Modeling the Earliest Stages of Gliomagenesis Using Human iPSC-derived NPCs in A Three-dimensional Alginatebased Matrix

Objective: The development of gliomas is believed to be triggered by isocitrate dehydrogenase (IDH1/2) mutations, but there is limited information on how IDH1/2 mutations trigger gliogenesis. This is because studies over the years have often used patient humor samples, transformed cells, or normal stem cells with driver mutations to explore the early stages of glioma development. In this study, we constructed a model to understand the specific effects of IDH1-R132H mutation alone by preparing an alginate-based 3D culture with NPCs and hence sought to avoid the effects of other driver mutations.
Methods: Human induced pluripotent stem cells were differentiated into neural progenitor cells (NPCs). NPCs embedded in an alginate-based 3D matrix were incubated in neural progenitor medium for 1 day (day 0). Neural Progenitor Medium was then removed from the NPC-alginate beads and incubated for 14 days with conditioned media from immortalized human astrocytes (IHAs) that produced doxycycline-induced wild-type IDH1 and mutant IDH1. On day 14, IHA-conditioned media were replaced with Neural Progenitor Medium and incubated for 3 days (day 17). RNA was isolated from NPCs on days 0 and 17, and key genes [Tet methylcytosine dioxygenase 1 (TET1) and Mesenchyme Homeobox 2 (MEOX2)] previously reported to be altered in IDH1-mutant gliomas, were evaluated by RT-qPCR.
esults: Optimal alginate-based 3D culture conditions were established using NPCs. Expression of key genes was examined on days 0 and 17. In the NPCalginate bead 3D culture model, TET1 was upregulated and MEOX2 tended to be downregulated after exposure to IHA-IDH1-R132H conditioned medium.
Conclusion: We have developed a novel NPC-alginate bead 3D culture model for the first time in the literature. By recapitulating the earliest stages of gliomagenesis, this model will allow the study of the effects of the IDH1-R132H mutation without confounding the effects of the other mutations.