Instructive starPEG-Heparin biohybrid 3D cultures for modeling human neural stem cell plasticity, neurogenesis, and neurodegeneration
Instructive starPEG-Heparin biohybrid 3D cultures for modeling human neural stem cell plasticity, neurogenesis, and neurodegeneration
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指导性明星 PEG-肝素生物混合 3D 培养物用于模拟人类神经干细胞可塑性、神经发生和神经变性
DOI：
10.1101/225243
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发表时间：
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影响因子：
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通讯作者：
Carsten Werne
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作者：
Christos Papadimitriou;Mehmet I Cosacak;Violeta Mashkaryan;Hilal Celikkaya;Laura Bray;Prabesh Bhattarai;Heike Hollak;Xin Chen;Shuijin He;Christopher L Antos;Alvin Thomas;Jens Friedrichs;Andreas Dahl;Yixin Zhang;Uwe Freudenberg;Carsten Werne
人类神经发育和神经退行性变的三维模型在以组织模拟方式探索基于干细胞的再生疗法时至关重要。然而，现有的3D培养系统不足以模拟神经干细胞的固有可塑性，由于其不明确的组成和缺乏可控性的物理特性。采用基于糖胺聚糖的细胞响应性水凝胶平台，我们刺激原代和诱导的人神经干细胞（NSC），以表现神经源性可塑性并在体外形成广泛的神经元网络。3D培养物表现出神经递质反应性、电生理活性和组织特异性细胞外基质（ECM）沉积。通过全转录组测序，我们确定了3D培养表达成熟的神经元标记物，并反映了与2D培养相比的成熟皮层神经元的体内形成。因此，我们的数据表明，我们建立的3D水凝胶培养支持人类神经元的组织模拟成熟。我们还通过用Aβ42肽处理培养物来示例性地模拟神经退行性疾病，并观察到阿尔茨海默病的已知人类病理影响，包括NSC增殖减少、神经元网络形成受损、突触丢失和ECM沉积失败以及Tau过度磷酸化升高和神经纤维缠结的形成。我们确定了Aβ42作用后原代和诱导的神经干细胞源性神经元转录组的变化，为进一步研究提供了有用的资源。因此，我们的基于水凝胶的人类皮质3D细胞培养物是研究神经发育和神经退行性各个方面的强大平台，例如Aβ42毒性和神经源性干细胞可塑性。重要意义神经干细胞（NSC）是人脑中新神经元的储存库，但它们在神经退行性后未能形成神经元。因此，了解神经干细胞的干细胞为基础的再生疗法的潜在用途，需要组织模拟人源化实验系统。我们报告了3D生物指导性水凝胶培养系统的适应性，其中人类神经干细胞形成神经元，这些神经元后来在受控的微环境中形成网络。我们还通过使用淀粉样蛋白-β 4肽（阿尔茨海默病的标志）对神经退行性毒性进行建模，观察到令人联想到人脑的表型，并确定神经元发育和退化期间的整体基因表达变化。因此，我们的还原论人性化的文化模式将是一个重要的工具，以解决NSC的可塑性，神经原性，和网络的形成在健康和疾病。
Three-dimensional models of human neural development and neurodegeneration are crucial when exploring stem-cell-based regenerative therapies in a tissue-mimetic manner. However, existing 3D culture systems are not sufficient to model the inherent plasticity of NSCs due to their ill-defined composition and lack of controllability of the physical properties. 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 networksin 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 thein vivomake-up 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. We also exemplarily modeled neurodegenerative conditions by treating the cultures with Aβ42 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 determined the changes in transcriptomes of primary and induced NSC-derived neurons after Aβ42, 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 Aβ42 toxicity and neurogenic stem cell plasticity.SignificanceNeural stem cells (NSC) are reservoir for new neurons in human brains, yet they fail to form neurons after neurodegeneration. Therefore, understanding the potential use of NSCs for stem cell-based regenerative therapies requires tissue-mimetic humanized experimental systems. We report the adaptation of a 3D bio-instructive hydrogel culture system where human NSCs form neurons that later form networks in a controlled microenvironment. We also modeled neurodegenerative toxicity by using Amyloid-beta4 peptide, a hallmark of Alzheimer’s disease, observed phenotypes reminiscent of human brains, and determined the global gene expression changes during development and degeneration of neurons. Thus, our reductionist humanized culture model will be an important tool to address NSC plasticity, neurogenicity, and network formation in health and disease.
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