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.