Tang Chunling, Wang Xinghui, Gentleman Eileen, Kurniawan Nicholas A
Department of Biomedical Engineering & Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands.
Centre for Craniofacial and Regenerative Biology, King's College London, London, UK.
Methods Mol Biol. 2024 Apr 23. doi: 10.1007/7651_2024_546.
Organoids have emerged as robust tools for unravelling the mechanisms that underly tissue development. They also serve as important in vitro systems for studying fundamentals of stem cell behavior and for building advanced disease models. During early development, a crucial step in the formation of the central nervous system is patterning of the neural tube dorsal-ventral (DV) axis. Here we describe a simple and rapid culture protocol to produce human neuroepithelial (NE) cysts and DV-patterned organoids from single human-induced pluripotent stem cells (hiPSCs). Rather than being embedded within a matrix, hiPSCs undergo a 5-day differentiation process in medium containing soluble extracellular matrix and are allowed to self-organize into 3D cysts with defined central lumen structures that express early neuroepithelial markers. Moreover, upon stimulation with sonic hedgehog proteins and all-trans retinoic acid, NE cysts further develop into NE organoids with DV patterning. This rapid generation of patterned NE organoids using simple culture conditions enables mimicking, monitoring, and longitudinal manipulation of NE cell behavior. This straightforward culture system makes NE organoids a tractable model for studying neural stem cell self-organization and early neural tube developmental events.
类器官已成为揭示组织发育潜在机制的强大工具。它们还作为重要的体外系统，用于研究干细胞行为的基本原理和构建先进的疾病模型。在早期发育过程中，中枢神经系统形成的关键步骤是神经管背腹（DV）轴的模式化。在这里，我们描述了一种简单快速的培养方案，可从单个人诱导多能干细胞（hiPSC）产生人神经上皮（NE）囊肿和具有DV模式的类器官。hiPSC不是嵌入基质中，而是在含有可溶性细胞外基质的培养基中经历5天的分化过程，并使其自组织成具有明确中央管腔结构的3D囊肿，这些囊肿表达早期神经上皮标志物。此外，在用音猬因子蛋白和全反式维甲酸刺激后，NE囊肿进一步发育成具有DV模式的NE类器官。使用简单的培养条件快速生成具有模式的NE类器官，能够模拟、监测和纵向操纵NE细胞行为。这种简单直接的培养系统使NE类器官成为研究神经干细胞自组织和早期神经管发育事件的易于处理的模型。
