Mesenchymal stem cells are the stem cells that replace the bone, fat and cartilage
tissues of the human body. In addition, these cells can form muscles, ligaments and
neurons. This wide multipotency has made mesenchymal stem cells of particular
interest in the fields of tissue engineering and regenerative medicine. Furthermore,
mesenchymal stem cells can modulate the immune system by reducing factors that
increase inflammation and immune recognition. This immune recognition
suppression has resulted in their application as part of bone marrow transplantation
in the prevention of 'graft versus host‘ disease. There are hundreds of on-going
clinical trials using these cells for the treatment of autoimmune diseases such as type
I diabetes, arthritis and multiple sclerosis. The increasing importance of these cells
has brought in to focus the culture methods used to for their expansion and
manipulation. Currently, animal derived components are used as surfaces for their
growth and as components in the culture media. This exposes these cells to animal
pathogens and antigens that can be passed to the recipients of these cells.
In the first part of this thesis, polymer microarrays were employed to identify
alternatives to the biological surfaces currently used for mesenchymal stem cell
culture. This platform allowed hundreds of polyacrylates/acrylamides and
polyurethanes to be simultaneously scrutinised to identify surfaces that could support
their growth and maintain their stem cell characteristics. Identified polymer surfaces
were monitored in long-term culture (10 passages) and were shown to retain the cell
phenotype and capacity to differentiate, thus providing chemically defined substrates
for long-term mesenchymal stem cell culture.
In the second part of this thesis, a 'smart‘ polymer microarray of hydrophilic cross-linked
polymers (hydrogels) were used to remove another key biological component
of culture, trypsin. These 'smart‘ hydrogels modulated their properties depending on
the temperature. Hydrogels that could trigger mesenchymal stem cell release after a
reduction in temperature were identified. A unique passaging system using a modest
temperature reduction for 1h was developed as a passaging method. Cells were
maintained and monitored for 10 passages using this novel enzyme free passaging
method. Analysis of the mesenchymal stem cell phenotype and differentiation
capacity revealed this method superior than conventional culturing methods.
In the final part of this thesis, a 'knowledge-based‘ small molecule library was
designed, which could potentially yield small molecules to manipulate/enhance the
mesenchymal stem cell state without the use of biological components. The key
protein pathways that control the stem cell state were examine with the
bioinformatics tool GeneGo was used to identify compounds that affected these
pathways, resulting in selection of 200 small molecules. The effect of the small
molecules on the mesenchymal phenotype was examined and 5 small molecules were
identified that enhanced the phenotype of these cells. The anti-inflammatory
properties associated with the hit compounds led to the investigation of their effects
on key surface proteins associated with the immune-modulatory state of the cells. In
this preliminary study, two of the small molecules, estriol and spermine, increased
the expression of a key mesenchymal stem cell marker STRO-1 and down regulated
ICAM-1, a critical component of the immune modulation capacity of this cell type