The Effect of HDAC Inhibitor MI192 on Stem Cell Behaviour- The Potential of Utilising MI192 for Bone Tissue Engineering

Controlling lineage specific differentiation of stem cells is crucial for functional tissue engineering, and current methods have drawbacks and limitations. Histone deacetlyase (HDAC) proteins are key to cellular epigenetics, and a number of studies have shown that their inhibitors (HDACis) can control stem cell fate. The aim of this study was to investigate the effect of a novel HDAC2 and HDAC3 selective inhibitor, MI192, on human adipose derived stem cells (ADSCs), exploring the potential of utilising MI192 in controlling the osteogenic differentiation of ADSCs, with the long term goal of improving the efficacy of bone tissue engineering. MI192 was synthesised in house and characterised with NMR analysis. The effect of MI192 was explored on commercially (Life Technologies) available ADSCs. It was found that 1 µM to 100 µM MI192 was cytotoxic and reduced ADSC viability, with a dose response observed. Using propidium iodide staining, it was demonstrated that MI192 halted the cell cycle in the G2M phase. FACs analysis showed that MI192 altered the stem cell marker expression profile of ADSCs; increasing expression of some markers (CD34 and CD146) and decreasing some (CD29, CD44, CD73, CD105, CD166). When utilised in a pre-treatment strategy in 2D models, MI192 improved the osteogenic differentiation of ADSCs, strongly increasing production of alkaline phosphatase protein, with 2 days of 30 µM pre-treatment being the optimal concentration and treatment length. MI192 pre-treatment also increased ADSC mineralisation (calcium accumulation stained with Alizarin Red and mineral nodule formation stained with Von Kossa) and increased osteogenic gene expression (BMP2, RUNX2, COL1 and ALP). The adipogenic differentiation of the ADSCs was inhibited by MI192 pre-treatment, with reduced lipid droplet accumulation (stained with Oil Red O) and adipogenic gene (PPARγ and ADIPOQ) expression seen. Increased osteogenic differentiation was further demonstrated in 3D models, using Am silk scaffolds. Cell attachment was assessed with live cell labelling, and collagen production, mineralisation and protein production analysed with histology and immunohistochemistry. Finally, TaqMan® Gene Expression Array Cards were utilised to investigate how two days of MI192 treatment altered the expression of 96 different genes. Some key Wnt related genes, as well as other key osteogenic genes such as BMP2 were up-regulated, providing some mechanistic explanation for the increase in osteogenic differentiation potential seen with MI192 pre-treatment. This thesis demonstrates the potential of utilising MI192 to improve bone tissue engineering strategies