Virology and Cell Technology Research Team, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani, Thailand.
Interdisciplinary Program in Genetic Engineering and Bioinformatics, Graduate School, Kasetsart University, Bangkok, Thailand.
Front Cell Infect Microbiol. 2023 Aug 25;13:1215205. doi: 10.3389/fcimb.2023.1215205. eCollection 2023.
Rabies is a highly fatal infectious disease that poses a significant threat to human health in developing countries. In vitro study-based understanding of pathogenesis and tropism of different strains of rabies virus (RABV) in the central nervous system (CNS) is limited due to the lack of suitable culture models that recapitulate the complex communication pathways among host cells, extracellular matrices, and viruses. Therefore, a three-dimensional (3D) cell culture that mimics cell-matrix interactions, resembling in vivo microenvironment, is necessary to discover relevant underlying mechanisms of RABV infection and host responses.
The 3D collagen-Matrigel hydrogel encapsulating hiPSC-derived neurons for RABV infection was developed and characterized based on cell viability, morphology, and gene expression analysis of neuronal markers. The replication kinetics of two different strains of RABV [wild-type Thai (TH) and Challenge Virus Standard (CVS)-11 strains] in both 2D and 3D neuronal cultures were examined. Differential gene expression analysis (DEG) of the neuropathological pathway of RABV-infected 2D and 3D models was also investigated via NanoString analysis.
The 3D hiPSC-derived neurons revealed a more physiologically interconnected neuronal network as well as more robust and prolonged maturation and differentiation than the conventional 2D monolayer model. TH and CVS-11 exhibited distinct growth kinetics in 3D neuronal model. Additionally, gene expression analysis of the neuropathological pathway observed during RABV infection demonstrated a vast number of differentially expressed genes (DEGs) in 3D model. Unlike 2D neuronal model, 3D model displayed more pronounced cellular responses upon infection with CVS-11 when compared to the TH-infected group, highlighting the influence of the cell environment on RABV-host interactions. Gene ontology (GO) enrichment of DEGs in the infected 3D neuronal culture showed alterations of genes associated with the inflammatory response, apoptotic signaling pathway, glutamatergic synapse, and trans-synaptic signaling which did not significantly change in 2D culture.
We demonstrated the use of a hydrogel-based 3D hiPSC-derived neuronal model, a highly promising technology, to study RABV infection in a more physiological environment, which will broaden our understanding of RABV-host interactions in the CNS.
狂犬病是一种具有高致死率的传染病，对发展中国家的人类健康构成重大威胁。由于缺乏能够重现宿主细胞、细胞外基质和病毒之间复杂通讯途径的合适培养模型，因此基于体外研究的对不同株狂犬病病毒（RABV）在中枢神经系统（CNS）中的发病机制和嗜性的理解受到限制。因此，需要一种模拟细胞-基质相互作用的三维（3D）细胞培养物，以重现体内微环境，从而发现 RABV 感染和宿主反应的相关潜在机制。
基于神经元标志物的细胞活力、形态和基因表达分析，开发并表征了用于 RABV 感染的 3D 胶原-Matrigel 水凝胶包封的 hiPSC 衍生神经元。在 2D 和 3D 神经元培养物中检查了两种不同株 RABV（野生型泰国（TH）和挑战病毒标准（CVS）-11 株）的复制动力学。还通过 NanoString 分析研究了 RABV 感染的 2D 和 3D 模型中神经病理学途径的差异基因表达分析（DEG）。
3D hiPSC 衍生神经元显示出更具生理相关性的神经元网络，并且比传统的 2D 单层模型具有更健壮和更长时间的成熟和分化。TH 和 CVS-11 在 3D 神经元模型中表现出不同的生长动力学。此外，RABV 感染期间观察到的神经病理学途径的基因表达分析表明，在 3D 模型中存在大量差异表达基因（DEG）。与 2D 神经元模型不同，与 TH 感染组相比，3D 模型在感染 CVS-11 时显示出更明显的细胞反应，突出了细胞环境对 RABV-宿主相互作用的影响。感染的 3D 神经元培养物中 DEG 的基因本体论（GO）富集显示，与炎症反应、凋亡信号通路、谷氨酸能突触和突触间信号转导相关的基因发生改变，而这些基因在 2D 培养物中没有显著变化。
我们展示了使用基于水凝胶的 3D hiPSC 衍生神经元模型，这是一种很有前途的技术，可在更生理的环境中研究 RABV 感染，从而加深我们对 CNS 中 RABV-宿主相互作用的理解。
