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Abstract
Background
Intracellular reactive oxygen species (ROS) have dual effects depending on their cellular level. ROS act as secondary messengers at a low concentration, although ROS exhaust the hematopoietic stem cell (HSC) compartment at a higher oxidized state. So, we investigated whether maintaining a low level of ROS could preserve the hematopoietic stem cell function according to the MnSOD over expression.
Methods
Human MnSOD cDNA was introdu ced into mouse HSCs and progenitor cells by using a MSCV-PGK-GFP retrovirus. The hematopoietic function of over-expressing MnSOD was evaluated in vitro on a colony-forming cell assay and in vivo in a competitive transplantation model. MnSOD-transduced, lineage negative, GFP+ B6.SJL (CD45.1+) mouse bone marrow cells were transplanted into lethally irradiated C57BL/6J (CD45.2+) mice in competition with CD45.1/45.2 double positive bone marrow mononuclear cells. We also measured the basal mRNA levels of antioxidants, including MnSOD, catalase and cellular glutathione peroxidase (GPx1), of C57BL/6J HSCs.
Results
On the colony-forming cell assay, a MnSOD over expression significantly preserved the CFU-M with irradiation as compared with the mice without irradiation. HSCs with an MnSOD over expression showed a tendency for higher engraftment ability on the competitive transplantation assay even after 200 cGy re-irradiation, and we observed a significantly higher myeloid differentiation potential after the second serial transplantation. The basal mRNA levels of MnSOD and catalase were less than 1∼2% and 2∼5%, respectively, in the long-term and short-term HSCs, respectively, and these cells didn't activate in spite of radiation stress.
References
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Fig. 2.
Schema of retroviral transduction into the mouse hematopoietic cells and the competitive transplantation.
Fig. 3.
Ectopic expression of human MnSOD in mouse cells. (A) Overexpression of MnSOD protein in the transduced AFT024 cells. Whole cell protein extracts were reacted with rabbit anti-human SOD and Actin antibodies. (B) Functional activity of overexpressed MnSOD protein in the transduced C57BL/6J bone marrow cells. Data are mean±sem of 2 independent experiments (∗P＜0.01).
Fig. 4.
In colony forming cell assay, MnSOD transduced HSCs significantly preserved CFU-M after 200cGy irradiation. CFCs were scored on day 8 in the non-irradiated plates (A) and 200cGy irradiated plates (B) after 4×104 per plate sorted GFP+ cells from MSCV-MnSOD-GFP or MSCV-PGK-GFP infected bone marrow cells were cultured in the Methocult M3434 medium. Data are mean±sem of 3 independent experiments (∗P＜0.05).
Fig. 5.
In vivo verification of human MnSOD gene in the CD45.1+GFP+ peripheral blood cells of transplanted mouse.
Fig. 6.
Competitive in vivo transplantation assay. (A) MnSOD overexpressed HSCs showed higher engraftment potential than those of control vector after 200cGy re-irradiation. Data represent mean±sem of relative CD45.1+ ratio of MnSOD to vector in the peripheral blood of 2 independent transplantation experiments (N=10 in vector group; N=11 in MnSOD group). (B) Maintain the repopulating capacity of MnSOD overexpressed HSCs in the serial transplantation. (C) Multilineage reconstitution showed preserving more myeloid differentiation in MnSOD overexpressed HSCs of second transplantation. Data are mean ±sem (N=6 in each group, ∗P＜0.05).
Fig. 7.
Baseline level and radiation effect of antioxidant mRNA expression of MnSOD (A), catalase (B), and cellular glutathione peroxidase (C) in the various hematopoietic cell compartments of C57BL/6J mouse by quantitative real-time RT-PCR. Results were normalized to the levels of β-actin. Data are meanratio±sem (N=3, ∗P＜0.05).
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