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: 8895296
• 负责人: Noriaki Ono
• 金额: $ 24.52万
• 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/8895296
• 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; 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 makeup; 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的细胞经历一系列步骤， 增殖和分化，并产生驱动骨生长的基质产生细胞。这个目标 研究项目是揭示软骨细胞和成骨细胞谱系中的干/祖细胞， 起源、分布、调节动力学和体内遗传谱。具体目标1。干样软骨细胞 出生后骺生长板软骨的顶部：在软骨内骨形成中， 称为生长板的特定区域在出生后继续增殖，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，存在自我更新的软骨细胞，它们是生长板中所有其他软骨细胞的来源 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。第二，标记保留细胞的遗传组成， 将基于cDNA微阵列表征生长板的顶部。软骨细胞特异性脉冲追踪 将进行实验以基于强力霉素可调节的Tet-off来鉴定缓慢复制的细胞 系统和组蛋白2B结合的EGFP（H2 B-EGFP）标记。标记保留和非标记保留软骨细胞 将通过荧光激活细胞分选（FACS）分离。在标记保留区特异表达的基因 软骨细胞将在发育过程中通过原位杂交检测其基因表达， 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。 具体目标2。成骨细胞谱系早期的早期细胞： 间充质干细胞 受转录因子Runx 2和Osterix（Osx）的调节，这些转录因子在定向成骨细胞后早期表达 脉Msx 2位于这两个转录因子的上游。Nestin最近被证明是一种 间充质干细胞的标志物。异源性、起源和自我更新 间充质干细胞 将通过基于双荧光标记的组合谱系追踪实验来研究体内群体。 系统使用Nestin-EGFP; Nestin-/Osx-/Runx 2-/Msx 2-CreERt; Rosa 26-CAG-tdTomato报告基因小鼠。双 将通过FACS分离阳性自我更新和单个阳性后代群体以分析 在每个群体中特异性上调的基因。具体目标3。干细胞/祖细胞 软骨细胞和成骨细胞谱系及其功能：诱导型CreERt BAC转基因小鼠，其中 CreERt表达受Aim 1和2的一种通常上调基因的启动子调控， 创造为了了解这些细胞在骨骼发育过程中的作用，将CreER t小鼠与 诱导型白喉毒素受体（iDTR）小鼠。白喉毒素将在不同的时间给予， 发育和正常骨骼发生的破坏将被监测，以阐明这些因素的作用。 体内的祖细胞。