3D Engineered Stem Cell Tumor Invasion

3D bioengineered constructs with healthy differentiated induced neural stem cells (hiNSCs) were generated using a similar methodology as described for tumor cultures and previously detailed for hiNSCs in the silk-scaffold system^{66 (link)}, with further optimization. The differentiating hiNSC constructs were transfected with a viral vector, AAVdj-hSyn-eYFP (Charu Ramakrishnan, Karl Deisseroth, Stanford University), such that the differentiated synapsin (Syn)-expressing neurons would express eYFP (Yellow fluorescent protein). GBM cells expanded as spheres from the patient-tumor were placed in close proximity to the 3D hiNSC constructs 7 mo post-differentiation. Before starting the co-cultures, the GBM cells were separately incubated with 5-aminolevulinic acid (5-ALA, Sigma–Aldrich A3785) for 48 h, for visualization following 5-ALA preferential accumulation. The invasion of healthy hiNSC cultures by the GBM cells and vice versa was observed 48 h post co-culture, by simultaneous live imaging of the differentiated hiNSCs by eYFP and GBM by 5-ALA.