Fundamental mechanisms causing pituitary stem cell aging in mice and humans

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Project Summary The pituitary gland produces hormones which maintain homeostasis of multiple target organs over the course of adult life, yet pituitary cells gradually display signs of reduced cellular and biological activity as animals age. Pituitary growth hormone production naturally declines with age in a process called the somatopause and is very low in humans over 60 years old. Adult pituitary stem cells expressing the stem marker SOX2 comprise a small proportion of adult pituitary cells and can proliferate and differentiate to regenerate nascent endocrine cells after organ damage or in response to physiologic demands. The number of pituitary stem cells declines over the course of adult life, and their ability to regenerate in response to organ damage rapidly declines with age, becoming very limited as early as middle-age at 8-months in mice. The fundamental mechanisms responsible for age-related loss of pituitary stem cell regenerative capacity are virtually unexplored. In this proposal, we will utilize cutting edge genomic and in vitro technologies to elucidate pituitary stem cell gene expression patterns over the course of aging and functionally determine which genes are required for pituitary stem cell-mediated regeneration. First, we will combine fluorescence activated cell sorting with single-cell RNA sequencing in order to build a sex-specific single-cell atlas of mouse pituitary stem cell transcriptomics between the ages of 2-month-old regenerative stem cells through to 8-month-old non-regenerative stem cells. This novel data will unveil the dynamic temporal changes in gene regulatory networks governing pituitary stem cell behavior throughout life and identify novel candidate genes causing loss of regeneration in old pituitary stem cells. Second, we will functionally validate the role of novel genes in pituitary stem cell proliferation and differentiation identified from our preliminary data and single-cell atlas. We will utilize an established small interfering knockdown assay to inhibit expression of the two candidates from our preliminary data that are young-stem cell specific for their ability to promote stem cell proliferation and/or differentiation. We will concurrently engineer a novel pituitary-specific fluorescent reporter gene in a human induced pluripotent stem cell line. This indicator of pituitary commitment in organoids will facilitate transitioning future functional studies of pituitary stem cell aging to a human in vitro model. Using these approaches, we will generate hypotheses and test candidate genes for regulation of age-dependent pituitary stem cell regeneration. This will uncover the molecular mechanisms enabling postnatal pituitary stem cell proliferation and differentiation. In the long term, this knowledge will aid efforts to prolong regeneration and combat aging of pituitary cells in the elderly.