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

Figure 3.

Figure 3

Conversion of astrocytes to mature neurons in vivo. Adult GFAP-Cre x Rosa-YFP mice were subjected to a focal ischemic insult to the right sensorimotor cortex and subjected to control and ND1 treatments. (A) Timeline of in vivo reprogramming experiments. Animals received sham, stroke control, or ND1 lentivirus injection to the peri-infarct region 7 days after stroke. empty control vector or ND1 lentivirus. Functional and psychological assessments were performed different days after stroke. Six weeks later, brain coronel sections were subjected to immunohistochemical staining with cell phenotype markers. (B) Representative images show cells expressing YFP (green, astrocytes), mCherry (red, ND1 infected cells), NeuN (light blue, mature neurons), and Hoechst 33342 (dark blue, nuclei of all cells). The enlarged merged image illustrates overlapped markers (yellow/orange), indicating astrocyte-converted iNeurons. (C) A high magnificent confocal 3-D image showing a converted iNeuron with overlaid markers mCherry (red, transfection marker), YFP (green, astrocytes origin), and NeuN (blue, mature neuronal marker). The converted cell adopted neuronal morphology of an extended axon. (D) Control experiment where the brain region was infected with empty control virus. Due to the lack of cell reprogramming and lineage change, the neuronal and astrocytes populations (NeuN of red color and YFP of green color) were distinctively located without co-localization. (E) Quantified data of the image analysis of mCherry/YFP/NeuN triple-positive cells. No such cell could be seen in sham and empty control vector cells. There were over 66.06% of mCherry/YFP/NeuN triple-positive cells among ND1-infected cells. N = 6 for sham or control, n = 8 for ND1 group. (F,G) At 3–4 weeks after ND1 transduction, the glutamatergic neuronal marker vGLUT (green) was detected and colocalized with mCherry (red; overlay color: yellow/orange) in the converted cells. The bar graph in (F) shows that more than 72.9% of converted cells expressed vGLUT. There was no vGLUT expression in empty vector control cells. N = 8 animals per group. (H,I) The dopaminergic neuronal marker tyrosine hydroxylase was detected via western blot in peri-infarct tissue from control and ND1 treated brains. The bar graph in (H) indicates a significant increase of the tyrosine hydroxylase (TH) level in the ND1 treated tissue compared to injection of the empty vector control. N = 3 independent brain samples for control and n = 7 for ND1. Unpaired t-test, *p < 0.05, t = 1.937, DF = 8; analysis of variance: F(2,6) = 1.433, p = 0.9279.