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一种用于神经分化为端脑类器官及有效调节FGF8信号传导的二维/三维混合方法

A Hybrid 2D/3D Approach for Neural Differentiation Into Telencephalic Organoids and Efficient Modulation of FGF8 Signaling.

作者信息

Bertacchi Michele, Maharaux Gwendoline, Studer Michèle

机构信息

Institut de Biologie Valrose (iBV), University Côte d'Azur (UniCA), CNRS, Inserm, Nice, France.

出版信息

Bio Protoc. 2025 Jun 20;15(12):e5354. doi: 10.21769/BioProtoc.5354.

Abstract

Human brain development relies on a finely tuned balance between the proliferation and differentiation of neural progenitor cells, followed by the migration, differentiation, and connectivity of post-mitotic neurons with region-specific identities. These processes are orchestrated by gradients of morphogens, such as FGF8. Disruption of this developmental balance can lead to brain malformations, which underlie a range of complex neurodevelopmental disorders, including epilepsy, autism, and intellectual disabilities. Studying the early stages of human brain development, whether under normal or pathological conditions, remains challenging due to ethical and technical limitations inherent to working with human fetal tissue. Recently, human brain organoids have emerged as a powerful in vitro alternative, allowing researchers to model key aspects of early brain development while circumventing many of these constraints. Unlike traditional 2D cultures, where neural progenitors and neurons are grown on flat surfaces, 3D organoids form floating self-organizing aggregates that better replicate the cellular diversity and tissue architecture of the developing brain. However, 3D organoid protocols often suffer from significant variability between batches and individual organoids. Furthermore, few existing protocols directly manipulate key morphogen signaling pathways or provide detailed analyses of the resulting effects on regional brain patterning. • To address these limitations, we developed a hybrid 2D/3D approach for the rapid and efficient induction of telencephalic organoids that recapitulate major steps of anterior brain development. Starting from human induced pluripotent stem cells (hiPSCs), our protocol begins with 2D neural induction using small-molecule inhibitors to achieve fast and homogenous production of neural progenitors (NPs). After dissociation, NPs are reaggregated in Matrigel droplets and cultured in spinning mini-bioreactors, where they self-organize into neural rosettes and neuroepithelial structures, surrounded by differentiating neurons. Activation of the FGF signaling pathway through the controlled addition of FGF8 to the culture medium will modulate regional identity within developing organoids, leading to the formation of distinct co-developing domains within a single organoid. Our protocol combines the speed and reproducibility of 2D induction with the structural and cellular complexity of 3D telencephalic organoids. The ability to manipulate signaling pathways provides an additional opportunity to further increase system complexity, enabling the simultaneous development of multiple distinct brain regions within a single organoid. This versatile system facilitates the study of key cellular and molecular mechanisms driving early human brain development across both telencephalic and non-telencephalic areas. Key features • This protocol builds on the method established by Chambers et al. [1] for generating 2D neural progenitors, followed by dissociation and reaggregation into 3D brain organoids. • For optimal growth and maturation, telencephalic organoids are cultured in spinning mini-bioreactors [2] or on orbital shakers. • The protocol enables the generation of telencephalic neural progenitors in 10 days and produces 3D telencephalic organoids containing neocortical neurons within one month of culture. • Addition of morphogens in the culture medium (example: FGF8) enhances cellular heterogeneity, promoting the emergence of distinct brain domains within a single organoid.

摘要

人类大脑发育依赖于神经祖细胞增殖与分化之间精确调控的平衡,随后是具有区域特异性身份的有丝分裂后神经元的迁移、分化和连接。这些过程由形态发生素梯度(如FGF8)精心编排。这种发育平衡的破坏会导致脑畸形,而脑畸形是一系列复杂神经发育障碍(包括癫痫、自闭症和智力残疾)的基础。由于使用人类胎儿组织存在固有的伦理和技术限制,研究人类大脑发育的早期阶段,无论是在正常还是病理条件下,仍然具有挑战性。最近,人类脑类器官已成为一种强大的体外替代方法,使研究人员能够模拟早期大脑发育的关键方面,同时规避许多这些限制。与传统的二维培养不同,在传统二维培养中神经祖细胞和神经元在平面上生长,三维类器官形成漂浮的自组织聚集体,能更好地复制发育中大脑的细胞多样性和组织结构。然而,三维类器官方案往往在批次和单个类器官之间存在显著差异。此外,现有的方案很少直接操纵关键的形态发生素信号通路,也很少对其对区域脑模式形成的影响进行详细分析。• 为了解决这些限制,我们开发了一种二维/三维混合方法,用于快速有效地诱导端脑类器官,该类器官概括了前脑发育的主要步骤。从人类诱导多能干细胞(hiPSC)开始,我们的方案首先使用小分子抑制剂进行二维神经诱导,以实现神经祖细胞(NP)的快速和均匀产生。解离后,NP在基质胶液滴中重新聚集,并在旋转微型生物反应器中培养,在那里它们自组织成神经玫瑰花结和神经上皮结构,周围是分化的神经元。通过向培养基中控制性添加FGF8激活FGF信号通路,将调节发育中类器官内的区域身份,导致在单个类器官内形成不同的共同发育区域。我们的方案结合了二维诱导的速度和可重复性以及三维端脑类器官的结构和细胞复杂性。操纵信号通路的能力提供了进一步增加系统复杂性的额外机会,使在单个类器官内同时发育多个不同的脑区成为可能。这个多功能系统有助于研究驱动端脑和非端脑区域早期人类大脑发育的关键细胞和分子机制。关键特征 • 本方案基于Chambers等人[1]建立的用于生成二维神经祖细胞的方法,随后解离并重新聚集形成三维脑类器官。• 为了实现最佳生长和成熟,端脑类器官在旋转微型生物反应器[2]或轨道振荡器上培养。• 该方案能够在10天内生成端脑神经祖细胞,并在培养一个月内产生包含新皮质神经元的三维端脑类器官。• 在培养基中添加形态发生素(例如FGF8)可增强细胞异质性,促进单个类器官内不同脑区的出现。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b11d/12222629/7b2823552a18/BioProtoc-15-12-5354-g001.jpg

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