Instruct: Given a scientific claim, retrieve passages that support or refute the claim.
Query: Osteocytes have an essential role in G-CSF induced HSPC mobilization.
The bone marrow (BM) niche comprises multiple cell types that regulate hematopoietic stem/progenitor cell (HSPC) migration out of the niche and into the circulation. Here, we demonstrate that osteocytes, the major cellular component of mature bone, are regulators of HSPC egress. Granulocyte colony-stimulating factor (G-CSF), used clinically to mobilize HSPCs, induces changes in the morphology and gene expression of the osteocytic network that precedes changes in osteoblasts. This rapid response is likely under control of the sympathetic nervous system, since osteocytes express the β2-adrenergic receptor and surgical sympathectomy prevents it. Mice with targeted ablation of osteocytes or a disrupted osteocyte network have comparable numbers of HSPCs in the BM but fail to mobilize HSPCs in response to G-CSF. Taken together, these results indicate that the BM/bone niche interface is critically controlled from inside of the bone matrix and establish an important physiological role for skeletal tissues in hematopoietic function.
The physiologic roles of angiopoietin-like proteins (Angptls) in the hematopoietic system remain unknown. Here we show that hematopoietic stem cells (HSCs) in Angptl3-null mice are decreased in number and quiescence. HSCs transplanted into Angptl3-null recipient mice exhibited impaired repopulation. Bone marrow sinusoidal endothelial cells express high levels of Angptl3 and are adjacent to HSCs. Importantly, bone marrow stromal cells or endothelium deficient in Angptl3 have a significantly decreased ability to support the expansion of repopulating HSCs. Angptl3 represses the expression of the transcription factor Ikaros, whose unregulated overexpression diminishes the repopulation activity of HSCs. Angptl3, as an extrinsic factor, thus supports the stemness of HSCs in the bone marrow niche.
Mesenchymal stem cells (MSCs) commonly defined by in vitro functions have entered clinical application despite little definition of their function in residence. Here, we report genetic pulse-chase experiments that define osteoblastic cells as short-lived and nonreplicative, requiring replenishment from bone-marrow-derived, Mx1(+) stromal cells with "MSC" features. These cells respond to tissue stress and migrate to sites of injury, supplying new osteoblasts during fracture healing. Single cell transplantation yielded progeny that both preserve progenitor function and differentiate into osteoblasts, producing new bone. They are capable of local and systemic translocation and serial transplantation. While these cells meet current definitions of MSCs in vitro, they are osteolineage restricted in vivo in growing and adult animals. Therefore, bone-marrow-derived MSCs may be a heterogeneous population with the Mx1(+) population, representing a highly dynamic and stress responsive stem/progenitor cell population of fate-restricted potential that feeds the high cell replacement demands of the adult skeleton.
Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3+ cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24- or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.
Mesenchymal stem cells (MSCs) and osteolineage cells contribute to the hematopoietic stem cell (HSC) niche in the bone marrow of long bones. However, their developmental relationships remain unclear. In this study, we demonstrate that different MSC populations in the developing marrow of long bones have distinct functions. Proliferative mesoderm-derived nestin(-) MSCs participate in fetal skeletogenesis and lose MSC activity soon after birth. In contrast, quiescent neural crest-derived nestin(+) cells preserve MSC activity, but do not generate fetal chondrocytes. Instead, they differentiate into HSC niche-forming MSCs, helping to establish the HSC niche by secreting Cxcl12. Perineural migration of these cells to the bone marrow requires the ErbB3 receptor. The neonatal Nestin-GFP(+) Pdgfrα(-) cell population also contains Schwann cell precursors, but does not comprise mature Schwann cells. Thus, in the developing bone marrow HSC niche-forming MSCs share a common origin with sympathetic peripheral neurons and glial cells, and ontogenically distinct MSCs have non-overlapping functions in endochondrogenesis and HSC niche formation.
The quiescent state is thought to be an indispensable property for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for adult hematopoiesis in the bone marrow (BM). Here, we demonstrate that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and antiapoptotic, and comprise a side-population (SP) of HSCs, which adhere to osteoblasts (OBs) in the BM niche. The interaction of Tie2 with its ligand Angiopoietin-1 (Ang-1) induced cobblestone formation of HSCs in vitro and maintained in vivo long-term repopulating activity of HSCs. Furthermore, Ang-1 enhanced the ability of HSCs to become quiescent and induced adhesion to bone, resulting in protection of the HSC compartment from myelosuppressive stress. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the BM niche.
Cells of the osteoblast lineage provide critical support for B lymphopoiesis in the bone marrow (BM). Parathyroid hormone (PTH) signaling in osteoblastic cells through its receptor (PPR) is an important regulator of hematopoietic stem cells; however, its role in regulation of B lymphopoiesis is not clear. Here we demonstrate that deletion of PPR in osteoprogenitors results in a significant loss of trabecular and cortical bone. PPR signaling in osteoprogenitors, but not in mature osteoblasts or osteocytes, is critical for B-cell precursor differentiation via IL-7 production. Interestingly, despite a severe reduction in B-cell progenitors in BM, mature B-lymphocytes were increased 3.5-fold in the BM of mice lacking PPR in osteoprogenitors. This retention of mature IgD(+) B cells in the BM was associated with increased expression of vascular cell adhesion molecule 1 (VCAM1) by PPR-deficient osteoprogenitors, and treatment with VCAM1 neutralizing antibody increased mobilization of B lymphocytes from mutant BM. Our results demonstrate that PPR signaling in early osteoblasts is necessary for B-cell differentiation via IL-7 secretion and for B-lymphocyte mobilization via VCAM1.
Pancytopenia is a major cause of morbidity in acute myeloid leukemia (AML), yet its cause is unclear. Normal osteoblastic cells have been shown to support hematopoiesis. To define the effects of leukemia on osteoblastic cells, we used an immunocompetent murine model of AML. Leukemic mice had inhibition of osteoblastic cells, with decreased serum levels of the bone formation marker osteocalcin. Osteoprogenitor cells and endosteal-lining osteopontin(+) cells were reduced, and osteocalcin mRNA in CD45(-) marrow cells was diminished. This resulted in severe loss of mineralized bone. Osteoclasts were only transiently increased without significant increases in bone resorption, and their inhibition only partially rescued leukemia-induced bone loss. In vitro data suggested that a leukemia-derived secreted factor inhibited osteoblastic cells. Because the chemokine CCL-3 was recently reported to inhibit osteoblastic function in myeloma, we tested its expression in our model and in AML patients. Consistent with its potential novel role in leukemic-dependent bone loss, CCL-3 mRNA was significantly increased in malignant marrow cells from leukemic mice and from samples from AML patients. Based on these results, we propose that therapeutic mitigation of leukemia-induced uncoupling of osteoblastic and osteoclastic cells may represent a novel approach to promote normal hematopoiesis in patients with myeloid neoplasms.
Although haematopoietic stem cells (HSCs) are commonly assumed to reside within a specialized microenvironment, or niche, most published experimental manipulations of the HSC niche have affected the function of diverse restricted progenitors. This raises the fundamental question of whether HSCs and restricted progenitors reside within distinct, specialized niches or whether they share a common niche. Here we assess the physiological sources of the chemokine CXCL12 for HSC and restricted progenitor maintenance. Cxcl12(DsRed) knock-in mice (DsRed-Express2 recombined into the Cxcl12 locus) showed that Cxcl12 was primarily expressed by perivascular stromal cells and, at lower levels, by endothelial cells, osteoblasts and some haematopoietic cells. Conditional deletion of Cxcl12 from haematopoietic cells or nestin-cre-expressing cells had little or no effect on HSCs or restricted progenitors. Deletion of Cxcl12 from endothelial cells depleted HSCs but not myeloerythroid or lymphoid progenitors. Deletion of Cxcl12 from perivascular stromal cells depleted HSCs and certain restricted progenitors and mobilized these cells into circulation. Deletion of Cxcl12 from osteoblasts depleted certain early lymphoid progenitors but not HSCs or myeloerythroid progenitors, and did not mobilize these cells into circulation. Different stem and progenitor cells thus reside in distinct cellular niches in bone marrow: HSCs occupy a perivascular niche and early lymphoid progenitors occupy an endosteal niche.
Endogenous glucocorticoids (GC) play an important role in the termination of the inflammatory response following infection and tissue injury. However, recent findings indicate that stress can impair the anti-inflammatory capacities of these hormones. Lipopolysaccharide (LPS)-stimulated splenocytes of mice that were repeatedly subjected to social disruption (SDR) stress were less sensitive to the immunosuppressive effects of corticosterone (CORT) as demonstrated by an increased production of pro-inflammatory cytokines and enhanced cell survival. Myeloid cells expressing the marker CD11b were shown to play a key role in this process. Here we investigated the role of the bone marrow as a potential source of the GC-insensitive cells. The study revealed that LPS-stimulated bone marrow cells, in the absence of experimental stress, were virtually GC-resistant and retained high levels of cell viability after treatment with CORT. Recurrent exposure to the acute stressor over a period of 2, 4 or 6 days led to an increase in the GC sensitivity of the bone marrow cells. This increase in GC sensitivity was associated with enhanced mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), an increase in the number of myeloid progenitors, and a decrease in the proportion of mature CD11b+ cells. The changes in the cellular composition of the bone marrow were accompanied by an increase in splenic CD11b+ cell numbers. Simultaneous assessment of the GC sensitivity in bone marrow and spleen revealed a significant negative correlation between both tissues suggesting that social stress causes the redistribution of GC-insensitive myeloid cells from the bone marrow to the spleen.
A rare set of hematopoietic stem cells (HSC) must undergo a massive expansion to produce mature blood cells. The phenotypic isolation of HSC from mice offers the opportunity to determine directly their proliferation kinetics. We analyzed the proliferation and cell cycle kinetics of long-term self-renewing HSC (LT-HSC) in normal adult mice. At any one time, approximately 5% of LT-HSC were in S/G2/M phases of the cell cycle and another 20% were in G1 phase. BrdUrd incorporation was used to determine the rate at which different cohorts of HSC entered the cell cycle over time. About 50% of LT-HSC incorporated BrdUrd by 6 days and >90% incorporated BrdUrd by 30 days. By 6 months, 99% of LT-HSC had incorporated BrdUrd. We calculated that approximately 8% of LT-HSC asynchronously entered the cell cycle per day. Nested reverse transcription-PCR analysis revealed cyclin D2 expression in a high proportion of LT-HSC. Although approximately 75% of LT-HSC are quiescent in G0 at any one time, all HSC are recruited into cycle regularly such that 99% of LT-HSC divide on average every 57 days.
The bone marrow is the primary site for neutrophil production and release into the circulation. Because the CXC chemokine receptor-4/stromal derived factor-1 (CXCR4/SDF-1) axis plays a central role in the interactions of hematopoietic stem cells, lymphocytes, and developing neutrophils in the marrow, we investigated whether reciprocal CXCR4-dependent mechanisms might be involved in neutrophil release and subsequent return to the marrow following circulation. Neutralizing antibody to CXCR4 reduced marrow retention of infused neutrophils (45.7% +/- 0.5% to 6.9% +/- 0.5%) and was found to mobilize neutrophils from marrow (34.4% +/- 4.4%). Neutrophil CXCR4 expression and SDF-1-induced calcium flux decreased with maturation and activation of the cells, corresponding to the decreased marrow homing associated with these characteristics in vivo. Infusion of the inflammatory mediator and CXCR2 ligand KC led to mobilization of neutrophils from marrow by itself and was augmented 3-fold by low doses of CXCR4-blocking antibody that otherwise had no mobilizing effect. Examination of KC and SDF-1 calcium signaling demonstrated that the effect of KC may, in part, be due to heterologous desensitization to SDF-1. These results suggest that the CXCR4/SDF-1 axis is critical in circulating neutrophil homeostasis and that it may participate in the rapid release of neutrophils from the marrow during inflammation through a novel interaction with inflammatory CXC chemokines.
Mouse bone marrow hematopoietic stem cells were isolated with the use of a variety of phenotypic markers. These cells can proliferate and differentiate with approximately unit efficiency into myelomonocytic cells, B cells, or T cells. Thirty of these cells are sufficient to save 50 percent of lethally irradiated mice, and to reconstitute all blood cell types in the survivors.
The forkhead O (FoxO) family of transcription factors participates in diverse physiologic processes, including induction of cell-cycle arrest, stress resistance, differentiation, apoptosis, and metabolism. Several recent studies indicate that FoxO-dependent signaling is required for long-term regenerative potential of the hematopoietic stem cell (HSC) compartment through regulation of HSC response to physiologic oxidative stress, quiescence, and survival. These observations link FoxO function in mammalian systems with the evolutionarily conserved role of FoxO in promotion of stress resistance and longevity in lower phylogenetic systems. Furthermore, these findings have implications for aging in higher organisms and in malignant stem cell biology, and suggest that FoxOs may play an important role in the maintenance and integrity of stem cell compartments in a broad spectrum of tissues.
Adipocytes are part of hematopoietic microenvironment, even though up to now in humans, their role in hematopoiesis is still questioned. We have previously shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. As expected, FLFCs constitutively produced macrophage colony stimulating factor and induced CD34(+) differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cell-to-cell contact but could be restored in transwell culture conditions, together with granulocyte colony stimulating factor production. Both functions were also recovered when FLFCs cultured in contact with CD34(+) cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. An inflammatory cytokine such as interleukin-1 beta or dexamethasone modulates FLFC properties to restore granulopoiesis. Our data provide the first evidence that primary adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes. Disclosure of potential conflicts of interest is found at the end of this article.
Hematopoietic stem cells (HSCs) sustain blood formation throughout life and are the functional units of bone marrow transplantation. We show that transient expression of six transcription factors Run1t1, Hlf, Lmo2, Prdm5, Pbx1, and Zfp37 imparts multilineage transplantation potential onto otherwise committed lymphoid and myeloid progenitors and myeloid effector cells. Inclusion of Mycn and Meis1 and use of polycistronic viruses increase reprogramming efficacy. The reprogrammed cells, designated induced-HSCs (iHSCs), possess clonal multilineage differentiation potential, reconstitute stem/progenitor compartments, and are serially transplantable. Single-cell analysis revealed that iHSCs derived under optimal conditions exhibit a gene expression profile that is highly similar to endogenous HSCs. These findings demonstrate that expression of a set of defined factors is sufficient to activate the gene networks governing HSC functional identity in committed blood cells. Our results raise the prospect that blood cell reprogramming may be a strategy for derivation of transplantable stem cells for clinical application.
The mammalian blood system, containing more than 10 distinct mature cell types, stands on one specific cell type, hematopoietic stem cell (HSC). Within the system, only HSCs possess the ability of both multipotency and self-renewal. Multipotency is the ability to differentiate into all functional blood cells. Self-renewal is the ability to give rise to HSC itself without differentiation. Since mature blood cells (MBCs) are predominantly short-lived, HSCs continuously provide more differentiated progenitors while properly maintaining the HSC pool size throughout life by precisely balancing self-renewal and differentiation. Thus, understanding the mechanisms of self-renewal and differentiation of HSC has been a central issue. In this review, we focus on the hierarchical structure of the hematopoietic system, the current understanding of microenvironment and molecular cues regulating self-renewal and differentiation of adult HSCs, and the currently emerging systems approaches to understand HSC biology.
Bone homeostasis is maintained by the balance between bone-forming osteoblasts and bone-degrading osteoclasts. Osteoblasts have a mesenchymal origin whereas osteoclasts belong to the myeloid lineage. Osteoclast and osteoblast communication occurs through soluble factors secretion, cell-bone interaction and cell-cell contact, which modulate their activities. CD200 is an immunoglobulin superfamilly member expressed on various types of cells including mesenchymal stem cells (MSCs). CD200 receptor (CD200R) is expressed on myeloid cells such as monocytes/macrophages. We assume that CD200 could be a new molecule involved in the control of osteoclastogenesis and could play a role in MSC-osteoclast communication in humans. In this study, we demonstrated that soluble CD200 inhibited the differentiation of osteoclast precursors as well as their maturation in bone-resorbing cells in vitro. Soluble CD200 did not modify the monocyte phenotype but inhibited the receptor activator of nuclear factor kappa-B ligand (RANKL) signaling pathway as well as the gene expression of osteoclast markers such as osteoclast-associated receptor (OSCAR) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Moreover, MSCs inhibited osteoclast formation, which depended on cell-cell contact and was associated with CD200 expression on the MSC surface. Our results clearly demonstrate that MSCs, through the expression of CD200, play a major role in the regulation of bone resorption and bone physiology and that the CD200-CD200R couple could be a new target to control bone diseases.
Maintaining hematopoietic stem cell (HSC) quiescence is a critical property for the life-long generation of blood cells. Approximately 75% of cells in a highly enriched long-term repopulating HSC (LT-HSC) pool (Lin(-)Sca1(+)c-Kit(hi)CD150(+)CD48(-)) are quiescent, with only a small percentage of the LT-HSCs in cycle. Transcription factor GATA-3 is known to be vital for the development of T cells at multiple stages in the thymus and for Th2 differentiation in the peripheral organs. Although it is well documented that GATA-3 is expressed in HSCs, a role for GATA-3 in any prethymic progenitor cell has not been established. In the present study, we show that Gata3-null mutant mice generate fewer LT-HSCs and that fewer Gata3-null LT-HSCs are in cycle. Furthermore, Gata3 mutant hematopoietic progenitor cells fail to be recruited into an increased cycling state after 5-fluorouracil-induced myelosuppression. Therefore, GATA-3 is required for the maintenance of a normal number of LT-HSCs and for their entry into the cell cycle.
Hematopoietic stem cells (HSCs) rely on instructive cues from the bone marrow (BM) niche to maintain their quiescence and adapt blood production to the organism's needs. Alterations in the BM niche are commonly observed in blood malignancies and directly contribute to the aberrant function of disease-initiating leukemic stem cells (LSCs). Here, we review recent insights into the cellular and molecular determinants of the normal HSC niche and describe how genetic changes in stromal cells and leukemia-induced BM niche remodeling contribute to blood malignancies. Moreover, we discuss how these findings can be applied to non-cell-autonomous therapies targeting the LSC niche.
Hematopoietic stem cells (HSCs) in adult marrow are believed to be derived from fetal liver precursors. To study cell kinetics involved in long-term hematopoiesis, we studied single-sorted candidate HSCs from fetal liver that were cultured in the presence of a mixture of stimulatory cytokines. After 8–10 d, the number of cells in primary cultures varied from 10,000 cells. Single cells in slow growing colonies were recloned upon reaching a 100–200 cell stage. Strikingly, the number of cells in subclones varied widely again. These results are indicative of asymmetric divisions in primitive hematopoietic cells in which proliferative potential and cell cycle properties are unevenly distributed among daughter cells. The continuous generation of functional heterogeneity among the clonal progeny of HSCs is in support of intrinsic control of stem cell fate and provides a model for the long-term maintenance of hematopoiesis in vitro and in vivo.
Studies of the identity and physiological function of mesenchymal stromal cells (MSCs) have been hampered by a lack of markers that permit both prospective identification and fate mapping in vivo. We found that Leptin Receptor (LepR) is a marker that highly enriches bone marrow MSCs. Approximately 0.3% of bone marrow cells were LepR(+) , 10% of which were CFU-Fs, accounting for 94% of bone marrow CFU-Fs. LepR(+) cells formed bone, cartilage, and adipocytes in culture and upon transplantation in vivo. LepR(+) cells were Scf-GFP(+), Cxcl12-DsRed(high), and Nestin-GFP(low), markers which also highly enriched CFU-Fs, but negative for Nestin-CreER and NG2-CreER, markers which were unlikely to be found in CFU-Fs. Fate-mapping showed that LepR(+) cells arose postnatally and gave rise to most bone and adipocytes formed in adult bone marrow, including bone regenerated after irradiation or fracture. LepR(+) cells were quiescent, but they proliferated after injury. Therefore, LepR(+) cells are the major source of bone and adipocytes in adult bone marrow.
Androgens are widely used for treating Fanconi anemia (FA) and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2(-/-) mice were used to assess the therapeutic efficacy of oxymetholone (OXM) and its mechanism of action. Eighteen-month-old Fancd2(-/-) mice recapitulated key human FA phenotypes, including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic OXM treatment significantly improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells. RNA-Seq analysis implicated downregulation of osteopontin as an important potential mechanism for the drug's action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic OXM therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure.
Antigen-presenting, major histocompatibility complex (MHC) class II-rich dendritic cells are known to arise from bone marrow. However, marrow lacks mature dendritic cells, and substantial numbers of proliferating less-mature cells have yet to be identified. The methodology for inducing dendritic cell growth that was recently described for mouse blood now has been modified to MHC class II-negative precursors in marrow. A key step is to remove the majority of nonadherent, newly formed granulocytes by gentle washes during the first 2-4 d of culture. This leaves behind proliferating clusters that are loosely attached to a more firmly adherent "stroma. " At days 4-6 the clusters can be dislodged, isolated by 1-g sedimentation, and upon reculture, large numbers of dendritic cells are released. The latter are readily identified on the basis of their distinct cell shape, ultrastructure, and repertoire of antigens, as detected with a panel of monoclonal antibodies. The dendritic cells express high levels of MHC class II products and act as powerful accessory cells for initiating the mixed leukocyte reaction. Neither the clusters nor mature dendritic cells are generated if macrophage colony-stimulating factor rather than granulocyte/macrophage colony-stimulating factor (GM-CSF) is applied. Therefore, GM-CSF generates all three lineages of myeloid cells (granulocytes, macrophages, and dendritic cells). Since > 5 x 10(6) dendritic cells develop in 1 wk from precursors within the large hind limb bones of a single animal, marrow progenitors can act as a major source of dendritic cells. This feature should prove useful for future molecular and clinical studies of this otherwise trace cell type.
Bone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, postnatally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypes.