Stem cells and mineralized tissue : characterization and disease modeling

Abstract

Deficits in mineralized tissue, as a result of abnormal development, trauma, disease or aging, are major medical problems. Consequently, new methods to regenerate stable and functional bone is a main focus in the field of tissue engineering. Cell therapy using stem cells that adopt a mineralizing phenotype holds great potential for regeneration of calcified tissues such as bone and teeth. Stem cells provide an unlimited number of cells that can be used for bone differentiation. The mechanisms governing stem cell bone formation are however complex, and involve many factors. In this context, knowledge of basic bone biology, handling of stem cells in in vitro culture systems and the complex molecular mechanisms associated with normal and impaired bone development is fundamental. The aim of this project is to enhance the understanding of stem cell differentiation into the osteoblastic/mineralizing lineage. In the first study we investigated the roles of some of the constituents of the extracellular matrix (ECM) of dentin in mouse teeth. Using structural techniques, we found that osteoadherin (OSAD), a member of the family of mineralization-related small leucine-rich proteoglycan (SLRP) proteins, was localized at the mineralization front, closely associated with collagen fibers. This, together with data obtained from a functional assay, emphasizes the importance of OSAD in bone matrix maturation and mineralization. Continuing with in vitro model systems for subsequent application in the field of stem cells/regenerative medicine of bone and dentin, we highlighted important aspects of stem cells handling in the laboratory. We found that there were neither epigenetic alternations in selected histone modifications or marks nor any changes in protein expression when different passaging techniques were used. However, gene expression was significantly decreased for pluripotent markers using enzymatic split with a ROCK inhibitor, an effect that could be reversed upon mechanical passaging. These findings underline the fact that passaging techniques have to be taken into account when comparisons are made between cells that have undergone various treatments under different experimental conditions. In the last study we have derived induced Pluripotent Stem Cells (iPSCs) from a family with a mutation in the PIGT gene, which in addition to severe CNS defects causes a number of craniofacial bone and tooth abnormalities. iPSC-based models have emerged as useful systems to model human disease, both to unravel disease mechanisms and to provide test assays for drugs. We determined transcriptional and epigenetic changes in the patient-derived cells as compared to healthy control cells, with a focus on gene networks associated with bone development. We found four important genes to be downregulated compared to health unrelated iPSC controls, OPN, MMP2, ACVR1, and MMP2 which all have shown to have important roles in in bone and skeletal development. Furthermore, they showed patterns of epigenetic regulation, highlighting the importance of histone modifications and DNA methylation in the disease. Regenerative therapy with autologous cells using the patients’ own reprogrammed cells to replace damaged tissue may reduce patient suffering and healthcare costs. This thesis presents findings that might be of future use in the development of such cell-based bone replacement strategies