Mesenchymal stem cells are functionally abnormal in patients with immune thrombocytopenic purpura.

BACKGROUND AIMS: Immune thrombocytopenic purpura (ITP) is a bleeding disorder characterized by an accelerated destruction of platelets as a result of the presence of autoreactive antibodies. Patients with ITP also display activated platelet-autoreactive T cells. Mesenchymal stem cells (MSC) inhibit both T- and B-cell activation and may have functional impairments in autoimmune disorders. METHODS: We analyzed the potential role of MSC in the pathogenesis of ITP. RESULTS: MSC from ITP showed an impaired proliferative capacity and a lower capability of inhibiting activated T-cell proliferation compared with healthy donors. While MSC from controls showed a decreased expression of p27 after stimulation with platelet-derived growth factor, this effect was not observed in MSC from patients. Furthermore, MSC from healthy donors down-regulated p16 upon exposure to platelet-released supernatant, while this effect was not observed for ITP. Interestingly, caspase 9 expression was higher in MSC from ITP. CONCLUSIONS: These abnormalities suggest a role of MSC malfunction in the physiopathology of the disease and may have therapeutic implications.

Comparison of ex vivo expansion culture conditions of mesenchymal stem cells for human cell therapy.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells. Based on their properties, several clinical trials have been designed to explore their potential therapeutic effect. Fetal calf serum (FCS, commonly used for in vitro expansion) is an undesirable source of xenogeneic antigens and bears the risk of transmitting contaminations. As an alternative for FCS, platelet lysate (PL) and both autologous and allogeneic human serum have been proposed. The aim of this study is to compare the culture of bone marrow (BM)-derived MSCs in the presence of different serum supplements to determine the effect on cell growth, differentiation potential, and immunologic function. STUDY DESIGN AND METHODS: MSCs from BM of healthy volunteer donors were grown in the presence of 10% FCS supplemented with 1 ng/mL basic fibroblast growth factor (bFGF), 10% human serum supplemented with 1 ng/mL bFGF, 5% PL, and PL 5% supplemented with 1 ng/mL bFGF (PL plus bFGF). RESULTS: MSCs that expanded in either medium showed a comparable morphology, phenotype, and proliferative and differentiation capacity. While the presence of MSCs in vitro significantly decreased CD3/CD28-mediated T-cell activation, this effect was significantly higher in MSCs cultured with human serum. Production of interferon-gamma was inhibited by cocultured media with MSCs while MSCs also induced a significant inhibition of cell cycle in T cells. DISCUSSION: In conclusion, PL or autologous serum could offer an alternative to the use of FCS in MSC expansion for clinical use maintaining the same growing potential, phenotype, immunomodulatory properties, and differentiation potential.

The effect of mesenchymal stem cells on the viability, proliferation and differentiation of B-lymphocytes.

BACKGROUND: Mesenchymal stem cells are multilineage non-hematopoietic progenitor cells that play a key role in supporting the lymphohematopoietic system. Their distribution in bone marrow and secondary lymphoid organs allows an intimate interaction with T- and B-lymphocytes. While their effect on T-lymphocytes has been extensively analyzed, data on the effect of mesenchymal stem cells on B cells are more limited. We analyzed the effects of mesenchymal stem cells on B-lymphocytes and the pathways involved in these effects. DESIGN AND METHODS: The effect of MSC on the proliferation and viability of B cells was evaluated using MTT assays, annexin/7-amino-actinomycin D and propidium iodide staining. The B-cell maturation pattern was established using flow cytometry based on the expression of different markers related to the differentiation of B cells, such as CD38, CD138, CD19 and CCR7, and to the expression of surface and intracellular immunoglobulins. Finally, western blot assays were used to identify the pathways involved in the effects of mesenchymal stem cells on B-lymphocytes. RESULTS: Mesenchymal stem cells increased viability and blocked the cell cycle of B-lymphocytes in the G(0)/G(1) phase. In vitro exposure of B cells to plasmacytoid dendritic cells induced B-cell differentiation as shown by an increased number of CD38(++)/CD138(++) cells, which also displayed higher levels of cytoplasmic immunoglobulin and lower levels of CD19, CCR7 and surface immunoglobulin. Interestingly, this maturation pattern was inhibited by adding mesenchymal stem cells to the culture. Finally, mesenchymal stem cells modified the phosphorylation pattern of the extracellular response kinase 1/2 and p38 pathways which are both involved in B-cell viability, proliferation and activation. CONCLUSIONS: Mesenchymal stem cells increase B-cell viability while inhibiting proliferation, arresting B-lymphocytes in the G(0)/G(1) phase of the cell cycle. The presence of mesenchymal stem cells blocked B-cell differentiation as assessed by flow cytometry. Finally, mesenchymal stem cells modified the activation pattern of the extracellular response kinase and the p38 mitogen-activated protein kinase pathways in B-lymphocytes.

Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification.

OBJECTIVE: Optimization of the mesenchymal stem cells (MSC) isolation and expansion method. MATERIALS AND METHODS: Mononuclear cells (MNC) from bone marrow aspirates were obtained by both density gradient centrifugation (standard method) and gravity sedimentation. Cells were cultured in standard conditions (10% fetal calf serum and normal oxygen tension [21% O(2)]) and expansion results compared to those obtained with the same culture conditions to which platelet lysate (PL) preparations were added; in addition, the 21% O(2) concentration was compared to a lower (5%) concentration (hypoxia) until the fourth cell passage. Time of expansion, number of cells obtained, morphology, cell surface markers, and differentiation potential were evaluated. RESULTS: MSC obtained by any of the different culture conditions expressed comparable immunophenotype and were able to differentiate into osteoblasts, adipocytes, and chondrocytes. When the number of MSC obtained at fourth passage was analyzed, the highest cell numbers were obtained with gravity sedimentation isolation and PL-supplemented culture and the expansion time was the shortest when cells were cultured under hypoxic conditions. CONCLUSION: MSC isolation by MNC gravity sedimentation together with culture medium supplementation with 5% of PL in a hypoxic atmosphere (5% O(2)) significantly improved MSC yield and reduced expansion time compared to the standard accepted protocols.

Multifunctional role of Erk5 in multiple myeloma.

Multiple myeloma is characterized by the accumulation of terminally differentiated B cells in the bone marrow, due to increased proliferation and restricted apoptosis of the myelomatous clone. Here we have studied the participation of a novel mitogen-activated protein kinase (MAPK) route, the extracellular signal-regulated kinase 5 (Erk5) pathway, in the regulation of myeloma cell proliferation and apoptosis. Erk5 was expressed in cells isolated from patients and in myeloma cell lines. The myeloma growth factor interleukin 6 (IL-6) activated Erk5, and this activation was independent of Ras and Src. Expression of a dominant-negative form of Erk5 restricted the proliferation of myeloma cells and inhibited IL-6-dependent cell duplication. This dominant-negative form also sensitized myeloma cells to the proapoptotic action of dexamethasone and PS341. The latter compound caused a profound decrease in the amount of endogenous Erk5 and was less effective in inducing apoptosis when the level of Erk5 was increased by transfection of Erk5. These results place the Erk5 route as a new regulatory signaling pathway that affects multiple myeloma proliferation and apoptosis.

Imatinib mesylate (STI571) inhibits multiple myeloma cell proliferation and potentiates the effect of common antimyeloma agents.

c-Kit has been shown to be mutated in several types of tumours, and its activity has been correlated with increased proliferation rates in a subset of multiple myeloma (MM) patients. We have investigated the effect of imatinib mesylate (STI571), an inhibitor of c-Kit, on MM cells. STI571 inhibited the proliferation of MM cells by arresting cell cycle progression. Western blotting of cell cycle proteins showed that STI571 increased the levels of p21 and p16. MM cells expressed abl, but its level of tyrosine phosphorylation was low and unaffected by treatment with STI571. c-Kit was also expressed in certain MM cell lines, and its phosphorylation was stimulated by stem cell factor. However, the failure to detect the receptor protein in other MM cell lines in which cell proliferation was inhibited by STI571 suggests that its effect on these c-Kit-negative MM cell lines might be caused by the action of the drug on yet unknown targets. STI571 inhibited the proliferation of MM cells resistant to dexamethasone or melphalan and had an additive effect when combined with dexamethasone. Efforts to understand the action of STI571 in MM cells may help to identify these potentially useful targets in the treatment of this and other disorders.