Frank Barry

Mesenchymal stem cells avoid allogeneic rejection.

Adult bone marrow derived mesenchymal stem cells offer the potential to open a new frontier in medicine. Regenerative medicine aims to replace effete cells in a broad range of conditions associated with damaged cartilage, bone, muscle, tendon and ligament. However the normal process of immune rejection of mismatched allogeneic tissue would appear to prevent the realisation of such ambitions. In fact mesenchymal stem cells avoid allogeneic rejection in humans and in animal models. These finding are supported by in vitro co-culture studies. Three broad mechanisms contribute to this effect. Firstly, mesenchymal stem cells are hypoimmunogenic, often lacking MHC-II and costimulatory molecule expression. Secondly, these stem cells prevent T cell responses indirectly through modulation of dendritic cells and directly by disrupting NK as well as CD8+ and CD4+ T cell function. Thirdly, mesenchymal stem cells induce a suppressive local microenvironment through the production of prostaglandins and interleukin-10 as well as by the expression of indoleamine 2,3,-dioxygenase, which depletes the local milieu of tryptophan. Comparison is made to maternal tolerance of the fetal allograft, and contrasted with the immune evasion mechanisms of tumor cells. Mesenchymal stem cells are a highly regulated self-renewing population of cells with potent mechanisms to avoid allogeneic rejection.

Interferon-γ does not break, but promotes the immunosuppressive capacity of adult human mesenchymal stem cells

The ability of mesenchymal stem cells (MSC) to suppress alloresponsiveness is poorly understood. Herein, an allogeneic mixed lymphocyte response was used as a model to investigate the mechanisms of MSC‐mediated immunomodulation. Human MSC are demonstrated to express the immunosuppressive cytokines hepatocyte growth factor (HGF), interleukin (IL)‐10 and transforming growth factor (TGF)‐β1 at concentrations that suppress alloresponses in vitro. MSC also express cyclooxygenase 1 and 2 and produce prostaglandin E2 constitutively. Blocking studies with indomethacin confirmed that prostaglandins contribute to MSC‐mediated allosuppression. The proinflammatory cytokine interferon (IFN)‐γ did not ablate MSC inhibition of alloantigen‐driven proliferation but up‐regulated HGF and TGF‐β1. IFN‐γ also induced expression of indoleamine 2,3, dioxygenase (IDO), involved in tryptophan catabolism. Use of an antagonist, 1‐methyl‐L‐tryptophan, restored alloresponsiveness and confirmed an IDO contribution to IFN‐γ‐induced immunomodulation by MSC. Addition of the tryptophan catabolite kynurenine to mixed lymphocyte reactions (MLR), blocked alloproliferation. These findings support a model where IDO exerts its effect through the local accumulation of tryptophan metabolites rather than through tryptophan depletion. Taken together, these data demonstrate that soluble factors, or products derived from MSC, modulate immune responses and suggest that MSC create an immunosuppressive microenvironment capable of modulating alloresponsiveness even in the presence of IFN‐γ.

Cell contact, prostaglandin E2 and transforming growth factor beta 1 play non-redundant roles in human mesenchymal stem cell induction of CD4+CD25Highforkhead box P3+ regulatory T cells

Adult human mesenchymal stromal or stem cells (MSC) can differentiate into a variety of cell types and are candidate cellular therapeutics in regenerative medicine. Surprisingly, these cells also display multiple potent immunomodulatory capabilities, including allosuppression, making allogeneic cell therapy a possibility. The exact mechanisms involved in regulatory T cell induction by allogeneic human MSC was examined, using purified CD4+ populations and well‐characterized bone marrow‐derived adult human MSC. Allogeneic MSC were shown to induce forkhead box P3 (FoxP3)+ and CD25+ mRNA and protein expression in CD4+ T cells. This phenomenon required direct contact between MSC and purified T cells, although cell contact was not required for MSC induction of FoxP3 expression in an unseparated mononuclear cell population. In addition, through use of antagonists and neutralizing antibodies, MSC‐derived prostaglandins and transforming growth factor (TGF)‐β1 were shown to have a non‐redundant role in the induction of CD4+CD25+FoxP3+ T cells. Purified CD4+CD25+ T cells induced by MSC co‐culture expressed TGF‐β1 and were able to suppress alloantigen‐driven proliferative responses in mixed lymphocyte reaction. These data clarify the mechanisms of human MSC‐mediated allosuppression, supporting a sequential process of regulatory T cell induction involving direct MSC contact with CD4+ cells followed by both prostaglandin E2 and TGF‐β1 expression. Overall, this study provides a rational basis for ongoing clinical studies involving allogeneic MSC.

Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy

Adult mesenchymal stem cells (MSCs), found in the bone marrow, have the potential to differentiate into multiple connective tissue types, including cartilage. In this study, we examined the potential of a porous gelatin sponge, Gelfoam, for use as a delivery vehicle for MSCs in cartilage regeneration therapy. Adult human MSCs (hMSCs) were seeded throughout the gelatin sponge after a 2-h incubation period. When cultured for 21 days in vitro in a defined medium supplemented with 10 ng/mL of TGF-beta 3, hMSC/Gelfoam constructs produced a cartilage-like extracellular matrix containing sulfated glycosaminoglycans (s-GAGs) and type-II collagen, as evident upon histologic evaluation. Constructs loaded with a cell suspension of 12 x 10(6) cells/mL produced an extracellular matrix containing 21 microg of s-GAG/microg of DNA after 21 days of culture. This production was more efficient than constructs loaded at higher or lower cell densities, indicating that the initial seeding density influences the ability of cells to produce extracellular matrix. When implanted in an osteochondral defect in the rabbit femoral condyle, Gelfoam cylinders were observed to be very biocompatible, with no evidence of immune response or lymphocytic infiltration at the site. Based on these observations we conclude that Gelfoam resorbable gelatin sponge is a promising candidate as a carrier matrix for MSC-based cartilage regeneration therapies.

Monocyte Chemotactic Protein-1 Secreted by Primary Breast Tumors Stimulates Migration of Mesenchymal Stem Cells

Purpose: Major barriers to effective adenovirus-based gene therapy include induction of an immune response and tumor-specific targeting of vectors. The use of mesenchymal stem cells (MSC) as systemic delivery vehicles for therapeutic genes has been proposed as a result of their combined ability to home in on the tumor site and evade the host immune response. This study is aimed at investigating factors mediating homing of human MSCs to breast cancer primary cultures and cell lines in vitro and in vivo. Experimental Design: Fluorescently labeled MSCs were given to mice bearing breast cancer xenografts, and tumor tissue was harvested to detect MSC engraftment. MSC migration in response to primary breast tumors in vitro was quantified, and chemokines secreted by tumor cells were identified. The role of monocyte chemotactic protein-1 (MCP-1) in cell migration was investigated using antibodies and standards of the chemokine. Serum MCP-1 was measured in 125 breast cancer patients and 86 healthy controls. Results: Engrafted MSCs were detected in metastatic breast tumors in mice after systemic administration. There was a significant increase in MSC migration in response to primary breast tumor cells in vitro (6-fold to 11-fold increase). Tumor explants secreted a variety of chemokines including GROα, MCP-1, and stromal cell–derived factor-1α. An MCP-1 antibody caused a significant decrease (37-42%) in MSC migration to tumors. Serum MCP-1 levels were significantly higher in postmenopausal breast cancer patients than age-matched controls (P < 0.05). Conclusions: These results highlight a role for tumor-secreted MCP-1 in stimulating MSC migration and support the potential of these cells as tumor-targeted delivery vehicles for therapeutic agents.

MESENCHYMAL STEM CELL SURFACE ANTIGEN SB-10 CORRESPONDS TO ACTIVATED LEUKOCYTE CELL ADHESION MOLECULE AND IS INVOLVED IN OSTEOGENIC DIFFERENTIATION

Bone marrow contains a rare population of mesenchymal stem cells (MSCs) capable of giving rise to multiple mesodermal tissues including bone, cartilage, tendon, muscle, and fat. The cell surface antigen recognized by monoclonal antibody SB‐10 is expressed on human MSCs but is lost during their developmental progression into differentiated phenotypes. Here we report on the immunopurification of the SB‐10 antigen and its identification as activated leukocyte‐cell adhesion molecule (ALCAM). Mass spectrometry establishes that the molecular mass of ALCAM is 80,303 ± 193 Da and that it possesses 17,763 ± 237 Da of N‐linked oligosaccharide substituents. Molecular cloning of a full‐length cDNA from a MSC expression library demonstrates nucleotide sequence identity with ALCAM. We also identified ALCAM homologs in rat, rabbit, and canine MSCs, each of which is over 90% identical to human ALCAM in their peptide sequence. The addition of antibody SB‐10 Fab fragments to human MSCs undergoing osteogenic differentiation in vitro accelerated the process, thereby implicating a role for ALCAM during bone morphogenesis and adding ALCAM to the group of cell adhesion molecules involved in osteogenesis. Together, these results provide evidence that ALCAM plays a critical role in the differentiation of mesenchymal tissues in multiple species across the phylogenetic tree.

Potential role of mesenchymal stem cells (MSCs) in the breast tumour microenvironment: stimulation of epithelial to mesenchymal transition (EMT)

Bone marrow-derived mesenchymal stem cells (MSCs) are known to specifically migrate to and engraft at tumour sites. Understanding interactions between cancer cells and MSCs has become fundamental to determining whether MSC-tumour interactions should be harnessed for delivery of therapeutic agents or considered a target for intervention. Breast Cancer Cell lines (MDA-MB-231, T47D & SK-Br3) were cultured alone or on a monolayer of MSCs, and retrieved using epithelial specific magnetic beads. Alterations in expression of 90 genes associated with breast tumourigenicity were analysed using low-density array. Expression of markers of epithelial–mesenchymal transition (EMT) and array results were validated using RQ-PCR. Co-cultured cells were analysed for changes in protein expression, growth pattern and morphology. Gene expression and proliferation assays were also performed on indirect co-cultures. Following direct co-culture with MSCs, breast cancer cells expressed elevated levels of oncogenes (NCOA4, FOS), proto-oncogenes (FYN, JUN), genes associated with invasion (MMP11), angiogenesis (VEGF) and anti-apoptosis (IGF1R, BCL2). However, universal downregulation of genes associated with proliferation was observed (Ki67, MYBL2), and reflected in reduced ATP production in response to MSC-secreted factors. Significant upregulation of EMT specific markers (N-cadherin, Vimentin, Twist and Snail) was also observed following co-culture with MSCs, with a reciprocal downregulation in E-cadherin protein expression. These changes were predominantly cell contact mediated and appeared to be MSC specific. Breast cancer cell morphology and growth pattern also altered in response to MSCs. MSCs may promote breast cancer metastasis through facilitation of EMT.

Mesenchymal stem cells in joint disease and repair.

Osteoarthritis (OA), a prevalent chronic condition with a striking impact on quality of life, represents an enormous societal burden that increases greatly as populations age. Yet no approved pharmacological intervention, biologic therapy or procedure prevents the progressive destruction of the OA joint. Mesenchymal stem cells (MSCs)—multipotent precursors of connective tissue cells that can be isolated from many adult tissues, including those of the diarthrodial joint—have emerged as a potential therapy. Endogenous MSCs contribute to maintenance of healthy tissues by acting as reservoirs of repair cells or as immunomodulatory sentinels to reduce inflammation. The onset of degenerative changes in the joint is associated with aberrant activity or depletion of these cell reservoirs, leading to loss of chondrogenic potential and preponderance of a fibrogenic phenotype. Local delivery of ex vivo cultures of MSCs has produced promising outcomes in preclinical models of joint disease. Mechanistically, paracrine signalling by MSCs might be more important than differentiation in stimulating repair responses; thus, paracrine factors must be assessed as measures of MSC therapeutic potency, to replace traditional assays based on cell-surface markers and differentiation. Several early-stage clinical trials, initiated or underway in 2013, are testing the delivery of MSCs as an intra-articular injection into the knee, but optimal dose and vehicle are yet to be established.

CHONDROGENIC DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS WITHIN AN ALGINATE LAYER CULTURE SYSTEM

SummaryHuman mesenchymal stem cells (hMSCs) derived from bone marrow have the capacity to differentiate along a number of connective tissue pathways and are an attractive source of chondrocyte precursor cells. When these cells are cultured in a three-dimensional format in the presence of transforming growth factor-β, they undergo characteristic morphological changes concurrent with deposition of cartilaginous extracellular matrix (ECM). In this study, factors influencing hMSC chondrogenesis were investigated using an alignate layer culture system. Application of this system resulted in a more homogeneous and rapid synthesis of cartilaginous ECM than did micromas cultures and presented a more functional format than did alginate bead cultures. Differentiation was found to be dependent on initial seeding density and was interrelated to cellular proliferation. Maximal glycosaminoglycan (GAG) synthesis defined an, optimal hMSC seeding density for chondrogenesis at 25×106 cells/ml. Inclusion of hyaluronan in the alginate layer at the initiation of cultures enhanced chondrogenic differentiation in a dose-dependent manner, with maximal effect seen at 100 μg/ml. Hyaluronan increased GAG synthesis at early time points, with greater effect, seen at lower cell densities, signifying cell-cell contact involvement. This culture system offers additional opportunities for elucidating conditions influencing chondrogenesis and for modeling cartilage homeostasis or osteoarthritic changes.

Mesenchymal stem cells as vehicles for gene delivery

Mesenchymal stem cells contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose, and marrow stroma. Transduction of mesenchymal stem cells from species other than humans is required for the development of disease models in which mesenchymal stem cells-based gene delivery is evaluated. Attempts to transduce mesenchymal stem cells from some species with amphotropic retroviral vectors were unsuccessful, leading to comparative mesenchymal stem cells transductions with xenotropic and gibbonape leukemia virus envelope-pseudotyped retroviral vectors. Human, baboon, canine, and rat mesenchymal stem cells were transduced optimally with amphotropic vector supernatants. In contrast, sheep, goat, and pig mesenchymal stem cells showed highest transduction levels with xenotropic retroviral vector supernatant, and rabbit mesenchymal stem cells were transduced optimally with gibbon-ape-enveloped vectors. Using a myeloablative canine transplantation model and gene-marked canine mesenchymal stem cells, the biodistribution of infused and ex vivo expanded mesenchymal stem cells were examined. The majority of transduced canine mesenchymal stem cells were found in the bone marrow samples. The current study shows the use of mesenchymal stem cells as a delivery vehicle for gene transfer studies, and validates the feasibility of delivering mesenchymal stem cells to the marrow compartment for stromal regeneration after cancer-associated cytotoxic therapies.