早期无引导人脑类器官神经血管生态位建模到允许的雏鸡胚绒毛膜尿囊膜中
1Instituto de Biología Celular y Neurociencias “Prof. E. De Robertis” (IBCN),CONICET – Universidad de Buenos Aires, 2Facultad de Medicina, Departamento de Biología Celular, Histología, Embriología y Genética,Universidad de Buenos Aires, 3Institute of Neurosciences, Pathology and Experimental Therapeutics Dept,University of Barcelona, 4Functional Neurogenomics Group, Neurodevelopmental Disorders,IDIBELL, L’Hospitalet de Llobregat, 5IBE, Institute of Evolutionary Biology (UPF-CSIC), Department of Medicine and Life Sciences,Universitat Pompeu Fabra, 6Institució Catalana de Recerca i Estudis Avançats (ICREA) and Universitat Pompeu Fabra, 7Center for Genomic Regulation (CRG),The Barcelona Institute of Science and Technology, 8BarcelonaBeta Brain Research Center,Pasqual Maragall Foundation, 9Instituto de Investigación en Biomedicina (IBioBA) – CONICET – Instituto Partner de la Sociedad Max Planck
Summary
在这里，我们提出了一种将处于多个成熟阶段的人脑类器官移植到鸡绒毛膜尿囊膜 （CAM） 中的方案。大脑类器官是按照无指导的标准化协议生长的。
Abstract
将类器官移植到模型动物的血管化组织中，例如免疫缺陷小鼠或鸡胚绒毛膜尿囊膜 （CAM），已被证明对新生血管形成建模有效。CAM是一种富含血管化的胚外膜，其免疫反应性有限，因此成为人源性细胞移植的优良宿主模型。
本文描述了将多个成熟阶段分化的人脑类器官移植到CAM中的策略。大脑类器官的细胞组成随时间而变化，反映了人类大脑发育的里程碑。我们将相关成熟阶段的脑类器官移植到胚胎日（E）7鸡胚胎的CAM中：神经上皮扩增（18 DIV），早期神经发生（60 DIV）和早期胶质生成（180 DIV）。5 天后收获移植的脑类器官并分析其组织学特征。
在移植的类器官中未检测到新生血管形成的组织学迹象或与移植物相邻的异常血管。此外，在移植类器官的细胞组成中观察到显着变化，即胶质纤维酸性蛋白阳性反应性星形胶质细胞的数量增加。然而，细胞结构的变化取决于类器官成熟阶段。总而言之，这些结果表明，大脑类器官可以在CAM中生长，并且它们根据移植时的成熟阶段显示出细胞结构的差异。
Introduction
人脑类器官是一种新兴技术，使我们能够在体外概括人脑的早期发育1,2,3。然而，该模型的主要局限性之一是缺乏血管化，血管化不仅在大脑稳态中起着不可或缺的作用，而且在大脑发育中也起着不可或缺的作用4.除了输送氧气和营养物质外，越来越多的证据表明，大脑的血管系统在发育过程中调节神经分化、迁移和突触发生 5,6。因此，迫切需要建立可靠的模型，为大脑类器官提供缺失的血管信号和结构，从而增强人脑类器官生成7的复杂性。
在所提出的血管化方法中，可以考虑两种主要的流线型：类器官移植到活生物体中，以及共培养内皮细胞和神经细胞的纯体外技术8,9,10,11,12。小鼠脑移植既昂贵又耗时，这使得其他技术与更简单的模型相关。雏鸡绒毛膜尿囊膜 （CAM） 测定已广泛用于研究血管生成 13,14,15。在过去的十年中，几个小组成功地将不同类型的类器官（包括肾脏16,17、心脏18 和肿瘤类器官19,20）移植到 CAM 中。然而，人们对将人脑类器官移植到 CAM 中的功效、毒性/排斥反应、生理效应和方法知之甚少。另一个有趣但尚未探索的方面是在CAM和类器官星形胶质细胞界面之间形成嵌合血脑屏障（BBB）。先前的开创性工作表明，通过移植星形胶质细胞和星形胶质细胞条件培养基在CAM中产生BBB的假定可行性21,22,23。然而，成熟的星形胶质细胞似乎无法达到这个24,25。因此，星形胶质细胞诱导的BBB形成仍然存在争议，移植人脑类器官将使我们能够阐明这一争议。
这篇视频文章描述了一种 将卵 内人脑类器官移植到 CAM 中的方案，该方案可促进生长、改善和血管化，从而产生包含组织学相容的 BBB 元件的类器官。在这里，我们提出了一个确保鸡胚胎存活的方案，并报告了CAM维持大脑类器官生长的允许性。
Protocol
白角鸡（Gallus gallus）胚胎按照美国国家研究委员会生命科学委员会实验动物资源研究所的实验动物护理和使用指南进行处理，实验由巴塞罗那大学实验动物护理和使用委员会批准。 1.非引导脑类器官制备 在室温（RT）下预热的mTESR1中维持H9人胚胎干细胞（hESCs），在6孔板中溶解在DMEM（1：40）中的溶解在5%CO2 培养箱中，溶解在DMEM（1：40）中?…
Representative Results
选择移植的胚胎成熟时间表当受精卵在38°C和60%相对湿度下孵化时，实验从D0开始。绒毛膜尿囊膜 （CAM） 是一种高度血管化的胚外膜，在卵子孵化后发育。它是由尿囊和绒毛膜融合而成的。在D1处，孵育24小时后，刺穿气室以防止CAM附着在内壳膜上。与后期穿刺 （D4） 相比，在 D1 处刺穿气室可提高气室的质量。CAM继续生长，直到D12，当它包裹整个卵子内容物并牢固地粘附在内壳膜?…
Discussion
在这项研究中，我们描述了一个包含许多关键步骤的详细方案，这些步骤在移植时提供人脑类器官的有利生长和发育，而不会干扰鸡胚胎的存活。我们建议在孵育24小时（第1天）后使用无菌针刺穿鸡蛋的气室。此外，我们还尝试在第 4 天进行穿刺（在用光检查蛋壳以测试脉管系统的发育以确保我们只使用健康的胚胎后）。然而，由于CAM血管靠近蛋壳，这导致胚胎活力下降。应该注意的是，施加过?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢 UB 的 Alcántara 博士和 Ortega 博士以及 Acosta 博士实验室的其他成员进行了富有洞察力的讨论。S.A.是巴塞罗那大学加泰罗尼亚将军学院的Serra-Hunter研究员助理教授。
Materials
| Anti-TUBB3 [Tuj1], mouse | BioLegend | 801201 | 1:1,000 |
| Anti-GFAP, rabbit | GeneTex | GTX108711 | 1:500 |
| Anti-rabbit AlexaFluor 488, goat. | Invitrogen | A-21206 | 1:1,000 |
| Anti-mouse AlexaFluor 594, goat | Jackson ImmunoResearch | 715-585-150 | 1:500 |
| Fertilized White Leghorn chicken (Gallus gallus) eggs | Granja Gibert (Cambrils, Spain) | ||
| DAPI | Invitrogen | D1306 | 1:10,000 |
| DPX | Sigma | 100579 | xylene-based mounting medium |
| Gentle Dissociation Solution | CreativeBiolabs | ITS-0622-YT187 | cell dissociation solution |
| Matrigel | BD Biosciences | 356234 | |
| Mowiol 4-88 mounting media | Merk | 81381 | |
| Paper towel, lab-grade | Sigma-Aldrich | Z188956 | |
| ROCK inhibitor Y27632 | Millipore | SCM075 | 10 nM |
| Sharp-Point Surgical Scissors | VWR | 470106-340 | |
| Superfrost Plus Adhesion Microscope Slides | Epredia | J1800AMNZ |
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Citar este artigo
Fiore, L., Arderiu, J., Martí-Sarrias, A., Turpín, I., Pareja, R. I., Navarro, A., Holubiec, M., Bianchelli, J., Falzone, T., Spelzini, G., Scicolone, G., Acosta, S. Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane. J. Vis. Exp. (204), e65710, doi:10.3791/65710 (2024).