Neural Regen Res. 2021 Dec 10
Tao Wang, Jian-Cheng Liao, Xu Wang, Qing-Song Wang, Kai-Ying Wan, Yi-Yi Yang, Qing He, Jia-Xuan Zhang, Gong Chen, and Wen Li
Products used in the paper Details Operation
AAV vector packaging For the in vivo experiments, single stranded adenovirusassociated viruses were used. A short version of the human glial fibrillary acidic protein (GFAP) promoter (681 bp, provided by PackGene® Biotech, LLC, Guangzhou, Guangdong, China) was used to construct pAAV GFAP::GFP and pAAV GFAP::NeuroD1-P2A-GFP. AAV serotype 9 (AAV9) was produced by PackGene® Biotech, LLC (Guangzhou, China). Iodixanol gradient (Merck/Sigma-Aldrich, St Louis, MO, USA) ultracentrifugation was used for AAV purification. All AAV used in this study was diluted in 0.001% Pluronic F-68 solution (Poloxamer 188 Solution, Caisson Laboratories, Smithfield, UT, USA). Request Quote

Research Field: CNS

Keywords: 5-bromo-2′-deoxyuridine, NeuroD1, astrocyte-to-neuron conversion, reprogramming, neural regeneration, reactive astrocytes, neurons, lineage tracing, fate mapping, neural stem cell

AAV Serotype: AAV9

Dose: As with the ET-1 injection, 1 μL of virus at a concentration of 5 × 10^11 GC/mL was injected into the parenchyma in each side 30 days after the ET-1 injection.

Routes of Administration: As with the ET-1 injection, 1 μL of virus at a concentration of 5 × 10^11 GC/mL was injected into the parenchyma in each side 30 days after the ET-1 injection.

Targeted organ: brain

Animal or cell line strain: Thirty adult male wild-type FVB/NJ mice (8–10 weeks old, ~25 g) were used for the in vivo experiments. Fifteen postnatal (P0–3) Sprague-Dawley rats were used for isolating primary astrocytes for in vitro experiments.


5-Bromo-2′-deoxyuridine (BrdU) is a halogenated pyrimidine that can be incorporated into newly synthesized DNA during the S phase of the cell cycle. BrdU is widely used in fate-mapping studies of embryonic and adult neurogenesis to identify newborn neurons, however side effects on neural stem cells and their progeny have been reported. In vivo astrocyte-to-neuron (AtN) conversion is a new approach for generating newborn neurons by directly converting endogenous astrocytes into neurons. The BrdU-labeling strategy has been used to trace astrocyte-converted neurons, but whether BrdU has any effect on the AtN conversion is unknown. Here, while conducting a NeuroD1-mediated AtN conversion study using BrdU to label dividing reactive astrocytes following ischemic injury, we accidentally discovered that BrdU inhibited AtN conversion. We initially found a gradual reduction in BrdU-labeled astrocytes during NeuroD1-mediated AtN conversion in the mouse cortex. Although most NeuroD1-infected astrocytes were converted into neurons, the number of BrdU-labeled neurons was surprisingly low. To exclude the possibility that this BrdU inhibition was caused by the ischemic injury, we conducted an in vitro AtN conversion study by overexpressing NeuroD1 in cultured cortical astrocytes in the presence or absence of BrdU. Surprisingly, we also found a significantly lower conversion rate and a smaller number of converted neurons in the BrdU-treated group compared with the untreated group. These results revealed an unexpected inhibitory effect of BrdU on AtN conversion, suggesting more caution is needed when using BrdU in AtN conversion studies and in data interpretation.

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