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List of figures 64 Fig.

List of figures 64 Fig. 11: Population doubling time (PDT) of normoxic and hypoxic cultured hMSCs. Cells cultured under standard conditions showed a highly inhomogeneous PDT until day 40 and did not increase their dividing time before senescence. A superior homogenous PDT was detected for hypoxic cells, while a high increase during senescence occurred. Dashed lines indicate the beginning of population senescence. ....................................................................................... 27 Fig. 12: WST-Assay to assign the metabolic activity of normoxic and hypoxic cultured hMSCs. Normoxic stem cells were set to 100%. Compared to the control group (red, 100.0 ± 3.2%), hypoxic cultured cells showed a significant higher metabolic activity (blue, 136.9 ± 37.05%, p=0.0012). Experiment was repeated three times in quintets. Mean & SD. ............................ 28 Fig. 13: Exemplary phase contrast images of normoxic and hypoxic cultured hMSCs. In the first quarter (4 days in culture) cells had a comparable distribution of the different subpopulations for both settings. The fraction of FC (*) cells continuously increased in normoxic conditions over the next quarters (2.Q: 16 days, 3.Q: 34 days in culture) until cell population went into senescence after 52 days (4.Q). Hypoxic cells instead showed a homogenous morphology with an extent of RS (§) and SS (#) cells over the second (24 days) and third (44 days) quarter. In the last quarter, directly before senescence, hypoxic cultured cells also displayed an increased fraction of FC cells. Bar: 200 µm. ....................................................................... 29 Fig. 14: Quantification of cell area and aspect ratio of normoxic and hypoxic cultured hMSCs. Within the first culture quarter, cells in both conditions consisted majory of small RS/SS cells and very low FC cells. The second quarter revealed an slight increase of FC cells for normoxic cells and nearly no changes for hypoxia. 100 cells were evaluated per culture condition and time point. RS: rapidly self-renewing cells, SS: spindle-shaped cells, FSS: flattened spindle-shaped cells, FC: flattened cells..................................................................................................................... 30 Fig. 15: Quantification of cell area and aspect ratio of normoxic and hypoxic cultured hMSCs. The third quarter ended with an additional cell area increase for normoxic cells, while hypoxic still keep their small and homogenous morphology. Directly before senescence the cells in both experimental settings had the typical flattended morphology. 100 cells were evaluated per culture condition and time point. RS: rapidly self-renewing cells, SS: spindle-shaped cells, FSS: flattened spindle-shaped cells, FC: flattened cells. ......................................................... 31 Fig. 16: Exemplary white light confocal images of normoxic or hypoxic cultured hMSCs on different substrates. Overall, normoxic cultured cells were higher on polystyrene surfaces (NUNC) and collagen I, fibronectin or laminins coated glass slides. The colour bar represents the cells height. Bar: 50 µm. ................................................................................................................... 33 Fig. 17: Cell volume measurement from white light confocal microscopy images. 25 cells per culture condition and substrate were evaluated. Normoxic as well as hypoxic cells showed some bigger outliers on polystyrene. Coating with collagen I, fibronectin or laminin decreased the volume distribution in both culture settings, whereat hypoxic cells appeared more homogenous. Median: dashed line; Mean: dotted line (dashed & dotted line: Median and Mean overlapping).............................................................................................................................. 35

List of figures 65 Fig. 18: Comparison of hMSC migration in normoxic and hypoxic environment on different proteins. Normoxic cells were assigned to 100%. At least 100 cells from two runs were included. Mean & SD, **

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