Abstract
The side population (SP) cells of the murine bone marrow (BM) contain primitive hematopoietic stem cells (HSCs). To understand the effect of irradiation on these cells, C57BL/6NHsd mice were irradiated to 10 Gy total body dose and sacrificed 24 hours later. The bone marrow was removed and stained with Hoechst dye, and Annexin V as an early marker of apoptosis. The SP cells were analyzed by flow cytometry. Following irradiation, there was no change in the frequency of SP cells in total nucleated BM cells between non-irradiated (0.081 ± 0.033%) and irradiated mice (0.093 ± 0.027%). In contrast, there was a decrease in total nucleated BM and SP cells. SP cells decreased by 60.1% from 44873 ± 22099 SP cells per harvest to 17550 ± 4805 SP cells per harvest 24 hours after 10 Gy. The number of lymphocytes in the irradiated BM decreased from 25.7% to 4.5% after irradiation. Since the majority of the SP cells are located in the lymphocyte sorting region, there was a 6-fold decrease in the percent of SP cells found in the live lymphocyte region following irradiation (0.469 ± 0.185% compared to 2.923 ± 1.913%).
Annexin V was used to stain for the appearance of phosphatidylserine (PS) on the cell surface as an early marker for apoptosis. The appearance of Annexin V positive cells did not increase significantly with irradiation in SP compared to non-irradiated SP cells (26 ± 8% and 25 ± 12%, respectively). In contrast, the percent of apoptotic cells in the non-SP fraction was significantly increased following irradiation compared to the non-irradiated cells (35 ± 8% and 25 ± 6%, respectively) (p=0.02). Our data indicate that irradiation was not inducing apoptosis in SP cells but in non-SP cells. It might suggest that SP cells could be more resistant to radiation damage. Future investigations will be performed to understand the functional activity of SP cells from lethally irradiated mouse BM and the molecular mechanism of those SP cells in resistance to irradiation damage.
Discrimination of the Hoechst side population in mouse bone marrow with violet and near-ultraviolet laser diodes