Dynamics and Regulation of Bone Stem Cells in vivo - Supplement Proposal

体内骨干细胞的动力学和调节 - 补充提案

• 批准号: 9895953
• 负责人: Noriaki Ono
• 金额: $ 22.87万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-07 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9895953
• 关键词: Adipocytes; Adult; Alleles; Behavior; Biological Assay; Bone Diseases; Bone Growth; Bone Marrow; Bone Marrow Cells; Cells; Characteristics; Child; Chondrocytes; Clinical; Collagen Type II; Data; Deformity; Development; Engineering; Epiphysial cartilage; Exhibits; Expression Profiling; Family; Gene Expression; Genes; Genetic; Growth; High Fat Diet; Hypertrophy; Immunodeficient Mouse; In Vitro; Knowledge; Label; Lead; Life; LoxP-flanked allele; Maintenance; Marrow; Mesenchymal; Modality; Modeling; Molecular; Natural regeneration; Operative Surgical Procedures; Osteoblasts; PTH gene; Patients; Physiologic Ossification; Process; Property; Publishing; Regulation; Reporter; Research; Rest; Reticular Cell; Role; Signal Transduction; Societies; Source; Stem cells; Stromal Cell-Derived Factor 1; Stromal Cells; Testing; Tibial Fractures; Transgenic Organisms; Transplantation; Well in self; adipocyte differentiation; base; beta catenin; bone; experimental study; improved; in vivo; intramembranous bone formation; novel; parathyroid hormone-related protein; postnatal; progenitor; regenerative; response to injury; retention rate; rosiglitazone; self-renewal; smoothened signaling pathway; stem; stromal progenitor; transcription factor; transcriptome sequencing; young adult

PROJECT SUMMARY/ABSTRACT Bone disorders and deformities are prevalent in children and young adults. Due to lack of effective modalities to regenerate growing bones, these young patients often undergo multiple surgical interventions, posing a significant burden on them, their family and society. During bone growth, chondrocytes and osteoblasts are continuously generated to make bones bigger and stronger. Endogenous bone stem cells that serve as the source of these cells have not been completely understood. Fundamental knowledge of how these bone stem cells coordinate the two processes of endochondral and intramembranous ossification is essential to develop a reliable approach to regenerate growing bones. In this project, the characteristics of distinct types of bone stem cells that actively promote bone growth will be identified. We hypothesize that a subset of resting chondrocytes in the postnatal growth plate behave as growth-associated bone stem cells, and become a source of mesenchymal stromal progenitor cells in bone marrow; these two types of bone stem/progenitor cells concertedly promote proper bone growth and maintenance. Identifying characteristics and molecular regulations of bone stem cells will facilitate our endeavor to reproduce these cells through regenerative engineering. In Aim1, we will identify molecular mechanisms regulating properties and fates of resting chondrocytes. The working hypothesis is that resting chondrocytes of the postnatal growth plate exhibit unique characteristics as growth-associated bone stem cells, whose properties and fates are regulated by Hedgehog signaling. We will first identify a self-renewing multipotent subpopulation of resting chondrocytes using in vitro colony-forming assays and in vivo transplantation of isolated growth plate cells. We will second identify the unique gene expression profiles of self-renewing colony-forming resting chondrocytes. We will further define roles of Hedgehog signaling in determining self-renewal and differentiation of resting chondrocytes by modulating its signaling components, while simultaneously tracing their behavior both in vivo and in vitro. In Aim2, we will define formation and fates of bone marrow mesenchymal stromal progenitors in growing bones. The working hypothesis is that growth plate chondrocytes undergo hypertrophy and transform into Cxcl12- abundant reticular (CAR) cells in bone marrow that behave as regional and reactive mesenchymal stromal progenitor cells. We will first define differentiation potentials of CAR cells into osteoblasts and adipocytes in vivo through intermittent PTH administration and a high-fat diet containing rosiglitazone. We will second determine CAR cells' response to injury using a semistabilized tibial fracture model. We will also identify effects of these manipulations on CAR cells' expression levels of key transcription factors that regulate cell fate choice. We will third define the properties of CAR cells as mesenchymal stromal progenitors through in vitro colony- forming assays and in vivo transplantation of isolated bone marrow cells. We will further define roles of β- catenin signaling as a cell fate determinant of osteoblast versus adipocyte differentiation using its floxed allele.

项目总结/摘要 骨骼疾病和畸形在儿童和年轻人中普遍存在。由于缺乏有效的方法， 为了再生生长中的骨骼，这些年轻患者经常接受多次手术干预， 给他们、他们的家庭和社会带来沉重负担。在骨生长过程中，软骨细胞和成骨细胞 使骨骼变得更大更强内源性骨干细胞作为 这些细胞的来源尚未完全了解。关于这些骨柄 细胞协调软骨内骨化和膜内骨化这两个过程， 可靠的方法来再生生长的骨骼。在本项目中，不同类型的骨柄的特征 将鉴定积极促进骨生长的细胞。我们假设一部分静止的软骨细胞 在出生后的生长板表现为生长相关的骨干细胞，并成为一个来源， 骨髓中的间充质基质祖细胞;这两种类型的骨干/祖细胞 协调地促进适当的骨骼生长和维持。鉴别特征和分子 骨干细胞的调控将有助于奋进通过再生来复制这些细胞， 工程.在Aim 1中，我们将确定调节静息细胞特性和命运的分子机制。 软骨细胞工作假设是，出生后生长板的静止软骨细胞表现出独特的 作为生长相关的骨干细胞的特性，其性质和命运由Hedgehog调节 发信号。我们将首先利用体外细胞培养技术鉴定一种具有自我更新能力的多能静息软骨细胞亚群， 集落形成测定和分离的生长板细胞的体内移植。我们将第二次确定 自我更新集落形成静息软骨细胞的独特基因表达谱。我们将进一步定义 Hedgehog信号在静息软骨细胞自我更新和分化中的作用 调节其信号成分，同时追踪它们在体内和体外的行为。在 目的2：明确骨髓间充质干细胞在骨生长过程中的形成和命运。 工作假设是生长板软骨细胞经历肥大并转化为Cxcl 12-。 骨髓中丰富的网状（CAR）细胞，表现为区域性和反应性间充质基质 祖细胞我们将首先确定CAR细胞向成骨细胞和脂肪细胞的分化潜能， 通过间歇性PTH给药和含罗格列酮的高脂饮食体内。我们将第二 使用半稳定化胫骨骨折模型确定CAR细胞对损伤的反应。我们还将确定影响 这些操作对CAR细胞的关键转录因子表达水平的影响，这些转录因子调节细胞命运的选择。 我们将第三次通过体外集落形成来定义CAR细胞作为间充质基质祖细胞的特性。 分离的骨髓细胞的形成测定和体内移植。我们将进一步定义β- 使用其floxed等位基因作为成骨细胞与脂肪细胞分化的细胞命运决定因子的连环蛋白信号传导。