Background-Potent stem/progenitor cells have been isolated from normal human dental pulps termed dental pulp stem cells (DPSCs). However, it is unknown whether these cells exist in inflamed pulps (IPs).

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 ...

Adult human mesenchymal stem cells (hMSCs) possess multilineage differentiation potential, and differentiated hMSCs have recently been shown to have the ability to transdifferentiate into other lineages. However, the molecular signature of hMSCs is not well-known, and the mechanisms regulating their self-renewal, differentiation, and transdifferentiation are not completely understood. In this study, we demonstrate that fully differentiated hMSCs could dedifferentiate, a likely critical step for transdifferentiation. By comparing the global gene expression profiles of undifferentiated, differentiated, and dedifferentiation cells in three mesenchymal lineages (osteogenesis, chondrogenesis, and adipogenesis), we identified a number of "stemness" and "differentiation" genes that might be essential to maintain adult stem cell multipotency as well as to drive lineagespecific commitment. These genes include those that encode cell surface molecules, as well as components of signaling pathways. These genes may be valuable for developing methods to isolate, enrich, and purify homogeneous population of hMSCs and/or maintain and propagate hMSCs as well as guide or regulate their differentiation for gene and cell-based therapy. Using small interfering RNA gene inactivation, we demonstrate that five genes (actin filamentassociated protein, frizzled 7, dickkopf 3, protein tyrosine phosphatase receptor F, and RAB3B) promote cell survival without altering cell proliferation, as well as exhibiting different effects on the commitment of hMSCs into multiple mesenchymal lineages.

Mesenchymal stem cells (MSCs) derived from adult tissues are an important candidate cell type for cell-based tissue engineering and regenerative medicine. Currently, clinical applications for MSCs require additional surgical procedures to harvest the autologous MSCs (i.e., from bone marrow) or commercial allogeneic alternatives. We have recently identified a population of mesenchymal progenitor cells (MPCs) in traumatized muscle tissue that has been surgically debrided from traumatic orthopaedic extremity wounds. The purpose of this study was to evaluate whether MPCs derived from traumatized muscle may provide a clinical alternative to bonemarrow MSCs by comparing their morphology, proliferation capacity, cell surface epitope profile and differentiation capacity. After digesting the muscle tissue with collagenase, the MPCs cells were enriched by a direct plating technique. The morphology and proliferation rate of the musclederived MPCs was similar to bone-marrow derived MSCs. Both populations expressed cell surface markers characteristic for MSCs (CD 73, CD 90 and CD105), and did not express markers typically absent on MSCs (CD14, CD34 and CD45). After 21 days in specific differentiation media, the histological staining and gene expression of the MPCs and MSCs was characteristic for differentiation into osteoblasts, chondrocytes and adipocytes but not into myoblasts. Our findings demonstrate that traumatized muscle-derived MPCs exhibit similar phenotype and resemble MSCs derived from the bone marrow. MPCs harvested from traumatized muscle tissue may be considered for applications in tissue engineering and regenerative medicine following orthopaedic trauma requiring circumferential debridement.

IntroductionBone marrow (BM) stroma currently represents the most common and investigated source of mesenchymal progenitor cells (MPCs); however, comparable adult progenitor or stem cells have also been isolated from a wide variety of tissues. This study aims to assess the functional similarities of MPCs from different tissues and to identify specific factor(s) related to their multipotency.MethodsFor this purpose, we directly compared MPCs isolated from different adult tissues, including bone marrow, tonsil, muscle, and dental pulp. We first examined and compared proliferation rates, immunomodulatory properties, and multidifferentiation potential of these MPCs in vitro. Next, we specifically evaluated activin A expression profile and activin A:follistatin ratio in MPCs from the four sources.ResultsThe multidifferentiation potential of the MPCs is correlated with activin A level and/or the activin A:follistatin ratio. Interestingly, by siRNA-mediated activin A knockdown, activin A was shown to be required for the chondrogenic and osteogenic differentiation of MPCs. These findings strongly suggest that activin A has a pivotal differentiation-related role in the early stages of chondrogenesis and osteogenesis while inhibiting adipogenesis of MPCs.ConclusionsThis comparative analysis of MPCs from different tissue sources also identifies bone marrow-derived MPCs as the most potent MPCs in terms of multilineage differentiation and immunosuppression, two key requirements in cell-based regenerative medicine. In addition, this study implicates the significance of activin A as a functional marker of MPC identity.

Objective We and others previously demonstrated that sirtuin 1 (SIRT-1) regulates apoptosis and cartilage-specific gene expression in human chondrocytes and mouse models. This study was undertaken to determine if SIRT-1 enzymatic activity plays a protective role in cartilage homeostasis in vivo, by investigating mice with SIRT-1 mutations to characterize their cartilage. Methods Articular cartilage was harvested from the paws and knees of 5- and 6-month-old wild-type (WT) mice and mice homozygous for SIRT-1tm2.1Mcby (SIRT-1y/y), an allele carrying a point mutation that encodes a SIRT-1 protein with no enzymatic activity (y/y mice). Mice ages 2 days old and 6–7 days old were also examined. Mouse joint cartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures. Results We found that articular cartilage tissue sections from y/y mice of up to 6 months of age contained reduced levels of type II collagen, aggrecan, and glycosaminoglycan compared to sections from WT mice. In contrast, protein levels of matrix metalloproteinase 8 (MMP-8), MMP-9, and MMP-13 were elevated in the cartilage of y/y mice. In addition, chondrocyte apoptosis was elevated in SIRT-1 mutant mice as compared to their WT littermates. Consistent with these observations, protein tyrosine phosphatase 1b was elevated in the y/y mice. Conclusion Our in vivo findings in this animal model demonstrate that mice with defective SIRT-1 also have defective cartilage, with elevated rates of cartilage degradation with age. Hence, normal cartilage homeostasis requires enzymatically active SIRT-1 protein.

All multicellular organisms keep a balance between sink and source activities by controlling nutrient transport at strategic positions. In most plants, photosynthetically produced sucrose is the predominant carbon and energy source, whose transport from leaves to carbon sink organs depends on sucrose transporters. In the model plant Arabidopsis thaliana, transport of sucrose into the phloem vascular tissue by SUCROSE TRANSPORTER 2 (SUC2) sets the rate of carbon export from source leaves, just like the SUC2 homologs of most crop plants. Despite their importance, little is known about the proteins that regulate these sucrose transporters. Here, identification and characterization of SUC2-interaction partners revealed that SUC2 activity is regulated via its protein turnover rate and phosphorylation state. UBIQUITIN-CONJUGATING ENZYME 34 (UBC34) was found to trigger turnover of SUC2 in a light-dependent manner. The E2 enzyme UBC34 could ubiquitinate SUC2 in vitro, a function generally associated with E3 ubiquitin ligases. ubc34 mutants showed increased phloem loading, as well as increased biomass and yield. In contrast, mutants of another SUC2-interaction partner, WALL-ASSOCIATED KINASE LIKE 8 (WAKL8), showed decreased phloem loading and growth. An in vivo assay based on a fluorescent sucrose analog confirmed that SUC2 phosphorylation by WAKL8 can increase transport activity. Both proteins are required for the up-regulation of phloem loading in response to increased light intensity. The molecular mechanism of SUC2 regulation elucidated here provides promising targets for the biotechnological enhancement of source strength.carbon allocation | phloem loading | sucrose transporter | posttranslational regulation | Arabidopsis

Fibroblast growth factor – 2 (FGF2) and interleukin – 1β IL-1β) stimulate the expression of matrix metalloproteinases (MMPs) in articular chondrocytes, which may contribute to cartilage degradation and development of osteoarthritis. Histone deacetylases (HDACs) have recently been implicated in the regulation of MMP gene expression. To investigate the functional involvement of HDACs in the signaling pathway of FGF2 and IL-1β, we examined the effects of HDAC inhibition on activities of FGF2 or IL-1β on gene expression of MMP-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs – 5 (ADAMTS5), collagen type II, and aggrecan. Human articular chondrocyte cultures were treated with FGF2 or IL-1β in the presence or absence of HDAC inhibitor (trichostatin A, TSA). Gene expression levels after treatments were assessed using quantitative real time PCR. Results showed that FGF2 and IL-1β both increased MMP-1 and -13 expression, while IL-1βalso increased MMP-3 mRNA levels. These effects were attenuated in the presence of TSA in a dose dependent manner. In contrast to the effects on MMPs, FGF2 decreased mRNA levels of ADAMTS–5, which was not affected by HDAC inhibition. FGF2, IL-1β, and TSA inhibited expression of aggrecan, while TSA also decreased mRNA levels of collagen type II. These findings showed that HDAC inhibition antagonized FGF2 and IL-1β induced MMP expression. Combination of FGF2 and the HDAC inhibitor decreases both anabolic and catabolic genes, which may slow the cartilage turnover and be beneficial for maintaining cartilage integrity.