在2D结构中模拟人类小脑体 外 发育
Summary
本协议解释了从诱导多能干细胞生成2D单层小脑细胞，用于研究小脑发育的早期阶段。
Abstract
小脑的精确和及时发育不仅对准确的运动协调和平衡至关重要，而且对认知也至关重要。此外，小脑发育的中断与许多神经发育障碍有关，包括自闭症、注意力缺陷多动障碍 （ADHD） 和精神分裂症。人类小脑发育的研究以前只能通过尸检或神经成像进行，但这些方法不足以了解早期 发育过程中体内 发生的分子和细胞变化，这是许多神经发育障碍的起源。从体细胞产生人类诱导多能干细胞（iPSC）的技术的出现以及进一步将iPSC重新分化为神经元的能力为早期大脑发育的 体外 建模铺平了道路。本研究为需要二维（2D）单层结构的应用提供了生成小脑细胞的简化步骤。代表早期发育阶段的小脑细胞通过以下步骤 来源 于人iPSC：首先，在三维（3D）培养中制造胚状体，然后用FGF2和胰岛素处理以促进小脑命运规范，最后，在聚l-鸟氨酸（PLO）/层粘连蛋白包被的底物上最终分化为单层。在分化 35 天时，iPSC 来源的小脑细胞培养物表达小脑标志物，包括 ATOH1、PTF1α、PAX6 和 KIRREL2，表明该方案产生谷氨酸能和 GABA 能小脑神经元前体，以及浦肯野细胞祖细胞。此外，分化的细胞显示出独特的神经元形态，并且对神经元身份的免疫荧光标志物（如TUBB3）呈阳性。这些细胞表达轴突引导分子，包括信号蛋白-4C、丛蛋白-B2和神经皮林-1，可以作为研究神经突生长和突触连接的分子机制的模型。该方法生成可用于下游应用的人类小脑神经元，包括需要 2D 单层格式的基因表达、生理和形态学研究。
Introduction
了解人类小脑发育和这一过程的关键时间窗口不仅对于解码神经发育障碍的可能原因很重要，而且对于确定治疗干预的新靶点也很重要。在体外模拟人类小脑发育一直具有挑战性，但随着时间的推移，已经出现了许多区分人类胚胎干细胞（hESC）或具有小脑谱系命运的iPSCs的方案1，2，3，4，5，6，7，8.此外，重要的是开发产生可重复结果的协议，相对简单（以减少错误），并且不承担沉重的货币成本。
小脑分化的第一个方案是从板状胚体（EB）的2D培养物中产生的，用类似于体内发育的各种生长因子诱导小脑命运，包括WNT，BMP和FGFs1，9。最近发表的方案主要在用FGF2和胰岛素诱导3D类器官培养中诱导分化，然后是FGF19和SDF1用于菱形唇样结构3，4，或使用FGF2，FGF4和FGF8的组合5。两种小脑类器官诱导方法都产生了相似的3D小脑类器官，因为两种方案都报告了在相同时间点相似的小脑标志物表达。Holmes和Heine扩展了他们的3D协议5，以表明2D小脑细胞可以从hESC和iPSCs产生，它们以3D聚集体的形式开始。此外，Silva等人7证明，代表2D成熟小脑神经元的细胞可以用与Holmes和Heine类似的方法生成，使用不同的时间点从3D切换到2D并延长生长和成熟时间。
目前的协议通过使用胰岛素和FGF2产生自由漂浮的拟胚体（EB），然后在第14天将EB接种在PLO /层粘连蛋白包被的培养皿上以进行2D生长和分化，从而诱导无饲养层iPSC中的小脑命运。到第35天，获得具有小脑身份的细胞。概括小脑发育早期阶段的能力，特别是在2D环境中，使研究人员能够回答需要单层结构实验的特定问题。该协议也适用于进一步的修改，例如微图案表面，轴突生长测定和细胞分选，以富集所需的细胞群。
Protocol
人类受试者研究获得了爱荷华大学机构审查委员会批准号201805995和爱荷华大学人类多能干细胞委员会批准号2017-02的批准。在获得书面知情同意后，从受试者那里获得皮肤活检。成纤维细胞在DMEM中用15%胎牛血清（FBS）和1%MEM-非必需氨基酸溶液在37 °C和5%CO2下培养。按照制造商的方案（参见 材料表）使用用于电穿孔的核苷载体，使用游离体重编程试剂盒对成纤维细胞进行重编程?…
Representative Results
3D 至 2D 小脑分化概述小脑细胞从iPSC开始产生。 图 1A 显示了整个工作流程以及为差异化添加的主要组件。在第0天，使用含有SB431542和Y-27632的CDM中的拉式玻璃移液管轻轻提起iPSC菌落（图1B）并放入超低附着板中，从而制备EB。在第 2 天添加 FGF2。在第7天，改变三分之一的培养基，并观察到EB形成（图1C）。在第14天，…
Discussion
在 体外模拟 人类小脑发育的能力对于疾病建模以及进一步了解正常大脑发育非常重要。不太复杂且具有成本效益的协议为可复制的数据生成和跨多个科学实验室的广泛实施创造了更多机会。这里使用Muguruma等人报告的生长因子使用不需要酶或解离剂的改进方法来描述小脑分化方案，该方法不需要酶或解离剂。图4 和改进的2D单层细胞生长方案类似于Holmes等人的方法<e…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢Jenny Gringer Richards在验证对照受试者方面的全面工作，我们从中生成了对照iPSC。这项工作得到了NIH T32 MH019113（致D.A.M.和K.A.K.），Nellie Ball Trust（致T.H.W.和A.J.W.），NIH R01 MH111578（致V.A.M.和J.A.W.），NIH KL2 TR002536（致A.J.W.）和Roy J. Carver慈善信托基金（致V.A.M.，J.A.W.和A.J.W.）的支持。这些数字是用 BioRender.com 创建的。
Materials
| 10 mL Serological pipette | Fisher Scientific | 13-678-26D | |
| 1-thio-glycerol | Sigma | M6145 | |
| 2 mL Serological pipette | Fisher Scientific | 13-678-26B | |
| 250 mL Filter Unit, 0.2 µm aPES, 50 mm Dia | Fisher Scientific | FB12566502 | |
| 35 mm Easy Grip Tissue Cluture Dish | Falcon | 353001 | |
| 4D Nucleofector core unit | Lonza | 276885 | Nucleofector |
| 5 mL Serological pipette | Fisher Scientific | 13-678-25D | |
| 60 mm Easy Grip Tissue Culture Dish | Falcon | 353004 | |
| 6-well ultra-low attachment plates | Corning | 3471 | |
| 9" Disposable Pasteur Pipets | Fisher Scientific | 13-678-20D | |
| Apo-transferrin | Sigma | T1147 | |
| Bovine serum albumin (BSA) | Sigma | A9418 | |
| Cell culture grade water | Cytiva | SH30529.02 | |
| Chemically defined lipid concentrate | Gibco | 11905031 | |
| Chroman 1 | Cayman | 34681 | |
| Class II, Type A2, Biological safety Cabinet | NuAire, Inc. | NU-540-600 | Hood, UV light |
| Costar 24-well plate, TC treated | Corning | 3526 | |
| Costar 6-well plate, TC treated | Corning | 3516 | |
| DAPI solution | Thermo Scientific | 62248 | |
| DMEM | Gibco | 11965092 | |
| DMEM/F12 | Gibco | 11320033 | |
| DMSO (Dimethly sulfoxide) | Sigma | D2438 | |
| DPBS+/+ | Gibco | 14040133 | |
| Emricasan | Cayman | 22204 | |
| Epi5 episomal iPSC reprogramming kit | Life Technologies | A15960 | |
| Essential 8-Flex | Gibco | A2858501 | PSC medium with heat-stable FGF2 |
| EVOS XL Core Imaging system | Life Technologies | AMEX1000 | |
| Fetal bovine serum – Premium Select | Atlanta Biologicals | S11150 | |
| FGF2 | Peprotech | 100-18B | |
| GlutaMAX supplement | Gibco | 35050061 | L-alanine-L-glutamine supplement |
| Ham's F12 Nutrient Mix | Gibco | 11765054 | |
| HERAcell VIOS 160i CO2 incubator | Thermo Scientific | 50144906 | |
| Human Anti-EN2, mouse | Santa Cruz Biotechnology | sc-293311 | |
| Human anti-Ki67/MKI67, rabbit | R&D Systems | MAB7617 | |
| Human anti-PTF1a, rabbit | Novus Biologicals | NBP2-98726 | |
| Human anti-TUBB3, mouse | Biolegend | 801213 | |
| IMDM | Gibco | 12440053 | |
| Insulin | Gibco | 12585 | |
| Laminin Mouse Protein | Gibco | 23017015 | |
| Matrigel Matrix | Corning | 354234 | Basement membrane matrix |
| MEM-NEAA | Gibco | 11140050 | |
| Mini Centrifuge | Labnet International | C1310 | Benchtop mini centrifuge |
| Monarch RNA Cleanup Kit (50 µg) | New England BioLabs | T2040 | Silica spin columns |
| Monarch Total RNA Miniprep Kit | New England BioLabs | T2010 | Silica spin columns |
| N-2 supplement | Gibco | 17502-048 | |
| Neurobasal medium | Gibco | 21103049 | |
| PBS, pH 7.4 | Gibco | 10010023 | |
| PFA 16% | Electron Microscopy Sciences | 15710 | |
| Polyamine supplement | Sigma | P8483 | |
| Poly-L-Ornithine (PLO) | Sigma | 3655 | |
| Potassium chloride | Sigma | 746436 | |
| SB431542 | Sigma | 54317 | |
| See through self-sealable pouches | Steriking | SS-T2 (90×250) | Autoclave pouches |
| Sodium citrate dihydrate | Fisher Scientific | S279-500 | |
| Syringe filters, sterile, PES 0.22 µm, 30 mm Dia | Research Products International | 256131 | |
| Trans-ISRIB | Cayman | 16258 | |
| TRIzol Reagent | Invitrogen | 15596018 | Phenol and guanidine isothiocyanate |
| TrypLE Express Enzyme (1x) | Gibco | 12604039 | Cell dissociation reagent |
| Vapor pressure osmometer | Wescor, Inc. | Model 5520 | Osmometer |
| Y-27632 | Biogems | 1293823 |
References
- Erceg, S., Lukovic, D., Moreno-Manzano, V., Stojkovic, M., Bhattacharya, S. S. Derivation of cerebellar neurons from human pluripotent stem cells. Current Protocols in Stem Cell Biology. , (2012).
- Erceg, S., et al. Efficient differentiation of human embryonic stem cells into functional cerebellar-like cells. Stem Cells and Development. 19 (11), 1745-1756 (2010).
- Muguruma, K. 3D culture for self-formation of the cerebellum from human pluripotent stem cells through induction of the isthmic organizer. Methods in Molecular Biology. 1597, 31-41 (2017).
- Muguruma, K., Nishiyama, A., Kawakami, H., Hashimoto, K., Sasai, Y. Self-organization of polarized cerebellar tissue in 3D culture of human pluripotent stem cells. Cell Reports. 10 (4), 537-550 (2015).
- Holmes, D. B., Heine, V. M. Streamlined 3D cerebellar differentiation protocol with optional 2D modification. Journal of Visualized Experiments: JoVE. (130), e56888 (2017).
- Holmes, D. B., Heine, V. M. Simplified 3D protocol capable of generating early cortical neuroepithelium. Biology Open. 6 (3), 402-406 (2017).
- Silva, T. P., et al. Maturation of human pluripotent stem cell-derived cerebellar neurons in the absence of co-culture. Frontiers in Bioengineering and Biotechnology. 8, 70 (2020).
- Nayler, S., Agarwal, D., Curion, F., Bowden, R., Becker, E. B. E. High-resolution transcriptional landscape of xeno-free human induced pluripotent stem cell-derived cerebellar organoids. Scientific Reports. 11, 12959 (2021).
- Salero, E., Hatten, M. E. Differentiation of ES cells into cerebellar neurons. Proceedings of the National Academy of Sciences of the United States of America. 104 (8), 2997-3002 (2007).
- Nie, Y., Walsh, P., Clarke, D. L., Rowley, J. A., Fellner, T. Scalable passaging of adherent human pluripotent stem cells. PLoS One. 9 (1), 88012 (2014).
- Chen, Y., et al. A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells. Nature Methods. 18 (5), 528-541 (2021).
- Machold, R., Fishell, G. Math1 is expressed in temporally discrete pools of cerebellar rhombic-lip neural progenitors. Neuron. 48 (1), 17-24 (2005).
- Wang, V. Y., Rose, M. F., Zoghbi, H. Y. Math1 expression redefines the rhombic lip derivatives and reveals novel lineages within the brainstem and cerebellum. Neuron. 48 (1), 31-43 (2005).
- Hoshino, M., et al. Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum. Neuron. 47 (2), 201-213 (2005).
- Mizuhara, E., et al. Purkinje cells originate from cerebellar ventricular zone progenitors positive for Neph3 and E-cadherin. Biologie du développement. 338 (2), 202-214 (2010).
- Ferreira, A., Caceres, A. Expression of the Class III β-tubulin isotype in developing neurons in culture. Journal of Neuroscience Research. 32 (4), 516-529 (1992).
- Sullivan, K. F., Cleveland, D. W. Identification of conserved isotype-defining variable region sequences for four vertebrate beta tubulin polypeptide classes. Proceedings of the National Academy of Sciences of the United States of America. 83 (12), 4327-4331 (1986).
- Joyner, A. L. Engrailed, Wnt and Pax genes regulate midbrain-hindbrain development. Trends in Genetics. 12 (1), 15-20 (1996).
- Joyner, A. L., Liu, A., Millet, S. Otx2, Gbx2 and Fgf8 interact to position and maintain a mid-hindbrain organizer. Current Opinion in Cell Biology. 12 (6), 736-741 (2000).
- Minaki, Y., Nakatani, T., Mizuhara, E., Inoue, T., Ono, Y. Identification of a novel transcriptional corepressor, Corl2, as a cerebellar Purkinje cell-selective marker. Gene Expression Patterns. 8 (6), 418-423 (2008).
- Aldinger, K. A., et al. Spatial and cell type transcriptional landscape of human cerebellar development. Nature Neuroscience. 24 (8), 1163-1175 (2021).
- Fossat, N., Courtois, V., Chatelain, G., Brun, G., Lamonerie, T. Alternative usage of Otx2 promoters during mouse development. Developmental Dynamics. 233 (1), 154-160 (2005).
- Akbarian, S., et al. The PsychENCODE project. Nature Neuroscience. 18 (12), 1707-1712 (2015).
- Aijaz, S., et al. Expression analysis of SIX3 and SIX6 in human tissues reveals differences in expression and a novel correlation between the expression of SIX3 and the genes encoding isocitrate dehyhrogenase and cadherin 18. Genomics. 86 (1), 86-99 (2005).
- Conte, I., Morcillo, J., Bovolenta, P. Comparative analysis of Six3 and Six6 distribution in the developing and adult mouse brain. Developmental Dynamics. 234 (3), 718-725 (2005).
- Andreasen, N. C., Pierson, R. The role of the cerebellum in schizophrenia. Biological Psychiatry. 64 (2), 81-88 (2008).
- Nopoulos, P. C., Ceilley, J. W., Gailis, E. A., Andreasen, N. C. An MRI study of cerebellar vermis morphology in patients with schizophrenia: Evidence in support of the cognitive dysmetria concept. Biological Psychiatry. 46 (5), 703-711 (1999).
- Jacobsen, L. K., et al. Quantitative morphology of the cerebellum and fourth ventricle in childhood-onset schizophrenia. American Journal of Psychiatry. 154 (12), 1663-1669 (1997).
- Saywell, V., Cioni, J. -. M., Ango, F. Developmental gene expression profile of axon guidance cues in Purkinje cells during cerebellar circuit formation. The Cerebellum. 13 (3), 307-317 (2014).
- Kim, D., Ackerman, S. L. The UNC5C netrin receptor regulates dorsal guidance of mouse hindbrain axons. Journal of Neuroscience. 31 (6), 2167-2179 (2011).
- Maier, V., et al. Semaphorin 4C and 4G are ligands of Plexin-B2 required in cerebellar development. Molecular and Cellular Neuroscience. 46 (2), 419-431 (2011).
- Telley, L., et al. Dual function of NRP1 in axon guidance and subcellular target recognition in cerebellum. Neuron. 91 (6), 1276-1291 (2016).
- Wang, S., et al. Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons. Scientific Reports. 5, 9232 (2015).
- Shabanipour, S., et al. Primary culture of neurons isolated from embryonic mouse cerebellum. Journal of Visualized Experiments. (152), e60168 (2019).
Citer Cet Article
Madencioglu, D. A., Kruth, K. A., Wassink, T. H., Magnotta, V. A., Wemmie, J. A., Williams, A. J. Modeling Human Cerebellar Development In Vitro in 2D Structure. J. Vis. Exp. (187), e64462, doi:10.3791/64462 (2022).