| Adipose-derived stem cells have recently emerged as a promising, abundant cell source for tissue replacement therapies for a wide range of applications, particularly in the area of musculoskeletal tissue engineering. In spite of recent research advancements towards developing stem cell therapies, there have been few successfully translated adult stem technologies to date. The goal of this research is to elucidate fundamental processes of human adipose-derived stem cell (hASC) differentiation, from the sub-cellular level, up to global changes in age and donor specific cell populations, with a particular focus on osteogenic differentiation. Though hASC are well established to differentiate into osteogenic cells types, exhibiting calcium accretion, an upregulation of osteogenic gene markers and an increase in alkaline phosphatase activity, the underlying mechanisms of these processes remain elusive. We hypothesized that an organelle known as the primary cilium plays an integral role in mediating hASC lineage specification. Primary cilia are composed of nine microtubule doublets arranged concentrically forming the axoneme of the cilium structure, however over 1000 proteins are estimated to be associated with the cilium. We have identified two particular cilia-associated proteins to play a role in hASC osteogenesis: polycystin-1 (PC1) and intraflagellar transport-88 (IFT88). Using siRNA knockdown techniques, we established that IFT88 expression was required to upregulate Runx2, an early gene marker of osteogenesis. Further, we established that knockdown of PC1 conferred reduced expression of later gene markers as well as a marked decrease in calcium accretion and endogenous alkaline phosphatase activity, indicators of osteogenic differentiation.;Following the study on chemically induced hASC differentiation, we generalized our hypothesis to explore the broader role of the primary cilium in hASC lineage specification. Previous reports from our group have identified 10% cyclic tensile strain to enhance hASC osteogenesis and we hypothesize the primary cilium functions in part as a mechanosensor in this process. To understand the cilium structure as a mechanosesnsor, we first wanted to analyze its morphology on the hASC population as well as on the osteogenically and adipogenically differentiated hASC populations. We found that in as few as three days of chemical stimulation, differentiating hASCs exhibited differential cilia expression. Cilia generally tended to be more prevalent in more committed cell types, however their expression was somewhat temporal during the differentiation process. Gene expression analysis of cilia-associated genes also suggested that cyclic tensile strain affects ciliary gene expression in addition to the frequency of expression of the cilia structure. In our efforts to uncover the fundamental mechanisms of hASC differentiation, we observed a persistent experimental challenge throughout a majority our studies: donor-to-donor variation. Behavior predictability is critical to the success of applying hASC technologies in the clinic, and without consistent cell behavior and predictable physiological profiles, hASC will not emerge as a reliable treatment method for tissue replacement therapies. The final study of this dissertation examines the degree of donor-to-donor variability within our lab's cell bank. Based on the observations in our cell lines, we hope that our findings may be extrapolated into the larger pool of information on hASCs. We highlight age-related changes in osteogenic and adipogenic differentiation and draw attention to important considerations for designing bench top experiments as well as creating clinically relevant technologies. Taken together, this dissertation addresses fundamental areas of research in adipose stem cell differentiation towards improving knowledge of their in vitro behavior for bench top studies, as well as furthering our knowledge of patient-specific information to facilitate their future clinical application. |
