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Simple fabrication and 3D-printing methods are used to generate tubes and filaments from self-assembled gels, which can be loaded in situ with gold nanoparticles, with the resulting gels encouraging stem cell proliferation.

We report the preparation of hybrid self-assembled microgel beads by combining the low molecular weight gelator (LMWG) DBS-CONHNH2 and the natural polysaccharide calcium alginate polymer gelator (PG). Microgel formulations based on LMWGs are extremely rare due to the fragility of the self-assembled networks and the difficulty of retaining any imposed shape. Our hybrid beads contain interpenetrated LMWG and PG networks, and are obtained by an emulsion method, allowing the preparation of spherical gel particles of controllable sizes with diameters in the mm or μm range. Microgels based on LMWG/alginate can be easily prepared with reproducible diameters, We report microgel beads with diameters of ca. 800 nm based on interpenetrating networks of a low-molecular-weight gelator and a polymer gelator, and demonstrate their use as heparin delivery vehicles to enhance stem cell growth.

Mesenchymal stromal cell (MSC) heterogeneity clouds biological understanding and hampers their clinical development. In MSC cultures most commonly used in research and therapy, we have identified an MSC subtype characterised by CD317 expression (CD317pos(29.77±3.00% of the total MSC population), comprising CD317dim(28.10±4.60%) and CD317bright(1.67±0.58%) MSCs) and a constitutive interferon signature linked to human disease. We demonstrate that CD317posMSCs induced cutaneous tissue damage when applied a skin explant model of inflammation, whereas CD317negMSCs had no effect. Only CD317negMSCs were able to suppress proliferative cycles of activated human T cellsin vitro, whilst CD317posMSCs increased polarisation towards pro-inflammatory Th1 cells and CD317negcell lines did not. Using anin vivoperitonitis model, we found that CD317negand CD317posMSCs suppressed leukocyte recruitment but only CD317negMSCs suppressed macrophage numbers. Using MSC-loaded scaffolds implanted subcutaneously in immunocompromised mice we were able to observe tissue generation and blood vessel formation with CD317negMSC lines, but not CD317posMSC lines. Our evidence is consistent with the identification of an immune stromal cell, which is likely to contribute to specific physiological and pathological functions and influence clinical outcome of therapeutic MSCs.

This paper reports simple strategies to fabricate self-assembled artificial tubular and filamentous systems from a low molecular weight gelator (LMWG). In the first strategy, tubular 'core-shell' gel structures based on the dibenzylidenesorbitol-based LMWG DBS-CONHNH

Novel biological therapies have revolutionised the management of Rheumatoid Arthritis (RA) but no cure currently exists. Mesenchymal stem cells (MSCs) immunomodulate inflammatory responses through paracrine signalling, including via secretion of extracellular vesicles (EVs) in the cell secretome. We evaluated the therapeutic potential of MSCs-derived small EVs in an antigen-induced model of arthritis (AIA).EVs isolated from MSCs cultured normoxically (21% O2, 5% CO2), hypoxically (2% O2, 5% CO2) or with a pro-inflammatory cytokine cocktail were applied into the AIA model. Disease pathology was assessed post-arthritis induction through swelling and histopathological analysis of synovial joint structure. Activated CD4+ T cells from healthy mice were cultured with EVs or MSCs to assess deactivation capabilities prior to application of standard EVs in vivo to assess T cell polarisation within the immune response to AIA.All EVs treatments reduced knee-joint swelling whilst only normoxic and pro-inflammatory primed EVs improved histopathological outcomes. In vitro culture with EVs did not achieve T cell deactivation. Polarisation towards CD4+ helper cells expressing IL17a (Th17) was reduced when normoxic and hypoxic EV treatments were applied in vitro. Normoxic EVs applied into the AIA model reduced Th17 polarisation and improved Th17:Treg homeostatic balance.Priming of MSCs in EV production can be applied to alter the therapeutic efficacy however normoxic EVs present the optimal strategy for broad therapeutic benefit. The varied outcomes observed in MSCs priming may promote EVs optimised for therapies targeted for specific therapeutic priorities. EVs present an effective novel technology with potential for cell-free therapeutic translation.

Rheumatoid arthritis (RA) is a debilitating and painful inflammatory autoimmune disease characterised by the accumulation of leukocytes in the synovium, cartilage destruction and bone erosion. The immunomodulatory effects of bone marrow derived mesenchymal stem cells (MSCs) has been widely studied and the recent observations that syndecan-3 (SDC3) is selectively pro-inflammatory in the joint led us to hypothesise that SDC3 might play an important role in MSC biology. MSCs isolated from bone marrow of wild type and Sdc3−/− mice were used to assess immunophenotype, differentiation, adhesion and migration properties and cell signalling pathways. While both cell types show similar differentiation potential and forward scatter values, the cell complexity in wild type MSCs was significantly higher than in Sdc3−/− cells and was accompanied by lower spread surface area. Moreover, Sdc3−/− MSCs adhered more rapidly to collagen type I and showed a dramatic increase in AKT phosphorylation, accompanied by a decrease in ERK1/2 phosphorylation compared with control cells. In a mouse model of antigen-induced inflammatory arthritis, intraarticular injection of Sdc3−/− MSCs yielded enhanced efficacy compared to injection of wild type MSCs. In conclusion, our data suggest that syndecan-3 regulates MSC adhesion and efficacy in inflammatory arthritis, likely via induction of the AKT pathway.

BackgroundNovel biological therapies have revolutionised the management of Rheumatoid Arthritis (RA) but no cure currently exists. Mesenchymal stem cells (MSCs) immunomodulate inflammatory responses through paracrine signalling via growth factors, cytokines, chemokines and extracellular vesicles (EVs) in the cell secretome; however, MSCs are still not available in the clinic. We evaluated the therapeutic potential of MSCs-derived EVs in an antigen-induced model of arthritis (AIA). MethodsEVs isolated from MSCs in normal (21% O2, 5% CO2) or hypoxic (2% O2, 5% CO2) culture or from MSCs pre-conditioned with a pro-inflammatory cytokine cocktail were applied into the AIA model. Disease pathology was assessed 3 days post arthritis induction through histopathological analysis of knee joints. Spleens and lymph nodes were collected and assessed for T cell polarisation within the immune response to AIA. Activated naïve CD4+ T cells from spleens of healthy mice were cultured with EVs or MSCs to assess deactivation capabilities. ResultsAll EV treatments significantly reduced knee-joint swelling and histopathological signs of AIA with enhanced responses to normoxic and pro-inflammatory primed EVs. Polarisation of T cells towards CD4+ helper cells expressing IL17a (Th17) was reduced when EV treatments from MSCs cultured in hypoxia or pro-inflammatory priming conditions were applied. ConclusionsHypoxically cultured EVs present a priming methodology that is as effective in reducing swelling, IL-17a expression, Th17 polarisation and T cell proliferation as pro-inflammatory priming. EVs present an effective novel technology for cell-free therapeutic translation in treating inflammatory arthritis and autoimmune disorders such as RA.

1AbstractRheumatoid arthritis (RA) is a debilitating and painful inflammatory autoimmune disease characterised by the accumulation of leukocytes in the synovium, cartilage destruction and bone erosion. The immunomodulatory effects of bone marrow derived mesenchymal stem cells (MSCs) has been widely studied and the recent observations that syndecan-3 (SDC3) is selectively pro-inflammatory in the joint led us to hypothesise that SDC3 might play an important role in MSC biology. MSCs isolated from bone marrow of wild type and Sdc3−/−mice were used to assess immunophenotype, differentiation, adhesion and migration properties and cell signalling pathways. While both cell types show similar differentiation potential and forward scatter values, the cell complexity in wild type MSCs was significantly higher than in Sdc3−/−cells and was accompanied by lower spread surface area. Moreover, Sdc3−/−MSCs adhered more rapidly to collagen type I and showed a dramatic increase in AKT phosphorylation, accompanied by a decrease in ERK1/2 phosphorylation compared with control cells. In a mouse model of antigen-induced inflammatory arthritis, intraarticular injection of Sdc3−/−MSCs yielded enhanced recovery compared to injection of wild type MSCs. In conclusion, our data suggest that syndecan-3 regulates MSC adhesion and efficacy in inflammatory arthritis, likely via induction of the AKT pathway.

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.

Aim: Human mesenchymal stem cells (hMSC) are multipotent progenitor cells. We propose the optimization of hMSC isolation and recovery using the application of a controlled hypoxic environment. Materials & Methods: We evaluated oxygen, glucose and serum in the recovery of hMSC from bone marrow (BMhMSC). Colony forming units-fibroblastic, cell numbers, tri-lineage differentiation, immunofluorescence and microarray were used to confirm and characterize BMhMSC. Results: In an optimized (2% O2, 4.5 g/l glucose and 5% serum) environment both colony forming units-fibroblastic (p = 0.01) and cell numbers (p = 0.0001) were enhanced over standard conditions. Transcriptional analysis identified differential expression of bone morphogenetic protein 2 (BMP2) and, putatively, chemokine (C-X-C motif) receptor 2 (CXCR2) signaling pathways. Conclusion: We have detailed a potential milestone in the process of refinement of the BMhMSC isolation process.

We evaluated the therapeutic potential of mesenchymal stem cell-conditioned medium (CM-MSC) as an alternative to cell therapy in an antigen-induced model of arthritis (AIA). Disease severity and cartilage loss were evaluated by histopathological analysis of arthritic knee joints and immunostaining of aggrecan neoepitopes. Cell proliferation was assessed for activated and naïve CD4+ T cells from healthy mice following culture with CM-MSC or co-culture with MSCs. T cell polarization was analysed in CD4+ T cells isolated from spleens and lymph nodes of arthritic mice treated with CM-MSC or MSCs. CM-MSC treatment significantly reduced knee-joint swelling, histopathological signs of AIA, cartilage loss and suppressed TNFα induction. Proliferation of CD4+ cells from spleens of healthy mice was not affected by CM-MSC but reduced when cells were co-cultured with MSCs. In the presence of CM-MSC or MSCs, increases in IL-10 concentration were observed in culture medium. Finally, CD4+ T cells from arthritic mice treated with CM-MSC showed increases in FOXP3 and IL-4 expression and positively affected the Treg:Th17 balance in the tissue. CM-MSC treatment reduces cartilage damage and suppresses immune responses by reducing aggrecan cleavage, enhancing Treg function and adjusting the Treg:Th17 ratio. CM-MSC may provide an effective cell-free therapy for inflammatory arthritis.

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.

Rheumatoid arthritis (RA) is the second most common form of arthritis and the most common inflammatory joint disorder in the UK, affecting more than 400,000 people with around 12,000 new cases diagnosed every year. The condition is chronic and degenerative, causing pain and swelling, stiffness and fatigue. The disorder affects females to males at a ratio of approximately 3:1 with an overall incidence of around 1 in 100. It is most commonly reported in people aged >40 years with most first diagnoses occurring at age

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.

Cartilage is poorly vascularised with a limited capacity for repair following damage. The poor vascularisation results in cartilage tissue having a low normoxic value. This study examined and compared the effects of physiological cartilage normoxia (2% O2), hypoxia (0.2% O2), and hyperoxia (21% O2) on human articular chondrocytes (hAC) during similar time courses to those prior to transplant in cell therapy procedures. hAC were isolated and maintained at 0.2% O2, 2% O2, or 21% O2. Population doublings (PDs), cell surface area, chondrogenic differentiation potential, RT-PCR, quantitative RT-PCR and immunohistochemistry (Collagen Type II) were used to confirm chondrogenic differentiation of micromass pellets in different O2. Isolation and maintenance of hAC at =2% O2 resulted in significant alterations in surface area (smaller), rate of proliferation (reduced), and chondrogenic differentiation potential (enhanced). Chondrogenic gene expression appeared largely insensitive to O2 concentration. A relationship was apparent between collagen type II protein presence and O2 concentration. Oxygen concentrations of 2% O2 or less promoted retention of a dedifferentiated hAC phenotype and enhanced stability of hAC chondrogenesis.

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.

Tendon injuries are common in either the workplace or sport activities, with some 3 to 5 million tendon and ligament injuries occurring annually worldwide. Management of tendon injury currently follows two routes: Conservative (rehabilitation and pain relief), or surgical. Irrespective of which of these primary treatment routes are followed, even if healing does occur, it may not result in a full gain of function. The inability of the tendon to self-repair and the relative inefficiency of current treatment regimens suggest that identifying alternative strategies is a priority. One such alternative is the use of stem cells to repair damage, either through direct application or in conjunction with scaffolding. We describe the current state of the art in terms of: (i) Molecular markers of tendon development, (ii) stem cell applicability to human tendon repair, (iii) scaffolding for in vitro tendon generation, and (iv) chemical/molecular approaches to both induce stem cell differentiation into tenocytes and maintain their proliferation in vitro.