Carolyn A. Meyers

Isolation and characterization of canine perivascular stem/stromal cells for bone tissue engineering

For over 15 years, human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC). The isolation of perivascular progenitor cells from human adipose tissue by a cell sorting strategy was first published in 2008. Since this time, the interest in using pericytes and related perivascular stem/stromal cell (PSC) populations for tissue engineering has significantly increased. Here, we describe a set of experiments identifying, isolating and characterizing PSC from canine tissue (N = 12 canine adipose tissue samples). Results showed that the same antibodies used for human PSC identification and isolation are cross-reactive with canine tissue (CD45, CD146, CD34). Like their human correlate, canine PSC demonstrate characteristics of MSC including cell surface marker expression, colony forming unit-fibroblast (CFU-F) inclusion, and osteogenic differentiation potential. As well, canine PSC respond to osteoinductive signals in a similar fashion as do human PSC, such as the secreted differentiation factor NEL-Like Molecule-1 (NELL-1). Nevertheless, important differences exist between human and canine PSC, including differences in baseline osteogenic potential. In summary, canine PSC represent a multipotent mesenchymogenic cell source for future translational efforts in tissue engineering.

BMP-2-induced bone formation and neural inflammation

Bone morphogenetic protein-2 (BMP-2), a potent osteoinductive cytokine from the transforming growth factor beta (TGF-β) family, is currently the most commonly used protein-based bone graft substitute. Although clinical use of BMP-2 has significantly increased in recent years, its prominence has also highlighted various adverse events, including induction of inflammation. This review will elucidate the relationship between BMP-2 and inflammation, with an emphasis on peripheral nerve inflammation and its sequelae. As well, we review the potential additive roles of nerve released factors with BMP2 in the context of bone formation.

NELL-1 induces Sca-1+ mesenchymal progenitor cell expansion in models of bone maintenance and repair

NELL-1 is a secreted, osteogenic protein first discovered to control ossification of the cranial skeleton. Recently, NELL-1 has been implicated in bone maintenance. However, the cellular determinants of NELL-1s bone-forming effects are still unknown. Here, recombinant human NELL-1 (rhNELL-1) implantation was examined in a clinically relevant nonhuman primate lumbar spinal fusion model. Prolonged rhNELL-1 protein release was achieved using an apatite-coated β-tricalcium phosphate carrier, resulting in a local influx of stem cell antigen-1-positive (Sca-1+) mesenchymal progenitor cells (MPCs), and complete osseous fusion across all samples (100% spinal fusion rate). Murine studies revealed that Nell-1 haploinsufficiency results in marked reductions in the numbers of Sca-1+CD45-CD31- bone marrow MPCs associated with low bone mass. Conversely, rhNELL-1 systemic administration in mice showed a marked anabolic effect accompanied by increased numbers of Sca-1+CD45-CD31- bone marrow MPCs. Mechanistically, rhNELL-1 induces Sca-1 transcription among MPCs, in a process requiring intact Wnt/β-catenin signaling. In summary, NELL-1 effectively induces bone formation across small and large animal models either via local implantation or intravenous delivery. NELL-1 induces an expansion of a bone marrow subset of MPCs with Sca-1 expression. These findings provide compelling justification for the clinical translation of a NELL-1-based therapy for local or systemic bone formation.

Combining Smoothened Agonist and NEL-Like Protein-1 Enhances Bone Healing

Background: Nonhealing bone defects represent an immense biomedical burden. Despite recent advances in protein-based bone regeneration, safety concerns over bone morphogenetic protein-2 have prompted the search for alternative factors. Previously, the authors examined the additive/synergistic effects of hedgehog and Nel-like protein-1 (NELL-1) on the osteogenic differentiation of mesenchymal stem cells in vitro. In this study, the authors sought to leverage their previous findings by applying the combination of Smoothened agonist (SAG), hedgehog signal activator, and NELL-1 to an in vivo critical-size bone defect model. Methods: A 4-mm parietal bone defect was created in mixed-gender CD-1 mice. Treatment groups included control (n = 6), SAG (n = 7), NELL-1 (n = 7), and SAG plus NELL-1 (n = 7). A custom fabricated poly(lactic-co-glycolic acid) disk with hydroxyapatite coating was used as an osteoinductive scaffold. Results: Results at 4 and 8 weeks showed increased bone formation by micro–computed tomographic analyses with either stimulus alone (SAG or NELL-1), but significantly greater bone formation with both components combined (SAG plus NELL-1). This included greater bone healing scores and increased bone volume and bone thickness. Histologic analyses confirmed a significant increase in new bone formation with the combination therapy SAG plus NELL-1, accompanied by increased defect vascularization. Conclusions: In summary, the authors’ results suggest that combining the hedgehog signaling agonist SAG and NELL-1 has potential as a novel therapeutic strategy for the healing of critical-size bone defects. Future directions will include optimization of dosage and delivery strategy for an SAG and NELL-1 combination product.

Sclerostin expression in skeletal sarcomas

Sclerostin (SOST) is an extracellular Wnt signaling antagonist which negatively regulates bone mass. Despite this, the expression and function of SOST in skeletal tumors remain poorly described. Here, we first describe the immunohistochemical staining pattern of SOST across benign and malignant skeletal tumors with bone or cartilage matrix (n=68 primary tumors). Next, relative SOST expression was compared to markers of Wnt signaling activity and osteogenic differentiation across human osteosarcoma (OS) cell lines (n=7 cell lines examined). Results showed immunohistochemical detection of SOST in most bone-forming tumors (90.2%; 46/51) and all cartilage-forming tumors (100%; 17/17). Among OSs, variable intensity and distribution of SOST expression were observed, which highly correlated with the presence and degree of neoplastic bone. Patchy SOST expression was observed in cartilage-forming tumors, which did not distinguish between benign and malignant tumors or correlate with regional morphologic characteristics. Finally, SOST expression varied widely between OS cell lines, with more than 97-fold variation. Among OS cell lines, SOST expression positively correlated with the marker of osteogenic differentiation alkaline phosphatase and did not correlate well with markers of Wnt/β-catenin signaling activity. In summary, SOST is frequently expressed in skeletal bone- and cartilage-forming tumors. The strong spatial correlation with bone formation and the in vitro expression patterns are in line with the known functions of SOST in nonneoplastic bone, as a feedback inhibitor on osteogenic differentiation. With anti-SOST as a potential therapy for osteoporosis in the near future, its basic biologic and phenotypic consequences in skeletal tumors should not be overlooked.

Early immunomodulatory effects of implanted human perivascular stromal cells during bone formation

Human perivascular stem/stromal cells (PSC) are a multipotent mesodermal progenitor cell population defined by their perivascular residence. PSC are most commonly derived from subcutaneous adipose tissue, and recent studies have demonstrated the high potential for clinical translation of this fluorescence-activated cell sorting-derived cell population for bone tissue engineering. Specifically, purified PSC induce greater bone formation than unpurified stroma taken from the same patient sample. In this study, we examined the differences in early innate immune response to human PSC or unpurified stroma (stromal vascular fraction [SVF]) during the in vivo process of bone formation. Briefly, SVF or PSC from the same patient sample were implanted intramuscularly in the hindlimb of severe combined immunodeficient (SCID) mice using an osteoinductive demineralized bone matrix carrier. Histological examination of early inflammatory infiltrates was examined by hematoxylin and eosin and immunohistochemical staining (Ly-6G, F4/80). Results showed significantly greater neutrophilic and macrophage infiltrates within and around SVF in comparison to PSC-laden implants. Differences in early postoperative inflammation among SVF-laden implants were associated with reduced osteogenic differentiation and bone formation. Similar findings were recapitulated with PSC implantation in immunocompetent mice. Exaggerated postoperative inflammation was associated with increased IL-1α, IL-1β, IFN-γ, and TNF-α gene expression among SVF samples, and conversely increased IL-6 and IL-10 expression among PSC samples. These data document a robust immunomodulatory effect of implanted PSC, and an inverse correlation between host inflammatory cell infiltration and stromal progenitor cell-mediated ossification.

WISP-1 drives bone formation at the expense of fat formation in human perivascular stem cells

The vascular wall within adipose tissue is a source of mesenchymal progenitors, referred to as perivascular stem/stromal cells (PSC). PSC are isolated via fluorescence activated cell sorting (FACS), and defined as a bipartite population of pericytes and adventitial progenitor cells (APCs). Those factors that promote the differentiation of PSC into bone or fat cell types are not well understood. Here, we observed high expression of WISP-1 among human PSC in vivo, after purification, and upon transplantation in a bone defect. Next, modulation of WISP-1 expression was performed, using WISP-1 overexpression, WISP-1 protein, or WISP-1 siRNA. Results demonstrated that WISP-1 is expressed in the perivascular niche, and high expression is maintained after purification of PSC, and upon transplantation in a bone microenvironment. In vitro studies demonstrate that WISP-1 has pro-osteogenic/anti-adipocytic effects in human PSC, and that regulation of BMP signaling activity may underlie these effects. In summary, our results demonstrate the importance of the matricellular protein WISP-1 in regulation of the differentiation of human stem cell types within the perivascular niche. WISP-1 signaling upregulation may be of future benefit in cell therapy mediated bone tissue engineering, for the healing of bone defects or other orthopaedic applications.

Frontal bone healing is sensitive to Wnt signaling inhibition via lentiviral encoded ß-catenin shRNA.

The Wnt/β-catenin signaling pathway plays an integral role in skeletal biology, spanning from embryonic skeletal patterning through bone maintenance and bone repair. Most experimental methods to antagonize Wnt signaling in vivo are either systemic or transient, including genetic approaches, use of small-molecule inhibitors, or neutralizing antibodies. We sought to develop a novel, localized model of prolonged Wnt/β-catenin signaling blockade by the application and validation of a lentivirus encoding β-catenin short hairpin RNA (shRNA). Efficacy of lentiviral-encoded β-catenin shRNA was first confirmed in vitro using bone marrow mesenchymal stromal cells, and in vivo using an intramedullary long bone injection model in NOD SCID mice. Next, the effects of β-catenin knockdown were assessed in a calvarial bone defect model, in which the frontal bone demonstrates enhanced bone healing associated with heightened Wnt/β-catenin signaling. Lentivirus encoding either β-catenin shRNA or random sequence shRNA with enhanced green fluorescent protein (control) was injected overlying the calvaria of NOD SCID mice and bone defects were created in either the frontal or parietal bones. Among mice treated with lentivirus encoding β-catenin shRNA, frontal bone defect healing was significantly reduced by all radiographic and histologic metrics. In contrast, parietal bone healing was minimally impacted by β-catenin shRNA. In aggregate, our data document the application and validation of a lentivirus encoding β-catenin shRNA model that represents an easily replicable tool for examining the importance of locoregional Wnt/β-catenin signaling in bone biology and regeneration.

WNT16 induces proliferation and osteogenic differentiation of human perivascular stem cells

Perivascular stem cells (PSC) are a progenitor population defined by their perivascular residence. Recent studies have examined the relative difference in Wnt ligands to induce PSC differentiation, including Wnt16. Here, we examine the role of Wnt16 in the proliferation and osteogenic differentiation of human PSC. Treatment of PSC with WNT16 significantly increased cell proliferation to a greater extent than did WNT3A. In addition, WNT16 showed a significant increase in osteogenic gene expression among PSC. These data demonstrate that WNT16 represents a combined mitogenic/pro-osteogenic stimulus that may play a functional role in human mesenchymal stem cell mediated bone repair.

Lineage-Specific Wnt Reporter Elucidates Mesenchymal Wnt Signaling during Bone Repair

β-Catenin-dependent Wnt signaling controls numerous aspects of skeletal development and postnatal bone repair. Currently available transgenic Wnt reporter mice allow for visualization of global canonical Wnt signaling activity within skeletal tissues, without delineation of cell type. This is particularly important in a bone repair context, in which the inflammatory phase can obscure the visualization of mesenchymal cell types of interest. To tackle the issue of tissue-specific Wnt signaling, we have generated and characterized a transgenic mouse strain [termed paired related homeobox 1 (Prx1)-Wnt-green fluorescent protein (GFP), by crossing a previously validated Prx1-Cre strain with a nuclear fluorescent reporter driven by T-cell factor/lymphoid enhancer factor activity (Rosa26-Tcf/Lef-LSL-H2B-GFP)]. Prx1-Wnt-GFP animals were subject to three models of long bone and membranous bone repair (displaced forelimb fracture, tibial cortical defect, and frontal bone defect). Results showed that, irrespective of bone type, locoregional mesenchymal cell activation of Wnt signaling occurs in a defined temporospatial pattern among Prx1-Wnt-GFP mice. In summary, Prx1-Wnt-GFP reporter animals allow for improved visualization, spatial discrimination, and facile quantification of Wnt-activated mesenchymal cells within models of adult bone repair.