Periosteal skeletal stem cells can migrate into the bone marrow and support hematopoiesis after injury

Abstract

Skeletal stem cells have been isolated from various tissues, including periosteum and bone marrow, where they exhibit key functions in bone biology and hematopoiesis, respectively. The role of periosteal skeletal stem cells in bone regeneration and healing has been extensively studied, but their ability to contribute to the bone marrow stroma is still under debate. In the present study, we characterized a whole bone transplantation model that mimics the initial bone marrow necrosis and fatty infiltration seen after injury. Using this model and a lineage tracing approach, we observed the migration of periosteal skeletal stem cells into the bone marrow after transplantation. Once in the bone marrow, periosteal skeletal stem cells are phenotypically and functionally reprogrammed into bone marrow mesenchymal stem cells that express high levels of hematopoietic stem cell niche factors such as Cxcl12 and Kitl. In addition, using ex vivo and in vivo approaches, we found that periosteal skeletal stem cells are more resistant to acute stress than bone marrow mesenchymal stem cells. These results highlight the plasticity of periosteal skeletal stem cells and their potential role in bone marrow regeneration after bone marrow injury.

Figure 1.. 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).

Figure 2.. Regenerating BM-MSCs are graft-derived and express HSC niche factors.

A. Schematic of a UBC-GFP femur transplanted into a Rosa^mT/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 Rosa^mT/mG mouse. C. Representative whole-mount confocal z-stack projections of a UBC-GFP bone transplanted into a Rosa^mT/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.

Figure 3.. 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. Arrowheads point at Nes-GFP⁺ cells within the bone and bone marrow. 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.

Figure 4.. 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.

Figure 5.. 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.

Figure 6.. 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-cre^ER;tdTomato femur into a WT recipient mouse. B. Representative whole-mount confocal z-stack projections of transplanted Postn-cre^ER;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-cre^ER;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.