Differentiation of induced pluripotent stem cells (iPSCs) and mesenchymal stromal cells (MSCs) remains a major challenge in regenerative medicine, while there is evidence, that this process can be influenced by mechanical cues. In this thesis, we investigated if differentiation of primary MSCs and generation of iPSC-derived MSCs (iMSCs) is supported by soft hydrogels. To this end, we compared in vitro culture on tissue culture plastic (TCP), on soft hydrogels, and three-dimensional (3D) culture within soft hydrogels. We demonstrate that human platelet lysate (hPL)-gel, composed of a fibrin network, supports isolation and culture expansion of primary MSCs with pronounced deposition of extracellular matrix proteins. However, hPL-gel hardly impacts on the in vitro differentiation potential or gene expression profiles of these cells. Furthermore, iPSCs can be effectively differentiated towards MSCs on hPL-gel. Unexpectedly, this complex differentiation process is not affected by the soft substrate: iMSCs generated on TCP or hPL-gel have the same morphology, immunophenotype, differentiation potential, and gene expression profiles. Moreover, global DNA methylation patterns of iMSCs generated on TCP or hPL-gel indicate that they are epigenetically alike. In contrast, 3D culture inside soft hydrogels strongly affects primary MSCs and differentiation of iPSCs towards MSCs. We demonstrate that culture inside hPL-gel enhances osteogenic and adipogenic differentiation, and changes gene expression profiles of primary MSCs. Moreover, 3D culture in fibrin gel hampers iPSC differentiation towards MSCs: in contrast to iMSCs generated in 2D culture on plastic and on fibrin gel, iPSC differentiation in fibrin gel gives rise to smaller cells with low expression of CD73 and CD105, and faint secretion of fibronectin. Furthermore, global gene expression and DNA methylation patterns of these cells were clearly distinct. Taken together, matrix elasticity alone does not influence differentiation of primary MSCs and iMSCs. On the other hand, 3D culture in an elastic matrix triggers transcriptomic changes in primary MSCs and hampers iPSC differentiation towards MSCs, indicating that the flat and stretched appearance on flat substrates supports the differentiation into this cell type
