Stem/progenitor cells of the chondrocyte and osteoblast lineage in vivo

描述(申请人提供)：在骨骼发育中，软骨细胞和成骨细胞谱系经历了一系列的增殖和分化步骤，并产生了驱动骨生长的基质产生细胞。本研究项目的目的是揭示软骨细胞和成骨细胞谱系中的干/祖细胞的来源、分布、调节动力学和体内的遗传学特征。具体目的1.生后生长板软骨顶部的干细胞样软骨细胞：在软骨内骨形成中，称为生长板的特定区域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，生长板中所有其他软骨细胞的来源是自我更新的软骨细胞的存在，将通过使用软骨细胞特异性诱导的CRERt和具有长追踪期的荧光报告系统的谱系跟踪实验来证明。其次，将基于cDNA微阵列来表征位于生长板顶部的标记保留细胞的遗传构成。将进行软骨细胞特异性脉冲追逐实验，以识别基于多西环素可调节的Tet-off系统和组蛋白2B结合的EGFP(H 2B-EGFP)标记的缓慢复制的细胞。标记保留和非标记保留的软骨细胞将通过荧光激活细胞分选(FACS)进行分离。通过使用微阵列实验中比较标记保留和快速增殖的软骨细胞中识别的探针，将通过原位杂交测试标记保留软骨细胞中特定表达的基因在发育过程中的基因表达。特定目的2.“间充质干细胞”成骨分化的早期细胞受转录因子Runx2和Osterix(OSX)的调控，这些转录因子在成骨细胞谱系中早期表达。MSX2可能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重荧光系统的联合谱系跟踪实验进行研究，该实验使用Nestin-EGFP；Nestin-/OSX-/Runx2-/MSX2-CreERt；rosa26-cag-td番茄报告小鼠。双阳性、自我更新和单阳性后代群体将被FACS分离，以分析在每个群体中特异上调的基因。特定目的3.软骨细胞和成骨细胞共同的干/祖细胞及其功能：可诱导的CreERt BAC转基因小鼠，其CreERt表达受Aim 1和Aim 2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中的作用，CreERt小鼠将与可诱导白喉毒素受体(IDTR)小鼠杂交。白喉毒素将在发育的不同时期给予，并将监测对正常骨骼形成的干扰，以阐明这些前体细胞在体内的作用。 公共卫生相关性：这一研究项目的前瞻性发现将对干细胞/祖细胞在骨骼发育中的性质和作用提供重要的见解。 这些信息将成为了解各种牙齿和颅面畸形的机制以及开发新的诊断类别和治疗方式的有价值的工具。

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.