Glioblastoma Multiforme (GBM) is an aggressive brain tumor with a high mortality rate. Direct reprogramming of glial cells to different cell lineages, such as induced neural stem cells (iNSCs) and induced neurons (iNeurons), provides genetic tools to manipulate a cell\'s fate as a potential therapy for neurological diseases. NeuroD1 (ND1) is a master transcriptional factor for neurogenesis and it promotes neuronal differentiation. In the present study, we tested the hypothesis that the expression of ND1 in GBM cells can force them to differentiate toward post-mitotic neurons and halt GBM tumor progression. In cultured human GBM cell lines, including LN229, U87, and U373 as temozolomide (TMZ)-sensitive and T98G as TMZ-resistant cells, the neuronal lineage conversion was induced by an adeno-associated virus (AAV) package carrying ND1. Twenty-one days after AAV-ND1 transduction, ND1-expressing cells displayed neuronal markers MAP2, TUJ1, and NeuN. The ND1-induced transdifferentiation was regulated by Wnt signaling and markedly enhanced under a hypoxic condition (2% O2 vs. 21% O2). ND1-expressing GBM cultures had fewer BrdU-positive proliferating cells compared to vector control cultures. Increased cell death was visualized by TUNEL staining, and reduced migrative activity was demonstrated in the wound-healing test after ND1 reprogramming in both TMZ-sensitive and -resistant GBM cells. In a striking contrast to cancer cells, converted cells expressed the anti-tumor gene p53. In an orthotopical GBM mouse model, AAV-ND1-reprogrammed U373 cells were transplanted into the fornix of the cyclosporine-immunocompromised C57BL/6 mouse brain. Compared to control GBM cell-formed tumors, cells from ND1-reprogrammed cultures formed smaller tumors and expressed neuronal markers such as TUJ1 in the brain. Thus, reprogramming using a single-factor ND1 overcame drug resistance, converting malignant cells of heterogeneous GBM cells to normal neuron-like cells in vitro and in vivo. These novel observations warrant further research using patient-derived GBM cells and patient-derived xenograft (PDX) models as a potentially effective treatment for a deadly brain cancer and likely other astrocytoma tumors.

Neural stem cells (NSCs) are multipotent cells that have the capacity for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. Valproic acid (VPA) is a widely prescribed drug for seizures and bipolar disorder in clinic. Previously, a number of researches have been shown that VPA has differential effects on growth, proliferation and differentiation in many types of cells. However, whether VPA can induce NSCs from embryonic cerebral cortex differentiate into neurons and its possible molecular mechanism is also not clear. Wnt signaling is implicated in the control of cell growth and differentiation during CNS development in animal model, but its action at the cellular level has been poorly understood. In this experiment, we examined neuronal differentiation of NSCs induced by VPA culture media using vitro immunochemistry assay. The neuronal differentiation of NSCs was examined after treated with 0.75 mM VPA for three, seven and ten days. RT-PCR assay was employed to examine the level of Wnt-3α and β-catenin. The results indicated that there were more β-tublin III positive cells in NSCs treated with VPA medium compared to the control group. The expression of Wnt-3α and β-catenin in NSCs treated with VPA medium was significantly greater compared to that of control media. In conclusion, these findings indicated that VPA could induce neuronal differentiation of NSCs by activating Wnt signal pathway.