Name: 作者聚焦：优化雏鸡胚胎中人脑类器官的神经血管发育
Uploaded: 2024-02-16T13:00:00
Description: Watch this Scientific Journal Video about Author Spotlight: Optimizing the Neurovascular Development of Human Brain Organoid in Chick Embryo at JoVE.com

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。

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
<ol><li>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?...

将人脑类器官嫁接到雏鸡胚胎的绒毛膜尿囊膜中

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+vascularized+brain+organoids+to+study+neurovascular+interactions.">Generation of vascularized brain organoids to study neurovascular interactions.</a> Elife. 11, e76707 (2022).</li><li>Paredes, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte+precursor+cell+specification+is+regulated+by+bidirectional+neural+progenitor-endothelial+cell+crosstalk.">Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor-endothelial cell crosstalk.</a> Nat Neurosci. 24 (4), 478-488 (2021).</li><li>Matsui, T. K., Tsuru, Y., Hasegawa, K., Kuwako, K. 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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+vascularized+brain+organoids.">Generation of human vascularized brain organoids.</a> Neuroreport. 29 (7), 588-593 (2018).</li><li>Cakir, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+human+brain+organoids+with+a+functional+vascular-like+system.">Engineering of human brain organoids with a functional vascular-like system.</a> Nat Methods. 16 (11), 1169-1175 (2019).</li><li>Shi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+human+cortical+organoids+(vorganoids)+model+cortical+development+in+vivo.">Vascularized human cortical organoids (vorganoids) model cortical development in vivo.</a> PLoS Biol. 18 (5), e3000705 (2020).</li><li>Mansour, A. 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(157), e60995 (2020).</li><li>Varzideh, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cardiomyocytes+undergo+enhanced+maturation+in+embryonic+stem+cell-derived+organoid+transplants.">Human cardiomyocytes undergo enhanced maturation in embryonic stem cell-derived organoid transplants.</a> Biomaterials. 192, 537-550 (2019).</li><li>Komatsu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cam+model+for+cic-dux4+sarcoma+and+its+potential+use+for+precision+medicine.">The cam model for cic-dux4 sarcoma and its potential use for precision medicine.</a> Cells. 10 (10), 2613 (2021).</li><li>Worsdorfer, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+complex+human+organoid+models+including+vascular+networks+by+incorporation+of+mesodermal+progenitor+cells.">Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells.</a> Sci Rep. 9 (1), 15663 (2019).</li><li>Janzer, R. C., Jaff, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+induce+blood-brain+barrier+properties+in+endothelial+cells.">Astrocytes induce blood-brain barrier properties in endothelial cells.</a> Nature. 325 (6101), 253-257 (1987).</li><li>Janzer, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier:+Cellular+basis.">The blood-brain barrier: Cellular basis.</a> J Inherit Metab Dis. 16 (4), 639-647 (1993).</li><li>Lobrinus, J. A., Juillerat-Jeanneret, L., Darekar, P., Schlosshauer, B., Janzer, R. 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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-TUBB3 [Tuj1], mouse&#160;</td>
			<td>BioLegend</td>
			<td>801201</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Anti-GFAP, rabbit</td>
			<td>GeneTex</td>
			<td>GTX108711</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Anti-rabbit AlexaFluor 488, goat.</td>
			<td>Invitrogen</td>
			<td>A-21206</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Anti-mouse AlexaFluor 594, goat</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-585-150</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Fertilized White Leghorn chicken (Gallus gallus) eggs</td>
			<td>Granja Gibert (Cambrils, Spain)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>1:10,000</td>
		</tr>
		<tr>
			<td>DPX</td>
			<td>Sigma</td>
			<td>100579</td>
			<td>xylene-based mounting medium&#160;</td>
		</tr>
		<tr>
			<td>Gentle Dissociation Solution</td>
			<td>CreativeBiolabs</td>
			<td>ITS-0622-YT187</td>
			<td>cell dissociation solution</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol 4-88 mounting media</td>
			<td>Merk</td>
			<td>81381</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towel, lab-grade</td>
			<td>Sigma-Aldrich</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y27632</td>
			<td>Millipore</td>
			<td>SCM075</td>
			<td>10 nM</td>
		</tr>
		<tr>
			<td>Sharp-Point Surgical Scissors</td>
			<td>VWR</td>
			<td>470106-340</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Adhesion Microscope Slides</td>
			<td>Epredia</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
	</tbody>
</table>

early unguided human brain organoid neurovascular niche modeling into the permissive chick embryo chorioallantoic membrane

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。

Human brain organoids are an emerging technique that allows us to recapitulate the early development of the human brain in vitro1,2,3. Nevertheless, one of the major limitations of this model is the lack of vascularization, which plays indispensable roles not only in brain homeostasis but also in brain development4. In addition to the delivery of oxygen and nutrients, accumulating evidence suggests that the vascular system of the brain regulates neural differentiation, migration, and synaptogenesis during development5,6. Therefore, there is an urgent need to establish reliable models that can provide the missing vascular signaling and structure to brain organoids, enhancing the complexity of human brain organoid generation7.
Among the proposed methods for vascularization, two main streamlines can be considered: organoid engrafting into a living organism and purely in vitro technologies co-culturing endothelial cells and neural cells8,9,10,11,12. Intracerebral transplantation in mice is costly and time-consuming, making other technologies relevant for simpler models. The chick chorioallantoic membrane (CAM) assay has been used extensively to study angiogenesis13,14,15. In the last decade, several groups have successfully engrafted different types of organoids, including kidney16,17, cardiac18, and tumor organoids19,20, into CAMs. Nevertheless, little is known about the efficacy, toxicity/rejection, physiological effect, and methods to engraft human brain organoids into the CAM. Another interesting and yet unexplored aspect is the formation of a chimeric blood-brain barrier (BBB) between the CAM and the organoid astrocytic interface. Previous pioneering work suggested the putative feasibility of generating a BBB in the CAM by transplanting astrocytes and astrocyte-conditioned medium21,22,23. However, mature astrocytes seem to be unable to achieve this24,25. Thus, the astrocyte-induced formation of the BBB remains debatable, and transplanting human brain organoids would allow us to shed light on this controversy.
This video article describes a protocol for an in ovo human brain organoid transplant into CAM that promotes growth, improvement, and vascularization, resulting in organoids that encompass histologically compatible BBB elements. Here, we present a protocol ensuring the survival of the chicken embryo and report on the permissivity of the CAM to sustain brain organoid growth.

作者聚焦：优化雏鸡胚胎中人脑类器官的神经血管发育

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

The key questions I aim to address revolve around the mechanisms and processes involving human brain formation in the context of normal development, as well as various pathological states. This includes investigating the factors influencing brain development, such as genetics, environmental, and epigenetic influences. Obtaining brain organoids that closely resemble the adult human brain, including vascularization, blood brain barrier functionality, the presence of microglia and cellular diversity is challenging.Overcoming this is essential for the development of more physiologically relevant brain organoid models that can efficiently mimic the adult human brain to enhance clinical applications. Through our research, we provide optimal conditions for transplanting human brain organoids into chorioallantoic membrane to ensure good development of the microglia. This clinical improvement is central for forthcoming efforts in the field to generate synthetic vascularized environment for brain organoids.The advantage of our protocol compared to other techniques lies in its high effectiveness and cost efficiency. We have achieved a high success rate in transplanting viable organoids using fertile eggs from farm chickens. In the future, our laboratory will focus on conducting functional assessments of neo vasculature associated with organoids, exploring potential use of this vascular design model for application of intravenous nanomedicine.Additionally, we aim to evaluate the biodistribution of these nanomedicines in the human central nervous system.

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?...

Watch this Scientific Journal Video about Author Spotlight: Optimizing the Neurovascular Development of Human Brain Organoid in Chick Embryo at JoVE.com

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。

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has ...

我旨在解决的关键问题围绕着正常发育背景下涉及人脑形成的机制和过程，以及各种病理状态。这包括调查影响大脑发育的因素，例如遗传学、环境和表观遗传影响。获得与成人大脑非常相似的大脑类器官，包括血管形成、血脑屏障功能、小胶质细胞的存在和细胞多样性是具有挑战性的。克服这一点对于开发更具生理相关性的大脑类器官模型至关重要，这些模型可以有效地模仿成人大脑以增强临床应用。通过我们的研究，我们为将人脑类器官移植到绒毛膜尿囊膜中提供了最佳条件，以确保小胶质细胞的良好发育。这种临床改进对于该领域即将为大脑类器官生成合成血管化环境的努力至关重要。与其他技术相比，我们的协议的优势在于其高效率和成本效益。我们在使用农场鸡的受精卵移植可行的类器官方面取得了很高的成功率。未来，我们的实验室将专注于对与类器官相关的新脉管系统进行功能评估，探索这种血管设计模型在静脉纳米医学应用中的潜在用途。此外，我们的目标是评估这些纳米药物在人类中枢神经系统中的生物分布。

early-unguided-human-brain-organoid-neurovascular-niche-modeling-into

Research

JoVE Journal

Developmental Biology

Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has proven efficient for neovascularization modeling. The CAM is a richly vascularized extraembryonic membrane, which shows limited immunoreactivity, thus becoming an excellent hosting model for human origin cell transplants.
This paper describes the strategy to engraft human brain organoids differentiated at multiple maturation stages into the CAM. The cellular composition of brain organoids changes with time, reflecting the milestones of human brain development. We grafted brain organoids at relevant maturation stages: neuroepithelial expansion (18 DIV), early neurogenesis (60 DIV), and early gliogenesis (180 DIV) into the CAM of embryonic day (E)7 chicken embryos. Engrafted brain organoids were harvested 5 days later and their histological features were analyzed.
No histological signs of neovascularization in the grafted organoids or abnormal blood vessels adjacent to the graftings were detected. Moreover, remarkable changes were observed in the cellular composition of the grafted organoids, namely, an increase in the number of glial fibrillary acidic protein-positive-reactive astrocytes. However, the cytoarchitectural changes were dependent on the organoid maturation stage. Altogether, these results suggest that brain organoids can grow in the CAM, and they show differences in the cytoarchitecture depending on their maturation stage at grafting.

Here, we present a protocol to engraft human brain organoids at multiple maturation stages into the chick chorioallantoic membrane (CAM). Brain organoids were grown following unguided standardized protocols.