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. 2021 Mar 26;13:612856. doi: 10.3389/fnagi.2021.612856

Figure 2.

Figure 2

Reprogramming induced cellular changes and functional activities in vitro. The ND1-mediated astrocytes reprogramming was monitored in cultures and functionally characterized. (A,B) Two weeks after ND1 virus or empty control vector transduction, astrocyte cultures were challenged by the scratch test to measure the motility of reprogrammed cells. From day 1 after the scratch insult, the damaged area recovered significantly faster by migrating cells in cultures infected by ND1 compared to the vector control culture. N = 3 cell cultures. Two-way ANOVA (Interaction: F(3,24) = 11.15, ***p < 0.0001; Time: F(3,24) = 18.66, ***p < 0.0001; ND1: F(1,8) = 53.87, ***p < 0.0001) followed by Holm–Sidak’s multiple comparisons test: *p < 0.05 and **p < 0.01 for ND1 vs. empty vector control. (C) Effect of the scratch test on the expression of migration factor focal adhesion kinase (FAK) in reprogrammed cells. In line with the increased motility 3 days after scratch, ND1-infected cells within 200 μm of the scratch expressed significantly higher immunofluorescence of phosphorylated FAK (p-FAK). N = 3 per group. One-way ANOVA, F(2,6) = 7.327, **p = 0.0245 followed by Holm–Sidak’s multiple comparisons test: *p < 0.05 vs. control. (D) mCherry-positive converted cells were subjected to whole-cell recordings 28 days after infection. In the current-clamp mode, membrane depolarization induced by current injections evoked depolarization generated action potentials. A hyperpolarization was observed upon the decay phase of the spikes, which is typical for neurons and suggestive of functional potassium channels in these cells. n = 10. (E) A longer membrane depolarization pulse evoked the firing of a chain of action potentials that are characteristics of functional neurons.