Objective

Without adult stem cells, mammalian tissues would not be able to maintain their natural homeostasis, as these cells play a vital part in the replenishment and repair processes, whereby old or damaged cells become replaced. With age, the regenerative capacity of tissues decreases significantly due to deterioration in number and/or function of tissue-specific adult stem cells, and alterations in the properties of stem cell niches and the regulatory cues they receive. One homeostatic mechanism shown to regulate adult stem cells is the circadian clock, which anticipates and synchronises vital tissue functions to the cyclic needs of the organism (food/fasting, activity/rest cycles). Research indicates that core clock proteins, such as Bmal1, bind to the promoters of various stem cell regulatory genes in an oscillatory manner and modulate their expression. In addition, several circadian cues have the ability to entrain stem cells. One novel entrainment cue is mechanical stimulation, however, whether or how mechanical cues entrain cellular and molecular clocks in adult stem cells and their progenitors is unknown. Our current hypothesis is that circadian entrainment mechanisms such as mechanical cues can be used to direct and control adult stem cell differentiation. To this end, our research aims to discover the most efficient mechanisms of entrainment in adult stem cells, as well as signalling pathways and downstream stem cell genes regulated by circadian timing. The methodology will include the culturing of adult stem cells from various tissue sources, including mesenchymal stem cells derived from bone marrow (BMMSCs), adipose (ADSCs) and dental pulp (DPSCs), and subjecting them to different forms of mechanical stimulation using a unique uniaxial mechanical stretch rig. The most efficient type of coating protein and stretch parameters used to optimise the system will also be investigated. Furthermore, different methods of circadian synchronisation will be explored to see if and how the different types of adult stem cell can be synchronised. The results thus far have shown that BMMSCs are capable of being synchronised by both chemical and mechanical methods. The more primitive DPSCs however, do not seem to show this capability for circadian entrainment. It has also been shown that the concentration and type of protein coating used on the silicone chambers substantially affects the cells undergoing the stretching process, and potentially affects their differentiation capabilities. These results imply significant roles for both the circadian clock cycle and mechanical stimulation in adult stem cell homeostasis and differentiation. This research shows the importance of the circadian clock cycle in regenerative medicine and cell therapies. The advantage to this approach is it negates the need to use chemical or thermal approaches to set the circadian clock in adult stem cells. Understanding circadian entrainment mechanisms of adult stem ce

Methods

The methodology will include the culturing of adult stem cells from various tissue sources, including mesenchymal stem cells derived from bone marrow (BMMSCs), adipose (ADSCs) and dental pulp (DPSCs), and subjecting them to different forms of mechanical stimulation using a unique uniaxial mechanical stretch rig. The most efficient type of coating protein and stretch parameters used to optimise the system will also be investigated. Furthermore, different methods of circadian synchronisation will be explored to see if and how the different types of adult stem cell can be synchronised.

Results

The results thus far have shown that BMMSCs are capable of being synchronised by both chemical and mechanical methods. The more primitive DPSCs however, do not seem to show this capability for circadian entrainment. It has also been shown that the concentration and type of protein coating used on the silicone chambers substantially affects the cells undergoing the stretching process, and potentially affects their differentiation capabilities. These results imply significant roles for both the circadian clock cycle and mechanical stimulation in adult stem cell homeostasis and differentiation.

Conclusion

This research shows the importance of the circadian clock cycle in regenerative medicine and cell therapies. The advantage to this approach is it negates the need to use chemical or thermal approaches to set the circadian clock in adult stem cells. Understanding circadian entrainment mechanisms of adult stem cells will help design future strategies to rescue or delay the age-related changes seen in tissue function and stem cell differentiation potential.