Figures and data
Heterogeneity of the periosteum at steady state
A. Experimental design. Nuclei were extracted from the periosteum of uninjured tibia and processed for single-nuclei RNAseq. B. Sorting strategy of nuclei stained with Sytox-7AAD for snRNAseq. Sorted nuclei are delimited by a red box. C. UMAP projection of color-coded clustering of the uninjured periosteum dataset. Six populations are identified and delimited by black dashed lines. D. Violin plots of key marker genes of the different cell populations.
Identification of periosteal SSPCs in the intact periosteum
A. UMAP projection of color-coded clustering of the subset of SSPC/fibroblasts. B. Feature plots of Prrx1 and Pdgfra in the subset of SSPC/fibroblasts. C. Feature plots of key marker genes of the different cell populations. D. Dot plot of the stemness markers Pi16, Ly6a (SCA1), Cd34, and Dpp4. E. Violin and feature plots of Cytotrace scoring in the subset of SSPC/fibroblasts, showing that SCA1+ SSPCs (cluster 0) are the less differentiated cells in the dataset. F. Experimental design: GFP+ SCA1+ and GFP+ SCA1- were isolated from uninjured tibia of Prx1Cre; R26mTmG mice and used for in vitro CFU assays or grafted at the fracture site of wild-type mice. G. In vitro CFU assay of murine periosteal Prx1-GFP+ SCA1+ and Prx1-GFP+ SCA1- cells. H. High magnification of SOX9 immunofluorescence of callus section 14 days post-fracture showing that GFP+SCA1+ cells contribute to the callus (white arrowheads) while GFP+ SCA1- cells are not contributing (n=3 per group).
Periosteal response to fracture at single-nuclei resolution
A. Experimental design. Nuclei were extracted from the periosteum of uninjured tibia of wild-type mice and from the injured periosteum and hematoma/callus at days 3, 5 and 7 post-tibial fracture and processed for single-nuclei RNAseq. B. UMAP projection of color-coded clustering of the integration of uninjured, day 3, day 5 and day 7 datasets. Eleven populations are identified and delimited by black dashed lines. C. Violin plots of key marker genes of the different cell populations. D. UMAP projection of the combined dataset separated by time-point. E. Percentage of cells in SSPC, injury-induced fibrogenic cell, osteoblast, chondrocyte, and immune cell clusters in uninjured, day 3, day 5 and day 7 datasets.
Cellular organization of the fracture callus
A. Picrosirius staining of the uninjured periosteum. B. Immunofluorescence and RNAscope on adjacent sections show the presence of SSPCs (Pi16-expressing cells) in the fibrous layer (fl), Postn-expressing cells in the cambium layer (cl), OSX+ osteoblasts, immune cells (CD45+) and endothelial cells (PECAM1+) in the periosteum (n=3 per group). C. Safranin’O staining of longitudinal callus sections at day 3-post tibial fracture. D. Immunofluorescence and RNAscope on adjacent sections show absence of SSPCs (Pi16+), and presence of IIFCs (Postn+) and immune cells (CD45+) in the activated periosteum and hematoma at day 3-post fracture. Chondrocytes (SOX9+,white arrowhead), osteoblasts (OSX+,white arrowhead), immune cells (CD45+) and endothelial cells (PECAM1+) are detected in the activated periosteum (n=3 per group). E. Safranin’O staining of longitudinal callus sections at day 5-post tibial fracture. F. Immunofluorescence and RNAscope on adjacent sections show IIFCs (Postn+), chondrocytes (SOX9+, white arrowhead), osteoblasts (OSX+,white arrowhead), immune cells (CD45+) and endothelial cells (PECAM1+) in the fibrosis, chondrocytes (SOX9+) in the cartilage and osteoblasts (OSX+), immune cells (CD45+,white arrowhead) and endothelial cells (PECAM1+) in the new bone. (n=3 per group). G. Safranin’O staining of longitudinal callus sections at day 7-post tibial fracture. H. Immunofluorescence and RNAscope on adjacent sections show IIFCs (Postn +), chondrocytes (SOX9+,white arrowhead), osteoblasts (OSX+,white arrowhead), immune cells (CD45+) and endothelial cells (PECAM1+) in the fibrosis, chondrocytes (SOX9+) in the cartilage and osteoblasts (OSX+), immune cells (CD45+,white arrowhead) and endothelial cells (PECAM1+) in the new bone (n=3 per group). Scale bars: A-B-E: 1mm, B-D-F: 100µm.
Periosteal SSPCs activate through a common fibrogenic state prior to undergoing osteogenesis or chondrogenesis
A. SSPCs, injury-induced fibrogenic cells (IIFCs), chondrocytes and osteoblasts from integrated uninjured, day 3, day 5 and day 7 post-fracture samples were extracted for a subset analysis. B. UMAP projection of color-coded clustering (left), color-coded sampling (middle) and monocle pseudotime trajectory (right) of the subset dataset. The four populations are delimited by black dashed lines. C. (top) Feature plots of the stem/progenitor, fibrogenic, chondrogenic and osteogenic lineage scores (middle) Scatter plot of the lineage scores along pseudotime. (bottom) Violin plot of the lineage score per time point. D. Distribution of the cells along the pseudotime per timepoint. E. Schematic representation of the activation trajectory of pSSPCs after fracture.
In vivo validation of pSSPC activation trajectory
A. (Top) Representative Safranin’O staining on longitudinal sections of the hematoma/callus at day 5 post-fracture. The callus is composed of fibrosis, cartilage (red dashed line) and bone (green dashed line). (Middle, box 1) Immunofluorescence on adjacent section shows decreased expression of POSTN (green) and increased expression of SOX9 (red) in the fibrosis-to-cartilage transition zone. (Bottom, box 2) Immunofluorescence on adjacent section shows decreased expression of POSTN (green) and increased expression of OSX (red) in the fibrosis-to-bone transition zone (n=3 per group). B. Experimental design: GFP+ SCA1+ SSPCs were isolated from uninjured tibia of Prx1Cre; R26mTmG mice and grafted at the fracture site of wild-type mice. Safranin’O staining of callus sections at day 5 post- fracture and high magnification of POSTN immunofluorescence of adjacent section showing that GFP+ cells contribute to the callus and that grafted SSPCs differentiate into POSTN+ IIFCS (white arrowheads) (n=4 per group). C. Experimental design: GFP+ IIFCs from periosteum and hematoma at day 3 post- fracture tibia were isolated from Prx1Cre; R26mTmG mice and grafted at the fracture site of wild-type mice. Safranin’O of callus sections at day 14 post-fracture and high magnification of OSX and SOX9 immunofluorescence of adjacent sections showing that GFP+ cells contribute to the callus and that grafted IIFCs differentiate into OSX+ osteoblasts (box 3, white arrowheads) and SOX9+ chondrocytes (box 4, white arrowheads) (n=4 per group). Scale bars: Low magnification: A: 500µm, B-C: 1mm. High magnification: 100µm.
Characterization of injury-induced fibrogenic cells
A. Gene ontology analyses of upregulated genes in IIFCs (clusters 2 to 6 of UMAP clustering from Fig. 5). B. Dot plot of ECM genes in UMAP clustering from Fig. 5. C. Feature plot per cluster and scatter plot along pseudotime of the mean expression of ECM genes. D. Gene regulatory network (GRN)-based tSNE clustering of the subset of SSPCs, IIFCs, chondrocytes and osteoblasts. E. Activation of Mta3, Six1, Sox9 and Sp7 regulons in SSPCs, IIFCs, chondrocytes and osteoblasts. Blue dots mark cells with active regulon. F. Number of regulons activated per cell in the SSPC, IIFC, osteoblast (Ob) and chondrocyte (Ch) populations. Statistical differences were calculated using one-way ANOVA. ***: p-value < 0.001. G. Heatmap of activated regulons in SSPC, IIFC, osteoblast (osteob) and chondrocyte (chondro) populations. H. Scatter plot of the activity of the combined fibrogenic regulons along monocle pseudotime from Fig. 5. I. Reactome pathway analyses of the fibrogenic regulons shows that the 3 most significant terms are related to Notch signaling (blue). J. Feature plot in Seurat clustering and scatter plot along monocle pseudotime of the Notch signaling score.
Gene regulatory network analyses identify gene cores driving fibrogenic to chondrogenic and osteogenic transitions.
A. Activation of Maf, Arntl, and Nfatc2 regulons in SSPCs, IIFCs, chondrocytes and osteoblasts. B. STRING interaction network of the chondro-core 1 and 2 transcription factors (blue and orange respectively). C. Feature plot of chondro-core 1 (top) and chondro-core 2 (bottom) activities in SSPCs, IIFCs, chondrocytes and osteoblasts in Seurat UMAP from Fig. 5. D. Scatter plot of chondro-core 1 (top) and chondro-core 2 (bottom) activities along monocle pseudotime and Acan expressing. E. Activation of Tcf7, Bclb11b and Tbx2 regulons in SSPCs, IIFCs, chondrocytes and osteoblasts. F. STRING interaction network of the osteo-core transcription factors (green) and their related genes shows that most of osteo-core related genes are involved in Wnt pathway (purple). G. Feature plot of the osteo- core activity in SSPCs, IIFCs, chondrocytes and osteoblasts in Seurat UMAP from Fig. 5. H. Scatter plot of osteo-core activity along monocle pseudotime and Ibsp expressing.
IIFCs are the main source of paracrine factors after fracture.
A. Outgoing interaction strengths of the different cell populations of the fracture environment determined using CellChat package. B. Comparison of incoming and outgoing interaction strengths across SSPC, IIFC, chondrogenic and osteogenic populations. C. Outgoing and incoming signaling from and to SSPCs, IIFCs, chondrocytes and osteoblasts. D. Cell-cell interactions identified between SSPCs, IIFCs, chondrocytes and osteoblasts. E. Violin plots of the score of BMP, TGFβ, PDGF, POSTN, PTN and ANGPTL signaling per timepoint. F. Scatter plot along pseudotime and violin plot per time point of the mean expression of the ligand and receptors involved in signaling from IIFCs. G. Circle plot of the interactions between SSPCs, IIFCs, chondrocytes and osteoblasts, showing that most signals received by SSPCs are coming for IIFCs. Ob: osteoblasts, Oc: osteoclasts, Ch: chondrocytes, SC: Schwann cells, Ad: Adipocytes.
Dot plot of marker genes of the populations from uninjured periosteum
Expression of known SSPC markers in the periosteum at steady state.
Feature plots of known markers of SSPCs in the SSPC/fibroblast subset in periosteum at steady state.
Heterogeneity and dynamics of the cell populations in the fracture environment.
A. Feature plots of the lineage score of the different cell populations in the combined fracture datasets. B. Percentage of cells in each cell population per time point.
Dot plot of marker genes of the populations from the combined fracture dataset
Periosteal SSPCs do not express Postn
A. RNAscope experiment showing the presence of Postn expressing cells in the inner cambium layer (cl) of the periosteum and Pi16-expressing cells in the fibrous layer (fl). B. Feature plots of Postn and Runx2 expression in the uninjured periosteum. B. Scatter plot of Runx2 and Postn expression in the uninjured periosteum dataset showing that Postn is mostly expressed by cells expressing Runx2. D. Feature plot of Postn expression in the subset of SSPCs, IIFCs, osteogenic and chondrogenic cells from Fig. 5B. E. Violin plot of Postn expression per time point.
Absence of SSPCs in the injured periosteum.
A. Feature plot of SSPC lineage score in the subset of SSPCs, IIFCs, osteoblasts and chondrocytes separated by time point from Fig. 5B. B. Violin plot of SSPC lineage score by time point.
UMAP projection highlighting the distribution of periosteal fibroblasts in the combined fracture dataset.
Validation of SSPC and IIFC sorting strategies.
A. UMAP projection of the clustering of the uninjured periosteum. B. Feature plots of Prxx1 and Ly6a expression in the uninjured dataset. C. UMAP projection of the clustering of the day 3 post-fracture periosteum and hematoma. D. Feature plots of Prxx1 and Mcam (CD146) expression in the day 3 post- fracture periosteum and hematoma dataset. E. Relative expression of cell population markers by Prx1+ SCA1+ SSPCs from uninjured periosteum and Prx1+ CD146- IIFCs from day 3 post-fracture periosteum and hematoma.
IIFCs are expressing ECM-related genes.
A. Violin plot of the extracellular matrix genes score in the integrated dataset. B. Feature plots of Aspn, Col3a1, Col5a1 and Col8a1 in the subset of SSPCs, IIFCs, osteoblasts and chondrocytes.
IIFCs do not undergo apoptosis.
A. Feature plot of the apoptosis score in the subset of SSPCs, IIFCs, osteoblast and chondrocytes. B. Violin plot of the apoptosis score separated by time points. C. Immunofluorescence of cleaved caspase 3 in callus fibrosis at day 3, 5 and 7 post-fracture. D. Percentage of cleaved caspase 3 positive cells in the fibrosis callus fibrosis at day 3, 5 and 7 post-fracture.
Regulon activity in the subset of SSPCs, IIFCs, osteoblasts and chondrocytes
A. Activity of Mta3, Six1, Sox9 and Sp7 regulons in the UMAP Seurat clustering of SSPCs, IIFCs, chondrocytes and osteoblasts. B. Activity of chondro-core 1 regulons in SCENIC tSNE clustering of SSPCs, IIFCs, chondrocytes and osteoblasts. C. Activity of chondro-core 2 regulons in SCENIC tSNE clustering of SSPCs, IIFCs, chondrocytes and osteoblasts. D. Activity of osteo-core regulons in SCENIC tSNE clustering of SSPCs, IIFCs, chondrocytes and osteoblasts.
Paracrine interactions from IIFCs.
A. Circle plots showing the interaction strengths between IIFCs in clusters 2 and 5 with the other cell populations. B. (left) Feature plot of the subset of SSPCs, IIFCs, chondrocytes and osteoblasts from Fig 5. (right) Outgoing and incoming interaction strengths of the subset of SSPCs, IIFCs, chondrocytes and osteoblasts. C. Dot plots of the expression of the ligands (underlined) and receptors of BMP, TGFβ, PDGF, POSTN, PTN and ANGPTL family involved in cell-cell interactions from IIFCs after fracture.
Lists of genes used for lineage score analyses of murine snRNAseq.
Lists of the regulons composing the cores.
