Effects on Chondrocyte Derived Induced Pluripotent Stem Cells Adhered to Nano Gradients Functionalized with Transforming Growth Factor Beta-1, -3 and Growth Differentiation Factor 5

Examensarbete för masterexamen
Master Thesis
Biotechnology (MPBIO), MSc
Andreasson, Linnea
Osteoarthritis (OA) is the most common joint disease, causing pain for the patient due to cartilage degeneration and a lot of costs to the society. Due to lack of knowledge regarding disease mechanisms, there is no existing drug based modifying therapy. In this project, the impact of TGFβ-1, TGFβ-3 and GDF5 signaling on the expression of aggrecan in chondrocytederived iPSCs, were elucidated by using a concentration gradient to gain information regarding OA disease mechanisms. The overall aim of the project was to use gold nanoparticle gradients and surfaces to optimize formation of cartilage producing cells from iPSCs where the focus was to induce a high aggrecan expression in the cells. Also, cells were stimulated on gradients and surfaces and then removed from the surfaces and formed to a 3D structure for further differentiation and visualization of the components. Particle gradients were produced by attaching gold nanoparticles to a glass surface with different spacing between particles and the gradients were then functionalized with signaling molecules and seeded with c-iPS cells, differentiated for 5 days and thereafter visualized using fluorescence imaging. Due to the protein concentration gradients, cells were stimulated differently over the gradient and by visualization of the cell behavior at the gradient, an area of interest was observed and new uniform surfaces were produced. No specific aggrecan expression was observed on surfaces differentiated for 5 days, especially not for GDF5 and TGFβ-1. TGFβ-3 indicated an elevated expression at high concentrations. Despite this result, 5 days of differentiation was found to be insufficient time and this result was also confirmed by the study were iPSCs were further differentiated for 6 weeks after removal from surfaces where there was a significant elevation of the aggrecan expression after 3 weeks of differentiation and it is suggested that the time should be prolonged in order to yield high aggrecan expressions. Even though the aim of the project to stimulate the cells to express a high expression of aggrecan was not fully fulfilled, other reactions of interest were observed on the surfaces. GDF5 stimulate cells tended to form a condensed structure where cells clustered together. In low concentrations of TGFβ-1, the effect in cells was indicated to be knocked out by laminin and higher concentrations were indicated to be sufficient for a cellular response of TGFβ-1. For cells on TGFβ-3 surfaces, aggrecan expression was indicated to be elevated in high TGFβ-3 concentrations. It can be concluded that the nanoparticle coated surfaces are effective substrates to use to study the effect of signaling molecules on cells and to optimize the differentiation process in stem cells.
Fysik , Physical Sciences
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