Nicotinamide alone accelerates the conversion of mouse embryonic stem cells into mature neuronal populations

Introduction
Vitamin B3 has been shown to play an important role during embryogenesis. Specifically, there is growing evidence that nicotinamide, the biologically active form of vitamin B3, plays a critical role as a morphogen in the differentiation of stem cells to mature cell phenotypes, including those of the central nervous system (CNS). Detailed knowledge of the action of small molecules during neuronal differentiation is not only critical for uncovering mechanisms underlying lineage-specification, but also to establish more effective differentiation protocols to obtain clinically relevant cells for regenerative therapies for neurodegenerative conditions such as Huntington’s disease (HD). Thus, this study aimed to investigate the potential of nicotinamide to promote the conversion of stem cells to mature CNS neurons.
Methods
Nicotinamide was applied to differentiating mouse embryonic stem cells (mESC; Sox1GFP knock-in 46C cell line) during their conversion towards a neural fate. Cells were assessed for changes in their proliferation, differentiation and maturation; using immunocytochemistry and morphometric analysis methods.
Results
Results presented indicate that 10 mM nicotinamide, when added at the initial stages of differentiation, promoted accelerated progression of ESCs to a neural lineage in adherent monolayer cultures. By 14 days in vitro (DIV), early exposure to nicotinamide was shown to increase the numbers of differentiated βIII-tubulin-positive neurons. Nicotinamide decreased the proportion of pluripotent stem cells, concomitantly increasing numbers of neural progenitors at 4 DIV. These progenitors then underwent rapid conversion to neurons, observed by a reduction in Sox 1 expression and decreased numbers of neural progenitors in the cultures at 14 DIV. Furthermore, GABAergic neurons generated in the presence of nicotinamide showed increased maturity and complexity of neurites at 14 DIV. Therefore, addition of nicotinamide alone caused an accelerated passage of pluripotent cells through lineage specification and further to non-dividing mature neurons.
Conclusions
Our results show that, within an optimal dose range, nicotinamide is able to singly and selectively direct the conversion of embryonic stem cells to mature neurons, and therefore may be a critical factor for normal brain development, thus supporting previous evidence of the fundamental role of vitamins and their metabolites during early CNS development. In addition, nicotinamide may offer a simple effective supplement to enhance the conversion of stem cells to clinically relevant neurons.

引言
已有研究表明，维生素B3（Vitamin B3）在胚胎发生过程中发挥重要作用。具体而言，越来越多的证据显示，烟酰胺（nicotinamide）——维生素B3的生物活性形式——作为形态发生素，在干细胞向包括中枢神经系统（central nervous system, CNS）在内的成熟细胞表型分化过程中扮演关键角色。对小分子在神经元分化过程中作用的深入认知，不仅有助于揭示谱系特化的潜在机制，还能助力建立更高效的分化方案，以获取临床相关细胞，用于亨廷顿病（Huntington’s disease, HD）等神经退行性疾病的再生治疗。因此，本研究旨在探究烟酰胺促进干细胞向成熟中枢神经系统神经元转化的潜力。

方法
本研究在小鼠胚胎干细胞（mouse embryonic stem cells, mESC；Sox1GFP敲入46C细胞系）向神经谱系转化的分化阶段，施加烟酰胺进行干预。通过免疫细胞化学与形态计量分析方法，评估细胞的增殖、分化及成熟状态变化。

结果
本研究结果显示，在分化初始阶段添加10毫摩尔烟酰胺，可促进贴壁单层培养体系中胚胎干细胞向神经谱系加速分化。在体外培养14天（days in vitro, DIV）时，早期接触烟酰胺可增加分化得到的βIII微管蛋白（βIII-tubulin）阳性神经元数量。在体外培养第4天（DIV），烟酰胺可降低多能干细胞的占比，同时提升神经前体细胞的数量。随后这些神经前体细胞可快速转化为神经元，具体表现为在体外培养第14天（DIV）时，Sox1的表达水平下调，培养体系中的神经前体细胞数量减少。此外，在烟酰胺存在下生成的GABA能神经元（GABAergic neurons），在体外培养第14天（DIV）时展现出更高的成熟度与更复杂的神经突起结构。综上，仅添加烟酰胺即可加速多能干细胞通过谱系特化阶段，并进一步转化为无增殖能力的成熟神经元。

结论
本研究结果表明，在最优剂量范围内，烟酰胺可单独且选择性地引导胚胎干细胞向成熟神经元转化，因此可能是正常脑发育的关键因子之一，这也佐证了此前关于维生素及其代谢产物在中枢神经系统早期发育中发挥基础作用的研究结论。此外，烟酰胺可作为一种简便高效的补充剂，用于提升干细胞向临床相关神经元的转化效率。