ReNcell VM cells were incubated under differentiation conditions

ReNcell VM cells were incubated under differentiation conditions for 1 day and 3 days in the presence and absence of EPO, respectively. During differentiation EPO caused a significant increase of metabolic activity after 1 day under normoxic condi tions from a concentration of 25 IU ml on and higher compared to control. A similar increase of the metabolic activity was observed at 3% O2, but higher EPO concentrations were needed for a significant change of activity. The signifi cant increase of the metabolic activity caused by EPO was not any longer present after 3 d of differentiation in both conditions normoxia and hypoxia as seen in Fig ure 4C and 4D.

By comparing the control values of both conditions, one can see a significant increase of the meta bolic activity at 3% oxygen at both time points of differ entiation, indicating a general influence of low oxygen on the cell metabolism which lasts for several days during differentiation. For comparison the Wst 1 assay at 1 d and 3 d of proliferating cells is shown in Fig ure 4F. Consistently, hypoxia increased the metabolic activity in this condition. Lowered oxygen promotes neuronal differentiation of NPCs Next, we investigated the effect of lowered oxygen on the neuronal differentiation of human NPCs. After the withdrawal of growth factors, ReNcell VM cells were either differentiated at 20% or 3% oxygen for 4 days. First we asked the question, whether the differences of the differentiation between 20% O2 and 3% O2 is caused by changes of the proportions of cells in each cell cycle phase.

Therefore Cilengitide we performed cell cycle measurements with flow cytometry, using the DNA binding dye propi dium iodide. Figure 5 shows the percentage of cells within the phases of the cell cycle within the first 24 h of differentiation. After 20 hours, 95% of the cells reached G1 G0 phase, both in normoxic as well as in hypoxic conditions. To verify neuronal differentiation, the expression of bIII tubulin was measured by FACS analysis. For these experiments we included additional culturing conditions. First, the cells proliferated at 20% oxygen and were dif ferentiated at either 20% or 3% oxygen. Sec ond, the cells were expanded at 3% and differentiated at 20% or 3% oxygen, respectively. In addition, EPO was applied at 10 IU ml and 100 IU ml with the onset of differentiation. As shown in Figure 5C, there is no difference in the percentage of bIII tubulin positive cells between 20% and 3% oxygen and also no influence of EPO until day 3 of differentiation. At this time point, the maximal number of neurons appears with an almost twofold increase of the percen tage of bIII tub cells under hypoxic conditions with 4. 51 0. 45% compared to 2. 61 0. 31%.

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