Discovery of a periosteal stem cell mediating intramembranous bone formation

Abstract

Bone consists of separate inner endosteal and outer periosteal compartments, each with distinct contributions to bone physiology and each maintaining separate pools of cells owing to physical separation by the bone cortex. The skeletal stem cell that gives rise to endosteal osteoblasts has been extensively studied; however, the identity of periosteal stem cells remains unclear^1-5. Here we identify a periosteal stem cell (PSC) that is present in the long bones and calvarium of mice, displays clonal multipotency and self-renewal, and sits at the apex of a differentiation hierarchy. Single-cell and bulk transcriptional profiling show that PSCs display transcriptional signatures that are distinct from those of other skeletal stem cells and mature mesenchymal cells. Whereas other skeletal stem cells form bone via an initial cartilage template using the endochondral pathway⁴, PSCs form bone via a direct intramembranous route, providing a cellular basis for the divergence between intramembranous versus endochondral developmental pathways. However, there is plasticity in this division, as PSCs acquire endochondral bone formation capacity in response to injury. Genetic blockade of the ability of PSCs to give rise to bone-forming osteoblasts results in selective impairments in cortical bone architecture and defects in fracture healing. A cell analogous to mouse PSCs is present in the human periosteum, raising the possibility that PSCs are attractive targets for drug and cellular therapy for skeletal disorders. The identification of PSCs provides evidence that bone contains multiple pools of stem cells, each with distinct physiologic functions.

Extended data 1:. Analysis of CTSK-mGFP cells in mouse femur.

a, CTSK-mGFP mesenchymal cells (green) were visualized in the mouse long bones at E14.5. Scale bar 200 μm. Enlarged images of areas marked by the dotted white boxes are provided in i and ii. DAPI for nuclei. b, Immunostaining for CD200 (magenta) confirmed co-localization (shown by yellow arrows) with CTSK-mGFP cells (green) in the periosteum. A separate pool of CD200+ cells are detected at the future primary ossification site (marked by dotted orange line). Nuclei stained with DAPI. Scale bar 20 μm. a-b, 3 independent experiments. c, CTSK-mGFP mesenchymal cells in the long bones of mice were detected by FACS at embryonic day 16.5. d, Visualization of CTSK-mGFP (green) cells in mouse long bones at E16.5. Nuclei stained with DAPI. Scale bar 500 μm. An enlarged view of the areas marked by dotted yellow boxes are shown in i and ii. CTSK-mGFP positive cells (green) were detected in the mouse periosteum (i and ii). e, CD200 (magenta) immunostaining confirmed co-localization with CTSK-mGFP cells (green) in the periosteum (top panel). CTSK-mGFP cells in the primary spongiosa morphologically consistent with osteoclasts stained negative for CD200 (bottom panel). Scale bar 20μm. c-e, 3 independent experiments. f, Visualization of CTSK-mGFP (green) cells in the periosteum (dotted white line) of mouse femur at post-natal day 6 (top) and day 12 (bottom). Nuclei shown by DAPI staining. Scale bar 20 μm. g, An enlarged view of osteocytes within the dotted white box is provided (i). CTSK-mGFP (green), nuclei (DAPI). Scale bar 20μm. 3 independent experiments. (ii) Quantification of total CTSK-mGFP labelled periosteal cells and mGFP labelled osteocytes in the mouse femur (***p= 6.95×10⁻¹⁶, mean ± S.E.M, n= 12 distinct areas of periosteum from 3 independent experiments. 2 tailed Student’s t-test). h, An enlarged view for main text Fig. 1e is provided. Representative images from 3 independent experiments. i, FACS plots showing expression of CD49f (left) and CD51 (right) in CTSK-mGFP cells isolated from long bones of 7-day old mice (Lineage - = Ter119−, CD45− and CD31−). 5 independent experiments. j, 8 week old Ctsk-cre mouse femurs were immunostained for RUNX2 (a, magenta, top), Alkaline phosphatase (ALPL) (b, magenta, middle), and Osteocalcin (c, magenta, bottom). CTSK-mGFP (green), nuclei stained by DAPI, co-localization shown by yellow arrows. Scale bar 20 μm. Representative images from 3 independent experiments. k, 12 day old Ctsk-cre mouse femurs were immunostained for Gremlin 1 (a, magenta, top) and Nestin (b, magenta, bottom). CTSK-mGFP (green), nuclei stained by DAPI, dotted white line indicates periosteum. Scale bar 20 μm. Representative images from 3 independent experiments.

Extended data 2:. FACS analysis on microdissected periosteal tissue and characterization of PSCs.

a, Flow cytometry of CTSK-mGFP cells microdissected from the periosteum of mouse (P7) long bones showing the distribution of PSC, PP1 and PP2 cells. b-c, Flow cytometry showing the distribution of PSCs, PP1 and PP2 cells in mouse long bones at day 15 (b) and day 32 (c) (Lineage - = Ter119−, CD45− and CD31−). a-c, Representative FACS plots from 10 independent experiments. d, Schematic representation of the strategy used for flow cytometry for analysis of periosteal PSC, PP1 and PP2 cell populations. e, FACS plot displaying the distribution of CD146 (i) and CD140α (ii) expression in bone marrow stromal cells. f, FACS plots displaying the distribution of CD140α (i) and CD146 (ii) in mouse periosteum obtained through periosteal microdissection. (Lineage - = Ter119−, CD45− and CD31−). e-f, Representative FACS plots from 5 independent experiments. g, Mouse bone marrow immunostained for CD146 (cyan) and CD140α (magenta). CTSK-mGFP (green), DAPI (blue) and Tomato red (red) to visualize stromal cells. Scale bar 100 μm. Representative images from 3 independent experiments. h, Clonogenic cells detected in the periosteum (top two panels, white arrows) and perichondrium region (bottom panel, white arrows) of mouse femur 2 weeks post induction of β-actin cre with tamoxifen. Enlarged view of areas marked by dotted white line are provided for each image. Scale bar 50 μm. Representative images from 3 independent experiments. i, FACS plots showing in vitro differentiation for PP1 (left plot) and PP2 (right plot) cells after 15 days of culture. Representative FACS plots from 3 independent experiments. j, Alizarin red staining (red) of bone 5 weeks after transplantation of non-CTSK MSCs (left) and PSCs (right) into the kidney capsule. Representative images from 5 independent experiments. k, Relative gene expression for bone (Runx2) and cartilage specific genes (Col2a1, Comp, Chad) 5 weeks after transplant of PSCs and non-CTSK MSCs. Non-CTSK MSC derived cells display significantly higher expression of cartilage specific genes than PSCs. (*p= 0.003 for Col2a1, *p= 0.002 for Comp, *p= 0.002 for Chad, mean ± S.D, n=3, 2 tailed Student’s t-test). l, CTSK-mGFP positive PSCs (green) were immunostained for RUNX2 (magenta, top panel) and Osteocalcin (magenta, middle and bottom panels) 3, 4 or 5 weeks after transplantation into the kidney capsule. Nuclei were stained with DAPI. Scale bar 20 μm. Representative images from 3 independent experiments.

Extended data 3:. Functional characterization of non-CTSK MSC cells, PSCs and its derivatives.

a, Total numbers of PSCs and non-CTSK MSCs in mouse femurs at postnatal day 7, day 15 and day 32. Significant decreases in PSCs are observed at day 15 (**p=0.006) and day 32 (**p=0.009) when compared to day 7. Significant decreases in non-CTSK MSCs are observed at day 15 (***p= 3.8×10⁻⁵) and day 32 (***p=0.0003) when compared to day 7 (mean ± S.D, n =3 independent experiments; 5 animals/group for day 7, day 15; 3 animals/group for day 32; 2-tailed Student’s t-test). b, μCT images of the bone formed by non-CTSK MSCs (left) and PSCs (right) 5 weeks after transplantation. Representative images from 5 independent experiments. c, Quantification of bone volume (BV) when equal numbers of non-CTSK MSCs and PSCs were transplanted into secondary hosts (p = non-significant (ns), mean ± S.E.M, n= 3 independent experiments, 2-tailed Student’s t-test). d, Clonal non-CTSK MSC colonies were split for differentiation into osteoblasts (Alizarin red staining; left) and adipocytes (Oil red O staining; middle; scale bar 20μm). Separately, chondrocyte differentiation potential was assayed (Alcian blue staining, right; scale bar 100μm). Representative images from 4 independent experiments. e, Clonal differentiation capacity of 10 colonies isolated from PSCs and non-CTSK MSCs after subsequent culture under osteogenic (left) and adipogenic (right) differentiation conditions. All 10 colonies from each population were equally multipotent (mean ± S.D, n= 3 independent experiments. f, Bright field images of primary (left), secondary (middle) and tertiary mesenspheres (right, scale bar 20 μm) derived from non-CTSK MSCs. Tomato red (red) expression is shown in the inserts. Representative images from 3 independent experiments. g, Quantification of % of PSCs and non-CTSK MSCs able to form mesenspheres. *p=0.02, one way ANOVA, Dunnett’s multiple comparison test, mean ± S.D, n = 3 independent experiments. h, FACS analysis of in vitro differentiation of non-CTSK MSCs (right and left plots) after 15 days of culture. Color coded boxes (red) indicates parent/daughter gates. i, FACS plots of non-CTSK MSC-derived cells after the first round of mammary fat pad transplantation (Lineage- = Ter119−, CD45− and CD31−). Color coded boxes (red and green) indicates parent/daughter gates. h-i, Representative FACS plots from 3 independent experiments. j, FACS for CD140α (i) and CD146 (ii) in PSCs after transplantation into the mammary fat pad. k, FACS for expression of GFP (i),CD140α (ii), and CD146 (iii) in non-CTSK MSCs after mammary fat pad transplantation. l, PP1 cells were transplanted into the mammary fat pad of primary hosts for 2.5 weeks and then analyzed by FACS (i-iii). Color coded boxes (green and magenta) indicate parent/daughter gates. m-n, PP2 cells were isolated by FACS and implanted into the mammary fat-pad of primary recipients. The PP2-derived cells in primary recipients were analyzed by FACS (m, i-iv), and PP2 cells were re-isolated for transplantation into secondary recipients. PP2-derived cells in secondary recipients were analyzed by FACS (n, i-iv). Color coded boxes (green, magenta and orange) indicate parent/daughter gates. j-n, Representative FACS plots from 3 independent experiments.

Extended data 4:. Differentiation of PSCs, PP1, PP2 cells when transplanted into kidney capsule of secondary hosts and FACS analysis of CTSK-mGFP calvarial cells.

a, CTSK-mGFP positive PSCs (green) were immunostained for Thy1.2 (magenta, top panel), 6C3 (magenta, middle panel) and CD105 (magenta, bottom panels) 3 weeks after transplantation into the kidney capsule of primary recipients. b, CTSK-mGFP positive PP1 cells (green) were immunostained for 6C3 (magenta, top panel) and CD105 (magenta, bottom panels) 3 weeks after transplantation into the kidney capsule. c, CTSK-mGFP positive PP2s (green) were immunostained for Thy1.2 (magenta, top panel), and CD105 (magenta, bottom panels) 3 weeks after transplantation into the kidney capsule. Nuclei were stained with DAPI, Tomato red (red). Scale bar 50 μm. a-c, Representative images from 3 independent experiments. d-e, FACS analysis of PSCs, PP1s and PP2s at P15 in mouse calvarial suture (d) and calvarial periosteum (e). f-g, FACS for THY in CTSK-mGFP cells isolated from calvarial suture (f i, g i) and calvarial periosteum (f ii, g ii) at day 15 (left plots) and day 32 (right plots). d-g, Representative FACS plots from 3 independent experiments. h, CD146 and SCA1 expression in CTSK-mGFP cells from the suture (i, ii) and periosteum (iii, iv) of P6 mouse calvarium. Representative FACS plots from 10 independent experiments. i-j, FACS for CD146 (i i, j i) and SCA1 (i ii, j ii) in calvarial periosteum of mice at day15 (top plots) and day 32 (bottom plots). Representative FACS plots from 3 independent experiments.

Extended data 5:. Functional characterization of CTSK-mGFP positive calvarial suture cells.

a, Bright field images of primary (left; scale bar 10μm) and secondary (right) mesenspheres derived from calvarial suture PSCs (top), PP1 (middle) and PP2 (bottom) cells. GFP (green) expression shown in the inserts. Representative images from 3 independent experiments. b, Quantification of the % of PSC, PP1 and PP2 cells able to form mesenspheres. Tertiary colony formation is significantly reduced in PSCs (**p=0.0034). Both PP1 and PP2 cells show significant reduction in secondary (***p=0.0002 for PP1and *p=0.016 for PP2) and tertiary mesensphere formation (****p=0.0001 for PP1, ****p=0.0001 for PP2). One way ANOVA, Dunnett’s multiple comparison test, mean ± S.D, n = 3 independent experiment). c, Clonal PSC colonies were split for differentiation into osteoblasts (Alizarin red staining; left) and adipocytes (Oil red O staining; middle; scale bar 10μm). Separately, chondrocyte differentiation potential was assayed (Alcian blue staining, right, scale bar 100μm). Representative images from 3 independent experiments. d, The amount of bone formed by PSCs, PP1, PP2 and non-CTSK MSCs 4 weeks after transplantation into the kidney capsule of secondary hosts was determined by μCT. e, Von Kossa staining (e i, dotted box in green) displaying bone organoid formation in the kidney capsule by PSCs (left panel, top), non-CTSK MSCs (right panel, top), PP1 (left panel, bottom) and PP2 (right panel, bottom) cells. Scale bar 50μm. Safranin O staining (e ii, dotted box in magenta) indicates an absence of cartilage formation (transplant area shown by dotted yellow line) after transplant of PSCs (left) and PP2 (right) cells. Scale bar 50μm f, In vitro osteogenic differentiation of PSCs (left image) and non-CTSK MSCs (right image) as determined by Alizarin red staining (red). d-f, Representative images from 3 independent experiments. g, Heatmap generated from quantitative real time PCR analysis shows differences in gene expression between calvarial suture PSCs and the progenitor populations, PP1 and PP2 cells. h, PSCs were transplanted into a mammary fat pad of primary hosts for 2.5 weeks and then analyzed by FACS (i-iii). Color coded boxes (green and magenta) indicate parent/daughter gates. i, FACS analysis of PP2 cells after transplantation into the mammary fat pad of primary hosts (i-iii).j, FACS analysis shows poor engraftment and loss of PP1 cells (as detected by GFP expression) when transplanted into the mammary fat pad of primary hosts. k-m, FACS plots demonstrating the in vitro differentiation of PSC (k), PP1 (l), and PP2 cells (m) when cultured for 2 weeks. Color coded boxes (magenta) indicate parent/ daughter gates for each analyzed cell population. h-m, Representative FACS plots from 3 independent experiments.

Extended data 6:. Gene expression analysis in CTSK-mGFP cells isolated from mouse femurs.

a, Bulk RNA sequencing was conducted in FACS isolated PSC, PP1, PP2 and non-CTSK mGFP MSCs from 6 day old mouse femurs. Hierarchical clustering analysis performed on RNA-seq data. b, Heatmap generated from bulk RNA sequencing of FACS sorted cells shows differences in gene expression between PSCs and the progenitor populations, PP1 and PP2 cells. c, Von Kossa staining indicates bone organoid formation by PSCs (left), PP1 (middle) and PP2 cells (right) 5 weeks after transplantation into the kidney capsule. Scale bar 20μm. Representative images from 3 independent experiments with 3 animals/group. d, Significantly reduced bone formation (BV) in PP1 (*p=0.04) and PP2 (*p=0.032) cells when compared to PSCs after transplantation. mean ± S.D, n= 3 independent experiments, 2 tailed Student’s t-test. e, Relative expression of Tnn (i) Tnmd (ii) Ifitm5 (iii) and Bglap (iv) among the four clusters (identified by 1–4) that were generated thorough analysis of 658 CTSK-mGFP positive mesenchymal cells using the Seurat package. Gene expression along the y-axis and Cell clusters (1–4) along x-axis. f, Expression of genes such as Bglap (i), Alpl (ii), Ifitm5 (iii), Tnn (iv), Tnmd (v) and Kera (vi) are shown by pseudcoloring of t-SNE plots. g, Heatmap generated from bulk RNA sequencing shows differences in gene expression between PSCs and the progenitor populations, PP1 and PP2 cells. h-k, Monocle analysis of Cell seq data. h, A brightfield image of a colony that was generated from single cell sorting of RAW 264.7 cells by FACS. Scale bar 20 μm. Representative images from 3 independent experiments. i, Graphs indicate the percentage of wells that received sorted cells by FACS (left graph) and the percentage of doublets detected in those wells (right graph) (mean ± S.D; n=4).j Data represents the total mRNA amount in the two 384-well plates (Plate 47 and Plate 48) that were sequenced by CELL-SEQ2.k, Pie chart displays that the analyzed CTSK-mGFP positive cells were mesenchymal in origin.

Extended data 7:. μCT, histomorphometric analysis and characterization of cells isolated from Osx ^flox; Ctsk-cre⁺ mouse femur.

a, μCT images of longitudinal sections of femurs from Osx^fl/fl; Ctsk-cre mice or littermate controls at 4-weeks of age. b, H and E staining showing growth plate morphology in Osx^fl/fl; Ctsk-cre mice or littermate controls. a-b, Representative images from 5 independent experiments. c, Graphs display measurement of bone length (i), midshaft along long axis (ii) and midshaft along short axis (iii). Osx ^fl/fl; Ctsk-cre⁺ mice show a significant reduction in bone length (i) compared to Osx ^fl/+; Ctsk-cre⁺ (*p= 0.039) and Osx^+/+ Ctsk-cre⁺ (*p= 0.034) (mean ± S.D, n = 6 animals per group, 2 tailed Student’s t-test). d, Quantification of bone volume/total volume (BV/TV) for trabecular bone (ns = non-significant, mean ± S.E.M, n = 4 animals per group, 2 tailed Student’s t-test). e, Quantification of histomorphometric parameters. Cortical mineral apposition rate (MAR; μm/day) (*p= 0.031; mean ± S.E.M, n=5 animals / group at 4 weeks of age; 2-tailed Student’s t-test). f, TRAP staining of osteoclasts in the femur of the indicated mice at 4 weeks of age. Trabecular bone area of mouse femurs. Scale bar 100μm. Representative images from 4 independent experiments. g, Quantification of osteoclast number/bone perimeter (No. Oc / B. Pm). (ns = non-significant, mean ± S.E.M, n = 4 animals per group, 2 tailed Student’s t-test). h, μCT images showing the amount of bone formed when periosteal PSCs (left column) and endosteal MSCs (right column) isolated from femurs of Osx^+/+ ; Ctsk-cre⁺ (top, i) and Osx^fl/fl ; Ctsk-cre⁺ mice (bottom, ii) were transplanted into the kidney capsule.i, Von Kossa staining (black) of bone organoids formed after transplantation of periosteal PSCs (left column) and endosteal MSCs (right column) isolated from Osx^+/+ ; Ctsk-cre⁺ (top, i) and Osx^fl/fl ; Ctsk-cre⁺ mice (bottom, ii) and transplanted into the kidney capsule. Scale bar 20μm.h-i, Representative images from 3 independent experiments.j, Alizarin red staining (red) of periosteal PSCs (left column) and endosteal MSCs (right column) isolated from the femur (i, ii) and calvarial sutures (iii, iv) of Osx^+/+ ; Ctsk-cre⁺ (top panel) and Osx^fl/fl ; Ctsk-cre⁺ mice (bottom panel) after culture under osteoblast differentiation conditions. Representative images from 3 independent experiments. k-l, FACS plots of contralateral unfractured femurs (k) and fractured femurs (l). Color coded boxes (red) indicate parent/daughter gates. Representative FACS plots from 3 independent experiments. m, A significant increase (*p= 0.019) is seen in non-CTSK MSCs in callus tissue 8-days post fracture. Values displayed represent the absolute number of cells isolated per fracture callus. mean ± S.D, n = 4 independent experiments, 4 animals/group; 2 tailed Student’s t-test. n, Graph displays significantly (*p= 0.017) higher fold amount of PSCs than non-CTSK MSCs in the fractured callus (mean ± S.D; n=3 independent experiment; 2-tailed Student’s t-test). Values displayed are normalized relative to the pre-fracture numbers of the same corresponding population to demonstrate the fold expansion after fracture. o-p, FACS plots on fractured callus after 3 (o) and 6 days (p) of fracture. Color coded boxes (green) indicate parent/daughter gates. Representative FACS plots from 3 independent experiments.

Extended data 8:. Ctsk-cre; mTmG mouse femur 6 days and 15 days post fracture.

a, Mouse femur 6 days post-fracture. An enlarged view of areas indicated by dotted white boxes is provided in inserts a, b, and c. CTSK-mGFP (green), Tomato red (red). Scale bar 500μm. Representative images from 3 independent experiments. b, Mouse femur 15 days post-fracture. An enlarged view of areas indicated by dotted white boxes is provided in inserts a, b, and c. CTSK-mGFP (green), Tomato red (red). Scale bar 500μm. Representative images from 3 independent experiments.

Extended data 9:. Characterization of CTSK-mGFP cells of mouse femur after fracture.

a, The periosteum of mouse femur 6 (left), 9 (middle) and 15 (right) days post-fracture. CTSK-mGFP (green), Tomato red (red). b, H and E staining of callus tissue 6 (top), 9 (middle) and 15 (bottom) days post-fracture. c, CD200 (magenta) immunostaining was conducted on femurs 6 (top two panels) and 9 (bottom panel) days post-fracture. CTSK-mGFP (green), DAPI for nuclei. d, Immunostaining for type 2 Collagen (magenta) was conducted 9 days post-fracture. e, TRAP staining (magenta) identifying osteoclasts in the bone callus (top) and bone marrow (middle) of fractured femurs. Few to no TRAP-positive cells were present in the periosteal region (bottom). CTSK-mGFP (green), DAPI for nuclei. a-e, Representative images from 3 independent experiments. f, PSCs isolated from fracture callus were transplanted into kidney capsule secondary hosts. μCT images of bone formation at 3 (left), 4 (middle) and 5 weeks (right) after PSC transplantation to the kidney capsule (i). Safranin O staining (red), and Von-Kossa staining (black) were performed on sectioned kidney samples to detect cartilage and bone at 3 (ii), 4 (iii) and 5 weeks (iv) after PSC transplantation. Scale bar 10 μm. H and E stains indicate that PSCs isolated from fracture callus are competent to recruit host-derived hematopoietic elements at the site of transplantation (yellow arrows, v). g, Immunostaining reveals co-localization of CTSK-mGFP cells (green) with cartilage specific markers such as Comp (magenta, top two panels) and Aggrecan (Acan, magenta, bottom panel) at 4 weeks after PSC transplantation. Scale bar 20μm. f-g, Representative images from 3 independent experiments.

Extended data 10:. Characterization of Osx ^flox; Ctsk-cre mice and human periosteal cells.

a, μ-CT images of Osx ^flox; Ctsk-cre mice 12 days post fracture. b, Safranin O staining was performed to detect cartilage in the callus 12 days after fracture. a-b, Representative images from 3 independent experiments. c, Significantly higher amounts of cartilage were detected in Osx^fl/fl ; Ctsk-cre⁺ mice when compared to Osx^fl/+ ; Ctsk-cre⁺ mice (*p=0.035) and Osx ^+/+; Ctsk-cre⁺ (*p=0.04) mice (mean ± S.D; n=3 independent experiment, 3 mice / group; 2-tailed Student’s t-test). d, H and E staining of callus tissue 12 days after fracture. Representative images from 3 independent experiments. e, Significantly lower bone volume (BV) was detected in Osx^fl/fl ; Ctsk-cre⁺ mice when compared to Osx^fl/+ ; Ctsk-cre⁺ mice (***p=0.0002) and Osx^+/+; Ctsk-cre⁺ (**p=0.002) mice (mean ± S.D; n=3 independent experiment, 3 animals / group; 2-tailed Student’s t-test). f, Safranin O staining for callus tissue 3 weeks after fracture. Representative images from 3 independent experiments. g, Significantly higher amounts of cartilage were detected in Osx^fl/fl ; Ctsk-cre⁺ mice when compared to Osx^fl/+ ; Ctsk-cre⁺ mice (***p=0.0005) and Osx ^+/+; Ctsk-cre⁺ (***p=0.0003) mice at 3 weeks post-fracture (mean ± S.D; n=3 independent experiment, 6 animals for control; 5 animals for het and knockout; 2-tailed Student’s t-test. h-i, FACS for CD49f, CD51 (h) Lepr (i) and CD146 (j) in human periosteal cells obtained from the femur. Representative FACS plots from 10 independent experiments. k, In vitro differentiation of h-PSCs (k, i-ii) and h-PP1 cells (k, iii-iv) after 3 weeks of culture. Color coded boxes (green) indicate parent/daughter gates for each cell type. Representative FACS plots from 3 independent experiments. l, Safranin O staining showing an absence of cartilage formation after h-PSC (left), h-PP1 (middle) and h-PP2 (right) transplantation into the kidney capsule of immunocompromised mice. The area containing the transplanted tissue is shown by the dotted yellow line. Representative images from 3 independent experiments.

Figure 1:. Cathepsin K-cre labels periosteal mesenchymal cells.

a, mGFP (green) signal in femur of Ctsk-cre; mTmG mice at postnatal day 10 (P10). Scale bar 500μm. Enlarged view of dotted white box. b, Endosteal CTSK-mGFP+ cells express the osteoclast marker TRAP (magenta). DAPI (blue) for nuclei.). a, b 5 independent experiments. c, Distribution of CTSK-mGFP cells in the endosteal digest (i, ii), periosteum (iii) and total bone digests (iv) by FACS. ****p=1.69×10⁻⁵ (v) (mean ± S.D; n=4; 2 tailed Student’s t-test). d-e, A subset of CTSK-mGFP (green) periosteal cells of the femur expresses type 1 Collagen (d, magenta; orange arrow, scale bar 25μm) and a subset expresses CD200 (e, magenta; yellow arrows, scale bar 200μm). DAPI (blue) for nuclei. Enlarged view of e, i-ii is provided in Extended data. 1h. 3 independent experiments. f-g, Flow cytometry of P7 mice to identify periosteal stem cells (PSC) and progenitor cells (PP1, PP2) in long bones. Color coded boxes (yellow, red, green) indicate parent/daughter gates. h, % of PSC, PP1 and PP2 populations over time in long bone digests. For PP1, **p = 0.0063 at day 15, ****p=0.0001 at day 32. PP2, **p = 0.0022 at day 15, ***p =0.0001 at day 32. One way ANOVA, Sidak’s multiple comparison test; mean ± S.D; n=5 for days 7 and 15, n=3 for day 32, representative of 3 independent experiments. i-k, Flow cytometry for LEPR (i), CD146 (j) and CD140α (k) versus CTSK-mGFP in long bones. 5 independent experiments. l-m, CTSK-mGFP cells display significantly fewer CD146+ (****p=0.0000029) and CD140α+ (**p= 0.0014) cells than non-CTSK mGFP cells (mean ± S.D; 3 independent experiments; 2-tailed Student’s t-test).

Figure 2:. Functional characterization of periosteal stem cells.

a, Brightfield images, primary (left; scale bar 20μm) and secondary (right; scale bar 10μm) mesenspheres derived from PSCs (top), PP1s (middle) and PP2s (bottom). GFP (green) in insert. 3 independent experiments. b, % of cells able to form spheres (top) and cell number/sphere (bottom) in PSCs, PP1s and PP2s. **p=0.0036 for % PSC tertiary mesenspheres, ****p=0.0001 for % PP1 secondary and tertiary mesenspheres, ***p= 0.0002 for % PP2 secondary, ****p= 0.0001 for % PP2 tertiary mesenspheres, (n=3 independent experiments). Dunnett’s multiple comparison test; mean ± S.D (top, bottom graphs) c, Immunostaining for CD200 (magenta) in primary (top) and secondary (bottom) PSC derived mesenspheres. DAPI (blue), scale bar 100μm. 3 independent experiments. d, Sorted PSCs cultured for 15 days and analyzed by flow (i, ii) 3 independent experiments. e, Single cell-derived PSC colonies were split for differentiation into osteoblasts (Alizarin red staining; left), adipocytes (Oil red O staining; middle; scale bar 50μm). Separately, chondrocyte differentiation potential was assayed (Alcian blue staining, right; scale bar 200μm). 3 independent experiments. f, Non-CTSK MSCs and CTSK-mGFP+ PSCs were transplanted into kidney capsule of secondary recipients (i, dotted black line). Donor origin of PSCs was confirmed with GFP retention (ii). Immunostaining for type 2 collagen (green in left box; magenta in right box) on 2 week non-CTSK MSC (red, left box) and PSC (green, right box)-derived organoids (iii). Safranin O (red) for cartilage and von Kossa (black) for mineralized bone on non-CTSK MSC (red box) and PSC (green box)-derived organoids 4 (top), 5 (middle) and 6 weeks (bottom) after transplantation (iv). Hematoxylin and eosin, non-CTSK MSC (top) and PSC (bottom)-derived organoids (v). 8 independent experiments. g, Schema of serial transplantation of PSCs into mouse mammary fat pad. h, FACS plots of PSC-derived cells after the first (i-iv) and second round of transplantation (v-viii). (Lineage- = Ter119−, CD45− and CD31−). Color coded boxes (green, magenta, orange) indicate parent/ daughter gates. 3 independent experiments.

Figure 3:. Periosteal stem cells in mouse calvarium and gene expressional analysis on mouse femoral periosteal cells.

a, CTSK-mGFP (green) cells in mouse calvarium at P15 (top) and P32 (bottom) (sagittal suture, white arrow; calvarial periosteum, blue arrow, scale bar 200μm). 3 independent experiments. b, THY expression in CTSK-mGFP cells from P6 suture (left) and calvarial periosteum (right).c, PSC, PP1 and PP2 in suture (i) and calvarial periosteum (ii) at P6. 10 independent experiments. d, Relative proportion of PSC, PP1and PP2 cell populations at day 6 in suture (green) and calvarial periosteum (purple). ***p = 2.7×10⁻⁵, *p=0.04, **p= 0.002 mean ± S.D; n=3 independent experiment, 5 animals pooled per group; 2-tailed Student’s t-test). e, Principal component analysis of RNA sequencing from FACS sorted PSC, PP1, PP2 and non-CTSK MSC populations from P6 mouse femurs. n=4 per group. f, Heatmap of gene expression in PSCs and non-CTSK MSCs. (g-k) 658 CTSK-mGFP+ mesenchymal cells isolated by FACS from P6 mouse femur and subjected to CEL-SEQ2. g, A two-dimensional representation (t-SNE) of global gene expression (Cluster 1= 276 cells, cluster 2=215 cells, cluster 3 = 145 cells, cluster 4=16 cells). h, Relative expression of Col2a1, Sox9, Kera, Alpl, Ly6a, Acta2 among the four clusters. Gene expression along y and clusters (1–4) along x-axes. i, Monocle analysis of CEL-SEQ2 data. Single cell trajectory obtained via unsupervised ordering of 658 CTSK-mGFP cells based on State (i) and Pseudotime (ii). Labelling of PSCs (iii), THY+ (iv) and SCA1+ (v) cells based on flow cytometry assessment of protein surface immunophenotype on the differentiation trajectory. j, Monocle analysis for relative gene expression versus pseudotime. k, Heatmap of differentially expressed genes across pseudotime.

Figure 4:. PSCs contribute to bone formation and fracture healing; human samples contain PSC-like cells.

a-b, μCT, skull (a) and femur (b; i-iv enlarged view), 4-week mice. Scale, 1mm. c, μCT, femur cortex. Cortical porosity, red arrows. d, H & E, mouse periosteum (*, insert). Scale, 20μm. n = 5 animals/group. e, Calcein labelling, mouse femur (i-iii), Scale, 10μm. (*p= 0.021; **p = 0.007, mean ± S.E.M, n=5 animals/group at 4 weeks old; 2-tailed Student’s t-test, 5 independent experiments). f, FACS in contralateral (i, ii) and fractured femurs (iii, iv). Green, parent/daughter gates.*p= 0.018, v; **p=0.009, vi in callus tissue 8-days post fracture (mean ± S.E.M; n=5; 2-tailed Student’s t-test). Ctsk-cre; mTmG mouse femurs 9-days post-fracture (vii). Enlarged views, white. CTSK-mGFP (green), mTomato (red), 3 independent experiments. g, Alizarin red, PSCs from fractured (bottom) or contralateral femur (top) (scale 200μm). 3 independent experiments. h, Safranin O (i, iii) and von-Kossa (ii, iv) 4 weeks after transplantation of PSCs from fractured (bottom) or contralateral femurs (top). 3 independent experiments. i, μCT, femur 3 weeks post fracture. n=8/group. j, H& E, fracture callus. Scale, 200μm. 3 independent experiments. k, Fracture non-union (Fisher’s exact test, *p = 0.025, n= 7, control and het n= 8, knockout). l, Callus bone volume. *p=0.042; mean ± S.E.M, n= 4, control and het; n=5, knockout, 2-tailed Student’s t-test). m, Immunohistochemistry, human femoral periosteum. H & E (left; scale bar 200μm), Cathepsin K (middle, red arrow), CD200 (right). High power insert (green). 4 independent experiments. n, FACS on human periosteum. n=10 experiments. o, Alizarin red (left, scale 200μm, low power insert), Oil red O (middle; white arrows; scale 50μm) and Alcian blue (right, scale 200μm) on cultured h-PSCs. n= 3. p, Von Kossa staining (black), bone organoids after h-PSC (left), h-PP1 (middle) and h-PP2 (right) xenograft. 3 independent experiments.