Bone marrow stromal cell-derived hepcidin has antimicrobial and immunomodulatory activities.

Bone marrow stromal cells (BMSCs) have immunomodulatory activities in numerous species and have been used in clinical trials. BMSCs also make antibacterial agents. Since hepcidin is known to have antimicrobial effects in fish, we wondered if it might also be used as an antimicrobial agent by mammalian BMSCs. In the present study, we show hepcidin expression in both mouse (mBMSC) and human BMSCs (hBMSC). We observed a hBMSC hepcidin-dependent degradation of ferroportin in HEK-293 reporter cells in vitro. In human and mouse bone marrows (BM) we detected hepcidin-positive BMSCs in close proximity to hematopoietic progenitors. The conditioned culture medium of hBMSCs significantly reduced bacterial proliferation that was partially blocked by a hepcidin-neutralizing antibody. Similarly, medium in which hepcidin-deficient (Hamp-/-) mouse BMSCs had been grown was significantly less effective in reducing bacterial counts than the medium of wild-type cells. In a zymosan-induced peritonitis mouse model we found that mBMSC-derived hepcidin reduced the number of invading polymorphonuclear (PMN) cells in the peritoneal cavity. Our results show that BMSC-derived hepcidin has antimicrobial properties in vitro and also reduces inflammation in vivo. We conclude that hepcidin should be added to the expanding arsenal of agents available to BMSCs to fight infections and inflammation.

Quantitative Craniofacial Analysis and Generation of Human Induced Pluripotent Stem Cells for Muenke Syndrome: A Case Report.

In this case report, we focus on Muenke syndrome (MS), a disease caused by the p.Pro250Arg variant in fibroblast growth factor receptor 3 (FGFR3) and characterized by uni- or bilateral coronal suture synostosis, macrocephaly without craniosynostosis, dysmorphic craniofacial features, and dental malocclusion. The clinical findings of MS are further complicated by variable expression of phenotypic traits and incomplete penetrance. As such, unraveling the mechanisms behind MS will require a comprehensive and systematic way of phenotyping patients to precisely identify the impact of the mutation variant on craniofacial development. To establish this framework, we quantitatively delineated the craniofacial phenotype of an individual with MS and compared this to his unaffected parents using three-dimensional cephalometric analysis of cone beam computed tomography scans and geometric morphometric analysis, in addition to an extensive clinical evaluation. Secondly, given the utility of human induced pluripotent stem cells (hiPSCs) as a patient-specific investigative tool, we also generated the first hiPSCs derived from a family trio, the proband and his unaffected parents as controls, with detailed characterization of all cell lines. This report provides a starting point for evaluating the mechanistic underpinning of the craniofacial development in MS with the goal of linking specific clinical manifestations to molecular insights gained from hiPSC-based disease modeling.

Differences in Steady-State Erythropoiesis in Different Mouse Bones and Postnatal Spleen.

Adult erythropoiesis is a highly controlled sequential differentiation of hematopoietic stem cells (HSCs) to mature red blood cells in the bone marrow (BM). The bones which contain BM are diverse in their structure, embryonic origin, and mode of ossification. This has created substantial heterogeneity in HSCs function in BM of different bones, however, it is not known if this heterogeneity influences erythropoiesis in different bones and different regions of the same bone. In this study, we examined steady state BM erythroid progenitors and precursors from different bones - the femur, tibia, pelvis, sternum, vertebrae, radius, humerus, frontal, parietal bone, and compared all to the femur. Trabecular and cortical regions of the femur were also compared for differences in erythropoiesis. In addition, mouse spleen was studied to determine at which age erythropoietic support by the spleen was lost postnatally. We report that total erythroid cells, and erythroid precursors in the femur are comparable to tibia, pelvis, humerus and sternum, but are significantly reduced in the vertebrae, radius, frontal, and parietal bones. Erythroid progenitors and multipotential progenitor numbers are comparable in all the bones except for reduced number in the parietal bone. In the femur, the epiphysis and metaphysis have significantly reduced number of erythroid precursors and progenitors, multipotential progenitors and myeloid progenitors compared to the diaphysis region. These results show that analysis of erythroid precursors from diaphysis region of the femur is representative of tibia, pelvis, humerus and sternum and have significant implications on the interpretation of the steady-state erythropoiesis finding from adult BM. Postnatal spleen supports erythroid precursors until 6 weeks of age which coincides with reduced number of red pulp macrophages. The residual erythroid progenitor support reaches the adult level by 3 months of age. In conclusion, our findings provide insights to the differences in erythropoiesis between different bones, between trabecular and cortical regions of the femur, and developmental changes in postnatal spleen erythropoiesis.

Lineage-specific differentiation of osteogenic progenitors from pluripotent stem cells reveals the FGF1-RUNX2 association in neural crest-derived osteoprogenitors.

Human pluripotent stem cells (hPSCs) can provide a platform to model bone organogenesis and disease. To reflect the developmental process of the human skeleton, hPSC differentiation methods should include osteogenic progenitors (OPs) arising from three distinct embryonic lineages: the paraxial mesoderm, lateral plate mesoderm, and neural crest. Although OP differentiation protocols have been developed, the lineage from which they are derived, as well as characterization of their genetic and molecular differences, has not been well reported. Therefore, to generate lineage-specific OPs from human embryonic stem cells and human induced pluripotent stem cells, we employed stepwise differentiation of paraxial mesoderm-like cells, lateral plate mesoderm-like cells, and neural crest-like cells toward their respective OP subpopulation. Successful differentiation, confirmed through gene expression and in vivo assays, permitted the identification of transcriptomic signatures of all three cell populations. We also report, for the first time, high FGF1 levels in neural crest-derived OPs-a notable finding given the critical role of fibroblast growth factors (FGFs) in osteogenesis and mineral homeostasis. Our results indicate that FGF1 influences RUNX2 levels, with concomitant changes in ERK1/2 signaling. Overall, our study further validates hPSCs' power to model bone development and disease and reveals new, potentially important pathways influencing these processes.

Mesenchymal stromal cells from infants with simple polydactyly modulate immune responses more efficiently than adult mesenchymal stromal cells.

Bone marrow-derived stromal cells or mesenchymal stromal cells (BMSCs or MSCs, as we will call them in this work) are multipotent progenitor cells that can differentiate into osteoblasts, adipocytes and chondrocytes. In addition, MSCs have been shown to modulate the function of a variety of immune cells. Donor age has been shown to affect the regenerative potential, differentiation, proliferation and anti-inflammatory potency of MSCs; however, the impact of donor age on their immunosuppressive activity is unknown. In this study, we evaluated the ability of MSCs derived from very young children and adults on T-cell suppression and cytokine secretion by monocytes/macrophages. MSCs were obtained from extra digits of children between 10 and 21 months and adults between 28 and 64 years of age. We studied cell surface marker expression, doubling time, lineage differentiation potential and immunosuppressive function of the MSCs. Young MSCs double more quickly and differentiate into bone and fat cells more efficiently than those from older donors. They also form more and dense colonies of fibroblasts (colony forming unit-fibroblast [CFU-F]). MSCs from both young and adult subjects suppressed T-cell proliferation in a mitogen-induced assay at 1:3 and 1:30 ratios. At a 1:30 ratio, however, MSCs from adults did not, but MSCs from infants did suppress T-cell proliferation. In the mixed lymphocyte reaction assay, MSCs from infants produced similar levels of suppression at all three MSC/T-cell ratios, but adult MSCs only inhibited T-cell proliferation at a 1:3 ratio. Cytokine analyses of co-cultures of MSCs and macrophages showed that both adult and young MSCs suppress tumor necrosis factor alpha (TNF-α) and induce interleukin-10 (IL-10) production in macrophage co-culture assay in a similar manner. Overall, this work shows that developing MSCs display a higher level of immunosuppression than mature MSCs.

Immunogenic potential of human bone marrow mesenchymal stromal cells is enhanced by hyperthermia.

BACKGROUND AIMS: Bone marrow-derived mesenchymal stromal cells (MSCs) have been reported to suppress T-cell proliferation and used to alleviate the symptoms of graft-versus-host disease (GVHD). MSCs are a mixed cell population and at this time there are no tools to isolate the cells responsible for the T-cell suppression. We wanted to find a way to enhance the immune-modulatory actions of MSCs and tried varying the temperature at which they were cultured. METHODS: We cultured human MSCs derived from healthy volunteers at different temperatures and tested their ability to switch macrophage character from pro-inflammatory to anti-inflammatory (M1 type to M2 type). Using an enzyme-linked immunosorbent assay (ELISA), we showed that when MSCs are cultured at higher temperatures their ability to induce co-cultured macrophages to produce more interleukin-10, (IL-10) (an anti-inflammatory cytokine) and less tumor necrosis factor alpha, (TNFα) (a pro-inflammatory cytokine) is increased. We performed Western blots and immunocytochemistry to screen for changes that might underlie this effect. RESULTS: We found that in hyperthermia the heat shock protein, HSF1, translocated into the nucleus of MSCs. It appears to induce the COX2/PGE2 (Cyclooxygenase2/Prostaglandin E2) pathway described earlier as a major mechanism of MSC-directed immune-suppression. CONCLUSION: Hyperthermia increases the efficacy of MSC-driven immune-suppression. We propose that changing the time of MSC administration to patients to mid-to-late afternoon when the body temperature is naturally highest might be beneficial. Warming the patient could also be considered.

Vasopressin stimulates the proliferation and differentiation of red blood cell precursors and improves recovery from anemia.

Arginine vasopressin (AVP) made by hypothalamic neurons is released into the circulation to stimulate water resorption by the kidneys and restore water balance after blood loss. Patients who lack this antidiuretic hormone suffer from central diabetes insipidus. We observed that many of these patients were anemic and asked whether AVP might play a role in red blood cell (RBC) production. We found that all three AVP receptors are expressed in human and mouse hematopoietic stem and progenitor cells. The AVPR1B appears to play the most important role in regulating erythropoiesis in both human and mouse cells. AVP increases phosphorylation of signal transducer and activator of transcription 5, as erythropoietin (EPO) does. After sublethal irradiation, AVP-deficient Brattleboro rats showed delayed recovery of RBC numbers compared to control rats. In mouse models of anemia (induced by bleeding, irradiation, or increased destruction of circulating RBCs), AVP increased the number of circulating RBCs independently of EPO. In these models, AVP appears to jump-start peripheral blood cell replenishment until EPO can take over. We suggest that specific AVPR1B agonists might be used to induce fast RBC production after bleeding, drug toxicity, or chemotherapy.

Transglutaminases factor XIII-A and TG2 regulate resorption, adipogenesis and plasma fibronectin homeostasis in bone and bone marrow.

Appropriate bone mass is maintained by bone-forming osteoblast and bone-resorbing osteoclasts. Mesenchymal stem cell (MSC) lineage cells control osteoclastogenesis via expression of RANKL and OPG (receptor activator of nuclear factor κB ligand and osteoprotegerin), which promote and inhibit bone resorption, respectively. Protein crosslinking enzymes transglutaminase 2 (TG2) and Factor XIII-A (FXIII-A) have been linked to activity of myeloid and MSC lineage cells; however, in vivo evidence has been lacking to support their function. In this study, we show in mice that TG2 and FXIII-A control monocyte-macrophage cell differentiation into osteoclasts as well as RANKL production in MSCs and in adipocytes. Long bones of mice lacking TG2 and FXIII-A transglutaminases, show compromised biomechanical properties and trabecular bone loss in axial and appendicular skeleton. This was caused by increased osteoclastogenesis, a cellular phenotype that persists in vitro. The increased potential of TG2 and FXIII-A deficient monocytes to form osteoclasts was reversed by chemical inhibition of TG activity, which revealed the presence of TG1 in osteoclasts and assigned different roles for the TGs as regulators of osteoclastogenesis. TG2- and FXIII-A-deficient mice had normal osteoblast activity, but increased bone marrow adipogenesis, MSCs lacking TG2 and FXIII-A showed high adipogenic potential and significantly increased RANKL expression as well as upregulated TG1 expression. Chemical inhibition of TG activity in the null cells further increased adipogenic potential and RANKL production. Altered differentiation of TG2 and FXIII-A null MSCs was associated with plasma fibronectin (FN) assembly defect in cultures and FN retention in serum and marrow in vivo instead of assembly into bone. Our findings provide new functions for TG2, FXIII-A and TG1 in bone cells and identify them as novel regulators of bone mass, plasma FN homeostasis, RANKL production and myeloid and MSC cell differentiation.

Factor XIII-A transglutaminase deficient mice show signs of metabolically healthy obesity on high fat diet.

F13A1 gene, which encodes for Factor XIII-A blood clotting factor and a transglutaminase enzyme, was recently identified as a potential causative gene for obesity in humans. In our previous in vitro work, we showed that FXIII-A regulates preadipocyte differentiation and modulates insulin signaling via promoting plasma fibronectin assembly into the extracellular matrix. To understand the role of FXIII-A in whole body energy metabolism, here we have characterized the metabolic phenotype of F13a1-/- mice. F13a1-/- and F13a1+/+ type mice were fed chow or obesogenic, high fat diet for 20 weeks. Weight gain, total fat mass and fat pad mass, glucose handling, insulin sensitivity, energy expenditure and, morphological and biochemical analysis of adipose tissue was performed. We show that mice lacking FXIII-A gain weight on obesogenic diet, similarly as wild type mice, but exhibit a number of features of metabolically healthy obesity such as protection from developing diet-induced insulin resistance and hyperinsulinemia. Mice also show normal fasting glucose levels, larger adipocytes, decreased extracellular matrix accumulation and inflammation of adipose tissue, as well as decreased circulating triglycerides. This study reveals that FXIII-A transglutaminase can regulate whole body insulin sensitivity and may have a role in the development of diet-induced metabolic disturbances.

Factor XIII-A transglutaminase acts as a switch between preadipocyte proliferation and differentiation.

Factor XIII-A (FXIII-A) transglutaminase (TG) was recently identified as a potential causative obesity gene in human white adipose tissue (WAT). Here, we have examined the role of TG activity and the role of protein crosslinking in adipogenesis. Mouse WAT and preadipocytes showed abundant TG activity arising from FXIII-A. FXIII-A was localized to the cell surface and acted as a negative regulator of adipogenesis by promoting assembly of fibronectin (FN) from plasma into preadipocyte extracellular matrix. This modulated cytoskeletal dynamics and maintained the preadipocyte state. FXIII-A-assembled plasma FN (pFN) matrix promoted preadipocyte proliferation and potentiated the proproliferative effects of insulin (INS) while suppressing the prodifferentiating INS signaling. FXIII-A-deficient mouse embryonic fibroblasts showed increased lipid accumulation and decreased proliferation as well as decreased pFN assembly into extracellular matrix. Thus, FXIII-A serves as a preadipocyte-bound proliferation/differentiation switch that mediates effects of hepatocyte-produced circulating pFN.

Circulating undercarboxylated osteocalcin and gingival crevicular fluid tumour necrosis factor-α in children.

BACKGROUND: Osteocalcin, a protein secreted by osteoblasts during bone formation, is negatively associated with adult periodontal disease. Little is known about this association in children. AIM: To examine the extent to which plasma undercarboxylated osteocalcin (ucOC) is associated with gingival crevicular fluid tumour necrosis factor-alpha (GCF TNF-α) - a potential marker of gingival inflammation - in children. METHODS: We used data from the Quebec Adipose and Lifestyle InvesTigation in Youth cohort, an ongoing longitudinal study on the natural history of obesity among Caucasian children with a family history of obesity in Quebec, Canada. This cross-sectional analysis from the baseline visit includes 120 children aged 8-10 years. Plasma ucOC and GCF TNF-α levels were determined by enzyme-linked immunosorbent assay. Linear regression analyses, adjusting for age, gender, family income, sexual maturity stage, daily physical activity, obesity, and fasting glucose were conducted, with TNF-α level as the dependent variable. RESULTS: A 1-ng/ml increase in ucOC was associated with a 0.96% decrease (95% confidence interval (CI): -1.69, -0.23) in GCF TNF-α level. CONCLUSION: A negative association between a marker of bone formation and a marker of gingival inflammation was observed as early as childhood among Caucasian children with a family history of obesity.

Transglutaminase activity arising from Factor XIIIA is required for stabilization and conversion of plasma fibronectin into matrix in osteoblast cultures.

Circulating plasma fibronectin (pFN), produced by hepatocytes, is a major component of the noncollagenous bone matrix where it was recently shown in vivo in mice to control the biomechanical quality as well as the mineral-to-matrix ratio in bone. FN fibrillogenesis is a process generally requiring FN binding to cellular integrins, and cellular tension to elongate and assemble the molecule. Whether soluble pFN undergoes cell-mediated assembly in bone is not fully established. FN is a well-known substrate for transglutaminases (TGs), which are protein-crosslinking enzymes capable of stabilizing macromolecular structures. The role of this modification regarding the function of FN in bone matrix has remained unknown. Osteoblasts express two TGs-transglutaminase 2 and Factor XIIIA-and we have shown that Factor XIIIA is the main TG active during osteoblast differentiation. In the present study, conducted using MC3T3-E1 osteoblast cultures and bone marrow stromal cells, we demonstrate that pFN requires a TG-mediated crosslinking step to form osteoblast matrix in vitro. This modification step is specific for pFN; cellular FN (EDA-FN) does not serve as a TG substrate. Inhibition of pFN assembly using a TG inhibitor, or depletion of pFN from cell culture serum, dramatically decreased total FN matrix assembly in the osteoblast cultures and affected both the quantity and quality of the type I collagen matrix, and decreased lysyl oxidase and alkaline phosphatase levels, resulting in decreased mineralization. Experiments with isozyme-specific substrate peptides showed that FXIIIA is responsible for the crosslinking of pFN. Addition of exogenous preactivated FXIIIA to osteoblast cultures promoted pFN assembly from the media into matrix. Exogenous TG2 had no effect. Analysis of pFN and EDA-FN fibrils by immunofluorescence microscopy demonstrated that they form distinct matrix network, albeit with minor overlap, suggesting different functions for the two FN forms. Further analysis using EDA-FN blocking antibody showed that it regulated preosteoblast proliferation whereas pFN depletion from the serum had no effect on this process. In conclusion, our study shows that pFN assembly into bone matrix in vitro requires FXIIIA transglutaminase activity making pFN assembly an active, osteoblast-mediated process.

Factor XIIIA transglutaminase expression and secretion by osteoblasts is regulated by extracellular matrix collagen and the MAP kinase signaling pathway.

Osteoblast differentiation is regulated by the presence of collagen type I (COL I) extracellular matrix (ECM). We have recently demonstrated that Factor XIIIA (FXIIIA) transglutaminase (TG) is required by osteoblasts for COL I secretion and extracellular deposition, and thus also for osteoblast differentiation. In this study we have further investigated the link between COL I and FXIIIA, and demonstrate that COL I matrix increases FXIIIA levels in osteoblast cultures and that FXIIIA is found as cellular (cFXIIIA) and extacellular matrix (ecmFXIIIA) forms. FXIIIA mRNA, protein expression, cellular localization and secretion were enhanced by ascorbic acid (AA) treatment and blocked by dihydroxyproline (DHP) which inhibits COL I externalization. FXIIIA mRNA was regulated by the MAP kinase pathway. Secretion of ecmFXIIIA, and its enzymatic activity in conditioned medium, were also decreased in osteoblasts treated with the lysyl oxidase inhibitor ß-aminopropionitrile, which resulted in a loosely packed COL I matrix. Osteoblasts secrete a latent, inactive dimeric ecmFXIIIA form which is activated upon binding to the matrix. Monodansyl cadaverine labeling of TG substrates in the cultures revealed that incorporation of the label occurred at sites where fibronectin co-localized with COL I, indicating that ecmFXIIIA secretion could function to stabilize newly deposited matrix. Our results suggest that FXIIIA is an integral part of the COL I deposition machinery, and also that it is part of the ECM-feedback loop, both of which regulate matrix deposition and osteoblast differentiation.

Plasma membrane factor XIIIA transglutaminase activity regulates osteoblast matrix secretion and deposition by affecting microtubule dynamics.

Transglutaminase activity, arising potentially from transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to 'block -and-track' enzyme(s) targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics.