Acceleration of Bone Repair in NOD/SCID Mice by Human Monoosteophils,Novel LL-37-Activated Monocytes

Background

An incomplete understanding of bone forming cells during wound healing and ectopic calcification has led to a search for circulating cells that may fulfill this function. Previously, we showed that monoosteophils, a novel lineage of calcifying/bone-forming cells generated by treatment of monocytes with the natural peptide LL-37, are candidates. In this study, we have analyzed their gene expression profile and bone repair function.

Methods and Findings

Human monoosteophils can be distinguished from monocytes, macrophages and osteoclasts by their unique up-regulation of integrin α3 and down-regulation of CD14 and CD16. Monoosteophils express high mRNA and protein levels of SPP1 (osteopontin), GPNMB (osteoactivin), CHI3L1 (cartilage glycoprotein-39), CHIT1 (Chitinase 1), MMP-7, CCL22 and MAPK13 (p38MAPKδ). Monocytes from wild type, but not MAPK13 KO mice are also capable of monoosteophil differentiation, suggesting that MAPK13 regulates this process. When human monoosteophils were implanted in a freshly drilled hole in mid-diaphyseal femurs of NOD/SCID mice, significant bone repair required only 14 days compared to at least 24 days in control treated injuries.

Conclusion

Human derived monoosteophils, characterized as CD45⁺α3⁺α3β⁺CD34⁻CD14⁻BAP (bone alkaline phosphatase)⁻ cells, can function in an animal model of bone injury.     

Complex dynamics of osteoclast formation and death in long-term cultures

Background

Osteoclasts, cells responsible for bone resorption, contribute to the development of degenerative, metabolic and neoplastic bone diseases, which are often characterized by persistent changes in bone microenvironment. We aimed to investigate the dynamics of osteoclast formation and death in cultures that considerably exceeded the length of standard protocol and to design a mathematical model describing osteoclastogenesis.

Methodology/Principal Findings

RAW 264.7 monocytic cells fuse to form multinucleated osteoclasts upon treatment with pro-resorptive cytokine RANKL. We have found that in long-term experiments (15–26 days), the dynamics of changes in osteoclast numbers was remarkably complex and qualitatively variable in different experiments. Whereas 19 of 46 experiments exhibited single peak of osteoclast formation, in 27 experiments we observed development of successive waves of osteoclast formation and death. Periodic changes in osteoclast numbers were confirmed in long-term cultures of mouse bone marrow cells treated with M-CSF and RANKL. Because the dynamics of changes in osteoclast numbers was found to be largely independent of monocytes, a two-species model of ordinary differential equations describing the changes in osteoclasts and monocytes was ineffective in recapitulating the oscillations in osteoclast numbers. Following experimental observation that medium collected from mature osteoclasts inhibited osteoclastogenesis in fresh cultures, we introduced a third variable, factor f, to describe osteoclast-derived inhibitor. This model allowed us to simulate the oscillatory changes in osteoclasts, which were coupled to oscillatory changes in the factor f, whereas monocytes changed exponentially. Importantly, to achieve the experimentally observed oscillations with increasing amplitude, we also had to assume that osteoclast presence stimulates osteoclast formation.

Conclusions/Significance

This study identifies the critical role for osteoclast autocrine regulation in controlling long-term dynamic of osteoclast formation and death and describes the complementary roles for negative and positive feedback mediators in determining the sharp dynamics of activation and inactivation of osteoclasts.     

MiR-133a in human circulating monocytes: a potential biomarker associated with postmenopausal osteoporosis

Background

Osteoporosis mainly occurs in postmenopausal women, which is characterized by low bone mineral density (BMD) due to unbalanced bone resorption by osteoclasts and formation by osteoblasts. Circulating monocytes play important roles in osteoclastogenesis by acting as osteoclast precursors and secreting osteoclastogenic factors, such as IL-1, IL-6 and TNF-α. MicroRNAs (miRNAs) have been implicated as important biomarkers in various diseases. The present study aimed to find significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis.

Methodology/Principal Findings

We used ABI TaqMan® miRNA array followed by qRT-PCR validation in circulating monocytes to identify miRNA biomarkers in 10 high and 10 low BMD postmenopausal Caucasian women. MiR-133a was upregulated (P=0.007) in the low compared with the high BMD groups in the array analyses, which was also validated by qRT-PCR (P=0.044). We performed bioinformatic target gene analysis and found three potential osteoclast-related target genes, CXCL11, CXCR3 and SLC39A1. In addition, we performed Pearson correlation analyses between the expression levels of miR-133a and the three potential target genes in the 20 postmenopausal women. We did find negative correlations between miR-133a and all the three genes though not significant.

Conclusions/Significance

This is the first in vivo miRNA expression analysis in human circulating monocytes to identify novel miRNA biomarkers underlying postmenopausal osteoporosis. Our results suggest that miR-133a in circulating monocytes is a potential biomarker for postmenopausal osteoporosis.     

Bone Marrow SSEA1+ Cells Support the Myocardium in Cardiac Pressure Overload

Rationale

Stage specific embryonic antigen 1+ (SSEA1+) cells have been described as the most primitive mesenchymal progenitor cell in the bone marrow. Cardiac injury mobilizes SSEA1+ cells into the peripheral blood but their in vivo function has not been characterized.

Objective

We generated animals with chimeric bone marrow to determine the fate and function of bone marrow SSEA1+ cells in response to acute cardiac pressure overload.

Methods and Results

Lethally irradiated mice were transplanted with normal bone marrow where the wild-type SSEA1+ cells were replaced with green fluorescent protein (GFP) SSEA1+ cells. Cardiac injury was induced by trans-aortic constriction (TAC). We identified significant GFP+ cell engraftment into the myocardium after TAC. Bone marrow GFP+ SSEA1 derived cells acquired markers of endothelial lineage, but did not express markers of c-kit+ cardiac progenitor cells. The function of bone marrow SSEA1+ cells after TAC was determined by transplanting lethally irradiated mice with bone marrow depleted of SSEA1+ cells (SSEA1-BM). The cardiac function of SSEA1-BM mice declined at a greater rate after TAC compared to their complete bone marrow transplant counterparts and was associated with decreased bone marrow cell engraftment and greater vessel rarefication in the myocardium.

Conclusions

These results provide evidence for the recruitment of endogenous bone marrow SSEA1+ cells to the myocardium after TAC. We demonstrate that, in vivo, bone marrow SSEA1+ cells have the differentiation potential to acquire endothelial lineage markers. We also show that bone marrow SSEA1+ deficiency is associated with a reduced compensatory capacity to cardiac pressure overload, suggesting their importance in cardiac homeostasis. These data demonstrate that bone marrow SSEA1+ cells are critical for sustaining vascular density and cardiac repair to pressure overload.     

Osteoclasts control osteoblast chemotaxis via PDGF-BB/PDGF receptor beta signaling

Background

Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding.

Methodology/Principal Findings

Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor β (PDGFR-β) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts.

Conclusions/Significance

We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-β signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.     

TNF inhibits production of stromal cell-derived factor 1 by bone stromal cells and increases osteoclast precursor mobilization from bone marrow to peripheral blood

Introduction

The objective of the present study was to investigate the role of the stromal cell-derived factor 1 (SDF-1)/CXCR4 axis in TNF-induced mobilization of osteoclast precursors (OCPs) from bone marrow.

Methods

OCPs were generated from bone marrow cells of TNF-transgenic mice or wild-type mice treated with TNF or PBS. The percentage of CD11b⁺/Gr-1^-/lo OCPs was assessed by fluorescence-activated cell sorting. OCP migration to the SDF-1 gradient and the osteoclast forming potency were assessed in chemotaxis/osteoclastogenic assays. SDF-1 expression was assessed by real-time RT-PCR, ELISA and immunostaining in primary bone marrow stromal cells, in the ST2 bone marrow stromal cell line, and in bones from TNF-injected mice.

Results

OCPs generated in vitro from wild-type mice migrated to SDF-1 gradients and subsequently gave rise to osteoclasts in response to RANKL and macrophage colony-stimulating factor. TNF reduced SDF-1 expression by ST2 cells. Bone marrow stromal cells from TNF-transgenic mice produced low levels of SDF-1. TNF treatment of wild-type mice decreased the SDF-1 concentration in bone marrow extracts and decreased the SDF-1 immunostaining of bone marrow stromal cells, and it also increased the circulating OCP numbers. The percentage of bone marrow CXCR4⁺ OCPs was similar in TNF-transgenic mice and wild-type littermates and in TNF-treated and PBS-treated wild-type mice.

Conclusion

Systemically elevated TNF levels inhibit bone marrow stromal cell production of SDF-1 and increase the release of bone marrow OCPs to the peripheral blood. Disruption of the SDF-1/CXCR4 axis by TNF may play an important role in mediating OCP mobilization from the bone marrow cavity in chronic inflammatory arthritis.     

HGF Expressing Stem Cells in Usual Interstitial Pneumonia Originate from the Bone Marrow and Are Antifibrotic

Background

Pulmonary fibrosis may result from abnormal alveolar wound repair after injury. Hepatocyte growth factor (HGF) improves alveolar epithelial wound repair in the lung. Stem cells were shown to play a major role in lung injury, repair and fibrosis. We studied the presence, origin and antifibrotic properties of HGF-expressing stem cells in usual interstitial pneumonia.

Methods

Immunohistochemistry was performed in lung tissue sections and primary alveolar epithelial cells obtained from patients with usual interstitial pneumonia (UIP, n = 7). Bone marrow derived stromal cells (BMSC) from adult male rats were transfected with HGF, instilled intratracheally into bleomycin injured rat lungs and analyzed 7 and 14 days later.

Results

In UIP, HGF was expressed in specific cells mainly located in fibrotic areas close to the hyperplastic alveolar epithelium. HGF-positive cells showed strong co-staining for the mesenchymal stem cell markers CD44, CD29, CD105 and CD90, indicating stem cell origin. HGF-positive cells also co-stained for CXCR4 (HGF+/CXCR4+) indicating that they originate from the bone marrow. The stem cell characteristics were confirmed in HGF secreting cells isolated from UIP lung biopsies. In vivo experiments showed that HGF-expressing BMSC attenuated bleomycin induced pulmonary fibrosis in the rat, indicating a beneficial role of bone marrow derived, HGF secreting stem cells in lung fibrosis.

Conclusions

HGF-positive stem cells are present in human fibrotic lung tissue (UIP) and originate from the bone marrow. Since HGF-transfected BMSC reduce bleomycin induced lung fibrosis in the bleomycin lung injury and fibrosis model, we assume that HGF-expressing, bone-marrow derived stem cells in UIP have antifibrotic properties.     

PlGF repairs myocardial ischemia through mechanisms of angiogenesis, cardioprotection and recruitment of myo-angiogenic competent marrow progenitors

Rationale

Despite preclinical success in regenerating and revascularizing the infarcted heart using angiogenic growth factors or bone marrow (BM) cells, recent clinical trials have revealed less benefit from these therapies than expected.

Objective

We explored the therapeutic potential of myocardial gene therapy of placental growth factor (PlGF), a VEGF-related angiogenic growth factor, with progenitor-mobilizing activity.

Methods and Results

Myocardial PlGF gene therapy improves cardiac performance after myocardial infarction, by inducing cardiac repair and reparative myoangiogenesis, via upregulation of paracrine anti-apoptotic and angiogenic factors. In addition, PlGF therapy stimulated Sca-1⁺/Lin⁻ (SL) BM progenitor proliferation, enhanced their mobilization into peripheral blood, and promoted their recruitment into the peri-infarct borders. Moreover, PlGF enhanced endothelial progenitor colony formation of BM-derived SL cells, and induced a phenotypic switch of BM-SL cells, recruited in the infarct, to the endothelial, smooth muscle and cardiomyocyte lineage.

Conclusions

Such pleiotropic effects of PlGF on cardiac repair and regeneration offer novel opportunities in the treatment of ischemic heart disease.     

Novel sulfated polysaccharides disrupt cathelicidins, inhibit RAGE and reduce cutaneous inflammation in a mouse model of rosacea

Background

Rosacea is a common disfiguring skin disease of primarily Caucasians characterized by central erythema of the face, with telangiectatic blood vessels, papules and pustules, and can produce skin thickening, especially on the nose of men, creating rhinophyma. Rosacea can also produce dry, itchy eyes with irritation of the lids, keratitis and corneal scarring. The cause of rosacea has been proposed as over-production of the cationic cathelicidin peptide LL-37.

Methodology/Principal Findings

We tested a new class of non-anticoagulant sulfated anionic polysaccharides, semi-synthetic glycosaminoglycan ethers (SAGEs) on key elements of the pathogenic pathway leading to rosacea. SAGEs were anti-inflammatory at ng/ml, including inhibition of polymorphonuclear leukocyte (PMN) proteases, P-selectin, and interaction of the receptor for advanced glycation end-products (RAGE) with four representative ligands. SAGEs bound LL-37 and inhibited interleukin-8 production induced by LL-37 in cultured human keratinocytes. When mixed with LL-37 before injection, SAGEs prevented the erythema and PMN infiltration produced by direct intradermal injection of LL-37 into mouse skin. Topical application of a 1% (w/w) SAGE emollient to overlying injected skin also reduced erythema and PMN infiltration from intradermal LL-37.

Conclusions

Anionic polysaccharides, exemplified by SAGEs, offer potential as novel mechanism-based therapies for rosacea and by extension other LL-37-mediated and RAGE-ligand driven skin diseases.     

Role of calcitonin gene-related peptide in bone repair after cyclic fatigue loading

Background

Calcitonin gene related peptide (CGRP) is a neuropeptide that is abundant in the sensory neurons which innervate bone. The effects of CGRP on isolated bone cells have been widely studied, and CGRP is currently considered to be an osteoanabolic peptide that has effects on both osteoclasts and osteoblasts. However, relatively little is known about the physiological role of CGRP in-vivo in the skeletal responses to bone loading, particularly fatigue loading.

Methodology/Principal Findings

We used the rat ulna end-loading model to induce fatigue damage in the ulna unilaterally during cyclic loading. We postulated that CGRP would influence skeletal responses to cyclic fatigue loading. Rats were fatigue loaded and groups of rats were infused systemically with 0.9% saline, CGRP, or the receptor antagonist, CGRP_8–37, for a 10 day study period. Ten days after fatigue loading, bone and serum CGRP concentrations, serum tartrate-resistant acid phosphatase 5b (TRAP5b) concentrations, and fatigue-induced skeletal responses were quantified. We found that cyclic fatigue loading led to increased CGRP concentrations in both loaded and contralateral ulnae. Administration of CGRP_8–37 was associated with increased targeted remodeling in the fatigue-loaded ulna. Administration of CGRP or CGRP_8–37 both increased reparative bone formation over the study period. Plasma concentration of TRAP5b was not significantly influenced by either CGRP or CGRP_8–37 administration.

Conclusions

CGRP signaling modulates targeted remodeling of microdamage and reparative new bone formation after bone fatigue, and may be part of a neuronal signaling pathway which has regulatory effects on load-induced repair responses within the skeleton.     

E-Selectin Mediated Adhesion and Migration of Endothelial Colony Forming Cells Is Enhanced by SDF-1α/CXCR4

Objective

Endothelial-colony forming cells (ECFCs) can be readily expanded from human umbilical cord blood and can facilitate repair of endothelial injury. E-selectin and SDF-1α are produced following endothelial injury and can regulate endothelial progenitor homing. Mechanisms of vascular repair specific to the mode of injury have not been well described in homogenous cell populations such as ECFCs and are needed for development of more effective vascular repair strategies.

Methods and Results

Lipopolysaccharide (LPS)-induced endotoxic injury to mature human umbilical vein endothelial cells (HUVEC) was compared with hypoxic and radiation injury. E-selectin expression in HUVEC cells is markedly increased (208-fold) following LPS-induced injury and facilitates increased ECFC adhesion and migration function in vitro. SDF-1α expression remains unchanged in LPS-treated HUVEC cells but increases more than 2 fold in fibroblasts undergoing similar endotoxic injury. SDF-1α induces expression of E-selectin ligands on ECFCs and facilitates greater E-selectin-mediated adhesion and migration of ECFCs in a CXCR4-dependent manner. Induction of E-selectin expression in HUVECs following hypoxic or radiation injury is negligible, however, while SDF-1α is increased markedly following hypoxia, highlighting injury-specific synergism between mediators of vascular repair.

Conclusion

E-selectin mediates adhesion and migration of ECFCs following endotoxic endothelial injury. SDF-1α augments E-selectin mediated ECFC adhesion and migration in a CXCR4-dependent manner.     

Preclinical evaluation of zoledronate using an in vitro mimetic cellular model for breast cancer metastatic bone disease

Background

The interactions between metastatic breast cancer cells and host cells of osteoclastic lineage in bone microenvironment are essential for osteolysis. In vitro studies to evaluate pharmacological agents are mainly limited to their direct effects on cell lines. To mimic the communication between breast cancer cells and human osteoclasts, a simple and reproducible cellular model was established to evaluate the effects of zoledronate (zoledronic acid, ZOL), a bisphosphonate which exerts antiresorptive properties.

Methods

Human precursor osteoclasts were cultured on bone-like surfaces in the presence of stimuli (sRANKL, M-CSF) to ensure their activation. Furthermore, immature as well as activated osteoclasts were co-cultured with MDA-MB-231 breast cancer cells. TRAP5b and type I collagen N-terminal telopeptide (NTx) were used as markers. Osteoclasts’ adhesion to bone surface and subsequent bone breakdown were evaluated by studying the expression of cell surface receptors and certain functional matrix macromolecules in the presence of ZOL.

Results

ZOL significantly suppresses the precursor osteoclast maturation, even when the activation stimuli (sRANKL and M-SCF) are present. Moreover, it significantly decreases bone osteolysis and activity of MMPs as well as precursor osteoclast maturation by breast cancer cells. Additionally, ZOL inhibits the osteolytic activity of mature osteoclasts and the expression of integrin β3, matrix metalloproteinases and cathepsin K, all implicated in adhesion and bone resorption.

Conclusions

ZOL exhibits a beneficial inhibitory effect by restricting activation of osteoclasts, bone particle decomposition and the MMP-related breast cancer osteolysis.

General significance

The proposed cellular model can be reliably used for enhancing preclinical evaluation of pharmacological agents in metastatic bone disease.     

Plasma L-cystine/L-glutamate imbalance increases tumor necrosis factor-alpha from CD14+ circulating monocytes in patients with advanced cirrhosis

Background and Aims

The innate immune cells can not normally respond to the pathogen in patients with decompensated cirrhosis. Previous studies reported that antigen-presenting cells take up L-Cystine (L-Cys) and secrete substantial amounts of L-Glutamate (L-Glu) via the transport system Xc- (4F2hc+xCT), and that this exchange influences the immune responses. The aim of this study is to investigate the influence of the plasma L-Cys/L-Glu imbalance observed in patients with advanced cirrhosis on the function of circulating monocytes.

Methods

We used a serum-free culture medium consistent with the average concentrations of plasma amino acids from patients with advanced cirrhosis (ACM), and examined the function of CD14+ monocytes or THP-1 under ACM that contained 0–300 nmol/mL L-Cys with LPS. In patients with advanced cirrhosis, we actually determined the TNF-alpha and xCT mRNA of monocytes, and evaluated the correlation between the plasma L-Cys/L-Glu ratio and TNF-alpha.

Results

The addition of L-Cys significantly increased the production of TNF alpha from monocytes under ACM. Monocytes with LPS and THP-1 expressed xCT and a high level of extracellular L-Cys enhanced L-Cys/L-Glu antiport, and the intracellular GSH/GSSG ratio was decreased. The L-Cys transport was inhibited by excess L-Glu. In patients with advanced cirrhosis (n = 19), the TNF-alpha and xCT mRNA of monocytes were increased according to the Child-Pugh grade. The TNF-alpha mRNA of monocytes was significantly higher in the high L-Cys/L-Glu ratio group than in the low ratio group, and the plasma TNF-alpha was significantly correlated with the L-Cys/L-Glu ratio.

Conclusions

A plasma L-Cys/L-Glu imbalance, which appears in patients with advanced cirrhosis, increased the TNF-alpha from circulating monocytes via increasing the intracellular oxidative stress. These results may reflect the immune abnormality that appears in patients with decompensated cirrhosis.     

miR-31 controls osteoclast formation and bone resorption by targeting RhoA

Introduction

Increased activity of osteoclasts is responsible for bone loss and joint destruction in rheumatoid arthritis. For osteoclast development and bone resorption activity, cytoskeletal organization must be properly regulated. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that suppress expression of their target genes. This study was conducted to identify crucial miRNAs to control osteoclasts.

Methods

miRNA expression in the bone marrow-derived macrophages (BMM) with or without receptor activator of nuclear factor κB ligand (RANKL) stimulation was analyzed by miRNA array. To examine the role of specific miRNAs in osteoclast formation, bone resorption activity and actin ring formation, the BMM were retrovirally transduced with miRNA antagomirs. To confirm whether the suppressive effects on osteoclastogenesis by miR-31 inhibition were mediated by targeting RhoA, osteoclast formation was analyzed in the presence of the RhoA inhibitor, exoenzyme C3.

Results

miR-31 was identified as one of the highly upregulated miRNAs during osteoclast development under RANKL stimulation. Inhibition of miR-31 by specific antagomirs suppressed the RANKL-induced formation of osteoclasts and bone resorption. Phalloidin staining of osteoclasts revealed that actin ring formation at the cell periphery was severely impaired by miR-31 inhibition, and clusters of small ringed podosomes were observed instead. In these osteoclasts, expression of RhoA, one of the miR-31 target genes, was upregulated by miR-31 inhibition in spite of the impaired osteoclastogenesis. Treatment with the RhoA inhibitor, exoenzyme C3, rescued the osteoclastogenesis impaired by miR-31 inhibition.

Conclusions

miR-31 controls cytoskeleton organization in osteoclasts for optimal bone resorption activity by regulating the expression of RhoA.     

Quantification of Cytokeratin 5 mRNA Expression in the Circulation of Healthy Human Subjects and after Lung Transplantation

Background

Circulating epithelial progenitor cells are important for repair of the airway epithelium in a mouse model of tracheal transplantation. We therefore hypothesized that circulating epithelial progenitor cells would also be present in normal human subjects and could be important for repair of the airway after lung injury. As lung transplantation is associated with lung injury, which is severe early on and exacerbated during episodes of infection and rejection, we hypothesized that circulating epithelial progenitor cell levels could predict clinical outcome following lung transplantation.

Methodology/Principal Findings

Quantitative Real Time PCR was performed to determine peripheral blood mRNA levels of cytokeratin 5, a previously characterized marker of circulating epithelial progenitor cells. Cytokeratin 5 levels were evaluated in healthy human subjects, in lung transplant recipients immediately post-transplant and serially thereafter, and in heart transplant recipients. All normal human subjects examined expressed cytokeratin 5 in their buffy coat in amounts that were not significantly influenced by age or gender. There was a profound, statistically significant decrease in cytokeratin 5 mRNA expression levels in lung transplant patients compared to healthy human subjects (p = 3.1×10⁻¹³) and to heart transplant recipients. There was a moderate negative correlation between improved circulating cytokeratin 5 mRNA levels in lung transplant recipients with recovering lung function, as measured by improved FEV1 values (rho = −0.39).

Conclusions/Significance

Levels of cytokeratin 5 mRNA, a proxy marker for circulating epithelial progenitor cells, inversely correlated with disease status in lung transplant recipients. It may therefore serve as a biomarker of the clinical outcome of lung transplant patients and potentially other patients with airway injury.     

Microenvironment Modulates Osteogenic Cell Lineage Commitment in Differentiated Embryonic Stem Cells

Background

Due to their self-renewal, embryonic stem cells (ESCs) are attractive cells for applications in regenerative medicine and tissue engineering. Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best. It is possible that the traditional culture methods, which have been used, are not optimal and that other approaches must be explored.

Methodology/Principal Findings

ESCs were differentiated into osteoblast lineage using a micro-mass approach. In response to osteogenic differentiation medium, many cells underwent apoptosis, while others left the micro-mass, forming small aggregates in suspension. These aggregates were cultured in three different culture conditions (adhesion, static suspension, and stirred suspension), then examined for osteogenic potential in vitro and in vivo. In adhesion culture, ESCs primed to become osteoblasts recommitted to the adipocyte lineage in vitro. In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification. In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells.

Conclusions/Significance

We demonstrate that microenvironment modulates cell fate and subsequent tissue formation during ESC differentiation. For effective tissue engineering using ESCs, it is important to develop optimized cell culture/differentiation conditions based upon the influence of microenvironment.     

Inositol Hexakisphosphate Inhibits Osteoclastogenesis on RAW 264.7 Cells and Human Primary Osteoclasts

Background

Inoxitol hexakisphosphate (IP6) has been found to have an important role in biomineralization and a direct effect inhibiting mineralization of osteoblasts in vitro without impairing extracellular matrix production and expression of alkaline phosphatase. IP6 has been proposed to exhibit similar effects to those of bisphosphonates on bone resorption, however, its direct effect on osteoclasts (OCL) is presently unknown.

Methodology/Principal Findings

The aim of the present study was to investigate the effect of IP6 on the RAW 264.7 monocyte/macrophage mouse cell line and on human primary osteoclasts. On one hand, we show that IP6 decreases the osteoclastogenesis in RAW 264.7 cells induced by RANKL, without affecting cell proliferation or cell viability. The number of TRAP positive cells and mRNA levels of osteoclast markers such as TRAP, calcitonin receptor, cathepsin K and MMP-9 was decreased by IP6 on RANKL-treated cells. On the contrary, when giving IP6 to mature osteoclasts after RANKL treatment, a significant increase of bone resorption activity and TRAP mRNA levels was found. On the other hand, we show that 1 µM of IP6 inhibits osteoclastogenesis of human peripheral blood mononuclear cells (PBMNC) and their resorption activity both, when given to undifferentiated and to mature osteoclasts.

Conclusions/Significance

Our results demonstrate that IP6 inhibits osteoclastogenesis on human PBMNC and on the RAW264.7 cell line. Thus, IP6 may represent a novel type of selective inhibitor of osteoclasts and prove useful for the treatment of osteoporosis.     

A Shift towards Pro-Inflammatory CD16+ Monocyte Subsets with Preserved Cytokine Production Potential after Kidney Transplantation

Background

The presence of monocyte-macrophage lineage cells in rejecting kidney transplants is associated with worse graft outcome. At present, it is still unclear how the monocyte-macrophage related responses develop after transplantation. Here, we studied the dynamics, phenotypic and functional characteristics of circulating monocytes during the first 6 months after transplantation and aimed to establish the differences between kidney transplant recipients and healthy individuals.

Methods

Phenotype, activation status and cytokine production capacity of classical (CD14++CD16−), intermediate (CD14++CD16+) and non-classical (CD14+CD16++), monocytes were determined by flow cytometry in a cohort of 33 healthy individuals, 30 renal transplant recipients at transplantation, 19 recipients at 3 months and 16 recipients at 6 months after transplantation using a cross-sectional approach.

Results

The percentage of both CD16+ monocyte subsets was significantly increased in transplant recipients compared to healthy individuals, indicative of triggered innate immunity (p≤0.039). Enhanced production capacity of tumor necrosis factor-α, interferon-γ and interleukin-1β was observed by monocytes at transplantation compared to healthy individuals. Remarkably, three months post-transplant, in presence of potent immunosuppressive drugs and despite improved kidney function, interferon-γ, tumor necrosis factor-α and interleukin-10 production capacity still remained significantly increased.

Conclusion

Our data demonstrate a skewed balance towards pro-inflammatory CD16+ monocytes that is present at the time of transplantation and retained for at least 6 months after transplantation. This shift could be one of the important drivers of early post-transplant cellular immunity.