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Novel Lineage Tracing System To Identify Site-specific Ectopic Skeletal Stem Cells
Chase A. Pagani, BA1, Amanda K. Huber, PhD1, Charles Hwang, BS1, Noelle Visser, MS1, Nicole Patel, BS1, Karen Kessel, BS1, Kaetlin Vasquez, MS1, Jane Y. Song, BS1, Deneen M. Wellik, PhD2, Benjamin Levi, MD1.
1University of Michigan Medical School, Ann Arbor, MI, USA, 2University of Wisconsin - Madison Stem Cell and Regenerative Medicine Center, Madison, WI, USA.

Purpose/Introduction: Traumatic heterotopic ossification (HO) is the formation of bone outside of the healthy skeletal system after traumatic injury causing decreased range of motion and discomfort in affected patients. Previous research has identified potential progenitor cells of HO, however, this is still contended. Better understanding of the cells contributing to HO will allow for targeted treatment to reduce HO in high-risk patients. It was recently discovered that cells expressing Hoxa11, an embryonic patterning transcription factor expressed exclusively in the zeugopod, is expressed by mesenchymal stem cells (MSCs) into adulthood and all skeletal lineages are marked downstream of these progenitors using a CreERT2 lineage tracing system. Currently, most lineage tracing systems label MSCs throughout the body, making it difficult to determine if genetic deletion is due to systemic or local effects. We hypothesize, using a mouse model of HO that Hoxa11+ MSCs will differentiate into site-specific HO-progenitor cells, and eventual form HO. Methods: Hoxa11-CreERT2;ROSA-TdTomato mice were treated with tamoxifen at 6 weeks of age, chased for one week, then injured with an established and reproducible HO-forming Achilles’ tenotomy with concurrent 30% total body surface area back burn. Injured and uninjured hind limb samples were harvested one week, three weeks, and nine weeks post-injury, sectioned for immunofluorescent histology, and imaged by confocal microscopy (Fig. 1A). Results: Examining the uninjured hindlimb of the Hoxa11-CreERT2;ROSA-TdTomato mouse, tdTomato-marked Hoxa11 lineage-positive cells were found throughout the zeugopod within tendon, enthesis, and bone (Fig. 1B). These cells were PDGFRα+ (not shown). Three weeks after injury, Hoxa11 lineage-positive cells are found at regions of heterotopic ossification in the burn/tenotomy model, specifically at the cut end of the tendon and calcaneus (Fig. 1B). After injury, Hoxa11 lineage+/PDGFRα+ cells express chondrocyte marker, SOX9, and pSMAD3, a downstream TGFβ1 signaling pathway transcription factor central to HO formation. Three weeks after injury, condensing chondrocytes within the HO anlagen are Hoxa11 lineage-positive and are colocalized with early bone marker, RUNX2. At nine weeks post-injury, SP7+ osteoblast and RUNX2+ pre-osteoblasts are marked with tdTomato. Hoxa11 cells were also able to differentiate into adipocytes and showed colocalization with perilipin and adipocyte morphology (Fig. 1C). Conclusions: Our results suggest that Hoxa11+ stem cells differentiate through endochondral ossification into heterotopic bone after injury, highlighting that local cells within the hind limb form the majority of HO bone, rather than circulating progenitor cells. Additionally, this lineage tracing system can be a useful tool to study skeletal stem cells and tendon pathology within the zeugopod.


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