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.

骨骼发育的摘要，软骨细胞和成骨细胞谱系的细胞经历了串行步骤 增殖和分化，并引起产生骨骼生长的基质细胞。目标的目标 研究项目旨在揭示软骨细胞和成骨细胞中的茎/祖细胞 在体内的起源，分布，调节动力学和遗传谱。特定的目标1。茎状的软骨细胞 产后骨出现生长板软骨的顶部：在软骨骨形成中，软骨细胞中的软骨细胞 所谓的生长板的特定区域继续在产后增殖，从而为骨骼延长提供了引擎。 在生长板顶部缓慢划分细胞可能具有产后干细胞的某些特征。 首先，存在自我更新软骨细胞，这些软骨细胞是生长板中所有其他软骨细胞的来源 将使用软骨细胞特异性诱导式Creert和A的谱系跟踪实验来证明 荧光记者系统具有较长的追逐时期。其次，在 生长板的顶部将根据cDNA微阵列进行表征。软骨细胞特异性脉搏练习 将进行实验以基于强力霉素调节的tet-off来识别缓慢复制的单元 系统和组蛋白2B结合的EGFP（H2B-EGFP）标签。保留标签和非标签的软骨细胞 将通过荧光活化的细胞分选（FACS）分离。特异性在标签ret的基因 软骨细胞将通过原位杂交在发育过程中测试其基因表达，并使用探针测试 在微阵列实验中鉴定出，比较了保留标签和快速增殖的软骨细胞。 特定目标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 由转录因子Runx2和Osterix（OSX）调节，在对成骨细胞的承诺后提早表示 血统。 MSX2是这两个转录因子的推测上游。 Nestin最近被证明是 间充质干细胞的标记。间充质干细胞的异质性，起源和自我更新 将通过基于双荧光的谱系跟踪实验研究体内人口 使用Nestin-EGFP的系统; nestin-/osx-/runx2-/msx2-creert; ROSA26-CAG-TDTOMATO记者小鼠。双倍的 FACS将隔离积极的自我更新和单一的阳性后代人群以分析 基因在每个人群中特别上调。特定目标3。常见的茎/祖细胞 软骨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AIM 1和2的通常上调基因之一的启动子的调节，将是 创建。为了了解这些细胞在骨骼发育中的作用，将与Creert小鼠交叉 可诱导的白喉毒素受体（IDTR）小鼠。白喉毒素将在不同时间进行 将监测开发和正常骨骼生成的破坏，以阐明这些作用 体内祖细胞。