丙戊酸 (VPA) 是一种临床批准的小分子，据报道可以激活 Wnt 信号传导，这对神经管的背腹 (DV) 模式至关重要。然而，人们对 VPA 对 DV 图案化过程的影响知之甚少。在这里，我们发现，尽管 VPA 对人类神经管类器官 (hNTO) 的早期形成有负面影响，但当在整个分化过程中添加 VPA 时，它会显着提高腹侧模式 hNTO 的效率。 RNA 测序和 RT-qPCR 分析表明 VPA 激活 hNTO 中的内源性 Wnt 信号传导。令人惊讶的是，转录组分析还发现了 GLI2 和 GLI3 蛋白降解基因的上调，其截短片段是 Shh 信号传导的转录抑制因子。蛋白质印迹分析证实了 VPA 处理后 GLI3R 蛋白的增加。因此，VPA可能通过激活Wnt（可以通过诱导GLI3表达来拮抗Shh信号传导）和/或通过诱导GLI蛋白降解来抑制Shh信号传导来增强hNTO的腹侧模式。我们进一步获得的结果表明，当VPA的起始时间被延迟并且其持续时间减少时，VPA仍然提高hNTO的图案化效率，对其早期形成影响微弱。总而言之，这项研究表明，VPA 增强了具有腹侧模式的更具可重复性的 hNTO 的生成，为 hNTO 在发育生物学和再生医学中的应用开辟了途径。© 2024 作者。北京干细胞与再生医学研究院和John Wiley联合出版的《细胞增殖》

Valproic acid (VPA), a clinically approved small molecule, has been reported to activate Wnt signalling that is critical for dorsal-ventral (DV) patterning of neural tube. However, little is known about the impact of VPA on DV patterning process. Here, we show that even though VPA has a negative impact on the early formation of human neural tube organoids (hNTOs), it significantly enhances the efficiency of ventrally patterned hNTOs, when VPA is added during the entire differentiation process. RNA sequencing and RT-qPCR analysis demonstrates VPA activates endogenous Wnt signalling in hNTOs. Surprisingly, transcriptome analysis also identifies upregulation of genes for degradation of GLI2 and GLI3 proteins, whose truncated fragment are transcriptional repressors of Shh signalling. The Western-blot analysis confirms the increase of GLI3R proteins after VPA treatment. Thus, VPA might enhance ventral patterning of hNTOs through both activating Wnt, which can antagonise Shh signalling by inducing GLI3 expression, and/or inhibiting Shh signalling by inducing GLI protein degradation. We further obtain results to show that VPA still increases patterning efficiency of hNTOs with a weak influence on their early formation when the initiation time of VPA is delayed and its duration is reduced. Taken together, this study demonstrates that VPA enhances the generation of more reproducible hNTOs with ventral patterning, opening the avenues for the applications of hNTOs in developmental biology and regenerative medicine.© 2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.