Skeletal Stem Cells Acquire Developmental Neural Crest Identity during Distraction Osteogenesis
Ryan C. Ransom, B.A., Ankit Salhotra, B.S., Ava C. Carter, BA, Ruth E. Jones, M.D., Deshka S. Foster, M.D., Howard Y. Chang, M.D., Ph.D., Derrick C. Wan, M.D., Michael T. Longaker, M.D., M.B.A..
Stanford University, Stanford, CA, USA.
PURPOSE: During development and tissue regeneration, changes in chromatin structure driven by master transcription factors lead to stimulus-responsive transcriptional programs. Accumulated evidence indicates that mechanical cues are transmitted to the nucleus directly or indirectly to orchestrate transcriptional activities that are crucial for tissue regeneration. We have developed a mouse model of mandibular distraction osteogenesis (DO) which allows for genetic dissection of mechanotransduction during bone formation. Specifically, we examine cell-type-specific responses to mechanical force within the mouse skeletal stem cell (SSC) hierarchy.
METHODS: To investigate the role of mechanotransduction via FAK in distraction osteogenesis, we inhibited FAK using the small molecule PF-573228 (Tocris), compared to DMSO controls. Micro-computed tomography (ÁCT) was performed to detect mineralization at the mid-point of consolidation (POD 29). To consider the cellular-level effects of FAKi under conditions of distraction, prospective FACS isolation was performed at the mid-point of distraction (POD 10). Conditional FAK knockout mice (Sox9::CreERT2;Ptk2fl/fl) were used to probe the effects of genetic ablation of the FAK pathway within the skeletal stem cell hierarchy. FAKcKO mice were placed in mandibular distraction and mandibular calluses were isolated for FACS at the point of mid- distraction (POD 10). We profiled the epigenetic state in DO under FAK inhibition (FAKi) using ATAC-seq and compared those cells to Fx and DO SSCs and BCSPs. To assess whether d-SSCs take on a more NCC-like identity, we compared their chromatin accessibility profiles to those from pre-migratory NCC precursors (NCC Prog) and post-migratory mandibular NCCs (NCC Md).
RESULTS: Using the assay for transposase-accessible chromatin (ATAC-seq), we show that these stem cell populations have a distinct pattern of chromatin accessibility during distraction, including within the FAK pathway (Fig. 1a). Bone formation under conditions of FAK inhibition (FAKi) during distraction was significantly diminished upon ÁCT analysis (Fig. 1b,c). Flow cytometry revealed that disruption of the FAK pathway, at both the genetic and protein level led to a disruption of the normal kinetics of skeletal stem and progenitor cells in response to distraction (Fig. 1d). Activation of this pathway induces a more primitive neural crest gene expression network (Fig. 1e), and the reversion of d-SSCs to a more developmental, NCC-like state is mechanoresponsive via FAK (Fig. 2a-d).
CONCLUSIONS: Here we provide evidence, for the first time, that controlled mechanical advancement of the lower jaw activates FAK signaling events which unlock gene regulatory programs normally active in cranial neural crest cells during facial morphogenesis, leading to an enhanced regenerative potential of tissue-resident SSCs in the adult mandible. Mechanotransduction via FAK in skeletal stem cells during distraction activates regulatory elements normally active in primitive neural crest cells. This reversion to a more developmental chromatin state underlies the robust tissue growth that facilitates stem cell-based regeneration of this adult tissue.
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