Doctoral Degrees (General Internal Medicine)

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Browsing Doctoral Degrees (General Internal Medicine) by browse.metadata.advisor "Ferris, William F."

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    The association between tissue non-specific alkaline phosphatase expression and differentiation of mesenchymal stromal cells
    (Stellenbosch : Stellenbosch University, 2017-03) Bartlett, Cara-Lesley; Ferris, William F.; Crowther, Nigel J.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. General Internal Medicine.
    ENGLISH SUMMARY: Diseases resulting from the dysregulation of adipocyte and osteoblast differentiation include diabetes type II and osteoporosis. Both adipocytes and osteoblasts are derived from the same progenitor cell type, known as mesenchymal stromal cells (MSCs), which may also differentiate into cells of several mesenchymal lineages. A more detailed understanding of the mechanisms involved in the differentiation of MSCs would provide valuable insight into the underlying causes of, as well as facilitate the development of improved treatments for, diseases related to MSC differentiation dysregulation. Tissue-nonspecific alkaline phosphatase (TNAP) is highly expressed in several tissues including bone tissue, where it has a well-established role in skeletal mineralisation. In recent years TNAP expression has been reported in adipocytes, and has been identified as identical to the stem cell marker, mesenchymal stem cell antigen-1 (MSCA-1). The above findings indicate that TNAP has diverse roles, and may be one of the factors involved in determining the differentiation pathway of MSCs. Previous studies have found that inhibition of TNAP in the mouse preadipocyte cell line, 3T3-L1 resulted in a decrease in lipid accumulation during in vitro adipogenic differentiation, suggesting that TNAP is involved in adipogenesis. In the present study, rat-derived primary MSCs were isolated from bone marrow (bmMSCs) as well as subcutaneous (scADSCs) and peri-renal visceral adipose (pvADSCs) depots, and differentiated in vitro towards either an adipocytic or osteoblastic phenotype. The expression of TNAP was assessed in rat-derived MSCs undergoing both adipogenic and osteogenic differentiation. TNAP expression levels were highest in bmMSCs, followed by scADSCs and pvADSCs, with higher alkaline phosphatase (ALP) activity observed during adipogenesis compared to osteogenesis in all three MSC types. The addition of the reported TNAP inhibitor, levamisole during osteogenesis prevented mineralisation in all MSC types, but had no significant effect on lipid accumulation during adipogenesis. Other reported inhibitors were also examined; Histidine was not successful in reducing lipid accumulation or mineralisation, while Lhomoarginine was able to significantly reduce lipid accumulation in all MSC types. The inhibitor results were not conclusive due to possible off target effects within the cells. Attempts to inhibit adipogenic differentiation by knockdown of TNAP expression in scADSCs using shRNA were not successful, as indicated by the presence of lipid droplets in cells where TNAP-specific shRNA was present. This study also revealed that ALP activity was localised to the membrane of intracellular lipid droplets characteristic of adipocytes, and that the same TNAP mRNA transcript type which is preferentially expressed in bone tissue is also preferably expressed during adipogenic differentiation of bmMSCs and scADSCs, while expression in pvADSCs was below detectable levels. TNAP isoforms differ from one another due to differences in posttranslational glycosylation pattern. Glycosylation differences were observed between bmMSCs differentiated from a naïve state towards an adipogenic, compared to an osteogenic, phenotype. Differences were also observed between scADSCs and bmMSCs when differentiated towards adipocytes. This may indicate that a distinct isoform of TNAP exists in adipocytes. In conclusion, this study confirms earlier findings on the presence of TNAP in adipocytes. Differences in TNAP expression from MSCs isolated from different tissue depots were also discovered. This study provides a characterisation of the role of TNAP in adipogenic differentiation; however, the exact mechanisms remain to be elucidated.