Dynamics and Regulations of Bone Stem Cells in Vivo

项目摘要/摘要 骨骼疾病和畸形在儿童和年轻人中很普遍。由于缺乏有效的模式 为了再生正在生长的骨骼，这些年轻的患者通常会接受多次手术治疗，形成一种 这给他们、他们的家庭和社会带来了沉重的负担。在骨骼生长过程中，软骨细胞和成骨细胞 不断产生，使骨骼变得更大更强壮。内源性骨干细胞作为 这些细胞的来源还没有完全弄清楚。基础知识这些骨干细胞是如何 细胞协调软骨内和膜内成骨两个过程，是发展成骨肉瘤所必需的 再生正在生长的骨骼的可靠方法。在这个项目中，不同类型的骨干的特征 将确定积极促进骨骼生长的细胞。我们假设一组静止的软骨细胞 在出生后的生长板中表现为生长相关的骨干细胞，并成为 骨髓中的间充质基质祖细胞；这两种类型的骨干/祖细胞 协同促进骨骼的正常生长和维护。识别特征和分子 骨干细胞的调控将有助于我们通过再生来繁殖这些细胞 工程学。在Aim1中，我们将确定调节休眠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 作为生长相关骨干细胞的特性，其特性和命运由刺猬调节 发信号。我们将首先在体外鉴定一组具有自我更新能力的静息软骨细胞亚群。 分离的生长板细胞的集落形成实验和体内移植。我们将第二次确定 自我更新集落形成休眠软骨细胞的独特基因表达谱。我们将进一步定义 Hedgehog信号在决定静止软骨细胞自我更新和分化中的作用 调节其信号成分，同时跟踪它们在体内和体外的行为。在……里面 AIM2，我们将确定骨髓间充质基质前体细胞在生长骨骼中的形成和命运。 工作假说是生长板软骨细胞肥大并转化为CXCL12- 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。我们将首先定义CAR细胞向成骨细胞和脂肪细胞的分化潜能 通过间歇性甲状旁腺素给药和含有罗格列酮的高脂饮食。我们将获得第二名 使用半稳定化的胫骨骨折模型确定CAR细胞对损伤的反应。我们还将确定影响 在这些操作中，CAR细胞表达调控细胞命运选择的关键转录因子的水平。 我们将第三次通过体外克隆来确定CAR细胞作为间充质基质前体细胞的特性。 分离的骨髓细胞的形成试验和体内移植。我们将进一步定义β的角色- 连环蛋白信号作为成骨细胞与脂肪细胞分化的决定因素。

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.