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Background. Mesenchymal stem cells (MSCs) have a therapeutic potential for the treatment of osteoarthritic (OA) joint pathology and pain. The aims of this study were to determine the influence of a passage number on the effects of MSCs on pain behaviour and cartilage and bone features in a rodent model of OA. Methods. Rats underwent either medial meniscal transection (MNX) or sham surgery under anaesthesia. Rats received intra-articular injection of either 1.5 × 106 late passage MSCs labelled with 10 μg/ml SiMAG, 1.5 × 106 late passage mesenchymal stem cells, the steroid Kenalog (200 μg/20 μL), 1.5 × 106 early passage MSCs, or serum-free media (SFM). Sham-operated rats received intra-articular injection of SFM. Pain behaviour was quantified until day 42 postmodel induction. Magnetic resonance imaging (MRI) was used to localise the labelled cells within the knee joint. Results. Late passage MSCs and Kenalog attenuated established pain behaviour in MNX rats, but did not alter MNX-induced joint pathology at the end of the study period. Early passage MSCs exacerbated MNX-induced pain behaviour for up to one week postinjection and did not alter joint pathology. Conclusion. Our data demonstrate for the first time the role of a passage number in influencing the therapeutic effects of MSCs in a model of OA pain.

Inflammation is characterised by the infiltration of leukocytes from the circulation and into the inflamed area. Leukocytes are guided throughout this process by chemokines. These are basic proteins which interact with leukocytes to initiate their activation and extravasation via chemokines receptors. This is enabled through chemokine immobilisation by glycosaminoglycans (GAGs) at the luminal endothelial surface of blood vessels. A specific stretch of basic amino acids on the chemokine, often at the C-terminus, interacts with the negatively charged GAGs which is considered an essential interaction for the chemokine function. Short chain peptides based on this GAG binding region of the chemokines CCL5, CXCL8 and CXCL12γ were synthesised using standard Fmoc chemistry. These peptides were found to bind to GAGs with high affinity which translated into a reduction of leukocyte migration across a cultured endothelial monolayer in response to chemokines. The leukocyte migration was inhibited upon removal of heparan sulphate (HS) from the endothelial surface and was found to reduce the ability of the chemokine and peptide to bind to endothelial cells in binding assays and to human rheumatoid arthritis (RA) tissue. Furthermore, control peptides lacking the basic amino acids showed reduced interaction with HS and no anti-chemotactic ability for leukocytes across an endothelial monolayer. The data suggest that the peptide competes with the wildtype (WT) chemokine for binding to GAGs such as HS and thereby reduces chemokine presentation and subsequent leukocyte migration. Furthermore, the lead peptide based on CXCL8 could reduce the disease severity and serum levels of the pro-inflammatory cytokine TNFα in a murine antigen induced arthritis model. Taken together, evidence is provided for interfering with the chemokine – GAG interaction as a relevant therapeutic approach. The use of site specific sequences of chemokines to target GAGs and compete with WT chemokines is a novel and promising avenue for the field.

Stem cell therapy may rely on delivery and homing through the vascular system to reach the target tissue. An optical tweezer model has been employed to exert different levels of shear stress on a single non-adherent human bone marrow–derived mesenchymal stem cell to simulate physiological flow conditions. A single-cell quantitative polymerase chain reaction analysis showed that collagen type 1, alpha 2 ( COL1A2), heat shock 70-kDa protein 1A ( HSPA1A) and osteopontin ( OPN) are expressed to a detectable level in most of the cells. After exposure to varying levels of shear stress, there were significant variations in gene transcription levels across human mesenchymal stem cells derived from four individual donors. Significant trend towards upregulation of COL1A2 and OPN gene expression following shear was observed in some donors with corresponding variations in HSPA1A gene expression. The results indicate that shear stress associated with vascular flow may have the potential to significantly direct non-adherent stem cell expression towards osteogenic phenotypic expression. However, our results demonstrate that these results are influenced by the selection process and donor variability.

Human embryonic stem cells (hESCs) hold great promise therapeutically. In order to deliver on this promise the correct defined conditions for long-term propagation must first be established. Researchers have now provided reports describing the benefits of culturing hESCs in physiologically approximate levels of oxygen. These physiological values fall in the range of 2 to 5% O2. Benefits include reduced spontaneous differentiation, enhanced chromosomal stability and increased clonality. Aims: The aim of our study was to examine the transcriptional consequences of culturing hESCs in physiological normoxia (2% O2) using microarray technology. Methods: Three karyoptically normal hESC lines (H1, H9 and RH1) were examined. At the initiation of this experiment, established hESC lines were redesignated as passage (p) 0 in 21% O2, then bifurcated into 21% O2 and 2% O2, and maintained for a further ten passages at which time samples were again collected. RNA was extracted from all sample points and subjected to microarray analysis using the Affymetrix U133 Plus 2.0 platform. Bioinformatic analysis was performed using dChip and GoStat. Results: We performed grouped analyses of gene expression of early (p0) versus late (p10) air-cultured cells. This revealed relative stability with six (air p0 baseline vs p10 experimental) and one (air p10 baseline vs p0 experimental) gene(s) displaying both greater than twofold and statistically significant upregulation. Conversely, we identified 302 gene upregulations and 56 downregulations when comparing 21% O2 (p0p10) with 2% O2 (p10). These significantly upregulated changes clustered into 82 over-represented and 9 under-represented ontology terms. These terms were indicative of signaling pathways, developmental potential and metabolism. Hierarchical clustering indicated a trend for 2% O2 cultured cells to cluster collectively with reduced heterogeneity when compared with 21% O2 cultured cells. Conclusions: The gene changes associated with 2% O2 culture may be predictive of novel cellular requirements for stable self-renewal, maintenance of pluripotency, and a reduction of hESC-line heterogeneity.