Department of Histology and Embryology, Faculty of Medicine in Hradec Králové, Charles University, Hradec Králové, Czech Republic.
Folia Biol (Praha). 2020;66(5-6):155-160. doi: 10.14712/fb2020066050155.
Early mouse neural stem cells (NSCs) first appear in embryonic day E5.5 and express pluripotency markers Oct4, Sox2, Nanog and early neural marker Sox1. Early NSCs are a good model for understanding the role of various pathways that control initial neural commitment. However, a protocol for differentiation of mouse embryonic stem cells (ESCs) into early NSCs by adherent monolayer culture has not yet been established. Hence, in this study, we identified the combination of growth factors and small molecules that differentiated mouse ESCs into early NSCs and supported their proliferation. Leukaemia inhibitory factor (LIF) was the first factor to be tested and it was found that ESCs can differentiate into early neurogenic lineage in the presence of LIF. However, we found that the induction is weaker in the presence of LIF as compared to cells differentiated in its absence. GSK-3 inhibitor, along with BMP and TGF-β pathway inhibitor (dual SMAD inhibition), are commonly used to sequentially direct ESCs towards NSCs. However, when we used this combination, mouse ESCs failed to differentiate into early NSCs. We observed that by adding Wnt inhibitor to the combination of GSK-3 inhibitor, BMP inhibitor, TGF-β inhibitor and LIF, it was possible to differentiate ESCs into early NSCs. qRT-PCR analysis of early NSCs illustrated that they expressed key pluripotency genes Oct4 and Nanog, albeit at levels lower than non-differentiated ESCs, along with early neural markers Sox1 and Pax6.
早期小鼠神经干细胞（NSC）最早出现在胚胎第 5.5 天，表达多能性标志物 Oct4、Sox2、Nanog 和早期神经标志物 Sox1。早期 NSC 是了解控制初始神经分化的各种途径作用的良好模型。然而，尚未建立通过贴壁单层培养将小鼠胚胎干细胞（ESC）分化为早期 NSC 的方案。因此，在本研究中，我们确定了将小鼠 ESC 分化为早期 NSC 并支持其增殖的生长因子和小分子组合。白血病抑制因子（LIF）是第一个被测试的因子，结果发现 LIF 存在时 ESC 可以分化为早期神经发生谱系。然而，我们发现与不存在 LIF 的细胞相比，LIF 存在时的诱导作用较弱。GSK-3 抑制剂与 BMP 和 TGF-β 通路抑制剂（双重 SMAD 抑制）一起常用于依次将 ESC 定向为 NSC。然而，当我们使用这种组合时，小鼠 ESC 未能分化为早期 NSC。我们观察到，通过向 GSK-3 抑制剂、BMP 抑制剂、TGF-β 抑制剂和 LIF 的组合中添加 Wnt 抑制剂，可以将 ESC 分化为早期 NSC。早期 NSC 的 qRT-PCR 分析表明，它们表达关键多能性基因 Oct4 和 Nanog，尽管水平低于未分化的 ESC，同时还表达早期神经标志物 Sox1 和 Pax6。
