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

Peer reviewedPublisher PD

Modulation of Human Mesenchymal Stem Cell Immunogenicity through Forced Expression of Human Cytomegalovirus US Proteins

BACKGROUND: Mesenchymal stem cells (MSC) are promising candidates for cell therapy, as they migrate to areas of injury, differentiate into a broad range of specialized cells, and have immunomodulatory properties. However, MSC are not invisible to the recipient's immune system, and upon in vivo administration, allogeneic MSC are able to trigger immune responses, resulting in rejection of the transplanted cells, precluding their full therapeutic potential. Human cytomegalovirus (HCMV) has developed several strategies to evade cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell recognition. Our goal is to exploit HCMV immunological evasion strategies to reduce MSC immunogenicity. METHODOLOGY/PRINCIPAL FINDINGS: We genetically engineered human MSC to express HCMV proteins known to downregulate HLA-I expression, and investigated whether modified MSC were protected from CTL and NK attack. Flow cytometric analysis showed that amongst the US proteins tested, US6 and US11 efficiently reduced MSC HLA-I expression, and mixed lymphocyte reaction demonstrated a corresponding decrease in human and sheep mononuclear cell proliferation. NK killing assays showed that the decrease in HLA-I expression did not result in increased NK cytotoxicity, and that at certain NK∶MSC ratios, US11 conferred protection from NK cytotoxic effects. Transplantation of MSC-US6 or MSC-US11 into pre-immune fetal sheep resulted in increased liver engraftment when compared to control MSC, as demonstrated by qPCR and immunofluorescence analyses. CONCLUSIONS AND SIGNIFICANCE: These data demonstrate that engineering MSC to express US6 and US11 can be used as a means of decreasing recognition of MSC by the immune system, allowing higher levels of engraftment in an allogeneic transplantation setting. Since one of the major factors responsible for the failure of allogeneic-donor MSC to engraft is the mismatch of HLA-I molecules between the donor and the recipient, MSC-US6 and MSC-US11 could constitute an off-the-shelf product to overcome donor-recipient HLA-I mismatch

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

Human bone marrow mesenchymal stem cells and chondrocytes promote and/or suppress the in vitro proliferation of lymphocytes stimulated by interleukins 2, 7 and 15

OBJECTIVES: To investigate whether human bone marrow-derived mesenchymal stem cells (BM-MSCs) and articular chondrocytes (ACs) affect the in vitro proliferation of T lymphocytes and peripheral blood mononuclear cells (PBMCs) driven by the homeostatic interleukin (IL)2, IL7 and IL15 cytokines binding to the common cytokine receptor gamma-chain (gamma(c)) in the absence of T cell receptor (TCR) triggering. METHODS: PBMCs, total T cells and T cell subsets (CD4+ and CD8+) were stimulated with IL2, IL7 or IL15 and exposed to cultured BM-MSCs and ACs at varying cell:cell ratio either in contact or in transwell conditions. Lymphocyte proliferation was measured by (3)H-thymidine uptake or by flow cytometry of carboxyfluorescein succinimidyl ester (CFSE)-labelled lymphocytes. RESULTS: MSCs and ACs enhanced and inhibited lymphocyte proliferation depending on the extent of lymphocyte baseline proliferation and on the MSC/AC to lymphocyte ratio. Enhancement was significant on poorly proliferating lymphocytes and mostly at lower MSC/AC to lymphocyte ratio. Suppression occurred only on actively proliferating lymphocytes and at high MSC/AC to lymphocyte ratio. Neither enhancement nor inhibition required cell-cell contact. CONCLUSIONS: There is a dichotomous effect of MSCs/ACs on lymphocytes proliferating in response to the homeostatic IL2, IL7 and IL15 cytokines likely to be encountered in homeostatic and autoimmune inflammatory conditions. The effect is determined by baseline lymphocyte proliferation, cell:cell ratio and is dependent on soluble factor(s). This should be taken into account when planning cellular therapy for autoimmune disease (AD) using stromal-derived cells such as MSCs

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

Bone marrow mesenchymal stromal cells (BM-MSCs) from healthy donors and auto-immune disease patients reduce the proliferation of autologous- and allogeneic-stimulated lymphocytes in vitro.

International audienceOBJECTIVES: To investigate the ability of bone marrow (BM)-derived mesenchymal stromal cells (BM-MSCs) in suppressing the proliferation of stimulated lymphocytes across a range of conditions including autologous BM-MSCs derived from autoimmune disease (AD) patients. METHODS: In vitro cultures of BM-MSCs from healthy donors and AD patients were established and characterized by their differentiation potential into adipocytes and osteoblasts, and their fibroblast-colony-forming unit (CFU-F) ability and phenotype by flow cytometry. BM-MSCs (irradiated and non-irradiated) from healthy and AD patients were tested for their ability to suppress the in vitro proliferation of autologous and allogeneic peripheral blood mononuclear cells (PBMC) (from healthy donors and patients suffering from various ADs) stimulated with anti-CD3epsilon antibody alone or in combination with anti-CD28 antibody. The anti-proliferative effect of the BM-MSCs from healthy donors was tested also on transformed B-cell lines as a model of non-antigen-stimulated lymphocytes. RESULTS: BM-MSCs from healthy donors and AD patients reduced the proliferation of autologous and allogeneic PBMCs by up to 90% in a cell dose-dependent fashion. The immunosuppression was independent of the proliferation of the BM-MSCs and was also effective on already proliferating cells. It was independent also of the clinical activity of AD. An MSC dose-dependent pattern of suppression of proliferation was observed also with transformed B-cell lines, similar to that observed with proliferating PBMC. CONCLUSIONS: The BM-MSCs exhibit extensive anti-proliferative properties against lymphocytes under different conditions. This property might offer a form of immunomodulatory cellular therapy for AD patients if further confirmed in animal models

Successes and failures of stem cell transplantation in autoimmune diseases

Over the past 15 years, more than 1500 patients have received HSCT, mostly autologous, as treatment for a severe autoimmune disease (AD). More than 1000 of these have been registered in the European Group for Bone Marrow Transplantation (EBMT) and European League Against Rheumatism (EULAR) combined database. A recent retrospective analysis of 900 patients showed that the majority had multiple sclerosis (MS; n = 345) followed by systemic sclerosis (SSc; n = 175), systemic lupus erythematosus (SLE; n = 85), rheumatoid arthritis (RA; n = 89), juvenile idiopathic arthritis (JIA; n = 65), and idiopathic cytopenic purpura (ITP; n = 37). An overall 85% 5-year survival and 43% progression-free survival was seen, with 100-day transplantation-related mortality (TRM) ranging between 1% (RA) and 11% (SLE and JIA). Approximately 30% of patients in all disease subgroups had a complete response, often durable despite full immune reconstitution. In many patients, such as in those with SSc, morphological improvement such as reduction of skin collagen and normalization of microvasculature was documented beyond any predicted known effects of intense immunosuppression alone. The high TRM was in part related to conditioning intensity, comorbidity, and age, but until the results of the 3 prospective randomized trials are known, an evidence-based modification of the conditioning regimen will not be possible.(1) In recent years, multipotent mesenchymal stromal cells (MSCs) have been tested in various AD, exploiting their immune-modulating properties and apparent low acute toxicity. Despite encouraging small phase 1/2 studies, no positive data from randomized, prospective studies are as yet available in the peer-reviewed literature