Neural lineage cells derived from dental pulp stem cells generate Schwann cells via oligodendrocyte progenitor cells in peripheral nerve regeneration

The current understanding is that Schwann cell transplantation is ideal strategy for peripheral nerve regeneration instead of autograft. It is difficult to obtain the required amount of Schwann cells which are best transplant condition, and central nervous cells have been gained attention in recent years, but its regenerative mechanism remain unknown. Neural stem/progenitor cells (NSPC) can generate various type of neural lineage cells (NLCs), and NSPCs derived from pluripotent stem cells are promising cells for cell therapy for neurodegenerative diseases. However, more safe and accessible cell source of NSPCs are required. In this study, we aim to provide NLCs derived from human dental pulp stem cells (DPSCs), and reveal the mechanism involved in regeneration after NLCs transplantation into peripheral nerve defect. Here, characterization of NLCs, paracrine effects for endothelial cells and Schwann cells, in xenotransplant for rat 10mm sciatic nerve defect, the differentiation, the survival, and outcome of nerve regeneration were investigated. Induced NLCs consisted of neuronal lineage cells, astrocyte lineage cells, oligodendrocyte lineage cells, and neural crest lineage cells. Considering retrospectively, NLCs were possible derived from NSPCs. Microarray analysis revealed neural markers of primary embryological development were up-regulated in induced NLCs compared to DPSCs. Moreover, NLCs enhanced the activity of endothelial cells and Schwann cells by paracrine effects in vitro. Two weeks after transplantation, many transplanted NLCs differentiated into platelet-derived growth factor receptor alpha (PDGFRa) + oligodendrocyte progenitor cells (OPCs), and PDGFRa+/p75 neurotrophin receptor + Schwann cells derived from OPCs were observed. Twelve weeks after transplantation, NLCs promoted functional repair of peripheral nerve. A few human Schwann cells survived, but did not myelinate axon. These findings suggest that some of mechanism promoting for peripheral nerve regeneration by transplanted NLCs. Transplantation of NLCs derived from DPSCs into partial peripheral nerve defect may be widely used for further experiments. Expression profiling of dental pulp stem cells and neural lineage cells derived from dental pulp stem cells.

当前学界普遍认为，施万细胞（Schwann cell）移植是替代自体移植（autograft）实现周围神经再生（peripheral nerve regeneration）的理想策略。但获取满足最佳移植条件所需数量的施万细胞存在技术难度，近年来中枢神经细胞逐渐受到研究关注，但其介导的神经再生机制仍未明确。
神经干细胞/祖细胞（Neural stem/progenitor cells, NSPC）可分化为多种神经谱系细胞（neural lineage cells, NLCs），而源自多能干细胞的NSPC在神经退行性疾病的细胞治疗中极具应用前景。不过目前仍需更安全、更易获取的NSPC来源。
本研究旨在获取源自人牙髓干细胞（human dental pulp stem cells, DPSCs）的神经谱系细胞，并阐明其移植至周围神经缺损部位后参与神经再生的相关分子机制。本研究针对大鼠10mm坐骨神经缺损模型开展异种移植（xenotransplant）实验，对神经谱系细胞的生物学特性、其对内皮细胞与施万细胞的旁分泌效应（paracrine effects）、移植后的细胞分化与存活情况，以及整体神经再生结局进行了系统探究。
诱导获得的神经谱系细胞包含神经元谱系细胞、星形胶质谱系细胞、少突胶质谱系细胞及神经嵴谱系细胞。回溯性分析显示，该神经谱系细胞或可由NSPC分化而来。基因芯片分析（microarray analysis）结果表明，与牙髓干细胞相比，诱导后的神经谱系细胞中胚胎发育早期的神经相关标志物表达显著上调。此外，体外实验证实，神经谱系细胞可通过旁分泌效应增强内皮细胞与施万细胞的生物学活性。
移植后2周，大量移植的神经谱系细胞分化为血小板衍生生长因子受体α（platelet-derived growth factor receptor alpha, PDGFRα）阳性的少突胶质细胞祖细胞（oligodendrocyte progenitor cells, OPCs），同时可观察到源自少突胶质细胞祖细胞的PDGFRα+/p75神经营养因子受体（p75 neurotrophin receptor）阳性施万细胞。
移植后12周，神经谱系细胞可显著促进周围神经的功能修复。仅少量人源施万细胞存活，且未形成轴突髓鞘。
上述研究结果提示，移植的神经谱系细胞可通过多种途径促进周围神经再生。将源自牙髓干细胞的神经谱系细胞应用于局限性周围神经缺损的修复，有望为后续相关实验提供广泛的应用基础。
本研究同时包含牙髓干细胞及其衍生神经谱系细胞的表达谱分析数据。