The role of osteoblast progenitors in response to bone anabolic agents

项目摘要-项目1 该项目的目标是了解骨质疏松症合成代谢疗法对 成骨祖细胞。该项目符合CORT在骨骼中的总体目标 干细胞（包括成骨细胞祖细胞）代表了一种重要的，但知之甚少的， 骨质疏松症合成代谢治疗靶点。具体而言，本项目将重点确定为什么 Romosozumab（抗硬化蛋白抗体）的骨合成代谢作用随时间迅速减弱。 为了实现这一转化研究的目标，将采取互补的方法 使用病人和老鼠的样本。首先，在目标1中，在前瞻性临床研究中，我们将获得 骨相关生物样本（经髂嵴芯活检、骨髓抽吸物和 外周血）的绝经后骨质疏松症妇女的过程中， romosozumab治疗。这些新采集的样本将与骨骼一起进行沿着分析 先前从用特立帕罗治疗的绝经后妇女获得的骨髓样品。 因此，我们将比较和对比这两种不同的骨质疏松症合成代谢的影响， 剂.在本项目中，将通过多色流式细胞术分析骨髓穿刺液， 和非造血基质细胞将使用一种抗体混合物进行研究， 表面表达的抗原，其细分骨髓基质以确定治疗效果 对人类骨髓基质成分的影响此外，非造血基质细胞将被 前瞻性分离用于单细胞RNA测序。这种分析将提供公正的 骨髓基质的分子异质性，骨质疏松症合成代谢的影响 治疗，以及解释romosozumab疗效随时间减弱的关键信息。到 为了使用人源性样本补充这些研究，在目标2中，我们将联合收割机谱系 在小鼠中用单细胞RNA测序追踪以评估sclerostin抗体对 成骨细胞前体的变化。在我们的方法中加入血统追踪将证明， 假定的祖细胞群成为成骨细胞，并建立跨物种的信心， 保守的基因调控网络，负责骨质疏松症的影响， 代理人随着时间该项目的结果将与 项目2以骨细胞为研究对象。生物信息学核心将在分析中发挥关键作用 跨物种的单细胞转录组数据。最终，这种合并分析将产生 关于骨干细胞生物学和骨反应的有力信息，新的假设 合成代谢剂。

Project Summary – Project 1 The goal of this project is to understand the effects of osteoporosis anabolic therapies on osteoblast progenitors. This project aligns with the overall goal of the CORT in that skeletal stem cells (including osteoblast progenitors) represent an important, yet poorly understood, target of osteoporosis anabolic therapy. Specifically, this project will focus on determining why the bone anabolic effect of romosozumab (anti-sclerostin antibody) wanes so quickly over time. To accomplish this translational research goal, complementary approaches will be pursued using patient and mouse samples. First, in Aim 1, in a prospective clinical study, we will obtain bone-relevant bio-samples (trans-iliac crest core biopsies, bone marrow aspirates, and peripheral blood) from post-menopausal women with osteoporosis during the course of romosozumab therapy. These newly-collected samples will be analyzed along with bone marrow samples previously obtained from post-menopausal women treated with teriparatide. Therefore, we will compare and contrast the effects of these two distinct osteoporosis anabolic agents. In this project, bone marrow aspirates will be analyzed by multi-color flow cytometry, and non-hematopoietic stromal cells will be studied using a cocktail of antibodies that detects surface-expressed antigens that sub-divide marrow stroma in order to define treatment effects on human marrow stromal fractions. In addition, non-hematopoietic stromal cells will be prospectively isolated for single cell RNA-sequencing. This analysis will afford unbiased perspectives on the molecular heterogeneity of marrow stroma, effects of osteoporosis anabolic treatments, and key information to explain the waning efficacy of romosozumab over time. To complement these studies using human-derived samples, in Aim 2 we will combine lineage tracing with single cell RNA-sequencing in mice to assess the effects of sclerostin antibody on osteoblast precursors over time. Incorporating lineage tracing into our approach will prove that putative progenitor populations become osteoblasts, and build confidence in cross-species, conserved gene regulatory networks that are responsible the effects of osteoporosis anabolic agents over time. Results from this project will be compared to comparable approaches in Project 2 which focus on osteocytes. The Bioinformatics Core will play a crucial role in analysis of cross-species single cell transcriptomic data. Ultimately, this merged analysis will generate powerfully-informed, novel hypotheses regarding bone stem cell biology and responses to bone anabolic agents.