Generation of cartilage from induced pluripotent stem cells: gene pathways and small molecules

Induced pluripotent stem cells (iPSCs) are a promising cell source for regenerative medicine including the treatment of articular cartilage defects and osteoarthritis (OA). There is currently an unmet clinical need for cell-based therapies and pharmacological treatments for OA and cartilage repair. Somatic cells are reprogrammed to iPSCs by the delivery of reprogramming transcription factors. Genome integrating transgenes increase the risk of tumorigenicity, therefore integration-free reprogramming approaches are preferred. iPSCs are commonly used for modelling specific diseases, and can be utilised in screening platforms for disease modifying drugs. The potential of iPSCs for clinical therapies is being explored, and their capacity for cartilage repair has been demonstrated in animal models in vivo. The primary focus is to generate iPSC derived articular cartilage for clinical applications. Numerous approaches to derive cartilage from iPSCs have been described with different levels of success. These include directed differentiation methods that recapitulate developmental signalling cues in vitro, indirect approaches such as embryoid body (EB) outgrowth and one-step media changes, or coculturing with articular chondrocytes (ACs). In general, iPSCs are induced to chondroprogenitor (iCP) or mesenchymal stromal cell (iMSC) intermediates that can generate cartilage tissue. A number of challenges associated with iPSC differentiation include cell heterogeneity, loss of chondrogenic potential during passaging, and the presence of hypertrophic and fibrous cartilage tissue. Furthermore iPSCs have variable induction efficiencies and differentiation propensities. iPSCs reprogrammed by episomal vectors were characterised and found to have unstable karyotypes by chromosomal microarray analysis. The differentiation efficiency of iPSCs toward chondrocytic and mesenchymal cells was assessed, following directed differentiation through a paraxial mesoderm or neural crest lineage. Two iPSC lines were found to have good induction efficiencies and chondrogenic propensities. Transcriptomic datasets were explored with Qiagen’s Ingenuity Pathway Analysis (IPA) causal networks analysis tool, to predict potential regulators of chondrogenesis. Molecules of interest were investigated by gene expression analysis in two BM-MSC lines with strong and weak chondrogenic propensities, during the first three days of chondrogenesis. USP18, SEMA4C, RCAN1, TEAD4, and FIBIN may have a role in early chondrogenic differentiation and require further investigation. A small molecule screening assay was developed using an automated liquid handling system, to screen for molecules enhancing the production of glycosaminoglycans (GAGs) in iPSC derived cells and BM-MSCs. Forskolin, sesamin, and baicalin enhanced GAG synthesis in iPSC derived cells, while no small molecules enhanced GAG synthesis in BM-MSCs any further than TGFb3 alone.

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University of Galway

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Attribution-NonCommercial-NoDerivatives 4.0 International

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University of Galway Theses (PhD Theses)