The Role of N-Acetylcysteine Supplementation on the Oxidative Stress Levels, Genotoxicity and Lineage Commitment Potential of Ex Vivo Murine Haematopoietic Stem/Progenitor Cells

Abstract

Objectives: The ex vivo maintenance of haematopoietic stem/progenitor cells (HSPCs) is crucial to ensure a sufficient supply of functional cells for research or therapeutic applications. However, when exposed to reactive oxygen species (ROS) in a normoxic microenvironment, HSPCs exhibit genomic instability which may diminish their quantity and quality. This study aimed to investigate the role of N-acetylcysteine (NAC) supplementation on the oxidative stress levels, genotoxicity and lineage commitment potential of murine haematopoietic stem/progenitor cells (HSPCs).

Methods: This study was carried out at the Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia, between June 2016 and July 2017. Bone marrow cells were isolated from nine mice and cultured in a growth medium. Various concentrations of NAC between 0.125-2 μM were added to the culture for 48 hours; these cells were then compared to non-supplemented cells harvested from the remaining three mice as the control group. A trypan blue exclusion test was performed to determine cell viability, while intracellular ROS levels and genotoxicity were determined by hydroethidine staining and comet assay, respectively. The lineage commitment potential of erythroid, myeloid and pre-B-lymphoid progenitor cells was evaluated via colony-forming cell assay.

Results: NAC supplementation at 0.25, 0.5 and 2 μM significantly increased cell viability (P <0.050), while intracellular ROS levels significantly decreased at 0.25 and 0.5 μM (P <0.050). Moreover, DNA damage was significantly reduced at all NAC concentrations (P <0.050). Finally, the potential lineage commitment of the cells was not significantly affected by NAC supplementation (P >0.050).

Conclusion: The findings of this study indicate that NAC supplementation may potentially overcome the therapeutic limitations of ex vivo-maintained HSPCs.

Keywords: Cell Lineage; DNA Damage; Hematopoietic Stem Cells; N-acetylcysteine; Reactive Oxygen Species.

Figure 1

Plot diagram showing the effect of 48-hour N-acetylcysteine (NAC) supplementation on the viability of ex vivo haematopoietic stem/progenitor cells. All values represent means ± standard error. NAC = N-acetylcysteine. *Significant difference in comparison to the control group (P <0.050).

Figure 2

Histogram showing the effect of 48-hour N-acetylcysteine supplementation on the level of reactive oxygen species in ex vivo haematopoietic stem/progenitor cells. All values represent means ± standard error. ROS = reactive oxygen species; NAC = N-acetylcysteine. *Significant difference in comparison to the control group (P <0.050).

Figure 3

Histograms showing the effect of 48-hour N-acetylcysteine (NAC) supplementation on (A) the percentage of tail DNA and (B) the tail moment of ex vivo haematopoietic stem/progenitor cells. Cells treated with 100 μM of hydrogen peroxide served as a positive control group for the purposes of assay validation. All values represent means ± standard error. H₂O₂ = hydrogen peroxide; NAC = N-acetylcysteine; AUs = arbitrary units. *Significant difference in comparison to the control group (P <0.050).

Figure 4

Histograms showing the effect of 48-hour N-acetylcysteine supplementation on the lineage commitment potential of ex vivo haematopoietic stem/progenitor cells. All values represent means ± standard error. CFU = colony-forming unit; M = macrophages; G = granulocytes; E = erythroid; Pre-B = lymphoid; NAC = N-acetylcysteine.