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

Figure 1.

Figure 1

In vitro direct reprogramming of astrocytes to induced neurons. Mouse astrocyte cultures were transduced with NeuroD1 (ND1) or empty vector control and stained for different cell markers. (A) Ten days after lentiviral transduction, successfully infected cells were monitored over time by the expression of the fluorescent mCherry reporter (red), implicating GFAP-ND1-IRES-Ubi-mCherry expression in these astrocytes. (B–D) Enlarged images of infected cells 14 days after transduction. Infected cells expressed the reporter gene mCherry (B), GFAP stain revealing an astrocyte-like morphology of astrocytes before reprogramming (C). In image (D), some cells develop the neuronal lineage marker Tuj1 (green) and adopted either a unipolar or bipolar morphology that is uncharacteristic of astrocytes. (E) A representative image of control culture with the empty vehicle virus showing the lack of Tuj-1 expression in infected cells (mCherry, red). There were also no alterations to the morphology of astrocytes. (F) Time course and reprogramming rate of the expression of the immature neuron marker Tuj1 in infected astrocytes up to 42 days after transduction. One-way ANOVA (F(5,36) = 77.91) followed by Holm–Sidak’s multiple comparisons test. N = 3 independent cell culture batches, ***p < 0.001. (G) Quantified cell counts of total cell numbers 10 and 28 days after transduction with ND1 or vector control. The reduced cell number implied attenuated cell proliferation after the ND1 transduction and neuronal conversion. Two-way ANOVA (interaction: F(1,10) = 2.377, p = 0.1542; time: F(1,10) = 0.174, p = 0.6854; treatment: F(1,10) = 358.9, ****p < 0.0001). (H) An immunostaining image shows coexpression of Tuj1 and GFAP in cultured astrocytes 6 weeks after transduction. (I) Confocal microscopy of an enlarged image from 6 weeks post-transduction, showing complex processes that are positive for Tuj-1 and synaptophysin colocalized with mCherry. Those cells under the culture condition did not show positivity to NeuN; however, they expressed the synaptic protein synaptophysin. (J) At 6 weeks after transduction, astrocytes undergoing direct reprogramming extended processes and developed nerve terminals positive to the pre-synaptic protein synapsin I (red). (K) The bar graph shows quantified data that at 6 weeks post-transduction, 58.7% of reprogrammed cells (mCherry-positive) expressed Tuj-1 and 17.4% of these cells also expressed synapsin 1. n = 3, paired t-test, *p < 0.05 vs. Tuj1 only cells. (L) Measurements of the length of extended processes from converted cells. There were drastically increased in the length of processes of cells received ND1 transduction. N = 3, two-way ANOVA (interaction: F(3,9) = 8.427, ***p < 0.001).