Abstract
Wnt proteins are powerful signalling molecules, involved in stem cell biology and tissue patterning in a wide range of organs and tissues. Their hydrophobic nature prevents their efficient diffusion, frequently resulting in highly localised and directed presentation to cells. Studies in simple model organisms have demonstrated a role for Wnts in polarising cells and inducing asymmetric cell divisions. The study of similar processes in mammalian systems is hampered by the difficulties involved in visualising Wnt proteins. Here, I use technology that covalently binds Wnt3a onto visible structures to observe and control their positioning relative to target cells. Previously, a novel 3D osteogenic culture of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) upon a Wnt3a-surface results in the formation of a patterned tissue in vitro, named the Wnt3a-induced Osteogenic Tissue Model (WIOTM). The tissue supports spatially separated populations of both self-renewed BM-hMSCs at the Wnt3a-surface, and a cascade of osteogenically differentiating cells that enter the 3D matrix. However, the cellular mechanisms by which this structure formed were unknown. In this thesis, I build upon preliminary results that the spindle is Wnt3a-orientated, and that components of the Wnt/β-catenin pathway are polarised in BM-hMSCs at metaphase, showing that this coincides with polarisation of polarity protein aPKCζ and markers of cell fate. The resulting daughter cells inherit different identities: this asymmetric cell division (ACD) maintains a self-renewed BM-hMSC that retains contact with Wnt3a-surface, and a more differentiated daughter cell that enters the 3D matrix and receives further osteogenic signals.I analyse whether these tissue engineering principles can influence osteogenesis in vivo in murine calvarial bone defects, a bone fracture model that is particularly conducive to the study of bone repair by intramembranous ossification. Through micro-Computed Tomography, I establish that transplantation of the entire engineered tissue as the WIOTM-bandage enhances bone repair in calvarial defects. Surprisingly, whilst implanted, human cells from the WIOTM-bandage maintain a similar pattern to the in vitro WIOTM, and contribute in vivo to cellular populations both in mineralised bone and the surrounding soft tissues.
In children, bone repair is a remarkably quick and regenerative process, but this capability can be lost in severe fractures and in the elderly. Further, bone-forming osteoblastic cells are derived from progenitor populations in the soft tissues that surround bones, yet the changes that occur in these tissues with aging are not well understood. This project uses mice throughout their lifespan, from juveniles to elderly mice which represent humans over 70 years old. I demonstrate age-related changes to tissue structure, vasculature, nuclear and cytoskeletal morphology, progenitor populations, and mitochondrial activity in the soft tissues that support parietal bone formation and repair. I show that even in early adulthood, some of these aspects begin to decline. Further, while young mice at the stage of rapid skeletal growth are capable of competent bone repair in calvarial defects, otherwise healthy adult mice already have a deficit in the innate capability for repair, but this can be overcome by the Wnt3a-bandage. In aged mice, the Wnt3a-bandage locally increases the number of CD90+ osteoprogenitor cells, but cannot significantly improve bone repair.
I reveal functional changes to calvarial skeletal stem/progenitor cell populations (SSPCs) during aging, through single cell transcriptomics and protein-level analyses, indicating shifts in the cellular energy supply, amongst other differences to Wnt/β-catenin pathway activation, proliferation and senescence. In order to address these deficiencies, I implement alternating-day intermittent fasting (IF) as a systemic metabolic intervention in aged mice, and demonstrate that this results in significant changes to tissue composition, and gene expression in SSPCs, in both the calvarial periosteum and suture mesenchyme. IF remarkably improves calvarial bone repair in elderly mice. To investigate the influence of systemic interventions on calvarial tissues further, and in the shorter term, I supplement aged mice with NMN (NAD+ precursor) and Akkermansia muciniphila (probiotic gut bacterium). I demonstrate that both interventions are capable of recapitulating the rejuvenating effects of IF on osteogenic tissues and bone repair in aged mice. The functional characterisation of the aged progenitor compartments, and finding methods for their rejuvenation, may prove critical for addressing bone repair in an aging society.
| Date of Award | 1 Sept 2024 |
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| Original language | English |
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| Supervisor | Shukry Habib (Supervisor) & Eileen Gentleman (Supervisor) |