Al Hosni, Rawiya Hosni Ali; (2020) Defining Biochemical and Biophysical Cues that Direct Stem Cell Behaviour. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The biochemical and biophysical cues that result in stem cell regulation in vivo are currently poorly understood. Although it is known that these cells reside within specialized environments known as niches, data on the contribution of cell-matrix, cellcell and cell-soluble mediators in maintaining quiescence, identity and activation is lacking. As such this PhD aims to develop technology that can be used to identify and optimise both biochemical and mechanical factors in relation to stem cell characteristics. Using RNA Sequencing and bioinformatic analysis, soluble mediators of human periosteal cell proliferation and potency were identified. This was achieved through the creation of transcriptional networks that reflected cell state in humanised culture conditions. The identification of upstream regulators and empirical analysis of their effect allowed the creation of a culture system based on 6 soluble factors that facilitated cell expansion whilst improving potency. Interestingly, these factors also promoted cell localization in a manner that replicated the organisation observed in native periosteum. To understand how matrix and mechanical cues can define cell identity and potency, hydrogels in combination with immortalized adipose stromal cells were used as a tuneable system to alter substrate stiffness. As matrix density contributes to oxygen diffusion, these hydrogels were additionally cultured in two different oxygen tensions. Culturing cells in a 3D environment composed of a soft hydrogel in physiological normoxia maintained long term stem cell gene expression. Interestingly, the presence of a dynamic culture with multiple matrix stiffness allowed for the identification of environments that defined stem cell identity. Periosteum-derived cells and adipose derived stromal cells were used within this thesis for their relevance in skeletal tissue engineering. However, it is envisaged that the technology described herein could be used to define biochemical and biophysical cues associated with stem cell potency and identity for any tissue resident stem cell population.

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