Fundamental mechanisms causing pituitary stem cell aging in mice and humans

项目摘要 脑下垂体产生激素，在整个过程中维持多个靶器官的体内平衡 然而，随着动物年龄的增长，垂体细胞逐渐显示出细胞和生物活性降低的迹象。 垂体生长激素的产生随着年龄的增长而自然下降，这一过程被称为体细胞周期， 在60岁以上的人群中非常低。表达干细胞标志物SOX 2的成体垂体干细胞包含一个表达干细胞标志物SOX 2的细胞。 小比例的成体垂体细胞，可增殖和分化以再生新生的内分泌 器官损伤后的细胞或对生理需求的反应。垂体干细胞数量下降 随着年龄的增长，它们对器官损伤的再生能力迅速下降， 年龄，在小鼠中早在8个月的中年时就变得非常有限。的基本机制 与年龄相关的垂体干细胞再生能力丧失的原因实际上还未被探索。在这 我们将利用最先进的基因组和体外技术来阐明垂体干细胞基因 在衰老过程中的表达模式和功能决定了哪些基因是垂体 干细胞介导再生首先，我们将联合收割机荧光激活细胞分选与单细胞RNA 测序，以建立小鼠垂体干细胞转录组学的性别特异性单细胞图谱 从2个月大的再生干细胞到8个月大的非再生干细胞。 这一新的数据将揭示垂体干基因调控网络的动态时间变化 细胞行为，并确定新的候选基因造成的损失再生旧垂体 干细胞其次，我们将在功能上验证新基因在垂体干细胞增殖中的作用， 从我们的初步数据和单细胞图谱中鉴定出的分化。我们将利用现有的小型 干扰敲低测定以抑制来自我们的初步数据的两种候选物的表达， 干细胞-对它们促进干细胞增殖和/或分化的能力特异的干细胞。我们将 在人诱导多能干细胞中同时工程化新垂体特异性荧光报告基因 细胞系这一垂体在类器官中的承诺指标将促进未来功能研究的过渡 垂体干细胞衰老的体外模型。使用这些方法，我们将生成假设 并测试调节年龄依赖性垂体干细胞再生的候选基因。这将揭开 促进垂体干细胞增殖和分化的分子机制。从长远 从长远来看，这些知识将有助于延长老年人垂体细胞的再生和对抗衰老。

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.