Lineage tracing of skeletal progenitor cells during postnatal bone formation

Abstract

INTRODUCTION: Fractures represent a common orthopaedic injury and create a large financial burden for the health care system. Non-union fractures often require surgery to assist the healing process. Understanding the origin of the postnatal skeletal stem cells will allow for locally delivered therapeutic treatments to be more exactly spatially targeted for fracture healing. Two forms of post-natal bone formation were studied, callus formation after fracture and ectopic bone growth. Both forms of bone formation closely follow the mechanisms of endochondral ossification. The Prx1 gene that is known to be expressed by skeletal stem/progenitor cells within the periosteal tissues was used to spatially follow this cell population during ectopic and fracture induced bone formation. OBJECTIVES: The purpose of this study was to define the cell lineages that arise from Prx1 expressing cells in fracture and ectopic bone models. METHODS: Prx1 expressing cells were tracked using Prx1CreER-GFP x RosaAi14 (dTomato indicator) and Prx1CreER-GFP x Ai14i (dTomato indicator) in the Rag1tm strain of transgenic mice. These mice strains respectively received a closed stabilized fracture or human demineralized bone matrix (DBM) that was surgically implanted onto the periosteal surface of the femur to initiate the development of ectopic bone. Tissue was collected at either day 10 or 14 post-fracture surgery or day 8 post-DBM implantation. Prx1CreER-GFP expression was induced by tamoxifen or control animals received no injection or corn oil. Three different tamoxifen induction protocols (30 days prior to surgery to allow for washout, three days prior to, or continuously after either fracture or ectopic bone induction were used. Fluorescent microscopy of the histological images were performed to assess the cell populations that expressed Prx1 and cell counting was used to quantify the percentages of Prx1 positive cells in specific regions of interest. qRT-PCR of Prx-1 mRNA expression was used to provide the relative gene expression of Prx1 in a variety of different tissues. RESULTS: Control animals that received corn oil or no Tamoxifen showed low levels (~5-15%) labeled cells, however there was a ~2 fold increase in labeling with Tamoxifen washout. Labeled cells were present within calvaria, femur, tibia, and forelimb bones, while very low amounts were located at the sternum rib junction but were absent in the vertebra. Up to 55-65% of the cells within fracture callus and ectopic bone models showed Prx1 labeled in both cartilage and bone cells. Most interestingly however was the increased presence of Prx1 positive cells throughout the skeletal muscle, growth plate, medial layers of blood vessels, and adipose tissue of appendiculum. Labeling was not in muscles of the chest and back. Upon investigation of several different organs very low levels of Prx1 positive cells in the kidney, aorta and brain.. However, no Prx1 cells were located in the heart, spleen or liver tissues. CONCLUSIONS: While a population of Prx1 cells was shown previously to be localized to the periosteum and contribute to fracture repair this study showed these cells in multiple tissue types throughout the appendiculum. Our results are the first in vivo demonstration to suggest that there is a multi-potential post natal mesenchymal stem population. This Prx1 positive cell population was not seen in ectopic bone that was induced outside the appendiculum indicating that these cells retained their original embryonic specification into the adult animal