Induced neural stem cells protect neuronal cells against apoptosis

Abstract

Background: Neuronal cells are vulnerable to many stresses that can cause apoptosis. Reprogramming of fibroblasts into induced neural stem cells (iNSCs) is a potentially unlimited source of neurons. Discovering agents that can provide neuronal protection against these apoptotic stimuli is important for developing therapeutic strategies for various brain diseases.

Material and methods: We investigated the therapeutic effects of iNSCs against apoptosis activator II (AAII)-induced apoptosis of cortical neuronal cells. Apoptosis was confirmed by double immunocytochemistry with NeuN and 4',6-diamidino-2-phenylindole using terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling. We performed Western blot analyses for activated caspase-3, Bcl-2, phosphorylated Akt, and phosphorylated extracellular signal-regulated protein kinase (ERK). The level of vascular endothelial growth factor (VEGF) was analyzed using enzyme-linked immunosorbent assays (P<0.05).

Results: Cortical neuronal cells cultured with iNSCs had fewer apoptotic cells than those cultured without iNSCs. We found that cells cultured with iNSCs had a significantly lower caspase-3 level and a significantly higher Bcl-2 level than cells cultured without iNSCs. Cells cultured with iNSCs had higher VEGF levels than cells cultured without iNSCs. The levels of phosphorylated Akt and phosphorylated ERK were significantly higher in cells cultured with iNSCs than in cells cultured without iNSCs.

Conclusions: Our findings suggest that iNSCs activate Akt and ERK, which are associated with the inhibition of neuronal apoptosis. Thus, treatment with iNSCs may help reduce neuronal loss in brain disease. Further studies aimed at proving this hypothesis might help establish therapeutic agents that can prevent neuronal cell death and help cure neurodegenerative diseases.

Figure 1

The differentiation potential of induced neural stem cells (iNSCs) in vitro. The differentiation potential of iNSCs was determined by immunocytochemistry with the antibodies neuronal marker (Nestin) and 4′,6-diamidino-2-phenylindole (DAPI), iNSC cultures contained NeuN-positive cells, indicating that fibroblasts were reprogrammed into an NSC-like state.

Figure 2

Dose-dependent neurotoxicity of apoptosis activator II (AAII) in cortical neuronal cells. Increasing concentrations of AAII were added to the culture media of cells, and the toxicity was estimated after 24 h using the WST-1 proliferation assay.

Figure 3

Effect of coculture with iNSCs on cortical neuronal cell proliferation. Cortical neuronal cells were cocultured with induced neuronal stem cells (iNSC). To assess potential effects of the iNSCs, a transwell culture system was used. The rates of proliferation of cortical neuronal cells cultured without iNSCs (A) or cocultured with iNSCs (B) was compared (×100).

Figure 4

Detection of apoptosis in cortical neuronal cells by terminal deoxynucleotidyl transferase mediated digoxigenin-dUTP-biotin nick-end labeling (TUNEL). To examine the apoptotic cells in the cortical neuronal cells, we performed TUNEL staining. The cultured group with induced neuronal stem cells (iNSC) had fewer apoptotic cells compared to the cultured group without iNSCs. Histograms show the average number of TUNEL-positive cells as percentage for each group. * p<0.05.

Figure 5

Detection of apoptosis in cortical neuronal cells by terminal deoxynucleotidyl transferase mediated digoxigenin-dUTP-biotin nick-end labeling (TUNEL) and neuronal nuclei (NeuN). To confirm the presence of neuronal apoptotic cells in the cortical neuronal cells, we performed double immunocytochemistry with TUNEL. Scale bar 20 um. Histograms show the average number of apoptotic cells as percentage for each group. * p<0.05.

Figure 6

Anti-apoptotic effects are mediated via activation of induced neural stem cells (iNSCs). To evaluate the anti-apoptotic effect, we performed Western blot analysis of cleaved caspase-3 and B-cell lymphoma 2 (Bcl-2). The cultured group with iNSCs induced a decrease in the level of caspase-3 and an increase in the level of Bcl-2. * p<0.05.

Figure 7

Anti-apoptotic proteins are elevated in supernatants of cocultured cells. To examine whether induced neural stem cells (iNSCs) coculture secrete VEGF for the repair, we performed enzyme-linked immunosorbent assays (ELISA). * p<0.05.

Figure 8

Anti-apoptotic effects are mediated via phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-related kinase (p-ERK). We performed Western blot analyses for p-Akt and p-ERK. The p-Akt and p-ERK were significantly higher in the cultured cells with induced neural stem cells (iNSCs). * p<0.05.