Hedgehog pathway permissive conditions allow generation of immortal cell lines from granule cells derived from cancerous and non-cancerous cerebellum.

Constantin Heil

doi:10.1098/rsob.180145

Abstract

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;Ptch1FL/FL 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.

Highlights

Cell culture technologies are used in biomedical research as well as developmental biology to conduct experimentation on controllable and scaleable systems
To confirm that vismodegib sensitivity is caused by its effect on the sonic hedgehog (SHH) pathway, the expression of NMYC and GLI1 proteins is assayed by western blot, both of which decreased in a dose-dependent manner
NIH3T3 cells do not belong to the neural lineage and require pre-treatment before being reactive to SHH pathway activation while primary granule cell progenitors (GCPs) cultures are transient and can constitute a bottleneck for generation of sample material

Summary

Introduction

Cell culture technologies are used in biomedical research as well as developmental biology to conduct experimentation on controllable and scaleable systems. Among the different biomedical fields, cancer research makes extensive use of cell cultures. The first human cells to be stably cultured were cancer cells and to this day most cell lines in use derive from human cancer [1]. Cancer cell lines are used extensively in pre-clinical research and are the basis for other technologies such as high-throughput screening or other biological assays [2,3]. Cells used in vitro can be distinguished based on their persistence in culture. Primary cells can generally be passaged a limited number of times before undergoing crisis [4], while transformed cells are characterized by autonomous unlimited proliferation.

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