Defining the developmental plasticity and differentiation potential of luminal stem cells in the mouse mammary gland during development and adult homeostasis – LumStemCell

The dynamic transcriptional networks that are developmentally coordinated to establish and maintain gene expression profiles in adult stem cells, ensuring their self-renewal throughout adult life, while allowing the proper differentiation of stem cell progeny during tissue homeostasis and remodeling, are still poorly defined. The proposed project, focused on mammary stem cells, will investigate the molecular signals regulating adult stem cell behavior in vivo using available mouse models, functional assays, transcriptomic analyses and organoid cultures. The mammary epithelium represents an ideal system to study stem cell plasticity, as it undergoes tremendous remodeling at each reproductive cycle, in the absence of injury. In addition, recent work has suggested the existence of unipotent mammary stem cells, showing long term memory of lineage commitment in vivo, that appear to be devoted to maintaining a single cell lineage in the postnatal and adult gland. Interestingly, these cells derive from multipotent embryonic stem cells and retain a high plasticity throughout life, as they can display multilineage differentiation capacity in transplantation assays. These experiments suggest that the fate and potential of stem cells can change, depending on whether a stem cell resides within its niche and responds to normal homeostatic cues, or whether it is removed from its niche and challenged to de novo tissue morphogenesis after transplantation. Importantly, reactivation of embryonic developmental programs in postnatal tissues underlies the observed plasticity of adult stem cells when dissociated from their original tissue, but it may also be responsible for disease onset. While elucidating the lineage hierarchies within the mammary epithelium is essential for understanding tissue morphogenesis and stem cell plasticity, the high cellular complexity of this tissue and the lack of robust markers have considerably limited the analysis of mammary epithelial lineages. We propose to define the transcriptional signals that define the lineage potential of different luminal mammary stem cells, utilizing a unique set of transgenic animals we generated, to mark and molecularly characterize defined cell populations in vivo. Through inducible genetic labeling approaches, we will study stem cell hierarchies and behavior in physiological conditions, and will also evaluate the extent to which such cells are dependent on specific signals to maintain their multipotent or lineage-restricted status, by testing if they can be “reprogrammed” upon conditional deletion or expression of selected mutations in vivo. The combination of genetic fate mapping experiments, followed by the isolation of these cells, the assessment of their plasticity in functional assays, and, finally, their targeting with selected mutations, should provide fundamental insights into the poorly understood mammary epithelial cell hierarchy and will help to unravel the molecular mechanisms implicated in balancing stem cell self-renewal and differentiation. This will improve our understanding of the molecular programs allowing the stem cell genome to remain refractory, or to become sensitive, to differentiation cues, a poorly explored and still largely unanswered question in adult tissue homeostasis. We expect that the accomplishment of the proposed studies will contribute to reveal the signals controlling stem cell plasticity and potentially, to rationalize the design of novel therapeutic drugs targeting defined signaling networks, as well as providing prognostic and diagnostic biomarkers of diseases.