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: 8279758
• 负责人: Noriaki Ono
• 金额: $ 13.8万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8279758
• 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

DESCRIPTION (provided by applicant): 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 o 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. PUBLIC HEALTH RELEVANCE: The prospective findings of this research project will give important insights into the property and the role of stem/progenitor cells in skeletal development. This information will become a valuable tool for understanding the mechanisms of various dental and craniofacial deformities and developing novel diagnosis categories and treatment modalities.

描述（由申请人提供）：在骨骼发育中，软骨细胞和成骨细胞谱系的细胞经历一系列增殖和分化步骤，并产生驱动骨生长的基质产生细胞。本研究项目的目标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。出生后骺生长板软骨顶部的干样软骨细胞：在软骨内骨形成中，称为生长板的特定区域中的软骨细胞在出生后继续增殖，为骨延长提供动力。生长板顶部缓慢分裂的细胞可能具有出生后干细胞的某些特征。首先，作为生长板中所有其他软骨细胞来源的自我更新软骨细胞的存在将通过使用软骨细胞特异性诱导型CreERt和具有长追踪期的荧光报告系统的谱系追踪实验来证明。其次，生长板顶部的标记保留细胞的遗传组成将基于cDNA微阵列进行表征。将进行软骨细胞特异性脉冲追踪实验，以基于多西环素可调节的Tet-off系统和组蛋白2B结合的EGFP（H2 B-EGFP）标记来鉴定缓慢复制的细胞。将通过荧光激活细胞分选（流式细胞仪）分离保留标记和非保留标记的软骨细胞。在标记保留软骨细胞中特异性表达的基因将通过原位杂交检测其在发育过程中的基因表达，使用在比较标记保留和快速增殖软骨细胞的微阵列实验中鉴定的探针。具体目标2。成骨细胞谱系早期的早期细胞“间充质干细胞”的成骨细胞分化受转录因子Runx 2和Osterix（Osx）调节，所述转录因子在定向成骨细胞谱系后早期表达。Msx 2位于这两个转录因子的上游。巢蛋白最近被证明是间充质干细胞的标志物。体内“间充质干细胞”群体的异质性、起源和自我更新将通过基于双荧光系统的组合谱系追踪实验来研究，所述双荧光系统使用Nestin-EGFP; Nestin-/Osx-/Runx 2-/Msx 2-CreERt; Rosa 26-CAG-tdTomato报告小鼠。将通过FACS分离双阳性自我更新和单阳性目的后代群体，以分析每个群体中特异性上调的基因。具体目标3。软骨细胞和成骨细胞谱系的共同干/祖细胞及其功能：将创建可诱导CreERt BAC转基因小鼠，其中CreERt表达受Aim 1和2的共同上调基因之一的启动子调控。为了了解这些细胞在骨骼发育过程中的作用，CreER t小鼠将与诱导型白喉毒素受体（iDTR）小鼠杂交。白喉毒素将在发育的不同时间给予，并将监测对正常骨骼发生的破坏，以阐明这些祖细胞在体内的作用。 公共卫生关系：本研究项目的前瞻性发现将对干/祖细胞在骨骼发育中的性质和作用提供重要的见解。 这些信息将成为一个有价值的工具，了解各种牙齿和颅面畸形的机制，并开发新的诊断类别和治疗方式。