Molecular Mechanisms of Cell Adhesion and Cytoskeletal Dynamics Underlying Human Mesenchymal Stem Cell Differentiation

Stem cell differentiation during tissue development and regeneration is a complex multistep process involving temporally and spatially orchestrated regulation. Mesenchymal stem cells (MSCs) are multipotent progenitor cells of mesodermal origin that differentiate into adipocytes and osteoblasts. The differentiation capacity of these adult stem cells marks them as intriguing candidates for soft and bone tissue regeneration and engineering. However, the molecular factors and mechanisms involved in the regulation of MSC differentiation have been studied using primarily animal models and human MSC differentiation remains understudied.

In this thesis, human MSCs harvested from adipose tissue (human adipose stem cell; hASC) or bone marrow (human bone marrow stem cell; hBMSC) were studied in vitro. Adipogenic or osteogenic differentiation of hMSCs was induced with biochemical agents in culture medium. Additionally, the cells were cultured under ions extracted from bioactive glass (BaG) to stimulate osteogenic differentiation. Central and evolutionarily conserved protein kinase pathways related to cell adhesion and cytoskeletal dynamics were studied in the context of directing hMSC differentiation potential. Focal adhesion kinase (FAK) is a key regulator of cell adhesion that mediates signaling through various targets, including mitogen- activated protein kinase (MAPK) pathways. Actin cytoskeleton dynamics are modulated by the Rho-associated coiled-coil kinase (ROCK) pathway. The ROCK downstream target, myocardin-related transcription factor A (MRTF-A) serves as an intriguing link between cytoskeletal dynamics and gene expression. The roles of these intracellular signaling pathways in hMSC differentiation were studied using small- molecule inhibitors of signal transduction.

The results demonstrate that cell adhesion mediated by FAK signaling acts as a molecular switch between adipogenic and osteogenic differentiation of hASCs. FAK pathway activity favored the osteogenic outcome of hASCs, but inhibition of cell adhesion by FAK inhibition resulted in enhanced adipogenesis. The activity of the FAK downstream target MAPK extracellular signal-regulated kinase (ERK) was found to be significant for both the adipogenic and osteogenic differentiation of hASCs. These results indicate that FAK and ERK pathways have distinct roles in the regulation of lineage commitment.

Based on the results, the ROCK pathway and MRTF-A are relevant regulators of the hASC lineage commitment through cytoskeletal modulation. We demonstrated that actin polymerization and contractility induced by ROCK activity are required for osteogenesis. ROCK inhibition enhances the number and size of intracellular lipid droplets, thus suggesting adipogenic maturation. Active MRTF-A signaling is important for the osteogenic course of hASCs. Inhibition of nuclear translocation of MRTF-A resulted in changes in actin cytoskeleton and enhanced adipogenic fate.

The results revealed that ions dissolved from the experimental boron-containing BaG are strong inducers of osteogenesis in hASCs and hBMSCs. A novel role in the context of hMSC osteogenesis was discovered for the MAPK family member p38/heat shock protein 27 (HSP27) signaling. Activation of the p38 MAPK/HSP27 pathway occurred early and temporally during BaG-induced osteogenesis, and inhibition of HSP27 phosphorylation decreased osteogenic differentiation. The results suggest that the cytoskeletal association of phosphorylated HSP27 could function as an early regulator in the osteogenic commitment of hMSCs.

The study results reported in this thesis increase the biological knowledge and understanding of hMSC differentiation and provide a background for knowledge- based solutions in tissue engineering and for the treatment of bone and adipose tissue-related disorders.