Takaoka Shohei, Uchida Fumihiko, Ishikawa Hiroshi, Toyomura Junko, Ohyama Akihiro, Matsumura Hideaki, Hirata Koji, Fukuzawa Satoshi, Kanno Naomi Ishibashi, Marushima Aiki, Yamagata Kenji, Yanagawa Toru, Matsumaru Yuji, Ishikawa Eiichi, Bukawa Hiroki
Department of Oral and Maxillofacial Surgery, University of Tsukuba Hospital, Tsukuba, Ibaraki, Japan.
Laboratory of Clinical Regenerative Medicine, Department of Neurosurgery, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Hum Cell. 2024 Nov;37(6):1638-1648. doi: 10.1007/s13577-024-01121-7. Epub 2024 Aug 29.
Techniques for triggering neural differentiation of embryonic and induced pluripotent stem cells into neural stem cells and neurons have been established. However, neural induction of mesenchymal stem cells, including dental pulp stem cells (DPSCs), has been assessed primarily based on neural-related gene regulation, and detailed studies into the characteristics and differentiation status of cells are lacking. Therefore, this study was aimed at evaluating the cellular components and differentiation pathways of neural lineage cells obtained via neural induction of human DPSCs. Human DPSCs were induced to neural cells in monolayer culture and examined for gene expression and mechanisms underlying differentiation using microarray-based ingenuity pathway analysis. In addition, the neural lineage cells were subjected to single-cell RNA sequencing (scRNA-seq) to classify cell populations based on gene expression profiles and to elucidate their differentiation pathways. Ingenuity pathway analysis revealed that genes exhibiting marked overexpression, post-neuronal induction, such as FABP7 and ZIC1, were associated with neurogenesis. Furthermore, in canonical pathway analysis, axon guidance signals demonstrated maximum activation. The scRNA-seq and cell type annotations revealed the presence of neural progenitor cells, astrocytes, neurons, and a small number of non-neural lineage cells. Moreover, trajectory and pseudotime analyses demonstrated that the neural progenitor cells initially engendered neurons, which subsequently differentiated into astrocytes. This result indicates that the aforementioned neural induction strategy generated neural stem/progenitor cells from DPSCs, which might differentiate and proliferate to constitute neural lineage cells. Therefore, neural induction of DPSCs may present an alternative approach to pluripotent stem cell-based therapeutic interventions for nervous system disorders.
已建立了将胚胎干细胞和诱导多能干细胞诱导分化为神经干细胞和神经元的技术。然而，间充质干细胞（包括牙髓干细胞，DPSCs）的神经诱导主要是基于神经相关基因调控进行评估的，而对细胞特性和分化状态的详细研究尚缺。因此，本研究旨在评估通过人牙髓干细胞的神经诱导获得的神经谱系细胞的细胞成分和分化途径。人牙髓干细胞在单层培养中被诱导为神经细胞，并使用基于微阵列的 Ingenuity 通路分析来检测基因表达和分化的潜在机制。此外，对神经谱系细胞进行单细胞 RNA 测序（scRNA-seq），以根据基因表达谱对细胞群体进行分类，并阐明其分化途径。 Ingenuity 通路分析显示，在神经元诱导后显著过表达的基因，如 FABP7 和 ZIC1，与神经发生相关。此外，在经典通路分析中，轴突导向信号显示出最大激活。scRNA-seq 和细胞类型注释揭示了神经祖细胞、星形胶质细胞、神经元和少量非神经谱系细胞的存在。此外，轨迹和伪时间分析表明，神经祖细胞最初产生神经元，随后分化为星形胶质细胞。这一结果表明，上述神经诱导策略从牙髓干细胞产生了神经干/祖细胞，这些细胞可能分化和增殖以构成神经谱系细胞。因此，牙髓干细胞的神经诱导可能为基于多能干细胞的神经系统疾病治疗干预提供一种替代方法。
