FGF2 induces RANKL gene expression as well as IL1ß regulated MHC class II in human bone marrow-derived mesenchymal progenitor stromal cells.

OBJECTIVE: Human bone marrow mesenchymal stromal cells (hBM-MSC) are being applied in tissue regeneration and treatment of autoimmune diseases (AD). Their cellular and immunophenotype depend on isolation and culture conditions which may influence their therapeutic application and reflect their in vivo biological functions. We have further characterised the phenotype induced by fibroblast growth factor 2 (FGF2) on healthy donor hBM-MSC focusing on the osteoimmunological markers osteoprotegerin (OPG), receptor activator of nuclear factor kB (RANK), RANK ligand (RANKL) and HLA-DR and their regulation of expression by the inflammatory cytokines IL1ß and IFNγ. METHODS: RANK, RANKL, OPG and HLA-DR expression in hBM-MSC expanded under specific culture conditions, were measured by RT-PCR and flow cytometry. MAPKs induction by FGF2, IL1ß and IFNγ in hBM-MSC was analysed by immunoblotting and RT-PCR. RESULTS: In hBM-MSC, OPG expression is constitutive and FGF2 independent. RANKL expression depends on FGF2 and ERK1/2 activation. IL1ß and IFNγ activate ERK1/2 but fail to induce RANKL. Only IL1ß induces P38MAPK. The previously described HLA-DR induced by FGF2 through ERK1/2 on hBM-MSC, is suppressed by IL1ß through inhibition of CIITA transcription. HLA-DR induced by IFNγ is not affected by IL1ß in hBM-MSC, but is suppressed in articular chondrocytes and lung fibroblasts. CONCLUSIONS: RANKL expression and IL1ß regulated MHC-class II, both induced via activation of the ERK1/2 signalling pathway, are specific for progenitor hBM-MSC expanded in the presence of FGF2. HLA-DR regulated by IL1ß and ERK1/2 is observed on hBM-MSC during early expansion without FGF2 suggesting previous in vivo acquisition. Stromal progenitor cells with this phenotype could have an osteoimmunological role during bone regeneration.

Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-ß.

Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor-associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial-to-mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT-related genes, downregulation of E-cadherin and acquisition of mesenchymal morphology. These effects required cell-to-cell contact and were mediated by surface-bound TGF-ß newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC-conditioned CRC cells were larger and characterized by higher vessel density, decreased E-cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC-conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT-related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells.

Clinical significance of coexisting antitopoisomerase I and anticentromere antibodies in patients with systemic sclerosis: a EUSTAR group-based study.

OBJECTIVES: To determine the clinical characteristics of simultaneous occurrence of antitopoisomerase (ATA) and anticentromere (ACA) autoantibodies in systemic sclerosis (SSc). METHODS: Data of patients (n=4,687) fulfilling the ACR criteria for SSc and followed in the EULAR Scleroderma Trials and Research (EUSTAR) cohort were analysed. Sera from patients with simultaneous ATA and ACA were reanalyzed centrally by indirect immunofluorescence, enzyme immunoassay, and immunoblot to confirm antibody status. RESULTS: A total of 29 patients (0.6%) had been documented double-positive for both ATA and ACA in the EUSTAR database. Sera of 14 cases were available for central analysis, of which 8 were confirmed to unequivocally contain both antibodies. The double-positive patients were on average 52.4 years of age, 87.5% were female, and 62.5% had diffuse cutaneous (dc) SSc. Compared with matched ACA single-positive disease, cutaneous and visceral complications were more prevalent in double-positive cases, but this prevalence did not differ significantly in comparison to ATA single-positives. CONCLUSIONS: Coexistence of ATA and ACA can be found at low prevalence in SSc. The clinical features of double-positive patients are not clearly dissimilar to those of patients harbouring only ATA. The data do not support a direct involvement of these antibodies in the pathogenesis of established SSc, but may lack statistical power.

Interleukin-1ß modulates endochondral ossification by human adult bone marrow stromal cells.

Inflammatory cytokines present in the milieu of the fracture site are important modulators of bone healing. Here we investigated the effects of interleukin-1ß (IL-1ß) on the main events of endochondral bone formation by human bone marrow mesenchymal stromal cells (BM-MSC), namely cell proliferation, differentiation and maturation/remodelling of the resulting hypertrophic cartilage. Low doses of IL-1ß (50 pg/mL) enhanced colony-forming units-fibroblastic (CFU-f) and -osteoblastic (CFU-o) number (up to 1.5-fold) and size (1.2-fold) in the absence of further supplements and glycosaminoglycan accumulation (1.4-fold) upon BM-MSC chondrogenic induction. In osteogenically cultured BM-MSC, IL-1ß enhanced calcium deposition (62.2-fold) and BMP-2 mRNA expression by differential activation of NF-κB and ERK signalling. IL-1ß-treatment of BM-MSC generated cartilage resulted in higher production of MMP-13 (14.0-fold) in vitro, mirrored by an increased accumulation of the cryptic cleaved fragment of aggrecan, and more efficient cartilage remodelling/resorption after 5 weeks in vivo (i.e., more TRAP positive cells and bone marrow, less cartilaginous areas), resulting in the formation of mature bone and bone marrow after 12 weeks. In conclusion, IL-1ß finely modulates early and late events of the endochondral bone formation by BM-MSC. Controlling the inflammatory environment could enhance the success of therapeutic approaches for the treatment of fractures by resident MSC and as well as improve the engineering of implantable tissues.

Fibroblast growth factor 2 and platelet-derived growth factor, but not platelet lysate, induce proliferation-dependent, functional class II major histocompatibility complex antigen in human mesenchymal stem cells.

OBJECTIVE: To document the specificity and the mechanism of induction of a novel class II major histocompatibility complex (MHC) antigen by mitogenic growth factors in human mesenchymal stem cells (MSCs) expanded in vitro for translational applications. METHODS: Expression of class II MHC molecules was measured in human MSCs and differentiated cells expanded in the presence of fibroblast growth factor 2 (FGF-2), platelet-derived growth factor BB (PDGF-BB), human platelet lysate, or interferon-γ (IFNγ). The roles of cell proliferation and growth factor-induced signaling pathways were investigated as well as the class II MHC assembly machinery and functional capacity. RESULTS: FGF-2 and, to a lesser extent, PDGF-BB induced in adult human MSCs the expression of HLA-DR (normally induced by inflammatory cytokines), which was able to stimulate CD4+ T cells via superantigen binding. In contrast to IFNγ, FGF induced HLA-DR expression only in human MSCs proliferating under its mitogenic effect and not in mouse MSCs or in differentiated human cells. Although it induced cell proliferation, human platelet lysate did not cause HLA-DR expression in human MSCs. HLA-DR expression occurred following FGF-specific binding to its receptor(s), mainly FGF receptor 1, without inducing IFNγ or tumor necrosis factor α expression. Both MAPK/ERK-1/2 and phosphatidylinositol 3-kinase/Akt controlled cell proliferation and HLA-DR expression, but only MAPK/ERK-1/2 controlled the induction of the class II MHC transcription activator protein CIITA, the major determinant of HLA-DR transcription. CONCLUSION: The induction of functional HLA-DR in proliferating progenitor MSCs is a property of human MSCs that have been expanded with mitogenic growth factors. This has potential biologic significance in the regulation and/or protection of progenitor cell subpopulations under sustained mitogenic proliferation and needs to be taken into account when expanding MSCs for use in in vivo applications.

The survey on cellular and engineered tissue therapies in Europe in 2011.

Following the coordinated efforts of five established scientific organizations, this report describes the "novel cellular therapy" activity (i.e., cellular treatments excluding hematopoietic stem cells [HSC] for the reconstitution of hematopoiesis) in Europe for the year 2011. Two hundred forty-six teams from 35 countries responded to the cellular therapy survey, 126 teams from 24 countries provided data on 1759 patients using a dedicated survey and 120 teams reported no activity. Indications were musculoskeletal/rheumatological disorders (46%; 99% autologous), cardiovascular disorders (22%; 100% autologous), hematology/oncology, predominantly including the prevention or treatment of graft-versus-host disease (18%; 2% autologous), neurological disorders (2%; 83% autologous), gastrointestinal (1%; 68% autologous), and other indications (12%; 77% autologous). Autologous cells were used predominantly for musculoskeletal/rheumatological (58%) and cardiovascular (27%) disorders, whereas allogeneic cells were used mainly for hematology/oncology (84%). The reported cell types were mesenchymal stem/stromal cells (56%), HSC (23%), chondrocytes (12%), dermal fibroblasts (3%), keratinocytes (2%), and others (4%). In 40% of the grafts, cells were delivered following ex vivo expansion, whereas cells were transduced or sorted, respectively, in 3% and 10% of the reported cases. Cells were delivered intraorgan (42%), intravenously (26%), on a membrane or gel (16%), or using 3D scaffolds (16%). Compared to last year, the number of teams participating in the dedicated survey doubled and, for the first time, all European Group for Blood and Marrow Transplantation teams reporting information on cellular therapies completed the extended questionnaire. The data are compared with those collected since 2008 to identify trends in the field. This year's edition specifically focuses on cardiac cell therapy.

The survey on cellular and engineered tissue therapies in Europe in 2010.

Following the coordinated efforts of five established scientific organizations, this report describes the novel cellular therapy activity in Europe for the year 2010. One hundred six teams from 27 countries responded to the cellular therapy survey, 69 teams from 21 countries provided data on 1010 patients using a dedicated survey; 37 teams reported no activity. These data were combined with an additional 260 records reported by 37 teams in 15 countries to the standard European group for Blood and Marrow Transplantation (EBMT) database. Indications were graft-vs.-host-disease (GvHD; 26%; 11% autologous), musculoskeletal disorders (25%; 93% autologous), cardiovascular disorders (20%; 100% autologous), epithelial disorders (16%; 44% autologous), autoimmune diseases (11%; 55% autologous), and neurological disorders (2%; 62% autologous). Autologous cells were predominantly used for musculoskeletal (39%) and cardiovascular (32%) disorders, whereas allogeneic cells were mainly used for GvHD (58%) and epithelial disorders (23%). The reported cell types were mesenchymal stem/stromal cells (MSC; 49%), hematopoietic stem cells (28%), chondrocytes (10%), dermal fibroblasts (4%), keratinocytes (1%), and others (8%). In 63% of the grafts, cells were delivered following ex vivo expansion, whereas cells were transduced or sorted respectively in 10% or 28% of the reported cases. Cells were delivered intraorgan (45%), intravenously (31%), on a membrane or gel (20%) or using 3D scaffolds (4%). Compared with last year, the number of teams adopting the dedicated survey was 1.25-fold higher and, with few exceptions, the collected data confirmed the captured trends. This year's edition specifically discusses scientific, clinical, regulatory, and commercial aspects related to the use of cell therapy for the repair of cartilage defects.

Close interactions between mesenchymal stem cells and neuroblastoma cell lines lead to tumor growth inhibition.

Mesenchymal stem cells (MSCs) have attracted much interest in oncology since they exhibit marked tropism for the tumor microenvironment and support or suppress malignant cell growth depending on the tumor model tested. The aim of this study was to investigate the role of MSCs in the control of the growth of neuroblastoma (NB), which is the second most common solid tumor in children. In vivo experiments showed that systemically administered MSCs, under our experimental conditions, did not home to tumor sites and did not affect tumor growth or survival. However, MSCs injected intratumorally in an established subcutaneous NB model reduced tumor growth through inhibition of proliferation and induction of apoptosis of NB cells and prolonged the survival of hMSC-treated mice. The need for contact between MSCs and NB cells was further supported by in vitro experiments. In particular, MSCs were found to be attracted by NB cells, and to affect NB cell proliferation with different results depending on the cell line tested. Moreover, NB cells, after pre-incubation with hMSCs, acquired a more invasive behavior towards CXCL12 and the bone marrow, i.e., the primary site of NB metastases. In conclusion, this study demonstrates that functional cross-talk between MSCs and NB cell lines used in our experiments can occur only within short range interaction. Thus, this report does not support the clinical use of MSCs as vehicles for selective delivery of antitumor drugs at the NB site unless chemotherapy and/or radiotherapy create suitable local conditions for MSCs recruitment.

The survey on cellular and engineered tissue therapies in Europe in 2009.

Thanks to the coordinated efforts of four major scientific organizations, this report describes the "novel cellular therapy" activity in Europe for the year 2009. Fifty teams from 22 countries reported data on 814 patients using a dedicated survey, which were combined to additional 328 records reported by 55 teams to the standard European Blood and Marrow Transplantation (EBMT) database. Indications were cardiovascular (37%; 64% autologous), graft-vs.-host disease (27%; 7% autologous), musculoskeletal (17%; 98% autologous), epithelial/parenchymal (8%; 73% autologous), autoimmune (9%; 84% autologous), or neurological diseases (3%; 50% autologous). Autologous cells were used predominantly for cardiovascular (42%) and musculoskeletal (30%) disorders, whereas allogeneic cells were used mainly for graft-vs.-host disease (58%) and cardiovascular (30%) indications. Reported cell types were mesenchymal stem/stromal cells (MSC) (46%), hematopoietic stem cells (27%), chondrocytes (7%), keratinocytes (5%), dermal fibroblast (13%), and others (2%). In 59% of the grafts, cells were delivered after expansion; in 2% of the cases, cells were transduced. Cells were delivered intraorgan (46%), on a membrane or gel (29%), intravenously (16%) or using 3D scaffolds (8%). As compared to last year, the number of teams adopting the dedicated survey was 1.7-fold higher, and, with few exceptions, the collected data confirmed the captured trends. This year's edition specifically describes and discusses the use of MSC for the treatment of autoimmune diseases, due to the scientific, clinical, and economical implications of this topic.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications.

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Platelet lysate as a serum substitute for 2D static and 3D perfusion culture of stromal vascular fraction cells from human adipose tissue.

Fetal bovine serum (FBS) and fibroblast growth factor (FGF)-2 are key supplements for the culture of stromal vascular fraction (SVF) cells from adipose tissue, both for typical monolayer (2D) expansion and for streamlined generation of osteogenic-vasculogenic grafts in 3D perfusion culture. The present study investigates whether factors present in human platelet lysate (PL) could substitute for FBS and FGF-2 in 2D and 3D culture models of SVF cells from human lipoaspirates. SVF cells were grown in medium supplemented with 10% FBS+FGF-2 or with 5% PL. In 2D cultures, PL initially supported SVF cell proliferation, but resulted in growth arrest shortly after the first passage. Freshly isolated SVF cells cultured with both media under perfusion for 5 days within 3D ceramic scaffolds induced bone formation after subcutaneous implantation in nude mice. However, blood vessels of donor origin were generated only using FBS+FGF-2-cultured cells. This was unexpected, because the proportion of CD34+/CD31+ endothelial lineage cells was significantly higher with PL than that of FBS+FGF-2 (33% vs. 3%, respectively). These results support the use of PL as a substitute of FBS+FGF-2 for short-term culture of human SVF cells, and indicate that more specific serum-free formulations are required to maintain a functionally vasculogenic fraction of SVF cells expanded under 3D perfusion.

Immunomodulatory properties of mesenchymal stem cells: a review based on an interdisciplinary meeting held at the Kennedy Institute of Rheumatology Division, London, UK, 31 October 2005.

Multipotent mesenchymal stromal cells isolated from bone marrow and other sites are currently being studied to determine their potential role in the pathogenesis and/or management of autoimmune diseases. In vitro studies have shown that they exhibit a dose-dependent antiproliferative effect on T and B lymphocytes, dendritic cells, natural killer cells and various B cell tumour lines--an effect that is both cell contact and soluble factor dependent. Animal models of autoimmune disease treated with multipotent mesenchymal stromal cells have mostly exhibited a positive clinical response, as have a limited number of patients suffering from acute graft versus host disease. This review summarizes the findings of a 1-day meeting devoted to the subject with the aim of coordinating efforts.

Haematopoietic stem cell transplantation for vasculitis including Behcet's disease and polychondritis: a retrospective analysis of patients recorded in the European Bone Marrow Transplantation and European League Against Rheumatism databases and a review of the literature.

OBJECTIVE: To evaluate the feasibility of haematopoietic stem cell transplantation (HSCT) in vasculitis. METHODS: This is a retrospective analysis of patients who had received HSCT for vasculitic diseases and have been reported to the European League Against Rheumatism autoimmune disease or European Bone Marrow Transplantation ProMISe databases. Information about the disease and outcome was obtained by a questionnaire sent to the referring centres. Response of the disease to HSCT was defined as partial or complete responses according to the ability to reduce immunosuppression after HSCT. In addition, the Medline database was searched for reports on HSCT in patients with vasculitis. RESULTS: Detailed information was obtained for 15 patients, whose median age at HSCT was 37 years. The diagnoses were cryoglobulinaemia in four patients, Behçet's disease in three patients, Wegener's granulomatosis in three patients, and undifferentiated vasculitis, Churg-Strauss angiitis, polychondritis, Takayasu arteritis and polyarteritis nodosa in one patient each. 14 patients received autologous HSCT and 1 an allogeneic HSCT as the first transplant. In three patients, further transplantation was given because of relapse. The overall response, including all consecutive transplantations (HSCT/patient, n = 1-3, median 1.3) to HSCT, was 93%, with 46% complete responses and 46% partial responses; median (range) duration of response at the time of reporting was 45 (16-84) months. Three patients died, one from advanced disease, one from cancer and one from graft-versus-host disease. The Medline search showed five other patients who were effectively treated with HSCT for vasculitic diseases. CONCLUSION: This retrospective study suggests that autologous HSCT is feasible for vasculitis. Its value remains to be tested in prospective controlled studies.

Human articular chondrocytes suppress in vitro proliferation of anti-CD3 activated peripheral blood mononuclear cells.

OBJECTIVE: To investigate whether mature human articular chondrocytes (AC) exhibit an antiproliferative effect on activated peripheral blood mononuclear cells (PBMC) and to compare this effect with other cells of mesenchymal origin. METHODS: AC from healthy cadaveric cartilage were grown for different passages, in the absence (control) or presence of factors enhancing cell de-differentiation (transforming growth factor (TGF)beta1, fibroblast growth factor (FGF)-2, and platelet derived growth factor (PDGF)bb-TFP medium). Cell ability to suppress PBMC proliferation driven by anti-CD3 antibody was measured by tritiated thymidine uptake following incubation for 48 h at different PBMC:AC ratios and expressed as percent of residual proliferation (RP). AC antiproliferative effect was compared to that of control dermal fibroblasts (DF) and bone marrow stromal cells (BMSC). RESULTS: AC exhibited a cell number-dependent antiproliferative effect. The strongest effect (up to 2% RP) was measured using the least expanded AC cultures. The use of TFP medium for AC expansion resulted in a significantly lower antiproliferative effect, in the range of that induced by BMSC (up to 18% RP). Also DF induced a marked antiproliferative effect (up to 11% RP). CONCLUSION: We report for the first time that human AC have a marked antiproliferative effect on anti-CD3 stimulated PBMC, which is reduced upon culture in medium-inducing extensive cell de-differentiation. These results reflect the immunosuppressive properties observed for other different mesenchymal cell types and raise the question of a potential common physiological role in local tissue protection.