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: 8418734
• 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/8418734
• 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.

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，并产生驱动骨骼生长的基质生成细胞。该研究项目的目标是揭示软骨细胞和成骨细胞谱系中的干/祖细胞的起源、分布、调节动力学和体内遗传谱。具体目标 1. 出生后骨骺生长板软骨顶部的干状软骨细胞：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，使用软骨细胞特异性诱导型 CreERt 和具有长追踪周期的荧光报告系统进行谱系追踪实验，证明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。其次，生长板顶部标记保留细胞的基因组成将基于 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 (H2B-EGFP) 标签来识别缓慢复制的细胞。将通过荧光激活细胞分选（FACS）分离标记保留和非标记保留软骨细胞。将使用在比较保留标记和快速增殖的软骨细胞的微阵列实验中鉴定的探针，通过原位杂交来测试在保留标记的软骨细胞中特异表达的基因的基因表达。具体目标 2. 成骨细胞谱系早期的早期细胞 “间充质干细胞”的成骨细胞分化受到转录因子 Runx2 和 Osterix (Osx) 的调节，这些转录因子在定型为成骨细胞谱系后早期表达。 Msx2 被认为是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。将通过基于使用 Nestin-EGFP 的双荧光系统的联合谱系追踪实验，研究体内“间充质干细胞”群体的异质性、起源和自我更新； Nestin-/Osx-/Runx2-/Msx2-CreERt； Rosa26-CAG-tdTomato 报告小鼠。将通过FACS分离感兴趣的双阳性自我更新和单阳性后代群体，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常见干/祖细胞及其功能：将创建可诱导的 CreERt BAC 转基因小鼠，其中 CreERt 表达受到目标 1 和 2 中常见上调基因之一的启动子的调节。为了了解这些细胞在骨骼发育过程中的作用，CreERt 小鼠将与诱导白喉毒素受体 (iDTR) 小鼠杂交。将在发育的不同时期施用白喉毒素，并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。

项目成果

Osteopontin deficiency enhances parathyroid hormone/ parathyroid hormone related peptide receptor (PPR) signaling-induced alteration in tooth formation and odontoblastic morphology.

• DOI: 10.1016/j.tice.2011.02.003
• 发表时间: 2011-06
• 期刊: TISSUE & CELL
• 影响因子: 2.6
• 作者: Morishita, Maki;Ono, Noriaki;Miyai, Kentano;Nakagawa, Tomomi;Hanyu, Ryo;Nagao, Masashi;Kamolratanakul, Paksinee;Notomi, Takuya;Rittling, Susan R.;Denhardt, David T.;Kronenberg, Henry M.;Ezura, Yoichi;Hayata, Tadayoshi;Nakamoto, Tetsuya;Noda, Masaki
• 通讯作者: Noda, Masaki

Occlusal hypofunction causes periodontal atrophy and VEGF/VEGFR inhibition in tooth movement.

• DOI: 10.2319/011712-45.1
• 发表时间: 2013-01
• 期刊: The Angle orthodontist
• 作者: Usumi-Fujita R;Hosomichi J;Ono N;Shibutani N;Kaneko S;Shimizu Y;Ono T
• 通讯作者: Ono T

Diverse contribution of Col2a1-expressing cells to the craniofacial skeletal cell lineages.

• DOI: 10.1111/ocr.12168
• 发表时间: 2017-06
• 期刊: Orthodontics & craniofacial research
• 影响因子: 3.1
• 作者: Sakagami N;Ono W;Ono N
• 通讯作者: Ono N

The fate of Osterix-expressing mesenchymal cells in dental root formation and maintenance.

在牙齿根形成和维持中表达卵形质细胞的命运。

• DOI: 10.1111/ocr.12167
• 发表时间: 2017-06
• 期刊: Orthodontics & craniofacial research
• 影响因子: 3.1
• 作者: Takahashi A;Ono N;Ono W
• 通讯作者: Ono W

Constitutively active parathyroid hormone receptor signaling in cells in osteoblastic lineage suppresses mechanical unloading-induced bone resorption

• DOI: 10.1074/jbc.m610782200
• 发表时间: 2007-08-31
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Ono, Noriaki;Nakashima, Kazuhisa;Noda, Masaki
• 通讯作者: Noda, Masaki