Whole bone transplantation is a good model to study bone marrow regeneration.

A. Schematic and picture of the bone transplantation procedure. B. Fold difference quantification of graft femur/host femur cellularity normalized to mean host femur cellularity. Total graft bone marrow cells, BM-MSCs and HSCs were analyzed monthly until 5 months after bone transplantation (BT) (n=3). Ordinary one-way ANOVA with Dunnett multiple comparisons was used to determine statistical significance. C. Schematic illustration of the non-competitive repopulating assay after bone transplantation. D. Donor HSC contribution of graft and host recipients at 4 weeks after bone marrow transplantation (n=10). E. Quantification of tri-lineage (myeloid, B lymphoid, and T lymphoid cells) engraftment 4 weeks post transplantation (n=10). This figure was created using BioRender.com.

Regenerating BM-MSCs are graft-derived and express HSC niche factors.

A. Schematic of a UBC-GFP femur transplanted into a RosamT/mG mouse. B. Representative FACS plots showing the gating strategy to determine the origin of the different cell fractions in the graft 5 months after transplantation of a UBC-GFP femur into a RosamT/mG mouse. C. Representative whole-mount confocal z-stack projections of a UBC-GFP bone transplanted into a RosamT/mG recipient 5 months after transplantation. Vascularization was stained with anti-CD31 and anti-CD144 antibodies. Scale bar = 100µm (n=2 mice). D. Origin of graft BM-MSCs, endothelial cells (EC) and hematopoietic cells (Hemato) analyzed by flow cytometry 5 months after bone transplantation (n=2). E. Schematic of the Nes-GFP femur transplantation into a Nes-GFP mouse recipient. F. Quantitative RT-PCR analysis of mRNA expression of Cxcl12 and Kitl expression relative to Gapdh in graft Nes-GFP+ BM-MSCs compared to steady-state Nes-GFP+ BM-MSCs at multiple time points after transplantation (n= 2-4 mice per time point). One-way ANOVA with Dunnett multiple comparisons was used to determine statistical significance. Data are represented as the mean ± SEM. Unless otherwise noted, statistical significance was determined using unpaired two-tailed Student’s t test. *p<0.05. ** p<0.01. *** p<0.001. ****p<0.0001. This figure was created using BioRender.com.

P-SSCs remain viable and expand after bone transplantation, in contrast to BM-MSCs.

A. Flow cytometric quantification of fold difference of total graft bone marrow and periosteum cellularity to total steady state cellularity. Different time points early after transplantation were analyzed (n=2-8). One-way ANOVA with Dunnett multiple comparisons was used to determine statistical significance. B. Absolute number of CD45-Ter119-CD31-CD51+CD200+ P-SSCs at steady state and 1-, 8- and 15-days post transplantation (n=3-4 mice per time point). One-way ANOVA with Dunnett multiple comparisons was used to determine statistical significance. C. Representative whole-mount confocal z-stack projections of Nes-GFP+ bone graft at steady state, three-, eight-, and fifteen-days post transplantation. Three independent experiments yielded similar results. Scale bar = 100µm D. Total bone marrow cellularity and BM-MSC absolute number 5 months after transplantation of bones with or without intact periosteum (n=3-4 mice per group). Data are represented as the mean ± SEM. Unless otherwise noted, statistical significance was determined using unpaired two-tailed Student’s t test. *p<0.05. ** p<0.01. *** p<0.001. ****p<0.0001.

Periosteal SSCs have a metabolic profile conferring a resistance to stress

A. Gene set enrichment analysis (GSEA) plots comparing P-SSCs versus BM-MSCs at steady state (n=3 per group). B. Quantitative RT-PCR analysis of mRNA expression of Cdkn1a, Cdkn1c, Cdk4 relative to Actb in sorted CD45-Ter119-CD31-CD51+CD200+ BM-MSCs and P-SSCs (n=3-6 per group). C. Flow cytometric analysis of glucose uptake at steady state in CD45-Ter119-CD31-CD51+CD200+ BM-MSCs and P-SSCs (n=5 per group). D. Quantification of cellular ROS at steady state in CD45-Ter119-CD31-CD51+CD200+ BM-MSCs and P-SSCs (n=8 per group). E. Quantitative RT-PCR analysis of mRNA expression of Sod1, Gls and Gpx1 relative to Actb in sorted CD45-Ter119-CD31-CD51+CD200+ BM-MSCs and P-SSCs (n=3-7 per group). F. Schematic illustration of the protocol for the in vitro apoptosis assay. BM-MSCs and P-SSCs were isolated and digested before plating in a 10cm dish. At near confluence, cells underwent CD45 lineage depletion and plated into multi-well plates. At near confluence, medium was switched from 20% FBS to 0% FBS. Cells were analyzed at the time of medium switch and 12 hours. G. Percentage of apoptotic BM-MSCs and P-SSCs cultured under 5% O2 at baseline and 12 hours after being in 0% FBS serum conditions (n=11-12 per group). Two-way ANOVA with Tukey’s multiple comparisons test was used to determine statistical significance. Data are represented as the mean ± SEM. Unless otherwise noted, statistical significance was determined using unpaired two-tailed Student’s t test. *p<0.05. ** p<0.01. *** p<0.001. ****p<0.0001. This figure was created using BioRender.com.

Periosteal SSCs migrate into the bone marrow and support stromal regeneration after bone transplantation.

A. Schematic of the transplantation of a WT bone enwrapped with periosteum from a UBC-GFP mouse donor into a WT recipient mouse. B. Pictures illustrating the transplantation of a WT bone enwrapped with periosteum from a UBC-GFP mouse donor into a WT recipient mouse. C. Representative whole-mount confocal z-stack projections of wild-type bone graft enwrapped with periosteum from a UBC-GFP mouse donor into a WT recipient mouse 5 months after transplantation. Three independent experiments yielded similar results. Right panel: arrows pointing to GFP+ periosteum located perivascularly. Scale bar = 50µm (left panel) and 20µm (right panel) D. Quantification of Cxcl12 and Kitl mRNA levels relative to Gapdh in sorted control CD45-Ter119-CD31-Nestin-GFP+ BM-MSCs, CD45-Ter119-CD31-CD51+CD200+ P-SSCs, and CD45-Ter119-CD31-CD51+CD200+GFP+ periosteum-derived graft BM-MSCs (n = 3-4 per group). One-way ANOVA with Tukey’s multiple comparisons was used to determine statistical significance. Data are represented as the mean ± SEM. Unless otherwise noted, statistical significance was determined using unpaired two-tailed Student’s t test. *p<0.05. ** p<0.01. *** p<0.001. ****p<0.0001. This figure was created using BioRender.com.

Periosteum-derived graft BM-MSCs adopt characteristics of baseline BM-MSCs, including the expression of HSC niche factors.

A. Schematic illustration of the transplantation of a Postn-creER;tdTomato femur into a WT recipient mouse. B. Representative whole-mount confocal z-stack projections of transplanted Postn-creER;tdTomato femurs into a WT recipient 8-, 15-, and 21-days after transplantation. Two-three independent experiments yielded similar results. Scale bar = 100 µm C. Percentage of graft periosteum-derived BM-MSCs labeled Tomato+ five months after transplantation of a bone from a Postn-creER;tdTomato mouse into a WT recipient (n=5). D. Heat map expression level of selected genes defined by previous studies for HSC niche cells and extracellular matrix genes (n=3-4). E. Volcano plot of P-SSCs compared to graft BM-MSCs showing higher expression of HSC niche-associated genes in graft BM-MSCs. Data are represented as the mean ± SEM. Unless otherwise noted, statistical significance was determined using unpaired two-tailed Student’s t test. *p<0.05. ** p<0.01. *** p<0.001. ****p<0.0001. This figure was created using BioRender.com.