Discussion Standard cultivation conditions for cell cultures com prise the use of 20% oxygen, nevertheless exactly a number of studies have described an enhanced proliferation in low ered oxygen. Reducing oxygen can have a number of different effects such as the increase of proliferation as shown by Zhao et al. and Studer et al. for rat embryonic mesencephalic cells, or conversely a decrease of proliferation as described by Chen et al. who showed that long term proliferation in hypoxia was not beneficial for hESC with short splitting intervals. Studer et al. investigated the proliferation and differentiation of embryonic mesencephalic rat cells and came to the conclusion that hypoxia was beneficial for the cells in culture and that EPO could mimic this effect under nor moxic oxygen levels.

Recently Santilli et al. described an increased proliferation though the cell cycle remained unaffected as well as an increased neuro nal differentiation and decreased cell death of human neural stem cells caused by mild hypoxia. The effects of lowered oxygen on the proliferation of stem and pro genitor cells are not limited to the central nervous sys tem. More physiological culturing conditions are also favoured by other cell types like bone marrow stro mal cells and mesenchymal cells. As a first step in this study we verified the expression of HIF 1a and the EpoR. The sen sibility of the hNPCs to hypoxic conditions is indicated by the expression of HIF 1a. A similar effect was observed by Zhou and Miller, Zhao et al. and Zhang et al. ranging from 30 minutes to 24 hours after the onset of hypoxia.

HIF 1 is activated under hypoxic conditions in a variety of cell types and the HIF 1 targeted genes play an important role in maintaining cellular homeostasis in response to hypoxia. To investigate the EpoR we chose western blot ting as the currently available antibodies lead to incon clusive results obtained by immunocytochemistry. The EpoR expression level was not altered by culturing the cells under EPO application or hypoxic conditions, the latter being in line with the absence of a hypoxic EPO effect. Even though this is contrary to Theus et al. where hypoxia led to an increase in the EpoR expression, Milosevic et al. likewise observed that hypoxia does not affect EPO signaling. This inconsis tency could be due to different culturing conditions or cell types. The effect of EPO on the metabolic activity and apoptosis is independent from the regulation of expression of its receptor since the expression levels are not altered between different stages of proliferation or differentiation, as well as EPO treated cells. In summary, we conclude that the differentiation of the human NPCs used in this study as a model system is hypoxia GSK-3 sensitive and EPO responsive.