Department of Molecular Medicine, University of Rome , 'La Sapienza', Rome , Italy.
Open Biol. 2019 Jan 31;9(1):180145. doi: 10.1098/rsob.180145.
Cerebellar granule cell progenitors (GCPs) undergo proliferation in the post-natal cerebellum that is dependent on sonic hedgehog (SHH) signalling. Deregulated SHH signalling leads to type 2 medulloblastoma (MB). In this work, a novel cell culture protocol is described, which is suitable for the establishment and long-term maintenance of GCP-derived cells. This method is first applied to SHH pathway active MB cells from Atoh1-cre; Ptch1 tumours, which leads to the generation of neurosphere-like cell lines expressing GCP markers and an active SHH signalling pathway. These cells also show high sensitivity to the Smoothened inhibitor vismodegib, therefore recapitulating the SHH pathway requirement for survival shown by type 2 MB. Analysis of culture supplements reveals that bFGF and fetal bovine serum act as inhibitors of the SHH pathway and therefore preclude generation of cell lines that are relevant to the study of the SHH pathway. Consequently, these insights are transferred from the context of MB to non-transformed, post-natal day 7 cerebellum-derived cellular explants. In contrast to other, previously used methods, these GCP cultures proliferate indefinitely and depend on SHH pathway activation, either by means of the small molecule SAG or through genetic ablation of Ptch1. This culture method therefore leads to the generation of immortal neurosphere-like cell lines, that are named murine SAG-dependent spheres (mSS). Despite long-term culture, mSS cells remain dependent on continuous stimulation of the SHH pathway. Further, mSS cells maintain their lineage after extensive periods in vitro, as demonstrated by their differentiation towards the neural lineage. Herein a simple method for the generation of immortal cell lines from murine cerebella is defined. These lines can be maintained indefinitely through hedgehog pathway activation and maintain the GCP lineage.
小脑颗粒细胞祖细胞 (GCP) 在出生后的小脑内进行增殖，这依赖于 sonic hedgehog (SHH) 信号。SHH 信号的失调导致 2 型髓母细胞瘤 (MB)。在这项工作中，描述了一种新的细胞培养方案，该方案适合建立和长期维持 GCP 衍生细胞。该方法首先应用于 Atoh1-cre;Ptch1 肿瘤中的 SHH 通路激活的 MB 细胞，导致表达 GCP 标志物和活跃 SHH 信号通路的神经球样细胞系的产生。这些细胞对 Smoothened 抑制剂 vismodegib 也表现出高度敏感性，因此再现了 2 型 MB 中显示的 SHH 通路对生存的要求。对培养补充剂的分析表明，bFGF 和胎牛血清作为 SHH 通路的抑制剂，因此阻止了与 SHH 通路研究相关的细胞系的产生。因此，这些见解从 MB 的背景转移到非转化的、出生后第 7 天的小脑源性细胞外植体。与其他先前使用的方法相反，这些 GCP 培养物无限期增殖，并依赖于 SHH 通路的激活，无论是通过小分子 SAG 还是通过 Ptch1 的基因缺失。因此，这种培养方法导致产生永生的神经球样细胞系，命名为小鼠 SAG 依赖性球体 (mSS)。尽管长期培养，mSS 细胞仍然依赖于 SHH 通路的持续刺激。此外，mSS 细胞在体外长时间保持其谱系，如向神经谱系分化所证明的那样。本文定义了一种从鼠脑中产生永生细胞系的简单方法。这些细胞系可以通过 hedgehog 通路的激活无限期维持，并保持 GCP 谱系。
