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Date of Award
Doctor of Philosophy (PhD)
Articular cartilage allows for near frictionless joint movement; however, when damaged the tissue has very little capacity for self-repair. Tissue engineering can be used to both repair damaged articular cartilage and as an in vitro model for joint disease. Commonly used cell sources for tissue engineering are adipose-derived and bone marrow-derived mesenchymal stem cells (ASCs and MSCs) because they can be patient matched, expand rapidly in culture, and have multipotent differentiation potential. However, donor-to-donor variability of differentiation potential can mask the results of in vitro experiments and ASCs and MSCs only retain their multipotency for a limited number of passages. To overcome the issues of limited culture lifespan and donor-to-donor variability we created an immortalized ASC line for optimized cartilage tissue engineered to be used as a model system for in vitro experiments. This ASC line was previously immortalized with the overexpression of human telomerase. However, it had limited chondrogenic potential as measured by production of cartilage extracellular matrix proteins. To increase its chondrogenic potential, we overexpressed the master transcription factor for cartilage development, SOX9. This overexpression greatly increased the production of cartilage extracellular matrix protein type II collagen and glycosaminoglycans during chondrogenic differentiation. We further characterized the response of these cells to mechanical and inflammatory stimuli such that they could be used for in vitro experiments involving cartilage development, homeostasis and inflammatory disease. The response of these cells to both mechanical and inflammatory stimuli was found to be similar to that of native chondrocytes, displaying the usability of this cell line as a model for cartilage tissue engineering. In addition to creating a model cell line, we also wanted to understand how MSCs change with passage and why that limits their differentiation at later passages. For this purpose, we used single cell RNA sequencing to investigate the transcriptome of MSCs from three donors at a single cell resolution over the course of eight passages. As these MSCs were not sorted at harvest, we expected there to be a heterogenous population of cells at the earlier passages, which was not found. Instead, the MSCs were fairly homogenous in their gene expression at passage 1, and their transcriptome subtly changed with each passage, with one of the three donors having a more substantial shift in gene expression around passage 6. From this data, we identified potential regulators of late passage phenotype including loss of IGFBP2 expression, decrease in JUN activity, and increase in CREB3L1 activity. The data collected in this project can be used along with other available datasets to better understand donor-to-donor variability, the largest source of unpredictability between MSC populations. For the purposes of understanding inflammatory joint disease and the stability of tissue engineered cartilage for clinical repair, we were interested in the regulation of the protein lubricin, and its gene PRG4. In healthy cartilage, the protein lubricin is produced by the superficial zone chondrocytes and plays a role in both the mechanics of joint motion as well as having an anti-inflammatory effect. However, the pathway of regulation of lubricin and its gene PRG4 in response to inflammatory stimuli had not previously been elucidated. By using MSC-derived tissue engineered cartilage as a model, we determined that when challenged with IL-1β, PRG4 expression increases, but lubricin content is unchanged. The increase in PRG4 expression was shown to be NFAT-mediated, a regulation pathway independent of the mechanical regulation of PRG4. Additionally, we found that PRG4 expression could be upregulated by using CRISPR activation. This increase did not prohibit IL-1β challenge from further upregulating the gene expression. These intriguing responses help us better understand progression of inflammatory joint disease as well as informing us as to the potential therapeutic effect, or lack thereof, of lubricin in tissue engineered cartilage when implanted into an inflammatory environment. Overall, this set of work increases our understanding of adult stem cells in expansion and for tissue engineering purposes. Furthermore, by creating cell lines and a knowledge base, this work helps build the foundation for future studies to investigate the progression of joint disease and develop therapies involving adult stem cells.
Spencer Lake, M. Farooq Rai, Jai Rudra, Simon Tang,
Available for download on Saturday, August 20, 2022