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了增殖和分化的串行步骤，并引起产生基质的细胞，这些细胞驱动骨骼生长。该研究项目的目的是揭示软骨细胞和成骨细胞谱系中的茎/祖细胞的起源，分布，调节动力学和体内的遗传谱。特定的目标1。茎状的软骨细胞在产后周围生长板软骨的顶部：在软骨骨形成中，所谓的特定区域的软骨细胞在称为生长板的特定区域继续在产后增殖，为骨延长提供了发动机。慢慢地将细胞在顶部o o生长板可能具有产后干细胞的某些特征。首先，使用谱系特异性诱导型creert和荧光细胞的荧光细胞特异性诱导者和荧光报告，将证明是生长板中所有其他软骨细胞的来源的自我更新软骨细胞的存在。其次，基于cDNA微阵列将表征生长板顶部的标记细胞的遗传组成。将进行软骨细胞特异性脉冲练习实验，以基于强力霉素调节的TET-OFF系统和2B结合的EGFP（H2B-EGFP）标记来鉴定缓慢复制细胞。荧光活化的细胞分选（FACS）将分离出标记和非标签的软骨细胞。使用与微阵列实验中鉴定的探针进行原位杂交在发育过程中，将在发育过程中特异性表达的基因在发育过程中的基因表达进行测试，该基因表达与微阵列实验中鉴定的探针进行了比较，并比较了保留标签的保留和快速增殖的软骨细胞。特定的目标2。早期细胞在成骨细胞谱系成骨细胞的成骨细胞分化“间充质干细胞”的分化受转录因子Runx2和Osterix（OSX）的调节，后者在承诺对成骨细胞谱系的承诺后表达早期表达。 MSX2是这两个转录因子的推测上游。最近已证明Nestin是间充质干细胞的标志物。将使用Nestin-EGFP基于双荧光系统的组合谱系跟踪实验来研究体内“间充质干细胞”种群的异质性，起源和自我更新； nestin-/osx-/runx2-/msx2-creert; ROSA26-CAG-TDTOMATO记者小鼠。 FACS将隔离双重阳性自我更新和单个阳性后代人群，以分析每个人群中专门上调的基因。具体目标3。软骨细胞的常见茎/祖细胞和成骨细胞谱系及其功能：可诱导的Creert BAC转基因小鼠，其中Creert表达受到AIM 1和2的常见上调基因之一的启动子的调节，将产生AIM 1和2的启动子。为了了解这些细胞在骨骼发育中的作用，将与可诱导的白喉受体（IDTR）小鼠交叉Creert小鼠。白喉毒素将在各个发育时期进行给药，并将监测正常骨骼生成的破坏，以阐明这些祖细胞在体内的作用。 公共卫生相关性：该研究项目的前瞻性发现将对STEM/祖细胞在骨骼发育中的特性和作用进行重要见解。 这些信息将成为理解各种牙科和颅面畸形的机制，并开发新的诊断类别和治疗方式的宝贵工具。