Name: 著者スポットライト:ニワトリ胚におけるヒト脳オルガノイドの神経血管発達の最適化
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

UBのAlcántara博士とOrtega博士、そしてAcosta博士の研究室の他のメンバーには、洞察に満ちた議論をしてくれたことに感謝します。S.A.は、バルセロナ大学カタルーニャ総合研究所のセラ・ハンター・フェロー助教授です。

ホワイトレグホーンニワトリ(Gallus gallus)の胚は、米国国立研究評議会生命科学委員会実験動物資源研究所の実験動物のケアと使用に関するガイドに従って処理され、実験はバルセロナ大学の実験動物のケアと使用のための評議会によって承認されました。
1. 非誘導脳オルガノイド調製
<ol><li>37°Cの5%CO2 インキュベーターでDMEM(1:40)に溶解した?...

ヒト脳オルガノイドのニワトリ胚の絨毛尿膜への移植

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+makes+organoids+good+models+of+human+neurogenesis.">What makes organoids good models of human neurogenesis.</a> Front Neurosci. 16, 872794 (2022).</li><li>Sun, X. 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. 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=An+in+vivo+model+of+functional+and+vascularized+human+brain+organoids.">An in vivo model of functional and vascularized human brain organoids.</a> Nat Biotechnol. 36 (5), 432-441 (2018).</li><li>Revah, O., 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=Maturation+and+circuit+integration+of+transplanted+human+cortical+organoids.">Maturation and circuit integration of transplanted human cortical organoids.</a> Nature. 610 (7931), 319-326 (2022).</li><li>Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+chorioallantoic+membrane+angiogenesis+model.">Chicken chorioallantoic membrane angiogenesis model.</a> Methods Mol Biol. 843, 47-57 (2012).</li><li>Nowak-Sliwinska, P., Segura, T., Iruela-Arispe, M. 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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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M., Stewart, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-evaluating+the+role+of+astrocytes+in+blood-brain+barrier+induction.">Re-evaluating the role of astrocytes in blood-brain barrier induction.</a> Dev Dyn. 197 (1), 14-25 (1993).</li><li>Giandomenico, S. L., Sutcliffe, M., Lancaster, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+long-term+culture+of+advanced+cerebral+organoids+for+studying+later+stages+of+neural+development.">Generation and long-term culture of advanced cerebral organoids for studying later stages of neural development.</a> Nat Protoc. 16 (2), 579-602 (2021).</li><li>Wagner-Amos, K., Seymour, R. 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この研究では、ニワトリ胚の生存を乱すことなく、移植時にヒト脳オルガノイドの良好な成長と発達を提供する多数の重要なステップを含む詳細なプロトコルについて説明します。24時間の孵化後(1日目)に卵の空気室に穴を開けるために滅菌針の使用を推奨しました。さらに、4日目に穿刺を試みました(卵の殻を光でチェックして血管系の発達をテストし、健康な胚のみで作業していることを...

ヒト脳オルガノイドは、ヒト脳の初期発生をin vitroで再現することを可能にする新しい技術です1,2,3。しかし、このモデルの大きな限界の1つは、脳の恒常性だけでなく、脳の発達にも不可欠な役割を果たす血管新生の欠如です4。酸素と栄養素の供給に加えて、脳の血管系が発達中の神経分化、移動、シナプス形成を調節していることを示唆する証拠が蓄積されています5,6。したがって、欠落している血管シグナル伝達と構造を脳オルガノイドに提供し、ヒト脳オルガノイド第7世代の複雑さを高めることができる信頼性の高いモデルを確立することが急務です。
血管新生のために提案された方法の中で、2つの主要な合理化ラインを考慮することができます:生体へのオルガノイド生着と、内皮細胞と神経細胞を共培養する純粋なin vitro技術8,9,10,11,12。マウスの脳内移植はコストと時間がかかるため、より単純なモデルに関連する他の技術があります。ニワトリ絨毛膜(CAM)アッセイは、血管新生の研究に広く使用されています13,14,15。過去10年間で、いくつかのグループが、腎臓16,17、心臓18、腫瘍オルガノイド19,20など、さまざまなタイプのオルガノイドをCAMに生着させることに成功しました。それにもかかわらず、ヒト脳オルガノイドをCAMに生着させる有効性、毒性/拒絶反応、生理学的効果、および方法についてはほとんど知られていません。また、CAMとオルガノイド星状細胞界面の間にキメラ血液脳関門(BBB)が形成されるという、興味深い点もあります。以前の先駆的な研究は、アストロサイトとアストロサイトコンディショニング培地を移植することにより、CAMでBBBを生成することの推定上の実現可能性を示唆しました21,22,23。しかし、成熟したアストロサイトはこれを達成できないようです24,25。したがって、アストロサイトによるBBBの形成については議論の余地があり、ヒト脳オルガノイドを移植することで、この論争に光を当てることができます。
このビデオ記事では、成長、改善、血管新生を促進し、組織学的に適合するBBB要素を含むオルガノイドをもたらすCAMへの卵 子ヒト 脳オルガノイド移植のプロトコルについて説明します。ここでは、ニワトリ胚の生存を保証するプロトコルを提示し、脳オルガノイドの成長を維持するためのCAMの許容性について報告します。

<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

免疫不全マウスやニワトリ胚の絨毛膜膜(CAM)などのモデル動物の血管新生組織にオルガノイドを生着させることは、血管新生モデリングに有効であることが証明されています。CAMは血管新生した胚外膜であり、免疫反応性が限られているため、ヒト由来の細胞移植の優れた宿主モデルとなっています。
本稿では、複数の成熟段階で分化したヒト脳オルガノイドをCAMに生着させる戦略について述べる。脳オルガノイドの細胞組成は、ヒトの脳発達のマイルストーンを反映して、時間とともに変化します。神経上皮拡大(18 DIV)、初期神経新生(60 DIV)、および初期神経膠形成(180 DIV)の関連する成熟段階の脳オルガノイドを、胚日(E)7ニワトリ胚のCAMに移植しました。生着した脳オルガノイドを5日後に採取し、その組織学的特徴を分析した。
移植されたオルガノイドまたは移植片に隣接する異常な血管における血管新生の組織学的兆候は検出されませんでした。.さらに、移植されたオルガノイドの細胞組成に顕著な変化、すなわちグリア線維性酸性タンパク質陽性反応性アストロサイトの数の増加が観察されました。しかし、細胞構造の変化はオルガノイドの成熟段階に依存していました。これらの結果は、脳オルガノイドがCAM内で増殖できることを示唆しており、移植時の成熟段階に応じて細胞構造に違いがあることを示しています。

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.

著者スポットライト:ニワトリ胚におけるヒト脳オルガノイドの神経血管発達の最適化

寛容なニワトリ胚絨毛膜への初期の非誘導ヒト脳オルガノイド神経血管ニッチモデリング

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.

移植のための胚成熟スケジュールの選択 実験は、受精卵を38°C、相対湿度60%で孵化させるD0から始まります。絨毛膜(CAM)は、卵の孵化後に発達する高度に血管新生した胚外膜です。それは尿膜と絨毛膜の融合によって形成されます。D1では、24時間のインキュベーション後、CAMが内殻膜に付着しないように空気室に穴を開けます。D1で空気室に穴を開けると、後の段階での穿刺(D4)...

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

ここでは、複数の成熟段階にあるヒト脳オルガノイドをニワトリの絨毛膜膜(CAM)に生着させるプロトコルを紹介します。脳オルガノイドは、ガイドなしで標準化されたプロトコルに従って増殖しました。

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

私が取り組むべき重要な問題は、正常な発達の文脈における人間の脳形成のメカニズムとプロセス、およびさまざまな病理学的状態を中心に展開しています。これには、遺伝学、環境、エピジェネティックな影響など、脳の発達に影響を与える要因の調査が含まれます。血管新生、血液脳関門機能、ミクログリアの存在、細胞の多様性など、成人の脳によく似た脳オルガノイドを得ることは困難です。これを克服することは、成人の脳を効率的に模倣して臨床応用を強化できる、より生理学的に関連性のある脳オルガノイドモデルの開発に不可欠です。私たちの研究を通じて、ヒト脳オルガノイドを絨毛膜に移植し、ミクログリアの良好な発達を確実にするための最適な条件を提供します。この臨床的改善は、脳オルガノイドの合成血管新生環境を生成するためのこの分野での今後の取り組みの中心となるものです。他の技術と比較した私たちのプロトコルの利点は、その高い有効性と費用対効果にあります。私たちは、農場の鶏の受精卵を使用して、生存可能なオルガノイドを移植することに高い成功率を達成しました。今後は、オルガノイドに関わる血管新生の機能評価に注力し、この血管設計モデルを用いたナノ医療への応用の可能性を探っていきます。さらに、ヒト中枢神経系におけるこれらのナノ医薬品の生体内分布を評価することを目指しています。

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.