Stem/progenitor cells of the chondrocyte and osteoblast lineage in vivo

体内软骨细胞和成骨细胞谱系的干细胞/祖细胞

• 批准号: 8848446
• 负责人: Noriaki Ono
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8848446
• 关键词: Binding; Bone Development; Bone Growth; Bone Lengthening; Cartilage; Categories; Cell Separation; Cells; Characteristics; Chondrocytes; Deformity; Dental; Development; Diagnosis; Diphtheria Toxin; Doxycycline; Epiphysial cartilage; Gene Expression; Genes; Genetic; Goals; Heterogeneity; Histones; In Situ Hybridization; Kinetics; Label; Mentors; Mesenchymal; Mesenchymal Stem Cells; Modality; Monitor; Mus; Mutant Strains Mice; Osteoblasts; Osteogenesis; Phase; Physiologic pulse; Population; Proliferating; Property; Reporter; Research Project Grants; Role; Scientist; Skeletal Development; Source; Stem cells; System; Tamoxifen; Testing; Tetanus Helper Peptide; Time; Transgenic Mice; base; cDNA Arrays; cell type; craniofacial; diphtheria toxin receptor; genetic profiling; in vivo; insight; interest; nerve stem cell; nestin protein; novel; osteoblast differentiation; postnatal; progenitor; promoter; prospective; research study; self-renewal; skeletogenesis; stem; stem cell population; tool; transcription factor

SUMMARY In skeletal development, cells of the chondrocyte and osteoblast lineage undergo serial steps of proliferation and differentiation, and give rise to matrix-producing cells that drive bone growth. The goal of this research project is to reveal stem/progenitor cells in the chondrocyte and osteoblast lineage in terms of their origin, distribution, regulated kinetics and genetic profiles in vivo. Specific Aim 1. Stem-like chondrocytes at the top of the postnatal epiphyseal growth plate cartilage: In endochondral bone formation, chondrocytes in the specific regions termed growth plates continue to proliferate postnatally, providing engines for bone lengthening. Slowly dividing cells at the top of the growth plate probably share some characteristics of postnatal stem cells. First, existence of self-renewing chondrocytes that are the sources of all other chondrocytes in the growth plate will be demonstrated by a lineage-tracking experiment using a chondrocyte-specific inducible CreERt and a fluorescent reporter system with a long chase period. Second, the genetic make-up of label-retaining cells at the top of the growth plate will be characterized based on cDNA microarrays. A chondrocyte-specific pulse-chase experiment will be performed to identify slowly replicating cells based on a doxycycline-regulatable Tet-off system and a histone 2B-bound EGFP (H2B-EGFP) label. Label-retaining and non-label-retaining chondrocytes will be isolated by fluorescent activated cell sorting (FACS). Genes specifically expressed in label-retaining chondrocytes will be tested for their gene expression during development by in situ hybridization, using probes identified in microarray experiments comparing the label-retaining and rapidly proliferating chondrocytes. Specific Aim 2. Early cells early in the osteoblast lineage: Osteoblast differentiation of mesenchymal stem cells is regulated by transcription factors Runx2 and Osterix (Osx) expressed early after commitment to the osteoblast lineage. Msx2 is putatively upstream of these two transcription factors. Nestin has been recently shown to be a marker of mesenchymal stem cells. Heterogeneity, origin and self-renewal of the mesenchymal stem cell population in vivo will be investigated by a combined lineage-tracking experiment based on a double fluorescent system using Nestin-EGFP; Nestin-/Osx-/Runx2-/Msx2-CreERt; Rosa26-CAG-tdTomato reporter mice. Double positive self-renewing and single positive descendant populations of interest will be isolated by FACS to analyze genes specifically upregulated in each population. Specific Aim 3. Common stem/progenitor cells of the chondrocyte and the osteoblast lineage and their function: Inducible CreERt BAC transgenic mouse in which CreERt expression is regulated by the promoter of one of the commonly upregulated genes of Aim 1 and 2 will be created. To understand the role of these cells during skeletal development, the CreERt mice will be crossed with inducible diphtheria toxin receptor (iDTR) mice. Diphtheria toxin will be administered at various times of development, and disruption on normal skeletogenesis will be monitored to elucidate the role of these progenitors in vivo.

在骨骼发育中，软骨细胞和成骨细胞谱系经历了一系列的步骤 增殖和分化，并产生基质产生细胞，驱动骨生长。这样做的目的是 研究项目是揭示软骨细胞和成骨细胞谱系中的干/祖细胞 在体内的起源、分布、调节动力学和遗传特征。具体目标1.干细胞样软骨细胞位于 生后生长板软骨顶端：软骨内成骨，软骨细胞 被称为生长板的特定区域在出生后继续增殖，为骨骼延长提供了引擎。 生长板顶部缓慢分裂的细胞可能具有出生后干细胞的一些特征。 首先，生长板中所有其他软骨细胞的来源是自我更新的软骨细胞的存在。 将通过使用软骨细胞特异性诱导的CreERt和 具有长追踪期的荧光报告系统。第二，标签保留细胞的遗传构成 生长板的顶部将基于cDNA微阵列进行表征。一种软骨细胞特异性的脉冲追逐器 将进行实验，以基于多西环素可调节的Tet-off来识别复制缓慢的细胞 系统和组蛋白2B结合的绿色荧光蛋白(H_2B-绿色荧光蛋白)标记。标记保留和非标记保留的软骨细胞 将通过荧光激活细胞分选(FACS)分离。标签保留中特异表达的基因 将使用探针通过原位杂交来测试软骨细胞在发育过程中的基因表达 在比较标记保留和快速增殖的软骨细胞的微阵列实验中鉴定。 特定目的2.成骨细胞谱系早期的早期细胞：间充质干细胞的成骨分化 是由转录因子Runx2和Osterix(OSX)调控的，这些转录因子在与成骨细胞接触后早期表达 血统。MSX2可能位于这两个转录因子的上游。巢蛋白最近被证明是一种 间充质干细胞的标记物。间充质干细胞的异质性、起源和自我更新 体内的种群将通过基于双荧光的组合血统追踪实验进行研究 系统使用nestin-EGFP；nestin-/osx-/runx2-/msx2-cret；rosa26-cag-td番茄报告小鼠。双倍 通过流式细胞仪分离出阳性的自我更新群体和单一的阳性后代群体进行分析 基因在每个种群中特别上调。具体目标3.儿童的共同干细胞/祖细胞 软骨细胞和成骨细胞系及其功能：可诱导的Creert BAC转基因小鼠 CreERt的表达是由目标1和目标2共同上调的基因之一的启动子调控的 已创建。为了了解这些细胞在骨骼发育过程中的作用，Creert小鼠将与 诱导型白喉毒素受体(IDTR)小鼠。白喉毒素将在不同的时间被注射 将监测发育和对正常骨骼形成的破坏，以阐明这些因素的作用 体内的祖细胞。