Stabilization of HIF 1a and EpoR expression levels in hNPCs The induction of HIF 1a, a key molecule of hypoxia, is a well characterized cellular response to lowered oxygen. Therefore dilution calculator HIF 1a expression in hNPCs cultured at 3% oxygen over a time course of 1 h, 3 h, 1 d, 2 d, 3 d and 4 d of differentiation was measured using western blot analysis. EPO treatment did not influence the expression levels of the protein. Although an early up regulation of HIF 1a could not be quantified, the consis tent expression of HIF 1a demonstrated that the system is HIF 1a sensitive. Western blot analysis of the EpoR were performed with proliferating as well as EPO treated cells differentiated for 3 days.
The quantifica tion of the data showed that the signal intensity is identi cal in all conditions tested, with no significant differences in the EpoR expression levels, indicating that any effect of EPO would not be mediated by an upregula tion of the EpoR, but by EPO itself. Influence of low oxygen and EPO on the proliferation rate of hNPCs To determine the effect of hypoxia on the proliferation, hNPCs were expanded either at 20% or 3% O2. In addi tion, EPO was added to proliferating cells at different concentrations and cell samples were collected every 24 h to verify the number of cells. At an oxygen level of 20%, EPO did not enhance cell proliferation of hNPCs compared to control cells. Consis tently, EPO did not change the proliferation levels of hNPCs at 3% oxygen. To investigate the effect of hypoxia on the proliferation of hNPCs, untreated cells from both conditions were compared and the number of cells ml was determined.
The prolif eration curves showed very similar results with no increase of the proliferation rate under hypoxic condi tions. The comparison of the doubling times of treated and untreated cells under normoxic and hypoxic conditions revealed no significant difference. Untreated cells cultured at 20% O2 showed a doubling time of 19. 48 1. 34 h and cells cultured at 3% O2 a doubling time of 20. 45 1. 53 h. In addition, no sig nificant difference of doubling times between the two groups could be detected with EPO treatment, 10 IU ml, 11. 76 2. 08 h versus 15. 12 1. 94 h, 50 IU ml, 17. 46 1. 78 h versus 19. 28 1. Dacomitinib 99 h, 100 IU ml, 18. 77 1. 57 h versus 19. 69 4. 15 h, 300 IU ml, 26. 38 5. 86 h versus 20. 57 2. 41 h. To verify the action of EPO, HCD 57 cells, an EPO dependent erythroleukemia cell line, were used. This cell line needs to be cultured with EPO for regular proliferation and stops proliferation when cultured with out EPO. The application of EPO resulted in a continuous proliferation of the cells, while the withdrawal of EPO stopped it.