Gudrun Stenbeck

Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells

The directed differentiation of human pluripotent stem cells offers the unique opportunity to generate a broad spectrum of human cell types and tissues for transplantation, drug discovery, and studying disease mechanisms. Here, we report the stepwise generation of bone-resorbing osteoclasts from human embryonic and induced pluripotent stem cells. Generation of a primitive streak-like population in embryoid bodies, followed by specification to hematopoiesis and myelopoiesis by vascular endothelial growth factor and hematopoietic cytokines in serum-free media, yielded a precursor population enriched for cells expressing the monocyte-macrophage lineage markers CD14, CD18, CD11b, and CD115. When plated in monolayer culture in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-kappaB ligand (RANKL), these precursors formed large, multinucleated osteoclasts that expressed tartrate-resistant acid phosphatase and were capable of resorption. No tartrate-resistant acid phosphatase-positive multinucleated cells or resorption pits were observed in the absence of RANKL. Molecular analyses confirmed the expression of the osteoclast marker genes NFATc1, cathepsin K, and calcitonin receptor in a RANKL-dependent manner, and confocal microscopy demonstrated the coexpression of the alphavbeta3 integrin, cathepsin K and F-actin rings characteristic of active osteoclasts. Generating hematopoietic and osteoclast populations from human embryonic and induced pluripotent stem cells will be invaluable for understanding embryonic bone development and postnatal bone disease.

Formation and function of the ruffled border in osteoclasts

Osteoclasts are multinucleated hematopoietic cells specialised for bone resorption. Dissolution of the inorganic fraction of the bone matrix is mediated by acidification of the bone surface in contact with the osteoclast whereas secreted lysosomal enzymes digest organic components. Through massive exocytosis, the plasma membrane in contact with the bone surface enlarges into the ruffled border, which has unusual features more similar to endosomal/lysosomal membranes. Maintenance of the ruffled border during resorption is achieved through a balance between exocytosis and endocytosis. Inactivation of proteins necessary for the extracellular acidification or of the proteases involved in matrix degradation leads to osteopetrosis; a disease characterised by dense bones.

Regulation of Osteoblast Differentiation by Pasteurella Multocida Toxin (PMT): A Role for Rho GTPase in Bone Formation†

The role of the Rho‐Rho kinase signaling pathway on osteoblast differentiation was investigated using primary mouse calvarial cells. The bacterial toxin PMT inhibited, whereas Rho‐ROK inhibitors stimulated, osteoblast differentiation and bone nodule formation. These effects correlated with altered BMP‐2 and −4 expression. These data show the importance of Rho‐ROK signaling in osteoblast differentiation and bone formation.

Endocytic trafficking in actively resorbing osteoclasts

Endocytosis and the subsequent intracellular trafficking of the endocytosed material are important determinants of cellular function. Osteoclasts, cells of the monocyte/macrophage family, are specialized for the internalization and processing of bone matrix. Transcytosis of endocytosed material has been observed in osteoclasts but the precise mechanism controlling this process is unclear. Here, we investigate the regulation of these trafficking events. To establish the directionality and kinetics of trafficking events in resorbing osteoclasts, we devised a system using fluorescent low-molecular-weight markers as probes to follow the route taken by the digested bone matrix. We demonstrate that this route is largely distinct from the pathway followed by proteins taken up by receptor-mediated endocytosis at the basolateral plasma membrane. Endocytosis and transcytosis from the ruffled border are fast processes, with a half-life of the endocytosed material inside the cells of 22 minutes. We demonstrate the crucial role of the microtubule network in transport from the ruffled-border area and provide evidence for a role of the cytoskeleton in the overall efficacy of trafficking. Moreover, we analyse the effect of the V-ATPase inhibitor bafilomycin A1 on endocytic uptake, which gives insight into the pH-dependent regulation of membrane trafficking and resorption in osteoclasts.

γ-COP, a coat subunit of non-clathrin-coated vesicles with homology to Sec21p

Constitutive secretory transport in eukaryotes is likely to be mediated by non‐clathrin‐coated vesicles, which have been isolated and characterized [(1989) Cell 58, 329–336; (1991) Nature 349, 215–220]. They contain a set of coat proteins (COPs) which are also likely to exist in a preformed cytosolic complex named coatomer [(1991) Nature 349, 248–250]. From peptide sequence and cDNA structure comparisons evidence is presented that one of the subunits of coatomer, γ‐COP, is a true constituent of non‐clathrin‐coated vesicles, and that γ‐COP is related to sec 21, a secretory mutant of the yeast Saccharomyces cervisiae.

Soluble NSF-attachment proteins

Soluble NSF-attachment proteins (SNAPs) are highly conserved proteins that participate in intracellular membrane fusion and vesicular trafficking. In mammals, there are three different isoforms of SNAPs, alpha-, beta- and gamma-SNAP. alpha- and gamma-SNAP are ubiquitously expressed, whereas beta-SNAP is the brain-specific isoform. SNAPs recruit NSF to the membrane after being bound to specific membrane receptors termed SNAREs, NSF, SNAPs and SNAREs form a heterooligomeric complex that is disrupted upon ATP hydrolysis by NSF, which is a prerequisite of membrane fusion. In addition, beta-SNAP interacts with the putative synaptic calcium sensor protein, synaptotagmin, and may be involved in calcium-regulated exocytosis. In the future, this property might be exploited for the development of new therapeutics for certain CNS pathologies.

Membrane topology of the 22 kDa integral peroxisomal membrane protein

In order to study the membrane topology and the possible function of the rat liver 22 kDa integral peroxisomal membrane protein (PMP 22) at a molecular level, we have cloned PMP 22 from a λgt11 expression library and sequenced its cDNA. Hydropathy analysis of the deduced primary structure indicates 4 putative transmembrane segments. The accessibility to exogenous aminopeptidase of PMP 22 in intact peroxisomes suggests that the N‐terminus faces the cytosol. A model of the topology of PMP 22 in the peroxisomal membrane is discussed. Homology studies revealed a striking similarity with the Mpv 17 gene product. Lack of this membrane protein causes nephrotic syndrome in mice.

Integrins and Other Cell Surface Attachment Molecules of Bone Cells

Publisher Summary Bone and cartilage cells express a wide variety of adhesion. Integrin expression has been studied extensively, but, generally, there is less information on expression of other adhesion molecule family members. Furthermore, there is also relatively little information on the expression and function of adhesion molecules of all classes during skeletal cell development, largely because currently, adequate markers to identify immature bone cells are not present, though the phenotypes of knockout mice are increasingly informative. Adhesion receptors fulfill many functions in the skeleton, and these are frequently linked to a variety of intracellular signaling pathways, leading to a central regulatory role for this class of molecules in bone metabolism. Knowledge of their role in bone resorption and cartilage integrity is extensive, although a function for cell adhesion receptors in bone formation has only been defined recently. Although no unique osteoblast, osteoclast, or chondrocyte adhesion molecule has been identified to date, therapeutic strategies based on selectively inhibiting highly expressed receptors, such as the αvβ3 integrin in osteoclasts, have proved to be successful in regulating excessive bone resorption. Better knowledge of the expression of adhesion molecules in bone and cartilage pathology is required, and elucidation of the role of cell–matrix interactions in the etiology of skeletal disease is likely to, therefore, remain a research challenge for the foreseeable future.

Innate immune humoral factors, C1q and factor H, with differential pattern recognition properties, alter macrophage response to carbon nanotubes

UNLABELLED Interaction between the complement system and carbon nanotubes (CNTs) can modify their intended biomedical applications. Pristine and derivatised CNTs can activate complement primarily via the classical pathway which enhances uptake of CNTs and suppresses pro-inflammatory response by immune cells. Here, we report that the interaction of C1q, the classical pathway recognition molecule, with CNTs involves charge pattern and classical pathway activation that is partly inhibited by factor H, a complement regulator. C1q and its globular modules, but not factor H, enhanced uptake of CNTs by macrophages and modulated the pro-inflammatory immune response. Thus, soluble complement factors can interact differentially with CNTs and alter the immune response even without complement activation. Coating CNTs with recombinant C1q globular heads offers a novel way of controlling classical pathway activation in nanotherapeutics. Surprisingly, the globular heads also enhance clearance by phagocytes and down-regulate inflammation, suggesting unexpected complexity in receptor interaction. FROM THE CLINICAL EDITOR Carbon nanotubes (CNTs) maybe useful in the clinical setting as targeting drug carriers. However, it is also well known that they can interact and activate the complement system, which may have a negative impact on the applicability of CNTs. In this study, the authors functionalized multi-walled CNT (MWNT), and investigated the interaction with the complement pathway. These studies are important so as to gain further understanding of the underlying mechanism in preparation for future use of CNTs in the clinical setting.

Role of vesicular trafficking in skeletal dynamics

Vesicular trafficking is critical for the function of bone cells, exemplified by bone diseases such as osteopetrosis, which frequently results from defects in this process. Recent work has further dissected the role of the endolysosomal system in both bone formation by osteoblasts and bone resorption by osteoclasts. This pathway also plays an important role in the communication between these and other cells in bone, through trafficking and degradation of growth factors and their receptors, and microvesicle release. In addition, a crucial role for autophagy in bone remodelling and bone disease is beginning to emerge. These insights into the molecular control of bone remodelling raise the possibility of developing novel therapeutics for bone diseases designed to target specific aspects of this process.