Investigation of brown and white adipose tissue differentiation of adipose derived mesenchymal stem cells on polyethyleneglycole dimethacrylate hydrogels

This study was financially supported by Hacettepe University Scientific Research Projects Coordination Unit with a graduate project entitled "Investigation of Brown and White Adipose Tissue Differentiation of Adipose Derived Mesenchymal Stem Cells on 3 Dimensional Hydrogels" (FYL-2018-17299). The aim of the present study is to investigate the differentiation potential of white adipose tissue derived mesenchymal stem cells (AdMSCs) to brown adipose tissue on 2 dimensional (2D) and 3 dimensional (3D) in vitro culture conditions. In the first part of study, PEG polymer was functionalizated with methacrylic anhydride (MA) by using microwave energy (1100 Watt, 5 min). Therefore, polyethylene glycole dimethacrylate (PEGDM) was synthesized with 99.4% methacrylation degree. Then, PEGDM was brought into solution with photo-cross-linking agent Irgacure2959 (0.1%, w/v) and exposed to 150 s UV light to obtain hydrogel structures to be used as a tissue scaffold for 3D cell cultures. In the next stage of the thesis, AdMSCs were cultured in brown adipogenic differentiation medium consisting of roziglitazone and triiodothyronine (T3) for 35 days on 2D and 3D culture conditions. While in 2D cultures cells were culturing in multi- well culture dishes, in 3D cultures cells were encapsulated in PEGDM hydrogels. Additionally, the effect of chronic cold exposure (for 2D cultures, every other day, 16°C, 15 min; for 3D cultures, every other day, 16°C, 1h) on in vitro adipogenic differentiation of AdMSCs was also investigated. In both culture conditions, 2D and 3D, lipid vacuoles in the differentiated cells were observed by optical microscope after staining with Oil Red O. The expression levels of UCP-1 and Cidea genes which are specific for brown adypocytes and PPAR-γ gene which is expressed from all adipocyte species were revealed by real time-polymerase chain reaction (RT-PCR). In addition, the precence of UCP-1 and PPAR-γ proteins in the differentiated cells was visualized with immunocytochemical and immunohistochemical analyzes. However, cold induction on in vitro cell culture conditions did not have a significant effect on the differentiation of AdMSCs to brown adipocytes. As a result, AdMSCs were differentiated to brown adipocytes on both culture conditions, 2D and 3D. Moreover, 3D cell cultures which obtained by encapsulation of AdMSCs into PEGDM hydrogels are proposed as a suitable system for obtaining brown adipocytes in vitro.