Mesenchymal Stem Cells from Wharton's Jelly and Amniotic Fluid

The discovery of mesenchymal stem cells (MSCs) in perinatal sources, such as the amniotic fluid (AF) and the umbilical connective tissue, the so-called Wharton's jelly (WJ), has transformed them into promising stem cell grafts for the application in regenerative medicine. The advantages of AF-MSCs and WJ-MSCs over adult MSCs, such as bone marrow-derived mesenchymal stem cells (BMMSCs), include their minimally invasive isolation procedure, their more primitive cell character without being tumourigenic, their low immunogenicity and their potential autologous application in congenital disorders and when cryopreserved in adulthood. This chapter gives an overview of the biology of AF-MSCs and WJMSCs, and their regenerative potential based on the results of recent preclinical and clinical studies. In the end, open questions concerning the use of WJ-MSCs and AF-MSCs in regenerative medicine will be emphasized.

In vitro analysis of osteogenic inhibition in non-union spinal fusion caused by nucleus pulposus cells.

Discectomy and spinal fusion is the gold standard for spinal surgery to relieve pain. However, fusion can be hindered for yet unknown reasons that lead to non-fusions with pseudo-arthrosis. Clinical observations indicate that presence of residual intervertebral disc (IVD) tissue might hinder the ossification. We hypothesize that BMP-antagonists are constantly secreted by IVD cells and potentially prevent the ossification process. Furthermore, L51P, the engineered BMP2 variant, stimulates osseo-induction of bone marrow-derived mesenchymal stem cells (MSC) by antagonizing BMP-inhibitors. Human MSCs, primary nucleus pulposus (NPC) and annulus pulposus cells (AFC) were isolated and expanded in monolayer cultures up to passage 3. IVD cells were seeded in 1.2% alginate beads (4Mio/mL) and separated by culture inserts from MSCs. MSCs were kept in 1:control medium, 2:osteogenic medium±alginate beads, 3:osteogenic medium+NPC (±L51P) and 4:osteogenic medium+AFC (±L51P) for 21 days. Relative gene expression of bone-related genes, alkaline phosphatase assay and histological staining were performed. Osteogenesis of MSCs was hindered as shown by reduced alizarin red staining in the presence of NPC. No such inhibition was observed if co-cultured with alginate only or in the presence of AFC. The results were confirmed on the RNA and protein level. Addition of L51Pto the co- cultures, however, induced mineralization of MSCs in presence of NPC. We demonstrated that NPC secrete BMP-antagonists that prevent osteogenesis of MSCs and L51P can antagonize BMP-antagonists and induce bone formation.

Conditioned medium from fresh and demineralized bone enhances osteoclastogenesis in murine bone marrow cultures

OBJECTIVES Osteoclasts rapidly form on the surface of bone chips at augmentation sites. The underlying molecular mechanism, however, is unclear. Soluble factors released from bone chips in vitro have a robust impact on mesenchymal cell differentiation. Whether these soluble factors change the differentiation of hematopoietic cells into osteoclasts remains unknown. METHODS Osteoclastogenesis, the formation of tartrate-resistant acid phosphatase-positive multinucleated cells, was studied with murine bone marrow cultures exposed to RANKL and M-CSF, and conditioned medium from fresh (BCM) and demineralized bone matrix (DCM). Histochemical staining, gene and protein expression, as well as viability assays were performed. RESULTS This study shows that BCM had no impact on osteoclastogenesis. However, when BCM was heated to 85°C (BCMh), the number of tartrate-resistant acid phosphatase-positive multinucleated cells that developed in the presence of RANKL and M-CSF approximately doubled. In line with the histochemical observations, there was a trend that BCMh increased expression of osteoclast marker genes, in particular the transcription factor c-fos. The expression of c-fos was significantly reduced by the TGF-β receptor I antagonist SB431542. DCM significantly stimulated osteoclastogenesis, independent of thermal processing. CONCLUSIONS These data demonstrate that activated BCM by heat and DBM are able to stimulate osteoclastogenesis in vitro. These in vitro results support the notion that the resorption of autografts may be supported by as yet less defined paracrine mechanisms.

Extracellular Iron is a Modulator of the Differentiation of Osteoclast Lineage Cells.

Osteoclasts originate from the hematopoietic stem cell and share a differentiation pathway with the cells of the monocyte/macrophage lineages. Development and activation of osteoclasts, and as a consequence regulation of bone resorption, depend on two growth factors: macrophage colony-stimulating factor and receptor activator of NF-κB ligand. Furthermore, cell development and activity are modulated by a microenvironment composed of cytokines and growth factors and of the extracellular matrix. Membrane transporters are a means for cells to interact with their environment. Within this study, the expression of proteins regulating cellular iron homeostasis in osteoclast-like cells grown from bone marrow-derived progenitors was compared to the expression of this set of proteins by monocyte/macrophage lineage cells. In differentiating osteoclasts, levels of transcripts encoding transferrin receptor 1 and divalent metal transporter 1 (Slc11A2) were increased, while levels of transcripts encoding ferroportin (Slc40A1) and natural resistance-associated macrophage protein 1 (Slc11A1) were decreased. Supplementation of the culture media with exogenous iron led to an increase in the proliferation of osteoclast progenitor cells and to the expression of a macrophage-like phenotype, while the development of osteoclasts was reduced. Upon transfer of mature OC onto a CaP substrate, iron depletion of the medium with the Fe(3+)-chelator Deferoxamine Mesylate decreased CaP dissolution by ~30 %, which could be restored by addition of exogenous iron. During the 24 h of the assay, no effects were observed on total TRAP activity. The data demonstrate transcriptional regulation of the components of cellular iron transporters during OC development and suggests that iron homeostasis may contribute to fine-tuning of the RANKL-induced OC development.

Cardiovascular changes after PMMA vertebroplasty in sheep: the effect of bone marrow removal using pulsed jet-lavage

Clinically, the displacement of intravertebral fat into the circulation during vertebroplasty is reported to lead to problems in elderly patients and can represent a serious complication, especially when multiple levels have to be treated. An in vitro study has shown the feasibility of removing intravertebral fat by pulsed jet-lavage prior to vertebroplasty, potentially reducing the embolization of bone marrow fat from the vertebral bodies and alleviating the cardiovascular changes elicited by pulmonary fat embolism. In this in vivo study, percutaneous vertebroplasty using polymethylmethacrylate (PMMA) was performed in three lumbar vertebrae of 11 sheep. In six sheep (lavage group), pulsed jet-lavage was performed prior to injection of PMMA compared to the control group of five sheep receiving only PMMA vertebroplasty. Invasive recording of blood pressures was performed continuously until 60 min after the last injection. Cardiac output and arterial blood gas parameters were measured at selected time points. Post mortem, the injected cement volume was measured using CT and lung biopsies were processed for assessment of intravascular fat. Pulsed jet-lavage was feasible in the in vivo setting. In the control group, the injection of PMMA resulted in pulmonary fat embolism and a sudden and significant increase in mean pulmonary arterial pressure. Pulsed jet-lavage prevented any cardiovascular changes and significantly reduced the severity of bone marrow fat embolization. Even though significantly more cement had been injected into the lavaged vertebral bodies, significantly fewer intravascular fat emboli were identified in the lung tissue. Pulsed jet-lavage prevented the cardiovascular complications after PMMA vertebroplasty in sheep and alleviated the severity of pulmonary fat embolism.

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single-stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll-like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin-6 (IL-6) and tumour necrosis factor-α secretion, but not interferon-α or IL-12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte-derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus-induced immunosuppression observed in infected pigs.

Influence of defective bone marrow osteogenesis on fracture repair in an experimental model of senile osteoporosis

http://www.ncbi.nlm.nih.gov/pubmed/20014309

Endocannabinoid content in fetal bovine sera - unexpected effects on mononuclear cells and osteoclastogenesis

The major endocannabinoids (ECs) arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) and related N-ethanolamines act as full and partial agonists at CB(1), CB(2), GPR55, PPAR and TRPV1 receptors to various degrees. These receptors are also expressed in immune cells like monocytes/macrophages where they regulate different cellular processes. In this study, potentially bioactive lipids in fetal bovine sera (FBS) were quantified by GC/MS. We found that several commercial FBS contain ECs and bioactive amounts of 2-AG (250-700 nM). We show that residual 2-AG from FBS can activate primary macrophages and increase migration and RANKL-stimulated osteoclastogenesis. Furthermore, 2-AG high-content sera specifically upregulated LPS-stimulated IL-6 expression in U937 cells. Polymyxin B beads may be used to selectively and efficiently remove 2-AG from sera, but not arachidonic acid and N-ethanolamines. In conclusion, 2-AG in cell culture media may significantly influence cellular experiments. CD14+ mononuclear cells which strongly express surface CB receptors may be particularly sensitive towards residual 2-AG from FBS. Therefore, the EC content in culture media should be controlled in biological experiments involving monocytes/macrophages.

Influence of pregnancy on the adipocytokine and peroxisome proliferator-activated receptor pathways in peripheral blood mononuclear cells from healthy donors and rheumatoid arthritis patients

To identify candidate genes that are regulated by human pregnancy and have the potential to modulate rheumatoid arthritis (RA) disease activity.

Differential modulation of ROS signals and other mitochondrial parameters by the antioxidants MitoQ, resveratrol and curcumin in human adipocytes

Abstract Mitochondrial reactive oxygen species (ROS) have been demonstrated to play an important role as signaling and regulating molecules in human adipocytes. In order to evaluate the differential modulating roles of antioxidants, we treated human adipocytes differentiated from human bone marrow-derived mesenchymal stem cells with MitoQ, resveratrol and curcumin. The effects on ROS, viability, mitochondrial respiration and intracellular ATP levels were examined. MitoQ lowered both oxidizing and reducing ROS. Resveratrol decreased reducing and curcumin oxidizing radicals only. All three substances slightly decreased state III respiration immediately after addition. After 24 h of treatment, MitoQ inhibited both basal and uncoupled oxygen consumption, whereas curcumin and resveratrol had no effect. Intracellular ATP levels were not altered. This demonstrates that MitoQ, resveratrol and curcumin exert potent modulating effects on ROS signaling in human adipocyte with marginal effects on metabolic parameters.

Migration of immature mouse DC across resting endothelium is mediated by ICAM-2 but independent of beta2-integrins and murine DC-SIGN homologues

Immature dendritic cells (DC) reside in tissues where they initiate immune responses by taking up foreign antigens. Since DC have a limited tissue half-life, the DC pool in tissues has to be replenished constantly. This implies that precursor/immature DC must be able to cross non-activated endothelium using as yet unknown mechanisms. Here we show that immature, but not mature bone marrow-derived murine DC migrate across resting endothelial monolayers in vitro. We find that endothelial intercellular adhesion molecule-2 (ICAM-2) is a major player in transendothelial migration (TEM) of immature DC, accounting for at least 41% of TEM. Surprisingly, the ICAM-2-mediated TEM was independent of beta2-integrins, the known ICAM-2 ligands, since neither blocking of beta2-integrins with antibodies nor the use of CD18-deficient DC affected the ICAM-2-specific TEM. In humans, the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) was shown to interact with ICAM-2, suggesting a similar role in mice. However, we find that none of the murine DC-SIGN homologues mDC-SIGN, murine DC-SIGN-related molecule-1 (mSIGN-R1) and mSIGN-R3 is expressed on the surface of bone marrow-derived mouse DC. Taken together, this study shows that ICAM-2 strongly supports transmigration of immature DC across resting endothelium by interacting with ligands that are distinct from beta2-integrins and DC-SIGN homologues.

Single-cell analysis divides bovine monocyte-derived dendritic cells into subsets expressing either high or low levels of inducible nitric oxide synthase

Dendritic cells (DC) are important cells at the interface between innate and adaptive immunity. DC have a key role in antigen processing and presentation to T cells. Effector functions of DC related to innate immunity have not been explored extensively. We show that bovine monocyte-derived DC (mDC) express inducible nitric oxide synthase (iNOS) mRNA and protein and produce NO upon triggering with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes (HKLM). An immunocytochemical analysis revealed that a sizeable subset (20-60%) copiously expresses iNOS (iNOShi) upon IFN-gamma/HKLM triggering, whereas the other subset expressed low levels of iNOS (iNOSlo). Monocyte-derived macrophages (mMphi) are more homogeneous with regard to iNOS expression. The number of cells within the iNOSlo mDC subset is considerably larger than the number of dead cells or cells unresponsive to IFN-gamma/HKLM. The large majority of cells translocated p65 to the nucleus upon triggering by IFN-gamma/HKLM. A contamination of mDC with iNOS-expressing mMphi was excluded as follows. (i) Cell surface marker analysis suggested that mDC were relatively homogeneous, and no evidence for a contaminating subset expressing macrophage markers (e.g. high levels of CD14) was obtained. (ii) iNOS expression was stronger in iNOShi mDC than in mMphi. The use of maturation-promoting stimuli revealed only subtle phenotypic differences between immature and mature DC in cattle. Nevertheless, these stimuli promoted development of considerably fewer iNOShi mDC upon triggering with IFN-gamma/HKLM. Immunocytochemical results showed that although a significant proportion of cells expressed iNOS only or TNF only upon triggering with IFN-gamma/HKLM, a significant number of cells expressed both iNOS and TNF, suggesting that TNF and iNOS producing (TIP) DC are present within bovine mDC populations obtained in vitro.

MRI characteristics and histology of bone marrow lesions in dogs with experimentally induced osteoarthritis

Signal changes within the bone marrow adjacent to osteoarthritic joints are commonly seen on magnetic resonance (MR) images in humans and in dogs. The histological nature of these lesions is poorly known. In this study, we describe the MR imaging of bone marrow lesions adjacent to the stifle joints of dogs with experimental osteoarthritis over 13 months. Histology of the proximal tibia at the end of the study was compared with the last MR imaging findings. In five adult dogs, the left cranial cruciate ligament was transected. Post-operatively, MR imaging was performed at 1, 2, 3, 4, 6, 8, and 13 months. Dogs were euthanised after 13 months and histological specimen of the proximal tibia were evaluated. Bone marrow edema like MR imaging signal changes were seen in every MR examination of all dogs in one or more locations of the proximal tibia and the distal femur. Lesions varied in size and location throughout the whole study with the exception of constantly seen lesions in the epiphyseal and metaphyseal region at the level of the tibial eminence. On histology, hematopoiesis and myxomatous transformation of the bone marrow and/or intertrabecular fibrosis without signs of bone marrow edema were consistent findings in the areas corresponding to the MR imaging signal changes. We conclude that within the bone marrow, zones of increased signal intensity on fat suppressed MR images do not necessarily represent edema but can be due to cellular infiltration. Contrary to humans, hematopoiesis is seen in bone marrow edema-like lesions in this canine model of osteoarthritis.