基于2D和3D人诱导多能干细胞的模型，用于剖析新皮质发育过程中原发性纤毛受累
1Imagine Institute, INSERM UMR 1163,Université de Paris, 2Imagine Institute, iPSC Core Facility, INSERM UMR U1163,Université de Paris, 3Necker Bio-image Analysis platform of the SFR Necker, 4Imagine Institute, Cell Imaging Platform, INSERM-US24-CNRS UMS 3633 Structure Fédérative de Recherche Necker, INSERM UMR U1163,Université de Paris, 5Fédération de Génétique, Hôpital Necker-Enfants Malades,Assistance Publique Hôpitaux de Paris, 6Pediatric Neurology,APHP- Necker Enfants Malades Hospital
Summary
我们提出了基于2D和3D人类诱导多能干细胞（hIPSC）的新皮质发育模型的生成和表征的详细方案，以及能够对原发性纤毛（PC）生物发生和功能进行定性和定量分析的补充方法。
Abstract
原代纤毛（PC）是非运动动态微管基细胞器，从大多数哺乳动物细胞表面突出。它们在细胞周期的G1 / G0阶段从较旧的中心粒中出现，而当细胞在G2 / M相边界重新进入细胞周期时，它们会分解。它们通过检测和转导对许多细胞过程至关重要的细胞外信号来充当信号中枢。与大多数细胞类型类似，所有新皮质神经干和祖细胞（NSPCs）已被证明携带PC，允许它们感知和转导正常大脑皮质发育所需的特定信号。在这里，我们提供详细的方案来生成和表征来自人类诱导多能干细胞（hIPSC）的二维（2D）和三维（3D）细胞模型，以进一步剖析PC在新皮质发育过程中的参与。特别是，我们提出了研究2D神经玫瑰花衍生NSPC中的PC生物发生和功能的方案，包括声波刺猬（SHH）途径的转导。为了利用大脑类器官的三维（3D）组织，我们描述了一种简单的方法，用于 对免疫染色 的大脑类器官进行3D成像。光学清除后，快速采集整个类器官可以检测整个类器官的新皮质祖细胞和神经元上的中心体和PC。最后，我们详细介绍了免疫染色和清除厚厚的自由漂浮类器官切片的程序，保留了大量3D空间信息，并允许对PC生物发生和功能进行详细的定性和定量分析所需的高分辨率采集。
Introduction
原代纤毛（PC）是基于微管的细胞器，可感知和转导来自细胞外环境的大量化学和机械线索。特别是，PC是脊椎动物中刺猬信号通路转导的中心细胞器1，2。虽然大多数神经细胞早已被证明含有PC，但这种细胞器在塑造中枢神经系统方面的贡献长期以来一直被低估。对新皮质发育的研究导致发现了多个神经干和祖细胞（NSPCs），它们都含有PC，其位置被认为对祖细胞命运的确定至关重要3，4，5，6，7。PC已被证明对于正常大脑皮层发育所需的细胞机制至关重要，包括NSPC扩增和承诺8，9，10，11，12 以及支持神经元迁移的桡神经胶质支架的心基底极性13。此外，在神经元切向皮质板迁移期间需要PC14，15。最后，已经提出了PC在大脑皮层中建立神经元突触连接中的作用16，17。总而言之，这些发现证明了PC在大脑皮质发育的主要步骤中起着至关重要的作用18，19 ，并提出了调查它们参与大脑皮质发育异常的病理机制的必要性。
最近的研究在很大程度上提高了我们对人类和动物模型中皮质发育之间重要细胞和分子差异的理解，强调了开发人类模型系统的必要性。根据这种观点，人类诱导多能干细胞（hIPSCs）代表了在相关遗传和细胞背景下研究疾病发病机制的一种有前途的方法。贴壁二维（2D）基于细胞的模型或神经玫瑰花含有类似于发育中大脑皮层中看到的NSPC，其组织成玫瑰花形结构，显示出正确的耳基极性20，21，22。此外，三维（3D）培养系统允许产生背前脑类器官，这些类器官概括了人类大脑皮质发育的许多特征23，24，25，26。这两种互补的基于细胞的建模方法提供了令人兴奋的视角，以剖析PC在大脑皮层正常和病理发育过程中的参与。
在这里，我们为神经玫瑰花和衍生的NSPC以及背前脑类器官的生成和表征提供了详细的方案。我们还提供了详细的方案，通过测试声波刺猬途径的转导并分析该途径中涉及的关键分子的动力学，来分析NSPC上存在的PC的生物发生和功能。为了利用大脑类器官的3D组织，我们还建立了一种简单且具有成本效益的方法，用于 对免疫 染色的大脑类器官进行3D成像，通过光学片显微镜可以快速采集整个类器官，具有高分辨率，能够可视化所有类型的新皮质祖细胞和整个类器官神经元上的PC。最后，我们在150μm自由浮动切片上调整了免疫组织化学，随后使用共振扫描共聚焦显微镜进行清除和采集，从而实现高分辨率图像采集，这是详细分析PC生物发生和功能所必需的。具体而言，3D成像软件允许对PC进行3D重建，随后分析形态参数，包括PC的长度，数量和方向，以及沿轴线测量纤毛成分的信号强度。
Protocol
1. 基于2D hIPS细胞的新皮质发育模型的生成 神经玫瑰花纹形成 从含有大型规则菌落的 hIPSC 培养开始，表现出小于 10% 的分化和不超过 80% 的汇合度。 用 2 mL PBS 冲洗 hIPSC。 加入2mL补充岩石抑制剂的NSPC诱导培养基（NIM + 10μM的Y-27632）。 使用针头从一个35毫米培养皿中手动解剖每个hIPSC菌落，以在水平和垂直方向上精确地切割每个菌落?…
Representative Results
基于 2D hIPS 细胞的模型，用于研究原发性纤毛生物发生和功能此处详述的方案改编自先前发表的研究20，21，22。该协议允许产生神经玫瑰花结构，其中包含类似于发育中的新皮层祖和神经元的新皮质祖和神经元。可以使用特定制造商进行常规免疫染色分析来执行详细验证3。例如，顶端祖细胞?…
Discussion
PC现在被认为是调节正常大脑皮质发育过程中关键步骤的关键细胞器18，19，31 包括NSPC扩增和承诺8，9，10，11，12 以及神经元迁移13，14 和突触发生<sup class="xref"…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家研究机构（ANR）对S.T.（ANR-17-CE16-0003-01）和N.B.B（ANR-16-CE16-0011和ANR-19-CE16-0002-01）的赠款的支持。LB由ANR在Investissments d’avenir计划（ANR-10-IAHU-01）和Bettencourt Schueller基金会（MD-PhD计划）下提供支持。Imagine Institute由ANR根据Investissments d’avenir计划（ANR-10-IAHU-01，CrossLab项目）获得国家资助，并作为第二个Investissd’Avenir计划（ANR-17-RHUS-0002）的一部分。
Materials
| 2-Mercaptoéthanol (50 mM) | ThermoFisher Scientific | 31350010 | |
| 6-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates | Corning | 3471 | |
| 96-well Clear Round Bottom Ultra-Low Attachment Microplate | Corning | 7007 | |
| B-27 Supplement (50X), minus vitamin A | ThermoFisher Scientific | 12587010 | |
| B-27 Supplement (50X), serum free | ThermoFisher Scientific | 17504044 | |
| CellAdhere Dilution Buffer | StemCell Technologies | 7183 | |
| DMEM/F-12, Glutamax | ThermoFisher Scientific | 31331028 | |
| DMSO | ATCC | 4-X | |
| Dorsomorphin | StemCell Technologies | 72102 | |
| Easy Grip 35 10mm | Falcon | 353001 | |
| EDTA | ThermoFisher Scientific | 15575020 | |
| EGF , 25µg | Thermofischer | PHG0315 | |
| FGF2 , 25µg | Thermofischer | PHG0264 | |
| Gentle Cell Dissociation Reagent | StemCell Technologies | 7174 | |
| Insulin | ThermoFisher Scientific | 12585014 | |
| KnockOut Serum | ThermoFisher Scientific | 10828028 | |
| Laminin (1mg) | Thermofischer | 23017015 | |
| LDN193189 | StemCell Technologies | 72147 | |
| Matrigel Growth Factor Reduced | Corning | 354230 | |
| MEM Non-Essential Amino Acids Solution (100X) | ThermoFisher Scientific | 11140050 | |
| Mowiol 4-88 | Sigma Aldrich | 81381-250G | |
| mTeSR1 | StemCell Technologies | 85850 | |
| Neural Basal Medium | Thermofischer | 21103049 | |
| Orbital shaker | Dutscher | 995002 | |
| PBS | ThermoFisher Scientific | 14190094 | |
| Penicillin-Streptomycin (10,000 U/mL) | ThermoFisher Scientific | 15140122 | |
| PFA 32% | Electron Microscopy Sciences | 15714 | |
| Poly-L-Ornithine (PO) | Sigma | P4957 | |
| Recombinant human BDNF 10 µg | Stem Cell Technologies | 78005 | |
| Recombinant Human FGF-basic | Peprotech | 100-18B | |
| rSHH | R&D Systems | 8908-SH | |
| SAG | Santa Cruz | Sc-202814 | |
| SB431542 | StemCell Technologies | 72232 | |
| Stembeads FGF2 | StemCulture | SB500 | |
| Sucrose | Sigma Aldrich | S7903-250G | |
| Superfrost Plus Adhesion Slides | Thermo Scientific | J1800AMNZ | |
| Supplément N2- (100X) | ThermoFisher Scientific | 17502048 | |
| TDE 2,2’-Thiodiethanol | Sigma Aldrich | 166782-500G | |
| Vitronectin | StemCell Technologies | 7180 | |
| Y-27632 | StemCell Technologies | 72304 | |
| Primary Antibodies | |||
| ARL13B | Abcam | Ab136648 | 1/200e |
| ARL13B | Proteintech | 17711-1-AP | 1/500e |
| CTIP2 | Abcam | Ab18465 | 1/500e |
| GLI2 | R&D Systems | AF3526 | 1/100 |
| GPR161 | Proteintech | 13398-1-AP | 1/100 |
| N-Cadherin | BD Transduction Lab | 610921 | 1/500e |
| P-Vimentin | MBL | D076-3 | 1/500e |
| PAX6 | Biolegend | PRB-278P | 1/200e |
| PCNT | Abcam | Ab4448 | 1/1000e |
| S0X2 | R&D Systems | MAB2018 | 1/200e |
| SATB2 | Abcam | Ab51502 | 1/200e |
| TBR2 | Abcam | Ab216870 | 1/400e |
| TPX2 | NovusBio | NB500-179 | 1/500e |
| γTUBULIN | Sigma Aldrich | T6557 | 1/500e |
| Secondary Antibodies | |||
| Donkey anti-rabbit AF488 | ThermoFisher Scientific | A21206 | 1/500e |
| Goat anti-mouse AF555 | ThermoFisher Scientific | A21422 | 1/500e |
| Goat anti-mouse AF647 | ThermoFisher Scientific | A21236 | 1/500e |
| Goat anti-rat AF555 | ThermoFisher Scientific | A21434 | 1/500e |
Referências
- Huangfu, D., et al. Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature. 426 (6962), 83-87 (2003).
- Goetz, S. C., Anderson, K. V. The primary cilium: a signalling centre during vertebrate development. Nature Reviews. Genetics. 11 (5), 331-344 (2010).
- Hansen, D. V., Lui, J. H., Parker, P. R. L., Kriegstein, A. R. Neurogenic radial glia in the outer subventricular zone of human neocortex. Nature. 464 (7288), 554-561 (2010).
- Lui, J. H., Hansen, D. V., Kriegstein, A. R. Development and evolution of the human neocortex. Cell. 146 (1), 18-36 (2011).
- Nonaka-Kinoshita, M., et al. Regulation of cerebral cortex size and folding by expansion of basal progenitors. The EMBO Journal. 32 (13), 1817-1828 (2013).
- Taverna, E., Götz, M., Huttner, W. B. The cell biology of neurogenesis: toward an understanding of the development and evolution of the neocortex. Annual Review of Cell and Developmental Biology. 30, 465-502 (2014).
- Fernández, V., Llinares-Benadero, C., Borrell, V. Cerebral cortex expansion and folding: what have we learned. The EMBO Journal. 35 (10), 1021-1044 (2016).
- Spear, P. C., Erickson, C. A. Apical movement during interkinetic nuclear migration is a two-step process. Biologia do Desenvolvimento. 370 (1), 33-41 (2012).
- Wilsch-Bräuninger, M., Florio, M., Huttner, W. B. Neocortex expansion in development and evolution – from cell biology to single genes. Current Opinion in Neurobiology. 39, 122-132 (2016).
- Anderson, C. T., Stearns, T. Centriole age underlies asynchronous primary cilium growth in mammalian cells. Current Biology: CB. 19 (17), 1498-1502 (2009).
- Paridaen, J. T. M. L., Wilsch-Bräuninger, M., Huttner, W. B. Asymmetric inheritance of centrosome-associated primary cilium membrane directs ciliogenesis after cell division. Cell. 155 (2), 333-344 (2013).
- Gabriel, E., et al. CPAP promotes timely cilium disassembly to maintain neural progenitor pool. The EMBO Journal. 35 (8), 803-819 (2016).
- Higginbotham, H., et al. Arl13b-regulated cilia activities are essential for polarized radial glial scaffold formation. Nature Neuroscience. 16 (8), 1000-1007 (2013).
- Baudoin, J. -. P., et al. Tangentially migrating neurons assemble a primary cilium that promotes their reorientation to the cortical plate. Neuron. 76 (6), 1108-1122 (2012).
- Higginbotham, H., et al. Arl13b in primary cilia regulates the migration and placement of interneurons in the developing cerebral cortex. Developmental Cell. 23 (5), 925-938 (2012).
- Kumamoto, N., et al. A role for primary cilia in glutamatergic synaptic integration of adult-born neurons. Nature Neuroscience. 15 (3), 399-405 (2012).
- Guadiana, S. M., et al. Arborization of dendrites by developing neocortical neurons is dependent on primary cilia and type 3 adenylyl cyclase. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 33 (6), 2626-2638 (2013).
- Thomas, S., Boutaud, L., Reilly, M. L., Benmerah, A. Cilia in hereditary cerebral anomalies. Biology of the Cell. 111 (9), 217-231 (2019).
- Hasenpusch-Theil, K., Theil, T. The multifaceted roles of primary cilia in the development of the cerebral cortex. Frontiers in Cell and Developmental Biology. 9, 630161 (2021).
- Shi, Y., Kirwan, P., Livesey, F. J. Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nature Protocols. 7 (10), 1836-1846 (2012).
- Boissart, C., et al. Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening. Translational Psychiatry. 3, 294 (2013).
- Chambers, S. M., et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnology. 27 (3), 275-280 (2009).
- Lancaster, M. A., Knoblich, J. A. Generation of cerebral organoids from human pluripotent stem cells. Nature Protocols. 9 (10), 2329-2340 (2014).
- Qian, X., et al. Generation of human brain region-specific organoids using a miniaturized spinning bioreactor. Nature Protocols. 13 (3), 565-580 (2018).
- Krefft, O., Jabali, A., Iefremova, V., Koch, P., Ladewig, J. Generation of standardized and reproducible forebrain-type cerebral organoids from human induced pluripotent stem cells. Journal of Visualized Experiments: JoVE. (131), (2018).
- Kadoshima, T., et al. Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex. Proceedings of the National Academy of Sciences of the United States of America. 110 (50), 20284-20289 (2013).
- Topol, A., Tran, N. N., Brennand, K. J. A guide to generating and using hiPSC derived NPCs for the study of neurological diseases. Journal of Visualized Experiments: JoVE. (96), e52495 (2015).
- Berg, S., et al. ilastik: interactive machine learning for (bio)image analysis. Nature Methods. 16 (12), 1226-1232 (2019).
- Hansen, J. N., et al. Multifocal imaging for precise, label-free tracking of fast biological processes in 3D. bioRxiv. , (2020).
- Pașca, S. P. The rise of three-dimensional human brain cultures. Nature. 553 (7689), 437-445 (2018).
- Andreu-Cervera, A., Catala, M., Schneider-Maunoury, S. Cilia, ciliopathies and hedgehog-related forebrain developmental disorders. Neurobiology of Disease. 150, 105236 (2021).
- Christensen, S. T., Morthorst, S. K., Mogensen, J. B., Pedersen, L. B. Primary cilia and coordination of Receptor Tyrosine Kinase (RTK) and Transforming Growth Factor β (TGF-β) signaling. Cold Spring Harbor Perspectives in Biology. 9 (6), (2017).
- Wheway, G., Nazlamova, L., Hancock, J. T. Signaling through the primary cilium. Frontiers in Cell and Developmental Biology. 6, 8 (2018).
- Sivitilli, A. A., et al. Robust production of uniform human cerebral organoids from pluripotent stem cells. Life Science Alliance. 3 (5), (2020).
- Quelennec, E., et al. Generation of two induced pluripotent stem cell lines IMAGINi004-A and IMAGINi005-A from healthy donors. Stem Cell Research. 48, 101959 (2020).
- Belle, M., et al. Tridimensional visualization and analysis of early human development. Cell. 169 (1), 161-173 (2017).
- Vigouroux, R. J., Belle, M., Chédotal, A. Neuroscience in the third dimension: shedding new light on the brain with tissue clearing. Molecular Brain. 10 (1), 33 (2017).
- Lallemant, L., Lebreton, C., Garfa-Traoré, M. Comparison of different clearing and acquisition methods for 3D imaging of murine intestinal organoids. Journal of Biological Methods. 7 (4), 141 (2020).
- Aoyagi, Y., Kawakami, R., Osanai, H., Hibi, T., Nemoto, T. A rapid optical clearing protocol using 2,2′-thiodiethanol for microscopic observation of fixed mouse brain. PloS One. 10 (1), 0116280 (2015).
Citar este artigo
Boutaud, L., Michael, M., Banal, C., Calderon, D., Farcy, S., Pernelle, J., Goudin, N., Maillard, C., Dimartino, C., Deleschaux, C., Dupichaud, S., Lebreton, C., Saunier, S., Attié-Bitach, T., Bahi-Buisson, N., Lefort, N., Thomas, S. 2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development. J. Vis. Exp. (181), e62667, doi:10.3791/62667 (2022).