Establishment and characterization of a human 3-D fat model - Adipogenesis of hASC in a spheroid model; 3-D cocultures of adipocytes and endothelial cells

Today, it is well recognized that white adipose tissue represents an important endocrine organ, which is part of a complex signaling network involved in the regulation of many physiological and pathophysiological processes. Hence, adipose tissue possesses great pharmacological and therapeutic potential and is increasingly considered as a promising drug discovery target. To make use of this potential, a thorough understanding of adipose tissue development and adipocyte functions is a prerequisite. However, current research in this field is largely based on conventional 2-dimensional (2 D) in vitro models, which cannot fully capture the complex microenvironment present in vivo, including the extensive cell-cell and cell-matrix interactions. Therefore, this thesis was focused on the establishment and characterization of a 3-dimensional (3 D) human adipose tissue model. Such a model system would not only be useful for basic research, but could ideally also be applied in tissue engineering and regenerative medicine, as well as in drug screening assays. In the first part of this work a spheroid model using human adipose-derived stem cells (ASC) was successfully established. With this 3-D culture system, it was possible to produce mechanically stable spheroids of defined size, in which ASC could be induced to undergo adipogenic differentiation. Culture conditions and protocols were optimized in order to ensure tissue homogeneity, to facilitate practical handling and subsequent analytics, to enhance flexibility, and to improve cost effectiveness for routine culture. Whereas it is well established that adipogenic differentiation involves a complex, highly regulated cascade of gene expression events, the role and influence of a tissue-like 3-D environment with respect to this process is still largely unknown. The newly developed ASC spheroid model was therefore applied to characterize the process of adipogenesis in a scaffold-free 3 D culture system on a functional and molecular level, with a special focus on the comparison to conventional 2-D culture. It was found that the application of a short-term induction protocol was sufficient to induce a strong adipogenic response in the 3 D spheroids, while lipid accumulation and the expression of marker genes were only minimal under the same conditions in 2-D. Similarly, both histology and gene expression data indicated that hormonal induction without either 3-isobutyl-methylxanthine (IBMX) or indomethacin was sufficient for the development of functional adipocytes in 3 D spheroids, but not in 2-D monolayers. These findings demonstrated that adipogenesis within the 3 D environment provided by the spheroids was less dependent on exogenous stimulation than in conventional 2-D monolayers. In vivo, adipogenesis is closely associated with angiogenesis. The reciprocal regulation and the intensive cellular crosstalk between (pre-)adipocytes and endothelial cells is of great importance for adipose tissue development. Up to now, however, almost all of our knowledge regarding the interdependence of adipogenesis and vascular growth is based on studies using various 2 D coculture systems. Therefore, it was the aim of the second part of this thesis to establish and characterize a 3 D coculture model of human ASC and human microvascular endothelial cells (MVEC) based on the monoculture spheroids developed in the first part of this work. In a first step, suitable culture conditions were established. The elimination of EGF from the culture medium as well as the application of a short-term adipogenic induction protocol led to a sufficient adipocyte conversion without affecting MVEC viability and proliferation. This coculture spheroid model was subsequently not only characterized with regard to cellular self-organization, but was also applied to investigate the cross-talk between the two cell types in a 3 D environment. The endothelial cells, which at first could be found preferentially on the surface of the constructs, began to accumulate in specific regions of the spheroids after several days. The formation of network-like MVEC structures could be detected in those parts of the spheroids via immunohistochemistry and confocal microscopy. Importantly, our data demonstrated that in the coculture spheroids adipogenesis was significantly impaired as compared to ASC monocultures. This was not only shown by a reduced triglyceride accumulation, but also on the molecular level, with many adipogenic marker genes being less strongly expressed in the cocultures than in the monocultures after hormonal induction. Finally, immunostaining of cryosections revealed that lipid accumulation was completely absent only in close proximity to the MVEC structures, but not in more distant regions, which suggests a local inhibitory effect of the endothelial cells on adipogenic differentiation.