ABSTRACT-cell population resident within collagenase-treated, culture-processed bone fragments, which upon migration established a homogeneous population of MPCs. Additionally, we have introduced a system of culturing these MPCs that best supports and maintains their optimal differentiation potential during long-term culture expansion. When cultured as described, the trabecular bone-derived cells display stem cell-like capabilities, characterized by a stable undifferentiated phenotype as well as the ability to proliferate extensively while retaining the potential to differentiate along the osteoblastic, adipocytic, and chondrocytic lineages, even when maintained in long-term in vitro culture.

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Adult human mesenchymal stem cells (MSCs) hold promise for an increasing list of therapeutic uses due to their ease of isolation, expansion, and multilineage differentiation potential. To maximize the clinical potential of MSCs, the underlying mechanisms by which MSC functionality is controlled must be understood. We have taken a deconstructive approach to understand the individual components in vitro, namely the role of candidate "stemness" genes. Our recent microarray gene expression profiling data suggest that interleukin-6 (IL-6) may contribute to the maintenance of MSCs in their undifferentiated state. In this study, we showed that IL-6 gene expression is significantly higher in undifferentiated MSCs as compared to their chondrogenic, osteogenic, and adipogenic derivatives. Moreover, we found that MSCs secrete copious amounts of IL-6 protein, which decreases dramatically during osteogenic differentiation. We further evaluated the role of IL-6 for maintenance of MSC "stemness", using a series of functional assays. The data showed that IL-6 is both necessary and sufficient for enhanced MSC proliferation, protects MSCs from apoptosis, inhibits adipogenic and chondrogenic differentiation of MSCs, and increases the rate of in vitro wound healing of MSCs. We further identified ERK1/2 activation as the key pathway through which IL-6 regulates both MSC proliferation and inhibition of differentiation. Taken together, these findings show for the first time that IL-6 maintains the proliferative and undifferentiated state of bone marrowderived MSCs, an important parameter for the optimization of both in vitro and in vivo manipulation of MSCs. KeywordsMesenchymal stem cells; Differentiation; Stemness; Interleukin-6 Three components are required for successful tissue engineering and regeneration: cells with regenerative potential, a biocompatible scaffold or matrix, and environmental and endogenous influences to drive these cells towards desired functional tissue neo-genesis. Adult human mesenchymal stem cells (MSCs) are a promising source of cells that can largely satisfy the first of our three necessary criteria. MSCs are tissue-resident stem cells [Pereira et al., 1995;Prockop, 1997]. They are commonly isolated from the bone marrow but can also be found in the perivascular regions of most tissues, such as adipose, skeletal muscle, and umbilical cord [Sarugaser et al., 2005;Zuk et al., 2002]. MSCs are capable of differentiation into several cell lineages, including osteoblasts, chondrocytes, adipocytes, and myoblasts [Jiang et al., 2002;Pittenger et al., 1999]. Owing to their ease of isolation, expansion, and multi-lineage differentiation capability, MSCs are a promising cell source for both tissue engineering and in vivo stimulation of other tissue-resident stem cells. In addition, MSCs possess immunosuppressive activities and have been successfully used to treat Graft vs. Host Disease [Le Blanc et al., 2004] and as a source for gene therapy (Osteogenesis Imperfecta) [Le Blanc et al., 2005].Although there is ...

Aggregation of the high affinity receptor for IgE (Fc⑀RI) leads to the phosphorylation of tyrosines on the ␤ and ␥ chains of the receptor by the Src family kinase Lyn. We have studied the interaction between Lyn and the Fc⑀RI in vivo using a transfection-based approach. Fc⑀RI were stably transfected into Chinese hamster ovary cells. The small amount of endogenous Src family kinase was sufficient to phosphorylate receptor tyrosines upon extensive aggregation of Fc⑀RI but not after addition of dimers of IgE. Upon stable co-transfection of Lyn kinase into the cells, dimers were now able to stimulate receptor phosphorylation and the response to more extensive aggregation was enhanced. In contrast, co-transfection with catalytically inactive Lyn inhibited the aggregation-induced phosphorylation by the endogenous kinase, and a quantitatively similar inhibition was observed in cells transfected with the SH4-containing unique domain of Lyn. Consistent with the results of others using alternative approaches, our additional studies using a yeast two-hybrid system detected a direct interaction between intact Lyn or its unique domain and the C-terminal cytoplasmic domain of the ␤ chain but not with the receptor's other cytoplasmic domains.The family of proteins known as the "multichain immune recognition receptors" includes the antigen receptors on B and T-lymphocytes and Fc receptors including the receptor with high affinity for IgE (Fc⑀RI) 1 (1). Highly homologous in structure, all these receptors utilize, at least in part, a common mechanism to initiate cellular responses; multivalent interactions with antigen lead to aggregation of the receptors and is followed by enhanced phosphorylation of tyrosines (in the "ITAM" motifs within the cytoplasmic domains) of the receptor itself by a receptor-associated Src family kinase (2). For Fc⑀RI, we recently presented direct evidence for a "transphosphorylation" mechanism that accounts for the earliest events (3, 4). The data showed that a small fraction of receptors are constitutively associated with the Src family kinase Lyn (4, 5) and that the enhanced phosphorylation that follows aggregation of the receptors is likely to result simply from the apposition of the kinase with its substrate. We have also shown that when the kinase available to the receptor is limited, shuttling of the enzyme between individual aggregates can regulate the intensity of the signal (6). 2The experiments described in this paper mainly explored the sites of interaction between Lyn kinase and Fc⑀RI. For the most part, the prior studies of others explored the interaction between Lyn and isolated portions of the receptor (7-10). The yeast two-hybrid system (11) used in some of our studies is an analogous approach. We also employed transfection techniques, which allowed us to examine the kinase-receptor interactions in a more physiological setting. The latter experiments also allowed us to test the effect of varying the level of Lyn on the responsiveness of the receptors to discrete stimuli, and thereby to t...

Continual loading and articulation cycles undergone by metallic (e.g., titanium) alloy arthroplasty prostheses lead to liberation of a large number of metallic debris particulates, which have long been implicated as a primary cause of periprosthetic osteolysis and postarthroplasty aseptic implant loosening. Long-term stability of total joint replacement prostheses relies on proper integration between implant biomaterial and osseous tissue, and factors that interfere with this integration are likely to cause osteolysis. Because multipotent mesenchymal stem cells (MSCs) located adjacent to the implant have an osteoprogenitor function and are critical contributors to osseous tissue integrity, when their functions or activities are compromised, osteolysis will most likely occur. To date, it is not certain or sufficiently confirmed whether MSCs endocytose titanium particles, and if so, whether particulate endocytosis has any effect on cellular responses to wear debris. This study seeks to clarify the phenomenon of titanium endocytosis by human MSCs (hMSCs), and investigates the influence of endocytosis on their activities. hMSCs incubated with commercially pure titanium particles exhibited internalized particles, as observed by scanning electron microscopy and confocal laser scanning microscopy, with time-dependent reduction in the number of extracellular particles. Particulate endocytosis was associated with reduced rates of cellular proliferation and cell-substrate adhesion, suppressed osteogenic differentiation, and increased rate of apoptosis. These cellular effects of exposure to titanium particles were reduced when endocytosis was inhibited by treatment with cytochalasin D, and no significant effect was seen when hMSCs were treated only with conditioned medium obtained from particulate-treated cells. These findings strongly suggest that the biological responses of hMSCs to wear debris are triggered primarily by the direct endocytosis of titanium particulates, and not mediated by secreted soluble factors. In this manner, therapeutical approaches that suppress particle endocytosis could reduce the bioreactivity of hMSCs to particulates, and enhance long-term orthopedic implant prognosis by minimizing wear-debris periprosthethic osteolysis. ß

The 8 isoform of protein kinase C is phosphorylated on tyrosine in response to antigen activation of the high-affinity receptor for immunoglobulin E. While protein kinase C-6 associates with and phosphorylates this receptor, immunoprecipitation of the receptor revealed that little, if any, tyrosine-phosphorylated protein kinase C-S is receptor associated. In vitro kinase assays with immunoprecipitated tyrosine-phosphorylated protein kinase C-S showed that the modified enzyme had diminished activity toward the receptor y-chain peptide as a substrate but not toward histones or myelin basic protein peptide. We propose a model in which the tyrosine phosphorylation of protein kinase C-S regulates the kinase specificity toward a given substrate. This may represent a general mechanism by which in vivo protein kinase activities are regulated in response to external stimuli.The activation of the mucosal mast cell model, rat basophilic leukemia (RBL) cells, by the interaction of antigen with cell-bound immunoglobulin E (IgE) initiates the rapid translocation and increased activity of protein kinase C (PKC) (1, 2). This PKC activation is critical to the effector function of the mast cell, namely exocytosis of mediators of the allergic response (3,4). Protein kinase C-8 associates with and phosphorylates the high-affinity receptor for IgE FcsRI (5) on these cells, while other PKC isozymes regulate phosphoinositide hydrolysis (6) and induce protooncogene expression (7).Recent studies have begun to suggest that PKC may regulate tyrosine kinases and is itself regulated by phosphorylation interactions with these kinases (8-10). Yao et al. (10) have shown a PKC-mediated inactivation of Bruton tyrosine kinase (BTK), an enzyme mutated in chromosome X-linked agammaglobulinemia patients. Moreover, BTK was found to associate with the f3I isoform of PKC in mast cells. Phosphorylation of serine/threonine kinases by tyrosine kinase activity has also been described (9, 11). Denning et al. (9) showed that PKC-8 was tyrosine phosphorylated in cells expressing oncogenic ras. Li et al. (12) reported that 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment of PKC-8 transfectants also led to tyrosine phosphorylation of this isozyme. More recent evidence from the latter group suggests that the tyrosine phosphorylation of PKC-8 can occur in response to platelet-derived growth factor receptor stimulation in PKC-8 transfected cells (13). Collectively, the latter studies document the phosphorylation of a serine/threonine kinase by a tyrosine kinase activity in response to a stimulus, although the physiological significance of tyrosine phosphorylation of PKC-8 is unclear.In the present study, we investigate whether tyrosine phosphorylation of PKC-8 occurs in response to FceRI aggregation and whether the tyrosine-phosphorylated form of PKC-8 is preferentially receptor associated. We also analyze the effect of tyrosine phosphorylation on the activity of PKC-8 toward various substrates, including the FcsRI y chain. MATERIALS AND METHODSCell Cu...

With the advent of recent protocols to isolate multipotent human mesenchymal stem cells (MSCs), there is a need for efficient transfection methodologies for these cells. Most standard transfection methods yield poor transfection efficiencies for MSCs (<1%). Here we have optimized a high-efficiency transfection technique for low passage MSCs derived from adult human bone marrow. This technique is an extension of electroporation, termed amaxa Nucleofection, where plasmid DNA is transfected directly into the cell nucleus, independent of the growth state of the cell. With this technique, we demonstrate up to 90% transfection efficiency of the viable population of MSCs, using plasmid construct containing a standard cytomegalovirus (CMV) early promoter driving expression of green fluorescent protein (GFP). Although little variation in transfection efficiency was observed between patient samples, a 2-fold difference in transfection efficiency and a 10-fold difference in expression levels per cell were seen using two distinct CMV-GFP expression plasmids. By fluorescence-activated cell sorting, the GFP expressing cells were sorted and subcultured. At 2 wk posttransfection, approx 25% of the population of sorted cells were GFP positive, and by 3 wk, nearly 10% of the cells still retained GFP expression. Transfection of these cells with plasmid containing either the collagen type I (Col1a1) promoter or the cartilage oligomeric matrix protein (COMP) promoter, each driving expression of GFP, produced a somewhat lower transfection efficiency (approx 40%), due in part to the lower activity of transcription from these promoters compared to that of CMV. Transfection with the collagen type II (Col2a1) promoter linked to GFP exhibited low expression, due to the fact that collagen type II is not expressed in these cells. Upon culturing of the Col2a1-GFP transfected cells in a transforming growth factor-beta3-containing medium known to induce mesenchymal chondrogensis, a significant enhancement of GFP level was seen, indicating the ability of the transfected cells to differentiate into chondrocytes and express cartilage-specific genes, such as Col2a1. Taken together, these data provide evidence of the applicability of this technique for the efficient transfection of MSCs.

When multivalent ligands attach to IgEs bound to the receptors with high affinity for IgE on mast cells, the receptors aggregate, tyrosines on the receptors become phosphorylated, and a variety of cellular responses are stimulated. Prior studies, confirmed here, demonstrated that the efficiency with which later events are generated from earlier ones is inversely related to the dissociation rate of the aggregating ligand. This finding suggests that the cellular responses are constrained by a ''kinetic proofreading'' regimen. We have now observed an apparent exception to this rule. Doses of the rapidly or slowly dissociating ligands that generated equivalent levels of tyrosine-phosphorylated receptors comparably stimulated a putatively distal event: transcription of the gene for monocyte chemoattractant protein 1. Possible explanations of this apparent anomaly were explored. W hen aggregated, the receptors with high affinity for IgE (Fc RI), like other members of the family of multichain immune recognition receptors (1), initiate cellular responses by promoting the phosphorylation of tyrosines in their own immunoreceptor tyrosine-based activation motifs (2). These esterifications are mediated by Lyn kinase (3). We and our collaborators, and others, are attempting to define the factors that are quantitatively most important in regulating these initiating events (4-8).In the 2H3 line of the rat basophilic leukemia tumor (9)-the cells in which the Fc RI have been studied most intensively-the amount of kinase available to the receptors appears to be limiting (5, 6). While exploring the consequences of this constraint, we also examined whether some of the cellular signaling pathways stimulated by Fc RI are subject to a kinetic proofreading regimen. In such a regime, the lifetime of the ligandreceptor complexes as well as their concentration determine the intensity of downstream signals (10,11). This proved to be the case with three homologous ligands we examined (12). Cells incubated with IgE directed toward the 2,4-dinitrophenyl (DNP) ligand were stimulated with a protein conjugated alternatively with multiple DNP, 2,4-dinitro-6-carboxyphenyl, or 2-nitrophenyl (NP) groups. These haptenic constituents have relative intrinsic affinities of 1:0.037:0.0006 for the IgE (12), and their complexes are expected to have progressively shorter lifetimes (13). § At doses stimulating comparable phosphorylation of the Fc RI, the more weakly binding derivatives were deficient in stimulating distal responses. Moreover, the synergy between the competition for limited kinase and the kinetic proofreading control of the signaling caused the low-affinity ligands to act as noncompetitive antagonists (12). Those studies were performed largely on cell suspensions. Because adherent cells are more responsive and maintain the level of certain activated components longer (14), we tested whether this difference in the experimental protocol would influence the kinetic proofreading. As reported here, quantitative differences were observed, but the p...