CD51 labels periosteal injury-responsive osteoprogenitors

The periosteum is a critical source of skeletal stem and progenitor cells (SSPCs) that form callus tissue in response to injury. There is yet to be a consensus on how to identify SSPCs in the adult periosteum. The aim of this study was to understand how potential murine periosteal SSPC populations behave in vivo and in response to injury. We evaluated the in vivo differentiation potential of Sca1−CD51+ and Sca1+CD51+ cells following transplantation. In vitro, the Sca1+CD51+ population appears to be more primitive multipotent cells, but after transplantation, Sca1−CD51+ cells showed superior engraftment, expansion, and differentiation into chondrocytes and osteoblasts. Despite representing a clear population with flow cytometry, we identified very few Sca1+CD51+ cells histologically. Using a periosteal scratch injury model, we successfully mimicked the endochondral-like healing process seen in unstable fractures, including the expansion and osteochondral differentiation of αSMA+ cells following injury. CD51+ cells were present in the cambium layer of resting periosteum and expanded following injury. Sca1+CD51− cells were mainly localized in the outer periosteal layer. We found that injury increased colony-forming unit fibroblast (CFU-F) formation in the periosteum and led to rapid expansion of CD90+ cells. Several other populations, including Sca1−CD51+ and CD34+ cells, were expanded by day 7. Mice with enhanced fracture healing due to elevated Notch signaling mediated by NICD1 overexpression showed significant expansion of CD51+ and CD34hi cells in the early stages of healing, suggesting these populations contribute to more rapid healing. In conclusion, we demonstrate that periosteal injury leads to the expansion of various SSPC populations, but further studies are required to confirm their lineage hierarchy in the adult skeletal system. Our data indicate that CD51+ skeletal progenitor cells are injury-responsive and show good engraftment and differentiation potential upon transplantation

Utilization of Transgenic Models in Evaluation of Osteogenic Differentiation of Embryonic Stem Cells

Previous studies reported that embryonic stem cells (ESCs) can be induced to differentiate into cells showing a mature osteoblastic phenotype by culturing them under osteo-inductive conditions. It is probable that osteogenic differentiation requires that ESCs undergo differentiation through an intermediary step involving a mesenchymal lineage precursor. Based on our previous studies indicating that adult mesenchymal progenitor cells express αSMA, we have generated ESCs from transgenic mice in which an αSMA promoter directs the expression of red fluorescent protein (RFP) to mesenchymal progenitor cells. To track the transition of ESC-derived MSCs into mature osteoblasts, we have utilized a bone-specific fragment of rat type I collagen promoter driving green fluorescent protein (Col2.3GFP). Following osteogenic induction in ESCs, we have observed expression of alkaline phosphatase and subsequent mineralization as detected by von Kossa staining. After one week of osteogenic induction, ESCs begin to express αSMARFP. This expression was localized to the peripheral area encircling a typical ESC colony. Nevertheless, these αSMARFP positive cells did not show activation of the Col2.3GFP promoter, even after 7 weeks of osteogenic differentiation in vitro. In contrast, Col2.3GFP expression was detected in vivo, in mineralized areas following teratoma formation. Our results indicate that detection of alkaline phosphatase activity and mineralization of ESCs cultured under osteogenic conditions is not sufficient to demonstrate osteogenic maturation. Our study indicates the utility of the promoter-visual transgene approach to assess the commitment and differentiation of ESCs into the osteoblast lineage

PDGF Modulates BMP2‐Induced Osteogenesis in Periosteal Progenitor Cells

ABSTRACT BMPs are used in various clinical applications to promote bone formation. The limited success of the BMPs in clinical settings and supraphysiological doses required for their effects prompted us to evaluate the influence of other signaling molecules, specifically platelet‐derived growth factor (PDGF) on BMP2‐induced osteogenesis. Periosteal cells make a major contribution to fracture healing. We detected broad expression of PDGF receptor beta (PDGFRβ) within the intact periosteum and healing callus during fracture repair. In vitro, periosteum‐derived progenitor cells were highly responsive to PDGF as demonstrated by increased proliferation and decreased apoptosis. However, PDGF blocked BMP2‐induced osteogenesis by inhibiting the canonical BMP2/Smad pathway and downstream target gene expression. This effect is mediated via PDGFRβ and involves ERK1/2 MAPK and PI3K/AKT signaling pathways. Therapeutic targeting of the PDGFRβ pathway in periosteum‐mediated bone repair might have profound implications in the treatment of bone disease in the future. © 2018 The Authors JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research

Lineage Tracing of Resident Tendon Progenitor Cells during Growth and Natural Healing

Unlike during embryogenesis, the identity of tissue resident progenitor cells that contribute to postnatal tendon growth and natural healing is poorly characterized. Therefore, we utilized 1) an inducible Cre driven by alpha smooth muscle actin (SMACreERT2), that identifies mesenchymal progenitors, 2) a constitutively active Cre driven by growth and differentiation factor 5 (GDF5Cre), a critical regulator of joint condensation, in combination with 3) an Ai9 Cre reporter to permanently label SMA9 and GDF5-9 populations and their progeny. In growing mice, SMA9 cells were found in peritendinous structures and scleraxis-positive (ScxGFP) cells within the tendon midsubstance and myotendinous junction. The progenitors within the tendon midsubstance were transiently labeled as they displayed a 4-fold expansion from day 2 to day 21 but reduced to baseline levels by day 70. SMA9 cells were not found within tendon entheses or ligaments in the knee, suggesting a different origin. In contrast to the SMA9 population, GDF5-9 cells extended from the bone through the enthesis and into a portion of the tendon midsubstance. GDF5-9 cells were also found throughout the length of the ligaments, indicating a significant variation in the progenitors that contribute to tendons and ligaments. Following tendon injury, SMA9 paratenon cells were the main contributors to the healing response. SMA9 cells extended over the defect space at 1 week and differentiated into ScxGFP cells at 2 weeks, which coincided with increased collagen signal in the paratenon bridge. Thus, SMA9-labeled cells represent a unique progenitor source that contributes to the tendon midsubstance, paratenon, and myotendinous junction during growth and natural healing, while GDF5 progenitors contribute to tendon enthesis and ligament development. Understanding the mechanisms that regulate the expansion and differentiation of these progenitors may prove crucial to improving future repair strategies

Preosteocytes/Osteocytes Have the Potential to Dedifferentiate Becoming a Source of Osteoblasts

Presently there is no clear evidence for the ability of mature osteogenic lineage cells to dedifferentiate. In order to identify and trace mature osteogenic lineage cells, we have utilized transgenic mouse models in which the dentin matrix protein 1 (Dmp1) promoter drives expression of GFP (active marker) or Cre recombinase (historic label) in preosteocytes/osteocytes. In long bone chip outgrowth cultures, in which cells on the bone surface were enzymatically removed, cells with previous activity of the Dmp1 promoter migrated onto plastic and down-regulated Dmp1-GFP expression. Dmp1Cre-labeled cells from these cultures had the potential to re-differentiate into the osteogenic lineage, while the negative population showed evidence of adipogenesis. We observed numerous Dmp1Cre-labeled osteoblasts on the surface of bone chips following their in vivo transplantation. Our data indicate that cells embedded in bone matrix are motile, and once given access to the extra bony milieu will migrate out of their lacunae. This population of cells is phenotypically and functionally heterogeneous in vitro. Once the preosteocytes/osteocytes leave lacunae, they can dedifferentiate, potentially providing an additional source of functional osteoblasts

Perivascular osteoprogenitors are associated with transcortical channels of long bones.

Bone remodeling and regeneration are dependent on resident stem/progenitor cells with the capability to replenish mature osteoblasts and repair the skeleton. Using lineage tracing approaches, we identified a population of Dmp1+ cells that reside within cortical bone and are distinct from osteocytes. Our aims were to characterize this stromal population of transcortical perivascular cells (TPCs) in their resident niche and evaluate their osteogenic potential. To distinguish this population from osteoblasts/osteocytes, we crossed mice containing inducible DMP1CreERT2/Ai9 Tomato reporter (iDMP/T) with Col2.3GFP reporter (ColGFP), a marker of osteoblasts and osteocytes. We observed iDMP/T+;ColGFP- TPCs within cortical bone following tamoxifen injection. These cells were perivascular and located within transcortical channels. Ex vivo bone outgrowth cultures showed TPCs migrated out of the channels onto the plate and expressed stem cell markers such as Sca1, platelet derived growth factor receptor beta (PDGFRβ), and leptin receptor. In a cortical bone transplantation model, TPCs migrate from their vascular niche within cortical bone and contribute to new osteoblast formation and bone tube closure. Treatment with intermittent parathyroid hormone increased TPC number and differentiation. TPCs were unable to differentiate into adipocytes in the presence of rosiglitazone in vitro or in vivo. Altogether, we have identified and characterized a novel stromal lineage-restricted osteoprogenitor that is associated with transcortical vessels of long bones. Functionally, we have demonstrated that this population can migrate out of cortical bone channels, expand, and differentiate into osteoblasts, therefore serving as a source of progenitors contributing to new bone formation

Preptin Deficiency Does Not Protect against High‐Fat Diet‐Induced Metabolic Dysfunction or Bone Loss in Mice

ABSTRACT Preptin is derived from the cleavage of the E‐peptide of pro‐insulin‐like growth factor (IGF)‐II and is an insulin secretagogue. Observational studies have linked elevated circulating preptin to metabolic dysfunction in humans; however, a causal role for preptin in metabolic dysfunction has not been established. Additionally, preptin can promote osteoblast proliferation and differentiation, suggesting a link with skeletal health. We previously described a global preptin knockout (KO) model. In this study, we sought to uncover the impact of preptin KO in mice on the response to a moderately high‐fat diet (HFD) and low‐fat diet (LFD). HFD groups had higher weight and fat mass gain, lower trabecular and cortical bone volume and fracture load, and higher liver triglycerides. In males, preptin deficiency led to lower blood glucose than wild‐type (WT) mice under LFD conditions. This was accompanied by differences in bone microarchitecture, including lower trabecular bone volume fraction, trabecular number, and lower cortical thickness. These differences were absent in female mice, although KO females had a HFD‐driven increase in fat mass and liver triglycerides that was absent in WT mice. Female WT mice had increased glucose‐stimulated insulin secretion under HFD conditions that was absent in female KO mice. Overall, preptin may have a detrimental impact on metabolism and a positive impact on bone health in male mice and may protect against liver fat storage in females while enabling islet compensation under HFD conditions. When we consider that serum preptin levels are elevated in humans of both sexes in pathological states in which insulin levels are elevated, the impact of preptin on comorbidity risk needs to be better understood. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research

Heterogeneity of murine periosteum progenitors involved in fracture healing

The periosteum is the major source of cells involved in fracture healing. We sought to characterize progenitor cells and their contribution to bone fracture healing. The periosteum is highly enriched with progenitor cells, including Sca1+ cells, fibroblast colony-forming units, and label-retaining cells compared to the endosteum and bone marrow. Using lineage tracing, we demonstrate that alpha smooth muscle actin (αSMA) identifies long-term, slow-cycling, self-renewing osteochondroprogenitors in the adult periosteum that are functionally important for bone formation during fracture healing. In addition, Col2.3CreER-labeled osteoblast cells contribute around 10% of osteoblasts but no chondrocytes in fracture calluses. Most periosteal osteochondroprogenitors following fracture can be targeted by αSMACreER. Previously identified skeletal stem cell populations were common in periosteum but contained high proportions of mature osteoblasts. We have demonstrated that the periosteum is highly enriched with skeletal progenitor cells, and there is heterogeneity in the populations of cells that contribute to mature lineages during periosteal fracture healing

SMA9+ progenitors in the expanded paratenon differentiate into ScxGFP+ cells at 2 weeks post-injury.

<p>SMA9+ cells (white arrows, D–F) at one week post-injury (A–J) are predominately negative for ScxGFP as seen in two photon image stacks in SMA9-ScxGFP mice. However, these cells differentiate into ScxGFP+ cells (yellow arrows, N–P,V) at 2 weeks (K–T) within the collagenous regions of the paratenon bridge while SMA9+ only cells still remain on the anterior surface (white arrows, K–M). By 5 weeks, there is reduced overlap in SMA9 and ScxGFP expression (V). J,T) Axial views depicting the 3 levels in the anterior views (A–J, K–T) of the defects at 1 and 2 weeks, respectively. U) Total area of SMA9 and ScxGFP positive signal at 1, 2, and 5 weeks. V) SMA9-ScxGFP double positive area normalized to either total SMA9 or ScxGFP area, respectively. Scale bars = 100 µm. *significantly different than 1 and 5 weeks, ∧significantly different than 1 week (p<0.05). Error bars denote ± SD. Full z-stacks can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096113#pone.0096113.s006" target="_blank">Video S1</a>.</p