Rapid isolation, expansion and differentiation of osteoprogenitors from full-term umbilical cord blood

There is an urgent clinical requirement for appropriate bone substitutes that can be used for the repair and regeneration of diseased or damaged skeletal tissues. Cell-sourcing limitations in particular have affected progress, largely because of the shortage of accessible tissues capable of yielding sufficient numbers of viable osteoprogenitor cells. Previous work has suggested that umbilical cord blood (UCB) contains circulating progenitor cells (mesenchymal stem cells) capable of osteogenic differentiation, although a comparable number of reports refute this claim. From a screen of more than 20 different culture conditions, we have identified an optimal, simple, and reliable technique to generate, from full-term human UCB, stromal cells with the ability to undergo rapid osteogenic differentiation. By comparing different sorting and culture strategies, we demonstrated that early exposure of mononuclear UCB cells to medium conditioned by osteoblastic cells in the presence of osteogenic supplements and human plasma, markedly increased the frequency of stromal cell growth, the rate of osteogenic differentiation, and their attachment to and spreading on calcium phosphate scaffolds. These findings suggest that full-term UCB may act as an appropriate source of osteoprogenitor cells, which will impact significantly on the development of autologous tissue- engineered bone constructs

NMDA receptor mediated regulation of human megakaryocytopoiesis

Identification of the regulatory inputs that direct megakaryocytopoiesis and platelet production is essential for the development of novel therapeutic strategies for the treatment of thrombosis and related hematologic disorders. We have previously shown that primary human megakaryocytes express the N-methyl-D-aspartate acid (NMDA) receptor 1 (NR1) subunit of NMDA-type glutamate receptors, which appear to be pharmacologically similar to those identified at neuronal synapses, responsible for mediating excitatory neurotransmission in the central nervous system. However, the functional role of NMDA receptor signaling in megakaryocytopoiesis remains unclear. Here we provide evidence that demonstrates the fundamental importance of this signaling pathway during human megakaryocyte maturation in vitro. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of RNA extracted from CD34+-derived megakaryocytes identified expression of NR2A and NR2D receptor subunits in these cells, as well as the NMDA receptor accessory proteins, Yotiao and postsynaptic density protein 95 (PSD-95). In functional studies, addition of a selective NMDA receptor antagonist, MK-801 inhibited proplatelet formation, without affecting proliferation or apoptosis. Exposure of CD34+ cells to MK-801 cultured for 14 days in the presence of thrombopoietin induced a decrease in expression of the megakaryocyte cell surface markers CD61, CD41a, and CD42a compared with controls. At an ultrastructural level, MK-801–treated cells lacked -granules, demarcated membranes, and multilobed nuclei, which were prominent in untreated mature megakaryocyte controls. Using immunohistochemistry on sections of whole tibiae from c-Mpl knockout mice we demonstrated that megakaryocytic NMDA receptor expression was maintained following c-Mpl ablation. These data support a fundamental role for glutamate signaling in megakaryocytopoiesis and platelet production, which is likely to be independent of thrombopoietin-mediated effects

Collagen–Poly(N-isopropylacrylamide) Hydrogels with Tunable Properties

NoThere is a lack of hydrogel materials whose properties can be tuned at the point of use. Biological hydrogels, such as collagen, gelate at physiological temperatures; however, they are not always ideal as scaffolds because of their low mechanical strength. Their mechanics can be improved through cross-linking and chemical modification, but these methods still require further synthesis. We have demonstrated that by combining collagen with a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAM), the mechanical properties can be improved while maintaining cytocompatibility. Furthermore, different concentrations of this polymer led to a range of hydrogels with shear moduli ranging from 105 Pa down to less than 102 Pa, similar to the soft tissues in the body. In addition to variable mechanical properties, the hydrogel blends have a range of micron-scale structures and porosities, which caused adipose-derived stromal cells (ADSCs) to adopt different morphologies when encapsulated within and may therefore be able to direct cell fate.EPSR

Wnt signalling in osteoblasts regulates expression of the receptor activator of NF?B ligand and inhibits osteoclastogenesis in vitro

Reports implicating Wnt signalling in the regulation of bone mass have prompted widespread interest in the use of Wnt mimetics for the treatment of skeletal disorders. To date much of this work has focused on their anabolic effects acting on cells of the osteoblast lineage. In this study we provide evidence that Wnts also regulate osteoclast formation and bone resorption, through a mechanism involving transcriptional repression of the gene encoding the osteoclastogenic cytokine receptor activator of NF{kappa}B ligand (RANKL or TNFSF11) expressed by osteoblasts. In co-cultures of mouse mononuclear spleen cells and osteoblasts, inhibition of GSK3ß with LiCl or exposure to Wnt3a inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated cells compared with controls. However, these treatments had no consistent effect on the differentiation, survival or activity of osteoclasts generated in the absence of supporting stromal cells. Activation of Wnt signalling downregulated RANKL mRNA and protein expression, and overexpression of fulllength ß-catenin, but not transcriptionally inactive ß-catenin {Delta}C(695-781), inhibited RANKL promoter activity. Since previous studies have demonstrated an absence of resorptive phenotype in mice lacking LRP5, we determined expression of a second Wnt co-receptor LRP6 in human osteoblasts, CD14+ osteoclast progenitors and mature osteoclasts. LRP5 expression was undetectable in CD14-enriched cells and mature human osteoclasts, although LRP6 was expressed at high levels by these cells. Our evidence of Wnt-dependent regulation of osteoclastogenesis adds to the growing complexity of Wnt signalling mechanisms that are now known to influence skeletal function and highlights the requirement to develop novel therapeutics that differentially target anabolic and catabolic Wnt effects in bone

Advancement of Mesenchymal Stem Cell Therapy in Solid Organ Transplantation (MISOT)

There is evolving interest in the use of mesenchymal stem cells (MSC) in solid organ transplantation. Pre-clinical transplantation models show efficacy of MSC in prolonging graft survival and a number of clinical studies are planned or underway. At a recent meeting of the MISOT consortium (MSC In Solid Organ Transplantation) the advances of these studies were evaluated and mechanisms underlying the potential effects of MSC discussed. Continued discussion is required for definition of safety and eventually efficacy endpoints for MSC therapy in solid organ transplantation.Nephrolog