A novel hydrogel-based instructive biohybrid 3D culture system for modeling human neural stem cell plasticity, neurogenesis, neurodevelopment, and neurodegeneration

Meaningful models of human neural development and neurodegeneration are extremely important when exploring stem-cell-based regenerative therapies. However, existing 3D cultures fall short of being highly defined, modular, and controllable. Adapting a glycosaminoglycan-based, cell-responsive hydrogel platform, we stimulated primary and induced human neural stem cells (NSCs) to manifest neurogenic plasticity and form extensive neuronal networks in vitro. The 3D cultures exhibited neurotransmitter responsiveness, electrophysiological activity, and tissue-specific extracellular matrix (ECM) deposition. By whole transcriptome sequencing, we identified that 3D cultures express mature neuronal markers, and reflect the in vivo genetic program of mature cortical neurons compared to 2D cultures. Thus, our data suggest that our established 3D hydrogel culture supports the tissue-mimetic maturation of human neurons in an unprecedented manner. We modeled neurodegenerative conditions by treating the cultures with A?42 peptide and observed the known human pathological effects of Alzheimer?s disease including reduced NSC proliferation, impaired neuronal network formation, synaptic loss and failure in ECM deposition as well as elevated Tau hyperphosphorylation and formation of neurofibrillary tangles. We also determined the changes in transcriptomes of primary and induced NSC-derived neurons after A?42, providing a useful resource for further studies. Thus, our hydrogel-based human cortical 3D cell culture is a powerful platform for studying various aspects of neural development and neurodegeneration, as exemplified for A?42 toxicity and neurogenic stem cell plasticity. Overall design: Primary (from Gestation week 21 of human fetus) and induced (adult human fibroblast-derived) neural stem cells are cultured in 2D and 3D. Next generation sequencing is performed at 3 weeks of culture.在探索基于干细胞的再生治疗策略时，构建可精准模拟人类神经发育与神经退行性病变的有效模型具有极其重要的研究意义。然而，现有的三维（3D）培养体系尚难以实现高度标准化、模块化与可控性。我们采用基于糖胺聚糖的细胞响应型水凝胶平台，诱导原代及诱导性人类神经干细胞（Neural Stem Cells, NSCs）展现神经源性可塑性，并在体外构建出大范围的神经元网络。该三维（3D）培养体系可表现出神经递质响应性、电生理活性，并能沉积组织特异性的细胞外基质（Extracellular Matrix, ECM）。通过全转录组测序分析，我们发现相较于二维（2D）培养体系，三维（3D）培养体系可表达成熟神经元标志物，并能模拟成熟皮层神经元的体内遗传表达程序。综上，本研究数据表明，我们构建的三维水凝胶培养体系可以前所未有的方式支持人类神经元实现模拟体内组织的成熟过程。我们通过向培养体系中添加Aβ42肽段构建神经退行性病变模型，成功复现了阿尔茨海默病已知的人类病理特征：包括神经干细胞增殖能力下降、神经元网络形成受损、突触丢失与细胞外基质沉积障碍，以及Tau蛋白过度磷酸化水平升高和神经原纤维缠结的形成。此外，我们还分析了Aβ42处理后原代及诱导性神经干细胞来源神经元的转录组变化，可为后续相关研究提供宝贵的参考资源。综上，我们构建的基于水凝胶的人类皮层三维细胞培养体系，是研究神经发育与神经退行性病变诸多方面的强大研究平台，本研究以Aβ42毒性及神经源性干细胞可塑性为例验证了这一点。实验整体设计：分别培养源自人类胎儿妊娠21周的原代神经干细胞，以及由成人成纤维细胞诱导得到的诱导性神经干细胞，分别进行二维（2D）及三维（3D）培养；于培养第3周时开展下一代测序（Next Generation Sequencing, NGS）。