Immobilization after injury alters extracellular matrix and stem cell fate
Amanda K. Huber, Nicole Patel, Chase A. Pagani, Simone Marini, Karthik R. Padmanabhan, Daniel L. Matera, Mohamed Said, Charles Hwang, Ginny Ching-Yun Hsu, Andrea A. Poli, Amy L. Strong, Noelle D. Visser, Joseph A. Greenstein, Reagan Nelson, Shuli Li, Michael T. Longaker, Yi Tang, Stephen J. Weiss, Brendon M. Baker, Aaron W. James, Benjamin Levi
Amanda K. Huber, Nicole Patel, Chase A. Pagani, Simone Marini, Karthik R. Padmanabhan, Daniel L. Matera, Mohamed Said, Charles Hwang, Ginny Ching-Yun Hsu, Andrea A. Poli, Amy L. Strong, Noelle D. Visser, Joseph A. Greenstein, Reagan Nelson, Shuli Li, Michael T. Longaker, Yi Tang, Stephen J. Weiss, Brendon M. Baker, Aaron W. James, Benjamin Levi
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Research Article Bone biology

Immobilization after injury alters extracellular matrix and stem cell fate

Abstract

Cells sense the extracellular environment and mechanical stimuli and translate these signals into intracellular responses through mechanotransduction, which alters cell maintenance, proliferation, and differentiation. Here we use a mouse model of trauma-induced heterotopic ossification (HO) to examine how cell-extrinsic forces impact mesenchymal progenitor cell (MPC) fate. After injury, single-cell (sc) RNA sequencing of the injury site reveals an early increase in MPC genes associated with pathways of cell adhesion and ECM-receptor interactions, and MPC trajectories to cartilage and bone. Immunostaining uncovers active mechanotransduction after injury with increased focal adhesion kinase signaling and nuclear translocation of transcriptional coactivator TAZ, inhibition of which mitigates HO. Similarly, joint immobilization decreases mechanotransductive signaling, and completely inhibits HO. Joint immobilization decreases collagen alignment and increases adipogenesis. Further, scRNA sequencing of the HO site after injury with or without immobilization identifies gene signatures in mobile MPCs correlating with osteogenesis, and signatures from immobile MPCs with adipogenesis. scATAC-seq in these same MPCs confirm that in mobile MPCs, chromatin regions around osteogenic genes are open, whereas in immobile MPCs, regions around adipogenic genes are open. Together these data suggest that joint immobilization after injury results in decreased ECM alignment, altered MPC mechanotransduction, and changes in genomic architecture favoring adipogenesis over osteogenesis, resulting in decreased formation of HO.

Authors

Amanda K. Huber, Nicole Patel, Chase A. Pagani, Simone Marini, Karthik R. Padmanabhan, Daniel L. Matera, Mohamed Said, Charles Hwang, Ginny Ching-Yun Hsu, Andrea A. Poli, Amy L. Strong, Noelle D. Visser, Joseph A. Greenstein, Reagan Nelson, Shuli Li, Michael T. Longaker, Yi Tang, Stephen J. Weiss, Brendon M. Baker, Aaron W. James, Benjamin Levi

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Figure 7

Immobilization results in genetic changes that alter MPC fate.

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Immobilization results in genetic changes that alter MPC fate. (A) Canon...
(A) Canonical correlation analysis of cells from the HO site of day 7 postinjury mobile and immobile mice defines 14 clusters, including an MPC subset. (B) MPCs from scATAC sequencing were identified based on RNA expression in the scRNA-seq results. Trajectory analysis of MPCs from (C) mobile and (D) immobile mice across pseudotime. (E) Adipogenic/osteogenic expression scores calculated on a per MPC basis from the clusters identified in the scRNA-seq analysis of day 0 naive, day 7 mobile, and day 7 immobilized mice. Chromatin accessibility in gene regions specific to the MPC cluster represented by heatmaps of the average log fold change differences in (F) mechanotransduction genes, (G) adipogenic genes, or (H) osteogenic genes compared with locations in other clusters. Heatmaps display the openness in 100 sampled cells from either mobile (Mob) or immobile (Imm) mice that contribute to the MPC cluster from the scATAC-seq analysis.