Hirzel, Estelle. Characterization of human bone marrow derived mesenchymal stem cells as a model for in vitro adipocytes studies. 2013, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_10618
To support research in this relevant field of our society, a good in vitro model for adipocytes and adipogenesis is necessary. However, most in vitro studies about adipocyte function are currently done with murine cell lines. These models have the disadvantages that they may not reflect the (patho-)physiologic situation in humans because of species differences. Another problem resides in major physiologic differences between immortalized and primary cells, particularly in view of their reaction to oxidative stress. As elevated oxidative stress is supposed to contribute to the development of type II diabetes mellitus (T2DM) in obese people, it might be advantageous to apply antioxidant treatments for these patients.
In this thesis two studies are presented. In the first study, human bone marrow derived mesenchymal stem cells (hBM-MSCs) are described and investigated as a model for (primary) adipocytes and adipogenesis. In the second study hBM-MSC derived adipocytes were treated with the well-characterized antioxidants MitoQ, resveratrol and curcumin and the effects of these substances on different mitochondrial parameters were determined.
In the first study, hBM-MSCs were grown to confluence and adipocyte differentiation was induced by differentiation medium. Cell number and protein content, fat accumulation, production of reactive oxygen species (ROS), cellular oxygen consumption, mitochondrial mass and morphology were assessed during a differentiation period of 22 days. The cell number did not change but the protein content increased markedly during adipogenesis, indicating that the cell mass increased over time. ROS production was measured with two different assays showing either oxidizing or reducing radicals. Both methods showed an increase in ROS, indicating that overall oxidative stress increased during adipocyte differentiation. Oxygen consumption of intact cells was measured with a Seahorse flux analyzer. Basal respiration as well as leak respiration and uncoupled oxygen consumption increased during adipogenesis. This result suggests an increase in mitochondrial mass. Therefore, mitochondrial content in a defined cytoplasmic volume was quantified using confocal microscopy. A distinct elevation of mitochondrial mass during adipocyte differentiation was observed. Mitochondrial morphology also changed during the differentiation process. During adipocyte differentiation mitochondria built a tight network while electron microscopy revealed that their inner morphology and cristae structure did not change.
In the second study, the effects of MitoQ, resveratrol and curcumin on ROS production, oxygen consumption, glycolysis and intracellular ATP content were assessed. All three substances were used at non-toxic concentrations. MitoQ lowered ROS production of oxidizing and reducing radicals, resveratrol and curcumin only decreased reducing and oxidizing radicals, respectively. The immediate effect of these antioxidants on oxygen consumption was measured in an Oxygraph; respiration after 24 h treatment was assessed with a Seahorse flux analyzer. Upon addition, all substances tested slightly decreased oxygen consumption. However, after 24 h only MitoQ inhibited the respiration. Intracellular ATP content did not change in response to any treatment.
In coclusion, hBM-MSC derived adipocytes are an interesting model for human fat cells in vitro. They efficiently differentiated into fat cells and shared many, but not all characteristics with the murine cells lines commonly used in this research. In fact, these cultured hBM-MSCs are one of the closest models to the human adipose tissue for in vitro studies.
MitoQ, resveratrol and curcumin acted as antioxidants in this cell type. In contrast to resveratrol and curcumin, MitoQ seemed to impair mitochondrial function but intracellular ATP levels did not change. Thus, all three antioxidants tested are interesting candidates for lowering oxidative stress and possibly preventing insulin resistance in obesity.
|Advisors:||Eberle, Alex N.|
|Committee Members:||Krähenbühl, Stephan|
|Faculties and Departments:||03 Faculty of Medicine > Bereich Medizinische Fächer (Klinik) > Ehemalige Einheiten Medizinische Fächer (Klinik) > Experimentelle Endokrinologie (Eberle)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||106 S.|
|Last Modified:||30 Jun 2016 10:54|
|Deposited On:||03 Feb 2014 07:43|
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