Stem cell-derived neural organoids as platforms to investigate glioblastoma invasion and migration: A systematic review

Figure 4 Overview of neural organoid generation protocols, glioblastoma multiforme co-culture strategies, and analytical methodologies. A: Top: Schematic representation of the main phases involved in the generation of neural organoids (NOs), including: (1) Embryoid body formation; (2) Neural induction; (3) Simultaneous embryoid body formation and neural induction; (4) Embedding in extracellular matrix; (5) Neural differentiation and maturation; and (6) Co-culture with 2D glioblastoma multiforme (GBM) cells or three-dimensional GBM spheroids. Most of the factors applied during these steps were specific to guided protocols designed to direct NOs toward a cortical identity, with the exception of the ROCK inhibitor, which was universally used in the early stages to enhance cell viability. Bottom: Temporal timeline (in days) of each phase as applied in the studies included in this review; B: Summary of methodologies used to assess GBM cell migration and invasion in NO models. Migration and invasion can be visualized using fluorescence or confocal microscopy, either by: (1) Tracking fluorescently labeled GBM cells on the surface of whole NOs; (2) Visualizing internal invasion using optical clearing techniques in whole NOs; and (3) Sectioning organoids into cryoslides for detailed imaging of GBM cells invasion. Alternatively, GBM cell invasion can be quantified by dissociating co-cultured NOs into single cells and analyzing them via flow cytometry, enabling the measurement of the proportion of GBM cells that infiltrated the organoids. ROCKi: ROCK inhibitor; bFGF: Basic fibroblasts growth factor; DM: Dorsomorphin; TGF: Transforming growth factor; BMPi: Bone morphogenetic protein inhibitor; EGF: Endothelial growth factor; BDNF: Brain-derived neurotrophic factor; GDNF: Glial cell line-derived neurotrophic factor; EB: Embryoid body; ECM: Extracellular matrix; EGFP: Enhanced green fluorescent protein; GFP: Green fluorescent protein; H2B-GFP: Histone-fused construct green fluorescent protein; RFP: Red fluorescent protein; NO: Neural organoid.