The dark side of BrdU in neural stem cell biology: detrimental effects on cell cycle, differentiation and survival

Abstract

5-Bromo-2'-deoxyuridin (BrdU) is frequently used in anaylsis of neural stem cell biology, in particular to label and to fate-map dividing cells. However, up to now, only a few studies have addressed the question as to whether BrdU labeling per se affects the cells to be investigated. Here, we focused on the potential impact of BrdU on neurosphere cultures derived from the adult rat brain and on proliferation of progenitors in vivo. In vitro, neurospheres were pulsed for 48 h with BrdU, and cell proliferation, cell cycle, differentiation, survival and adhesion properties were subsequently analyzed. BrdU inhibited the expansion of neural progenitors as assessed by MTS assay and increased the fraction of cells in the G0/G1-phase of the cell cycle. Moreover, BrdU increased cell death and dose-dependently induced adherence of NPCs. Cell adherence was accompanied by a reduced amount of active matrix-metalloproteinase-2 (MMP-2). Furthermore, BrdU repressed neuronal and oligodendroglial differentiation, whereas astroglial fate was not affected. In contrast to the in vitro situation, BrdU apparently did not influence endogenous proliferation of NPCs or neurogenesis in concentrations that are typically used for labeling of neural progenitors in vivo. Our results reveal so far uncharacterized effects of BrdU on adult NPCs. We conclude that, because of its ubiquitous use in stem cell biology, any potential effect of BrdU of NPCs has to be scrutinized prior to interpretation of data.

Effects of addictive drugs on adult neural stem/progenitor cells

“…False positive and negative results are possible, especially with fewer injections [5]. Moreover, BrdU may inhibit NSPC expansion by increasing the fraction of cells in the G 0 /G 1 -phase of the cell cycle and may also repress neuronal and oligodendroglial differentiation and increase cell death [47]. There are several ways to overcome these problems.…”

Distribution and fate of DCX/PSA-NCAM expressing cells in the adult mammalian cortex: A local reservoir for adult cortical neuroplasticity?

“…In addition, the side effects of BrdU labeling have been shown to compromise cell proliferation, cell migration, and influence the final cell position and number. BrdU is more toxic than tritiated thymidine, as demonstrated both in vitro in neural stem and progenitor cell cultures (Lehner et al, 2011) and in vivo in the rhesus monkey (Duque and Rakic, 2011).…”

Integration of the Neural Stem and Progenitor Cells into Existing Neuronal Circuitry and Adult Neurogenesis in the Dentate Gyrus of the Hippocampus

“…Although, generally BrdU infusion as an analog for thymidine nucleotide isused for evaluation of neurogenesis, birth dating, proliferation and cell tracking, but the effects of BrdU on the NSCs behavior have not been fully investigated [15][16][17][18]. For a long-lasting period it has been known that the long term exposure of the NSCs with BrdU promotes conformational changes in DNA structure, affects the balanced nucleotide pool in NSCs and subsequently alters cell cycle progression in which the majority of cells arrest in the G0/G1-phase of the cell cycle [19,23,44]. Also the recent studies have shown that the BrdU alters the DNA structure and stability, raises the risk of sister-chromatid exchange and mutations [18].…”

“…Even though, the BrdU has been widely used to label newly generated cells for birth dating, cell fate studies and specially neurogenesis as well as tracking of cells after transplantation but it has some limitations and pitfalls [15][16][17][18]. In the some experimental studies, it has been shown that the administration of the BrdU for a long time induces NSCs senescence, cell death and astroglial differentiation [19][20][21][22][23][24].Therefore, the present study aimed to investigate the potential neurogenesis and regenerative effects of the endogenous neural stem and progenitor cells by means of BrdU injection for labeling in a short time.…”