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Discussion 42 a

Discussion 42 a 1.5-times higher mitochondrial activity in hypoxic cultured cell compared to the normoxic control. Comparable results for amplified proliferative capacity were demonstrated by Grayson and colleagues who showed, that hypoxic cultivation increased mesenchymal cell numbers 30-fold compared to the normoxic group 98 and metabolic activity of neural progenitor cells was significantly higher 99 . To the more, comparable results could be obtained for neuronal stem cells 99,100 , human myogenic satellite cells 101 , umbilical cord cells 97 and various other cell types 102-104 . In contrast, early embryonic stem cell 105,106 or primary human myoblast proliferation 107 is inhibited by lower oxygen concentrations. These widely scattering facts indicate a specific oxygen microenvironment during development or for normal physiological function. These findings give hint that each specific cell type and development stage might rely on specific cell culture conditions. Coussons et. al. identified the transcription factor HIF1α as a key regulatory molecule telomerase reverse transcriptase (TERT) for long-term self-renewable capacity of embryonic stem cells 108,109 . Based on these results, hypoxic mid-term culture might be an interesting alternative to viral immortalisation with gene transfer 110 . Cell morphology Since the first discovery of at least two different subpopulations within in vitro cultured hMSCs 111 , numerous research groups examined differentiation potential of this subpopulations into the main lineages 112-115 . Haasters and Prall showed a correlation between cell morphology and cellular surface markers. Their results indicated that RS cells had all three positive markers expressed, whereas SS and FC lost at least one of their surface proteins 116 . Based on preliminary experiments, cell area and aspect ratio, which comprises a more specific definition 117 , were chosen to describe shape parameters. Our results revealed an aspect ratio greater 5 as an initial value for spindle-shaped cells. MSCs with a cell area of more than 10,000 µm 2 showed no dividing capacity. The studied donor persistently showed a heterogeneous cell population under standard normoxic conditions, whereas there was just a slight morphological shift into the last quarter of culture time. Interestingly hypoxic cells solely appeared as RS and SS cells until senescence. Through the combination of cum PD and morphological analysis, we hypothize that the selection of rapidly dividing cells by low oxygen levels might be a key factor for the increased proliferation. In relation to the expanding application spectrum for regenerative medicine a homogenous cell

Discussion 43 population with a high proliferation capacity provides various advantages, such as increased matrix and cytokine production. Prockop and colleagues identified the RS subpopulation as the main source for high differentiable cells 115,118-121 . Additionally, small cells indicate less F-actin stress fibres and thus might be in a more primitive, undifferentiated cell state 122,123 . Cell volume The cells volume is dependent on various factors, such as cell adhesion, cytoskeletal arrangement and locomotion 124 . In this context, filamentous actin (F-actin) plays the most pivotal role by arranging cells plasma membrane, including microvillis, stress fibres or filopodia/lamellipodia 59 which all result in volume changes. By confocal white light microscopy we were able to measure cell volumes on normal cell culture plastic faster compared to atomic force microscopy or impedance measurements. Moreover, glass slides were coated with the most noticeable proteins within the bone marrow, collagen I, fibronectin or laminin. Our results bare a slightly less volume for hypoxic cultured cells on plasma-treated polystyrene surface compared to the normoxic control. Collagen I coating provokes a volume reduction in both culture conditions, while hypoxic cells were stronger affected. Captivatingly, on fibronectin coated slides normoxic cells significantly increased their volume, whilst homogeneity decreased. However, hypoxic cells still had a homogenous, low volume appearance. Up to now, differences in cell volumes under different oxygen levels have not been studied yet. Nonetheless, vimentin - a major intermediate filament in mesenchymal cells and therefore influencing cell volume - is sensitively upregulated by lower oxygen concentrations within endothelial cells 125 . Furthermore, cytoskeletal rearrangement of F-actin in 2.2% oxygen for 24h was shown for mouse embryonic stem cells 126 . Moreover, hepatocytes showed a reorganisation of micro- and intermediate filaments 127 . Our results are the first hint, that oxygen differences in vitro and in vivo might affect cytoskeletal rearrangement of hMSCs. Time-Lapse analysis Cell migration is an essential biological phenomenon for physiological function as well as pathology in higher species. Endothelial cells during wound healing, leucocytes in inflammatory response or cancer cell migration during metastasis are just an

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