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

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

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

Haematopoietic stem cell transplantation for vasculitis including Behçet'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

Close Interactions between Mesenchymal Stem Cells and Neuroblastoma Cell Lines Lead to Tumor Growth Inhibition

<div><p>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. <em>In vivo</em> 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 <em>in vitro</em> 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.</p> </div

Invasiveness of neuroblastoma cells after contact with human mesenchymal stem cells.

<p>SH-SY5Y, Htla-230 and GI-LI-N NB cells incubated or not with hMSCs in a transwell system for 24 hours, were placed in the upper wells on Matrigel®-coated invasion chambers and separated by an 8 μm membrane from CXCL12 or bone marrow sample. After 24 hours, NB cells migrated into the lower chamber were stained and counted after May Grunwald-Giemsa. Panels A–C show the invasiveness of SH-SY5Y (Panel A), GI-LI-N (Panel B) and Htla-230 (Panel C) to CXCL12. Panels D shows the invasiveness of SH-SY5Y to CXCL12 in presence or absence of CXCR4 antagonist AMD3100 that was added to some wells. Panel E shows the invasiveness of SH-SY5Y to the bone marrow of an healthy donor. The data are expressed as mean values obtained from the count of 10 fields/well. Bars are SD. Statistical analysis was performed by Unpaired t test with Welch's correction.</p

Survival curves and immunohistochemical analysis of human Htla-230 neuroblastoma bearing mice treated with human mesenchymal stem cells.

<p>Panel A. Athymic mice (Nude-nu) were i.v. injected with ffLUC-Htla-230 (3×10⁶ cell/mouse; n = 15). Tumor establishment occurred 14 days after tumor cell inoculum as assessed by bioluminescence imaging. Panel B. Athymic mice (Nude-nu) were injected in the tail vein with Htla-230 (3×10⁶ cell/mouse) and treated i.v. with hMSCs (1×10⁶ cells/mouse, n = 6 or 3×10⁶ cells/mouse, n = 5) or saline solution (n = 6) 14 days after tumor cell inoculum. Survival curves were constructed by using the Kaplan–Meier method. Statistical analysis of different treatment groups was performed by Peto's log-rank test. Panel C. Representative hematoxylin-eosin-staining of tumors surgically removed from control and hMSC-treated Htla-230-bearing mice is shown. Original magnification 10x. Panel D. Representative immunohistochemical CD90 staining of lungs and tumors surgically removed from hMSC-treated Htla-230 bearing mice at different times after hMSC i.v. inoculum. Arrows indicate hMSCs positive for CD90. Original magnification 40x.</p

Human neuroblastoma cells attract human mesenchymal stem cells <i>in vitro</i>.

<p>Human MSCs, placed in the upper wells of transwell system, were separated by an 8 μm membrane from NB cells or NB cell conditioned medium that were dispensed in the lower well. Negative and positive controls were represented by serum-free medium (DMEM) and PDGF-BB at 100 ng/ml, respectively. Migration was quantified by counting migrated cells under the microscope after cell staining with May Grunwald. Panel A shows representative images of migrated hMSCs to DMEM, PDGF-BB, Htla-230 NB cells, Htla-230 conditioned medium. Original magnification 100x. Panel B shows quantification of migrated hMSC to different NB cells and NB conditioned medium. The data represent mean values obtained from the count of 10 fields/well. Bars represent SD. Panel C shows the results obtained by pooling all the data obtained from hMSC migration to NB cell lines and NB cell conditioned medium. Statistical analysis was performed by Unpaired t test with Welch's correction.</p

Effects of human mesenchymal stem cells on neuroblastoma cell proliferation.

<p>SH-SY5Y (Panel A) and Htla-230 (Panel B) and ACN (Panel C) NB cells were cultured in 96-well plates for 3 days in presence or absence of hMSCs at different ratio. After 3 days of co-culture, the cells were pulsed with ³H-thymidine for 18 hours and analyzed. The data are expressed as mean value ± SD from three different experiments. Statistical analysis was performed by Student's t test with Welch's correction.</p