Graduation Date

5-2013

Document Type

Master's Thesis

Degree Name

Master of Science

Department or Program

Biological Sciences

Department or Program Chair

Kiowa Bower, Ph.D.

First Reader

Michael Vellard, Ph.D.

Second Reader

Warren Hoeffler, Ph.D.

Abstract

Mucopolysaccharidosis IVA (MPS IVA; Morquio A), is a lysosomal storage disorder characterized by the deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), resulting in the accumulation of glycosaminoglycans (GAGs) such as keratan sulfate (KS) (Northover et al., 1996) and chondroitin-6-sulfate. There is no applicable animal model or appropriate cell lines currently available for the study of MPS IVA. Furthermore, patient-specific material is limited and difficult to obtain, rendering the study of MPSIVA pathogenesis challenging. Thus, the generation of an induced pluripotent stem cell (iPSC) line from patient-specific somatic cells shows potential for further examining the pathogenesis of MPS IVA. With this technology, differentiation protocols could be used to acquire desired cell types. In this study, an iPSC model was established for MPSIVA as well as an unaffected phenotype expressing pluripotency markers, rendering it capable for differentiations. A direct and a progressive approach were taken for the differentiation of iPSCs into a chondrogenic lineage. The direct differentiation of iPSCs into chondrocytes was unsuccessful due to inconsistent results. An alternate indirect route through the establishment of an intermediate mesenchymal stem cell (MSC) lineage before chondrogenesis was then investigated and reached. With the MSCs, it was shown that the severe disease clone not only has a decreased cell growth capacity, but also has larger lysosomes and altered cytoskeleton structure, suggesting a clear phenotype that could be altered upon treatment with rhGALNS. Furthermore, the MSCs can be loaded with KS to recapitulate accumulation within the cell and levels are corrected to the unaffected phenotype with rhGALNS. Thus a bioassay for measuring the intracellular activity of rhGALNS is introduced. Several attempts were made to further differentiate the MSCs into chondrocytes, a difficult endeavor with varying results. The established iPSC and MSC lines are a promising tool for the further development and understanding of MPSIVA as well of a functional bioassay for rhGALNS using the natural substrate, lumican, of the enzyme.

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