Terminal cell differentiation is a process essential for maintaining and regenerating tissues in all adult mammals and generates adipocytes (fat cells), neurons, skeletal muscle cells, and many other celltypes. Much evidence supports that in order to terminally differentiate, progenitor cells must first enter and then permanently exit the cell cycle. Failure of terminally differentiated cells to permanently exit from the cell cycle can lead to cancer and metabolic disorders1,2. Thus, proper cell cycle entry and exit is likely a critical early control point that determines whether or not a cell will correctly terminally differentiate. Whereas there has been much interest in using the cell cycle to control terminal cell differentiation, for example to prevent the differentiation of fat cells or to enhance differentiation of muscle and neuronal cells, efforts to do so have produced conflicting results and many unanswered questions. This is due to the challenge that the cell cycle both promotes and inhibits terminal cell differentiation and also due to the large heterogeneity in if and when cells proliferate and differentiate. To address both concerns, live tracking of individual cells is needed to understand cell cycle and differentiation dynamics which can only be indirectly inferred when using flow cytometry, RNAseq, or other single-cell snapshot methods. Live-cell imaging studies to understand terminal cell differentiation - where cells permanently exit the cell cycle - have not yet been made, and molecular mechanisms of the interplay between the cell cycle and differentiation are not well understood in any differentiation system. We will use mammalian adipogenesis as an in vitro and in vivo model to understand fundamental open questions about terminal differentiation. We will also use single-cell imaging approaches we developed to - for the first time - simultaneously track and quantitatively analyze both cell cycle and terminal differentiation live in thousands of single cells over the several-day long timecourse of adipogenesis. Our overarching hypothesis that we will test in this work is that the total number of differentiated cells can be maximized or minimized to control tissue size, health, and regeneration, by manipulating the relative strength of both differentiation and mitogen stimuli. The outcome of this work will be a framework how the cell cycle controls adipogenesis and how mitogen and adipogenic stimuli can be manipulated to ultimately control fat mass. Our results will likely have broad applicability not only to the maintenance of adipose tissue, but also more generally for the maintenance and regeneration of neuronal, muscle, and other terminally differentiated tissues.
Our overall goal is to understand the molecular mechanisms how the cell cycle and differentiation progression are linked and can be synergistically manipulated to regulate terminal cell differentiation of adipocytes and thus fat mass. Understanding how to regulate adipocyte differentiation has many immediate applications for treating the current worldwide epidemic of obesity. The results of this proposed work will likely have applicability to other terminal cell differentiation processes that are important for human health such as differentiation of neurons and skeletal muscle cells.
