Evaluation of human induced pluripotent stem cell-based cellular models for pathological investigation of genetic skeletal diseases
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The uniqueness of stem cells endows them with the capacity to be used as a valuable tool in the field of pathology, particularly the one from patients. Patient-specific cellular model can be generated by deriving donor-specific stem cells into disease-related cell type(s). Monitoring biological variation of the differentiated cells during ontogenesis offers a great opportunity to uncover an early cellular pathology. Importantly, such platform avoids the phenotypical uncertainty facing in studying of animal models. The discovery of induced pluripotent stem cells (iPSCs) further overcomes obstacles in regard to the deficiency in cell supplying due to the difficulties in acquiring and maintaining primary stem cells. With indefinite growth capacity and functional pluripotency, iPSCs allow researchers to produce infinite cell sources of interest that harbour the same hereditary traits as their donor counterpart. The benefits of iPSCs fundamentally boost the growth of using stem cell-based model in studying different human diseases. The work displayed in this dissertation specifically focuses on applying this new strategy for the pathological investigation of human skeletal diseases (SDs). The main content firstly sketches the current state in this field. The following results chapters describe the subprojects undertaken to expand the application of iPSC-based models for more human SDs and to evaluate the accuracy of current validation methods in identifying of the cell type derived from iPSCs, especially for establishing a mesenchymal stromal cell (MSC) from iPSCs. For the background and introduction, Chapter one (1) describes the technical principles and available approaches involved for cellular model creation, from iPSC generation to skeletal lineage differentiation; (2) lists all of the generated SDs-specific iPSC lines published in PubMed over the past decade, (3) summarizes the advantages and achievements of using these models in both SD-associated pathological and pharmaceutical studies and (4) finally, outlines the challenges, unsolved problems currently faced in this field. The result chapters comprise three papers, which were either published or reviewed. The study presented in each of these papers constitutes a result chapter. In chapter three, a monogenic chondrodysplasia, with the known genetic variation, is studied, called family osteochondritis dissecans (FOCD). It causes abnormalities of skeletal development and early-onset osteoarthritis (OA) in the affected family members. Cartilaginous models are generated using iPSCs and primary MSCs obtained from multiple FOCD patients, the application of which uncovers the cellular principles of FOCD and helps to gain the understanding of the role of the genetic component in OA development. Chapter four describes the research that was conducted to elucidate the phenomena with respect to the multipotent dissimilarity between MSCs derived from iPSC (iMSCs) and bone marrow (BM-MSCs). Confusingly, iMSCs that meet the minimal criteria for defining primary MSCs commonly show functional deficits, which are associated with inadequate chondrogenesis and adipogenesis. Such conflict between lineage determination and differentiation ability bring the uncertainty of using iMSCs not only for disease modelling but also in other MSC-related fields. Carefully comparing the gene expression profile between the two cells groups suggests that the functional dissimilarity of iMSCs is due to an imprecise lineage identification. Importantly, this result indicates that the regular standards used for defining MSCs are incapable of distinguishing certain mesodermal progenitors. In chapter five, iPSC model is generated for autosomal recessive osteopetrosis (ARO), a type of osteodysplasia, which induces abnormally increased bone density. This is the first reported human ARO iPSC model, which can overcome the shortage of patients’ cell supplying for research purpose. The dissertation concludes by integrating the learning across the three publications. Meanwhile, the plan of future study corresponding to each of the subprojects is discussed.