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Title: Factors that influence the chondrogenic differentiation of human embryonic stem cells
Author: Ridgway, Kimberley Elizabeth
ISNI:       0000 0004 2725 5775
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2011
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Native cartilage does not have the ability to heal effectively; therefore, there is a wide prevalence of articular cartilage disease and injury. Human embryonic stem cells provide unique opportunities for the study of differentiation events in vitro and for use in regenerative medicine therapies. Tissue engineering using these cells represents a promising treatment for cartilage lesions. Previous attempts were qualitative, highly variable and limited mainly to HI and H9 human embryonic stem cell lines. The current study aimed to examine the factors that influence the differentiation of the human embryonic stem cell line KCL002 towards the mesenchymal stem cell lineage and to utilise these cells in a cartilage tissue engineering modality. The studies investigated improving this process by choice of tissue engineering scaffold, coating protein and growth factor combination. KCL002 suggested expression of human embryonic stem cell-specific proteins and genes, including Oct4 and Nanog and by quantitative PCR was shown to contribute to trophectoderm, mesoderm and endoderm. KCL002 differentiation was directed towards the mesenchymal stem cell lineage using multiple protocols. A protocol based on harvesting residual auto differentiated cells led to deriving cells with characteristics of adult tissue-derived mesenchymal stem cells including fibroblastic morphology, plastic adherence, cell surface marker expression, and trilineage multipotential upon adipogenic, chondrogenic and osteogenic stimulation. These human embryonic stem cell-derived mesenchymal stem cells were then applied in a tissue engineering modality. Unique quantitative assays, for both protein and RNA, were used to assess the quality of matrix production by the derived cells to determine the best chondrogenic stimulation conditions. The derived cells seeded onto 3-D fibronectin-coated hyaluronic acid based Hyaff-l l scaffolds in the presence of TGF-β1 and BMP-7 gave the highest matrix production and quality of the conditions tested. In addition, members of the nuclear receptor superfamily were analysed for their stage-specific expression during KCL002 differentiation. Differentially expressed nuclear receptors were targeted with synthetic ligands to chemically control the differentiation of KCL002. A combination ofLE135, a RAR-β and RAR-α antagonist and GW647I, a PPAR-α antagonist, was found to enrich for the mesodermal phenotype in embryoid bodies although results failed to produce notable statistical significance. This small molecule analysis was also investigated for its use in aiding the tissue engineering protocol. In conclusion, this work demonstrated for the first time that it is possible to engineer cartilage from the KCL002 human embryonic stem cell line and this was achieved by improving growth factor combination as well as choice of scaffold and coating protein from the adult tissue-derived mesenchymal stem cell tissue engineering protocol. This is an important lesson for human embryonic stem cell research, not to just use the method to hand in the laboratory but to explore tissue engineering conditions beyond differentiation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available