These results indicate the supportive effect of lowered oxygen conditions for the differentia tion of hNPCs. In order to determine selleck chemicals the influence of hypoxia in detail, we cultured proliferating cells in low oxygen followed by a differentiation at 3% and 20% oxygen. In Figure 5D the percentage of neurons evalu ated by bIII tubulin expression is shown. Cells prolifer ated and differentiated at low oxygen levels displayed an increase of bIII tub cells at day 3 and at day 4 com pared to a proliferation of cells at 20% oxygen. Next we analysed whether EPO influenced neuronal differentia tion, but with both concentrations no change in the number of bIII tub cells was detected. Figure 5E shows a summary of 3 and 4 days differentiated hNPCs of all conditions tested.

At day 3 significant differences of neuronal differentiation have been found. The number of neurons was significantly increased up to 4. 51 0. 45% when differentiated at 3% oxygen, compared to 2. 95 0. 25% when differentiated at 20% O2. In addition, the expansion of cells at low oxygen increased the number of bIII tub cells. When differentiated at 3%, 5. 92 1. 66% of positive cells have been detected, when differentiated at 20%, 5. 20 0. 87% of positive cells have been found. This indicates that there seem to be two independent mechanisms of differentiation. First, a differentiation of human progeni tor cells in lowered oxygen increases the number of neurons and in addition, an expansion of cells in low ered oxygen influences the differentiation potential of hNPCs as well, independently of the culturing condi tions during differentiation.

Anti apoptotic effect of hypoxia and EPO on differentiated hNPCs Since differentiation of progenitor cells is associated with apoptosis and EPO is a well known anti apoptotic mediator, we investigated the amount of apoptotic cells during differentiation in normoxic and hypoxic condi tions. Again the cells differentiated up to 4 days and each day samples were taken from cells cul tured under normoxic and hypoxic conditions with and the amount of apoptotic cells in our cell population a TUNEL staining and consecutive FACS analysis was performed. Over time we observed a continuously rising apoptosis starting with 7. 78 3. 10% that culminated in 32. 43 4. 26% at day 4 in cells cultivated with normoxic oxygen levels.

During the first three days neither hypoxia nor EPO affected the apoptosis of the hNPCs. On day 4 of differentiation we remarkably observed that both in hypoxia and normoxic EPO treated cells the level of apoptotic cells was only half as high as in the normoxic control. There was no significant difference between EPO treated normoxic Anacetrapib cells and cells differentiated in hypoxia. Application of EPO under hypoxia did not lead to an additional effect 5 A western blot analysis was performed to measure the expression of the anti apoptotic protein Bcl 2 in cells differentiated up to 4 days.