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Discovery of a periosteal stem cell mediating intramembranous bone formation

Nature volume 562, pages 133–139 (2018)Cite this article

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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 unclear1,2,3,4,5. 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.

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Fig. 1: Cathepsin K-Cre labels periosteal mesenchymal cells.
Fig. 2: Functional characterization of periosteal stem cells.
Fig. 3: Periosteal stem cells in mouse calvarium and gene expressional analysis of mouse femoral periosteal cells.
Fig. 4: PSCs contribute to bone formation and fracture healing; human samples contain PSC-like cells.

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Data availability

RNA-seq and single-cell RNA-seq data have been deposited at the Gene Expression Omnibus under accession numbers GSE106237, linked to the subseries GSE106235 and GSE106236. Microscopy images are available from the corresponding author upon reasonable request.

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Acknowledgements

This project was funded by the Office of the Director of the NIH under award DP5OD021351 given to M.B.G. M.B.G. holds a Career Award for Medical Scientists from the Burroughs Welcome Foundation and a Basil O’Connor Award from the March of Dimes. This content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health. We thank D. Ballon, B. He, S. Mukherjee and the Flow Cytometry Core, Genomics Resources Core, Optical Microscopy Core and the Citigroup Biomedical Imaging Core at Weill Cornell Medicine for their technical support, and B. Sleckman and T. Evans for insightful comments on the manuscript.

Reviewer information

Nature thanks M. T. Longaker, M. Young and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA

    Shawon Debnath, Alisha R. Yallowitz, Sarfaraz Lalani, Ren Xu, Na Li, Mark Eiseman & Matthew B. Greenblatt

  2. Flow Cytometry Core Facility, Weill Cornell Medicine, New York, NY, USA

    Jason McCormick

  3. Genomics Resources Core Facility, Weill Cornell Medicine, New York, NY, USA

    Tuo Zhang

  4. Pathology and Laboratory Medicine Core Facility, Weill Cornell Medicine, New York, NY, USA

    Yifang Liu

  5. Department of Medicine, University of Massachusetts Medical School, North Worcester, MA, USA

    Yeon Suk Yang & Jae-Hyuck Shim

  6. Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Meera Hameed

  7. Orthopaedic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    John H. Healey

  8. Research Division, Department of Orthopaedic Surgery, Hospital for Special Surgery, New York, NY, USA

    Mathias P. Bostrom

  9. Division of Adult Reconstruction and Joint Replacement, Department of Orthopaedic Surgery, Hospital for Special Surgery, New York, NY, USA

    Mathias P. Bostrom

  10. Cancer Genomics and Evolutionary Dynamics, Weill Cornell Medicine, New York, NY, USA

    Dan Avi Landau

  11. New York Genome Center, New York, NY, USA

    Dan Avi Landau

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Contributions

S.D. initiated the study and M.B.G. supervised the project. S.D. and M.B.G. conceived the project. S.D. designed, conducted experiments and analysed data. A.R.Y. performed all mouse surgeries. J.M. supervised flow cytometry. S.L., T.Z. and D.A.L. performed data analysis on bulk RNA-seq and single-cell RNA-seq. R.X., M.E. and J.-H.S. performed cell culture, RT–PCR, immunostaining and μCT analysis. N.L., Y.L. and Y.S.Y. performed μCT, histology and cryosectioning of samples. M.H., M.P.B. and J.H.H. provided access to human samples, helped with sample processing and supervised human studies. S.D. and M.B.G. prepared the manuscript. All authors read and approved the manuscript.

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Correspondence to Matthew B. Greenblatt.

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Extended data figures and tables

Extended Data Fig. 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. 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). Scale bar, 20 µm. Images in a and b are representative of 3 independent experiments. c, CTSK–mGFP mesenchymal cells in the long bones of mice were detected by FACS at E16.5. d, Visualization of CTSK–mGFP cells (green) in mouse long bones at E16.5. Scale bar, 500 µm. An enlarged view of the areas marked by dotted yellow boxes are shown in i and ii. CTSK–mGFP 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 panels). CTSK–mGFP cells in the primary spongiosa morphologically consistent with osteoclasts stained negative for CD200 (bottom panels). Scale bar, 20 µm. Images in c–e are representative of 3 independent experiments. f, Visualization of CTSK–mGFP cells (green) in the periosteum (dotted white line) of mouse femur at postnatal day 6 (top) and 12 (bottom). Scale bar, 20 µm. g, CTSK–mGFP visualization shows rare mGFP+ osteocytes, an enlarged view of the dotted white box is provided (i). Scale bar, 20 µm. Image representative of 3 independent experiments. Quantification of total CTSK–mGFP-labelled periosteal cells and mGFP-labelled osteocytes in the mouse femur (ii). ***P = 6.95 × 10−16; two-tailed Student’s t-test. Data are mean ± s.e.m., n = 12 distinct areas of periosteum from 3 independent experiments. h, An enlarged view from Fig. 1e. 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. Representative plot from 5 independent experiments. j, Femurs from 8-week-old Ctskcre mice were immunostained for Runx2 (magenta, top), alkaline phosphatase (ALPL) (magenta, middle) and osteocalcin (magenta, bottom). Co-localization is shown by yellow arrows. Scale bar, 20 µm. Representative images from 3 independent experiments. k, Femurs of 12-day-old Ctskcre mice were immunostained for gremlin 1 (magenta, top) and nestin(magenta,bottom)).Dotted white line indicates periosteum. Scale bar, 20 µm. Representative images from 3 independent experiments.

Extended Data Fig. 2 FACS analysis of microdissected periosteal tissue and characterization of PSCs.

a, Flow cytometry of CTSK–mGFP cells microdissected from the periosteum of P7 mouse 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). Plots in a–c are representative of results from 10 independent experiments. d, Schematic representation of the strategy used for FACS analysis of periosteal PSC, PP1 and PP2 cell populations. e, FACS plot showing 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. Plots in e and f are representative of results from 5 independent experiments. g, Mouse bone marrow immunostained for CD146 (cyan) and CD140α (magenta). Scale bar, 100 µm. Representative images from 3 independent experiments. h, Clonogenic cells detected in the periosteum (top and middle, white arrows) and perichondrium region (bottom, white arrows) of mouse femur 2 weeks after induction of β-actin-Cre with tamoxifen. Enlarged views of outlined regions are shown. Scale bar, 50 µm. Representative images from 3 independent experiments. i, FACS plots showing in vitro differentiation for PP1 (left) and PP2 (right) 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. Scale bar, 50 µm. k, Relative gene expression for bone- (Runx2) and cartilage-specific genes (Col2a1, Comp, Chad) 5 weeks after transplantation of PSCs and non-CTSK MSCs. Non-CTSK MSC-derived cells display significantly higher expression of cartilage specific genes than PSCs. *P = 0.003 (Col2a1), *P = 0.002 (Comp), *P = 0.002 (Chad); two-tailed Student’s t-test. Data are mean ± s.d., n = 3. l, CTSK–mGFP+ PSCs (green) were immunostained for Runx2 (magenta, top) and osteocalcin (magenta, middle and bottom) 3, 4 or 5 weeks after transplantation into the kidney capsule. Scale bar, 20 µm. Representative images from 3 independent experiments.

Extended Data Fig. 3 Functional characterization of non-CTSK MSCs, PSCs and their 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 number of PSCs are observed at day 15 (**P = 0.006) and day 32 (**P = 0.009) 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) compared to day 7. Two-tailed Student’s t-test. Data are mean ± s.d., n = 3 independent experiments; 5 animals per group for day 7, day 15; 3 animals per group for day 32. 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 when equal numbers of non-CTSK MSCs and PSCs were transplanted into secondary hosts. Data are mean ± s.e.m., n = 3 independent experiments; two-tailed Student’s t-test. ns, not significant. d, Clonal non-CTSK MSC colonies were split for differentiation into osteoblasts (left, alizarin red staining) and adipocytes (middle, oil red O staining) (scale bar, 20 µm). Separately, chondrocyte differentiation potential was assayed (right, alcian blue staining; scale bar, 100 µm). 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. Data are mean ± s.d., n = 3 independent experiments. f, Bright-field images of primary (left), secondary (middle) and tertiary mesenspheres (right) derived from non-CTSK MSCs. Tomato red (red) expression is shown in the insets. Scale bar, 20 µm. Representative images from 3 independent experiments. g, The percentage of PSCs and non-CTSK MSCs able to form mesenspheres. *P = 0.02, one-way ANOVA, Dunnett’s multiple comparison test. Data are mean ± s.d., n = 3 independent experiments. h, FACS analysis of in vitro differentiation of non-CTSK MSCs after 15 days of culture. Red box indicates parent/daughter gate. i, FACS plots of non-CTSK MSC-derived cells after the first round of mammary fat pad transplantation. Colour-coded boxes (red and green) indicate parent/daughter gates. FACS plots in h and i are representative of 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 analysed by FACS (i–iii). Colour-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. PP2 derived cells in primary recipients were analysed by FACS (m, i–iv), and PP2 cells were re-isolated for transplantation into secondary recipients. PP2-derived cells in secondary recipients were analysed by FACS (n, i–iv). Colour-coded boxes (green, magenta and orange) indicate parent/daughter gates. Plots in j–n are representative of results from 3 independent experiments.

Extended Data Fig. 4 Differentiation of PSCs, PP1 and PP2 cells when transplanted into kidney capsule of secondary hosts and FACS analysis of CTSK–mGFP calvarial cells.

a, CTSK–mGFP+ PSCs (green) were immunostained for THY1.2 (magenta, top), 6C3 (magenta, middle) and CD105 (magenta, bottom) three weeks after transplantation into the kidney capsule of primary recipients. b, CTSK–mGFP+ PP1 cells (green) were immunostained for 6C3 (magenta, top) and CD105 (magenta, bottom) three weeks after transplantation into the kidney capsule. c, CTSK–mGFP+ PP2 cells (green) were immunostained for THY1.2 (magenta, top), and CD105 (magenta, bottom) three weeks after transplantation into the kidney capsule. Scale bar, 50 µm. Images in a–c are representative of 3 independent experiments. Scale bar, 50 µm. d, e, FACS analysis of PSCs, PP1s and PP2s at P15 in mouse calvarial suture (d) and calvarial periosteum (e). f, g, FACS for THY1.2 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). Plots in d–g are representative of results 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 day 15 (top plots) and day 32 (bottom plots). Representative FACS plots from 3 independent experiments.

Extended Data Fig. 5 Functional characterization of CTSK–mGFP+ calvarial suture cells.

a, Bright-field images of primary (left; scale bar, 10 µm) and secondary (right) mesenspheres derived from calvarial suture PSCs (top), PP1 (middle) and PP2 (bottom) cells. GFP (green) expression shown in the insets. Representative images from 3 independent experiments. b, Quantification of the percentage of PSC, PP1 and PP2 cells able to form mesenspheres. Tertiary colony formation is significantly reduced in PSCs (**P = 0.0034). PP1 and PP2 cells show significant reduction in both secondary (***P = 0.0002 for PP1 and *P = 0.016 for PP2) and tertiary mesensphere formation (****P = 0.0001 for PP1 and ****P = 0.0001 for PP2). One-way ANOVA, Dunnett’s multiple comparison test; mean ± s.d., n = 3 independent experiments. c, Clonal PSC colonies were split for differentiation into osteoblasts (alizarin red staining, left) and adipocytes (oil red O staining, middle; scale bar, 10 µm). Separately, chondrocyte differentiation potential was assayed (alcian blue staining, right; scale bar, 100 µm). 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 (top left), non-CTSK MSCs (top right), PP1 (bottom left) and PP2 (bottom right) cells. Scale bar, 50 µm. 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, 50 µm. f, In vitro osteogenic differentiation of PSCs (left) and non-CTSK MSCs (right) as determined by Alizarin red staining (red). Images in d–f are representative of 3 independent experiments. g, Heat map 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 analysed by FACS (i–iii). Colour-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. Magenta boxes indicate parent/daughter gates for each analysed cell population. Plots in h–m represent results from 3 independent experiments.

Extended Data Fig. 6 Gene expression analysis in CTSK–mGFP cells isolated from mouse femurs.

a, Bulk RNA-seq analysis of FACS-isolated PSC, PP1, PP2 and non-CTSK-mGFP MSCs from 6-day-old mouse femurs. Hierarchical clustering analysis was performed on RNA-seq data. b, Heat map generated from bulk RNA-seq 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, 20 µm. Representative images from 3 independent experiments with 3 mice per group. d, Significantly reduced bone formation (bone volume) in PP1 (*P = 0.04) and PP2 (*P = 0.032) cells compared to PSCs after transplantation. Two-tailed Student’s t-test. Data are mean ± s.d., n = 3 independent experiments. e, Relative expression of Tnn (i), Tnmd (ii), Ifitm5 (iii) and Bglap (iv) among the four clusters (identified by 1–4) that were generated through analysis of 658 CTSK–mGFP+ mesenchymal cells using the Seurat package. Cell clusters (1–4) along the x axis. f, Expression of genes such as Bglap (i), Alpl (ii), Ifitm5 (iii), Tnn (iv), Tnmd (v) and Kera (vi) are shown by pseudocolouring of t-SNE plots. g, Heat map generated from bulk RNA-seq shows differences in gene expression between PSCs and the progenitor populations, PP1 and PP2 cells. h–k, Monocle analysis of CEL-Seq2 data. h, Bright-field image of a colony that was generated from single-cell sorting of RAW264.7 cells by FACS. Scale bar, 20 µm. Representative image from 3 independent experiments. i, Graphs indicate the percentage of wells that received sorted cells by FACS (left) and the percentage of doublets detected in those wells (right). Data are mean ± s.d., n = 4. j, Data represent the total amount of mRNA in the two 384-well plates (plate 47 and plate 48) that were sequenced using CEL-Seq2. k, Pie chart, showing that the analysed CTSK–mGFP+ cells were mesenchymal in origin.

Extended Data Fig. 7 µCT, histomorphometric analysis and characterization of cells isolated from Osxfl/fl;Ctskcre mouse femur.

a, µCT images of longitudinal sections of femurs from Osxfl/fl;Ctskcre mice or littermate controls at 4 weeks of age. b, Haematoxylin and eosin staining showing growth plate morphology in Osxfl/fl;Ctskcre mice or littermate controls. Images in a and b are representative of 5 independent experiments. Scale bar, 100 µm. c, Bone length (i), midshaft along long axis (ii) and midshaft along short axis (iii). Osxfl/fl;Ctskcre mice show a significant reduction in bone length (i) compared to Osxfl/+;Ctskcre (*P = 0.039) and Osx+/+;Ctskcre (*P = 0.034). Two-tailed Student’s t-test. Data are mean ± s.d., n = 6 animals per group. d, Bone volume/total volume (BV/TV) for trabecular bone. Data are mean ± s.e.m., n = 4 animals per group, two-tailed Student’s t-test. e, Histomorphometric parameters. Cortical mineral apposition rate (MAR; µm day−1). *P = 0.031; two-tailed Student’s t-test; data are mean ± s.e.m., n = 5 animals per group at 4 weeks of age. f, TRAP staining of osteoclasts in the trabecular bone area of femurs of the indicated mice at 4 weeks of age. Scale bar, 100 µm. Representative images from 4 independent experiments. g, Quantification of osteoclast number/bone perimeter (No. Oc/B. Pm). Data are mean ± s.e.m., n = 4 animals per group, two-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+/+;Ctskcre (top) and Osxfl/fl;Ctskcre mice (bottom) were transplanted into the kidney capsule. Scale bar, 1 mm. i, Von Kossa staining (black) of bone organoids formed after transplantation of periosteal PSCs (left column) and endosteal MSCs (right column) isolated from Osx+/+;Ctskcre (top) and Osxfl/fl;Ctskcre mice (bottom) and transplanted into the kidney capsule. Scale bar, 20 µm. Images in h and i are representative of 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+/+;Ctskcre (top panel) and Osxfl/fl;Ctskcre mice (bottom panel) after culture under osteoblast differentiation conditions. Images are representative of 3 independent experiments. k, l, FACS plots of contralateral unfractured femurs (k) and fractured femurs (l). Colour-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. Data are mean ± s.d., n = 4 independent experiments, 4 animals/group; two-tailed Student’s t-test. n, Graph displays significantly (*P = 0.017) higher fold PSC count than non-CTSK MSCs in the fractured callus. Data are mean ± s.d.; n = 3 independent experiment; two-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 of cells from fractured callus after 3 (o) and 6 days (p) of fracture. Colour-coded boxes (green) indicate parent/daughter gates. Representative FACS plots from 3 independent experiments.

Extended Data Fig. 8 Ctskcre;mTmG mouse femur 6 days and 15 days after fracture.

a, Mouse femur 6 days after fracture (top). Bottom, enlarged view of areas indicated by dotted white boxes. CTSK–mGFP (green), mTomato red (red). Scale bar, 500 µm. Images are representative of 3 independent experiments. b, Mouse femur 15 days after fracture (top). Bottom, enlarged view of areas indicated by dotted white boxes. Scale bar, 500 µm. Images are representative of 3 independent experiments.

Extended Data Fig. 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 after fracture. b, Haematoxylin and eosin staining of callus tissue 6 (top), 9 (middle) and 15 (bottom) days after fracture. c, CD200 (magenta) immunostaining of femurs collected 6 (top and middle) and 9 (bottom) days after fracture. d, Immunostaining for type 2 collagen (magenta) 9 days after 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). Images in a–e are representative of 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 (iv) weeks after PSC transplantation. Scale bar, 10 µm. Haematoxylin and eosin staining indicates that PSCs isolated from fracture callus are competent to recruit host-derived haematopoietic 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 and middle) and aggrecan (magenta, bottom) 4 weeks after PSC transplantation. Scale bar, 20 µm. Images in f and g are representative of 3 independent experiments.

Extended Data Fig. 10 Characterization of Osxfl/fl;Ctskcre mice and human periosteal cells.

a, µCT images of Osxfl/fl;Ctskcre mice 12 days post-fracture. Scale bar 1 mm. b, Safranin O staining was performed to detect cartilage in the callus 12 days after fracture. Images in a and b are representative of 3 independent experiments. c, Significantly higher amounts of cartilage were detected in Osxfl/fl;Ctskcre mice compared to Osxfl/+;Ctskcre (*P = 0.035) and Osx+/+;Ctskcre (*P = 0.04) mice. Two-tailed Student’s t-test; data are mean ± s.d., n = 3 independent experiments, 3 mice per group. d, Haematoxylin and eosin staining of callus tissue 12 days after fracture. Representative images from 3 independent experiments. e, Significantly lower bone volume (BV) was detected in Osxfl/fl;Ctskcre mice compared to Osxfl/+;Ctskcre (***P = 0.0002) and Osx+/+;Ctskcre (**P = 0.002) mice. Two-tailed Student’s t-test; data are mean ± s.d., n = 3 independent experiments, 3 animals per group. 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 Osxfl/fl;Ctskcre mice compared to Osxfl/+;Ctskcre (***P = 0.0005) and Osx+/+;Ctskcre (***P = 0.0003) mice at 3 weeks after fracture. Two-tailed Student’s t-test; data are mean ± s.d., n = 3 independent experiments; 6 mice for control, 5 mice each for heterozygote and knockout. h–i, FACS using 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. Colour-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.

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Debnath, S., Yallowitz, A.R., McCormick, J. et al. Discovery of a periosteal stem cell mediating intramembranous bone formation. Nature 562, 133–139 (2018). https://doi.org/10.1038/s41586-018-0554-8

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