | ThermoFisher | |
| Insulin | 91077C | Sigma | |
| iPS DF6-9-9T.B | WiCell | ||
| Iso-pentane | PHR1661-2ML | Sigma | |
| L-Ascorbic acid | A-92902 | Sigma | |
| Matrigel | 354230 | Corning | basement membrane matrix |
| Monothioglycerol | M6154 | Sigma | |
| Mowiol | 475904 | Millipore | mounting medium |
| mTeSR1 | 85850 -500ml | Stem cell technologies | |
| N2 supplement | 17502048 | ThermoFisher | |
| Neurobasal | 12348017 | ThermoFisher | |
| Paraformaldehyde | 158127 | Sigma | |
| PBS-0.1 Single-Use Vessel | SKU: IA-0.1-D-001 | PBS Biotech | |
| PBS-MINI MagDrive Base Unit | SKU: IA-UNI-B-501 | PBS Biotech | |
| Recombinant human BDNF | 450-02 | Peprotech | |
| Recombinant human bFGF/FGF2 | 100-18B | Peprotech | |
| Recombinant human FGF19 | 100-32 | Peprotech | |
| Recombinant human GDNF | 450-10 | Peprotech | |
| Recombinant human SDF1 | 300-28A | Peprotech | |
| ROCK inhibitor Y-27632 | 72302 | Stem cell technologies | |
| SB431542 | S4317 | Sigma | |
| Sucrose | S7903 | Sigma | |
| SuperFrost Microscope slides | 12372098 | ThermoFisher | adhesion microscope slides |
| Tissue-Tek O.C.T. Compound | 25608-930 | VWR | |
| Tris-HCL 1M | T3038-1L | Sigma | |
| Triton X-100 | 9002-93-1 | Sigma | |
| Tween-20 | P1379 | Sigma | |
| UltraPure 0.5M EDTA, pH 8.0 | 15575020 | ThermoFisher |
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Cite This Article
Silva, T. P., Fernandes, T. G., Nogueira, D. E. S., Rodrigues, C. A. V., Bekman, E. P., Hashimura, Y., Jung, S., Lee, B., Carmo-Fonseca, M., Cabral, J. M. S. Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining. J. Vis. Exp. (160), e61143, doi:10.3791/61143 (2020).