Multilineage Differentiation of Mesenchymal Stem Cells for Cell-based Tissue Engineering

Adult mesenchymal stem cells (MSCs) can be differentiated towards tissue-forming cells and are used to recreate tissues in vitro. Yet, considerable challenges remain before the clinical goals of stem cell-based tissue engineering (TE) constructs can be achieved. One challenge is the design of scaffold materials that are able to keep stem cells in their undifferentiated functional phenotype and promote differentiation only after induction. We evaluated the utility of poly(ethylene glycol) diacrylate (PEGDA) superporous hydrogel (SPH) scaffolds for stem cell delivery in TE applications. Generally, flat PEGDA surfaces do not promote protein adsorption and are thought to be devoid of cell-matrix interactions. We observed a different scenario in the SPHs and showed for the first time that unmodified PEGDA SPHs can provide a microenvironment that enables stem cell-derived extracellular matrix (ECM) development and thus promotes stem cell survival. We further showed evidence of multilineage differentiation of hMSCs towards adipogenic, chondrogenic, endothelial and osteogenic lineages within PEGDA SPHs. No autodifferentiation was observed within uninduced control groups. We conclude that architecture and the physicochemical characteristics— interconnected pores ranging from 100 to 600 µm— of the scaffold hold properties that impact stem cell behavior. An appealing strategy to overcome insufficient oxygen and nutrient supply within the core of the implants is to create microvascular structures within the engineered scaffold itself. We showed that hMSCs gain some phenotypic and functional features of vessel-forming endothelial cells on matrigel and within collagen-filled SPHs. Another challenge in stem cell-based TE is the identification of reliable stem cell differentiation markers. Our observations within the SPHs indicated a discrepancy between mineralization and gene expression data. Thus, we evaluated putative adipogenic and osteogenic marker genes for their specificity towards the respective lineage. Adipogenic gene markers were significantly upregulated in hMSCs that underwent the adipogenic induction program but all osteogenic marker genes tested were also expressed in adipocyte-derived hMSCs. With alkaline phosphatase and osteopontin in particular being upregulated during adipogenic differentiation. In this dissertation, we identified a suitable scaffold for stem cell delivery, showed the potential of hMSCs in vascularization approaches, and demonstrated the need for more specific differentiation markers.