Fundamental Mechanisms Causing Pituitary Stem Cell Aging in Mice and Humans

导致小鼠和人类垂体干细胞衰老的基本机制

• 批准号: 10627790
• 负责人: Leonard Yan Ming Cheung
• 金额: $ 2.12万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10627790
• 关键词: Ablation; Adult; Age; Aging; Animals; Anterior Pituitary Gland; Atlases; Biological; Biological Assay; Biological Models; Candidate Disease Gene; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cells; Consumption; Data; Elderly; Endocrine; Engineering; Feedback; Female; Fluorescence; Fluorescence-Activated Cell Sorting; Future; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genomics; Goals; Homeostasis; Hormones; Human; In Vitro; Knowledge; Life; Maintenance; Mediating; Molecular; Molecular Analysis; Mus; Natural regeneration; Neurosecretory Systems; Organ; Organoids; Pathway interactions; Physiological; Pituitary Diseases; Pituitary Gland; Pituitary Hormones; Process; Production; Proliferating; Regenerative capacity; Reporter; Reporter Genes; Resolution; Role; Small Interfering RNA; Somatotropin; Specific qualifier value; Techniques; Technology; Testing; Time; Visual; age related; cell behavior; cell type; combat; gene regulatory network; genetic testing; genomic data; human model; human old age (65+); in vitro Model; in vitro activity; in vivo regeneration; induced pluripotent stem cell; insight; knock-down; male; middle age; molecular mass; novel; organ injury; organ regeneration; pituitary gland development; postnatal; regenerative; response; sex; single-cell RNA sequencing; stem; stem cell aging; stem cell genes; stem cell proliferation; stem cell self renewal; stem cells; tool; transcriptome; transcriptomics; virtual

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.

项目概要 垂体产生的激素可在整个过程中维持多个靶器官的稳态 然而，随着动物年龄的增长，垂体细胞逐渐表现出细胞和生物活性降低的迹象。 垂体生长激素的产生会随着年龄的增长而自然下降，这个过程称为躯体更年期，并且是 在 60 岁以上的人群中非常低。表达干标记 SOX2 的成体垂体干细胞包含 成人垂体细胞的一小部分，可以增殖和分化以再生新生的内分泌 器官损伤后或响应生理需求的细胞。垂体干细胞数量下降 在成年过程中，他们响应器官损伤的再生能力迅速下降 年龄方面，小鼠在 8 个月大的中年时期就变得非常有限。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尚未被探索。在这个 建议，我们将利用尖端的基因组和体外技术来阐明垂体干细胞基因 衰老过程中的表达模式和功能决定垂体所需的基因 干细胞介导的再生。首先，我们将荧光激活细胞分选与单细胞RNA结合起来 测序以构建小鼠垂体干细胞转录组学的性别特异性单细胞图谱 介于 2 个月大的再生干细胞和 8 个月大的非再生干细胞之间。 这些新数据将揭示控制垂体干的基因调控网络的动态时间变化 整个生命周期的细胞行为，并识别导致旧垂体再生丧失的新候选基因 干细胞。其次，我们将从功能上验证新基因在垂体干细胞增殖和 从我们的初步数据和单细胞图谱中鉴定出分化。我们将利用已建立的小型 干扰敲低测定以抑制我们初步数据中两个候选者的表达 年轻干细胞具有促进干细胞增殖和/或分化的能力。我们将 同时在人类诱导多能干细胞中设计一种新型垂体特异性荧光报告基因 细胞系。类器官中垂体承诺的这一指标将有助于未来功能研究的转变 垂体干细胞衰老的人体体外模型。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 促进出生后垂体干细胞增殖和分化的分子机制。在漫长的 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。