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

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

Therapeutic potential of allogeneic mesenchymal stromal cells transplantation for lupus nephritis

Animal and human studies have suggested the potential of mesenchymal stromal cells (MSCs) to treat systemic lupus erythematosus (SLE). Here, we present the results of compassionate MSC treatments for three SLE patients to provide the proof of concept for a randomized and controlled clinical trial. Three patients of different ethnicities who suffer from chronic SLE, and who presented with class IV active proliferative nephritis confirmed by biopsy, were treated with allogeneic MSCs from healthy donors. Ninety million cells were infused intravenously into each patient during high and very high activity disease flare-ups and follow-up was continued for 9 months. Multi-organic affectation was quantified by the SLE disease activity index (SLEDAI), and indicators of lupus nephritis activity, such as proteinuria, as well as lymphocyte and monocyte antigens and anti-HLA antibodies were measured at 1, 3, 6, and 9 months after treatment. Proteinuria levels improved dramatically during the 1st month after treatment and the ameliorations were sustained throughout the follow-up period. SLEDAI scores revealed early, durable, and substantial remissions that were complete for two patients and partial for the third patient and that permitted medication doses to be reduced 50-90%. These favourable outcomes support completion of the randomized and controlled MSC trial for SLE.Peer reviewe

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% O2]) and expansion results compared to those obtained with the same culture conditions to which platelet lysate (PL) preparations were added; in addition, the 21% O2 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% O2) significantly improved MSC yield and reduced expansion time compared to the standard accepted protocols. © 2008 ISEH - Society for Hematology and Stem Cells.S. Carrancio was supported by a grant from Junta de Castilla y León (FPI Grant EDU/1878/2006). V Barbado is supported by a research grant from Federación de Cajas de Ahorros de Castilla y León, Spain.Peer Reviewe

Multifunctional role of Erk5 in multiple myeloma

7 FiguresMultiple 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.From the Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Spain; Hospital Universitario de Salamanca, Spain; and Centro de Investigaciones Biológicas, Madrid, Spain.Peer reviewe

Biocompatibility of intravitreal injection of human mesenchymal stem cells in immunocompetent rabbits

[Purpose] To evaluate the feasibility, safety, and biocompatibility of intravitreal injection of human mesenchymal stem cells (MSCs) in immunocompetent pigmented rabbits.[Materials and methods] Thirty-two pigmented rabbits (24 females, 8 males; Chinchilla-New Zealand White) were divided into 8 groups of 4 animals. Commercially prepared human MSCs were injected (0.05 ml) into the post-lens vitreous of the right eyes. Groups 1 and 4 received isotonic medium (Ringer lactate-based), groups 2, 5, 7, and 8 received a low dose of 15 × 106 cells/ml. Groups 3 and 6 received a high dose of 30 × 106 cells/ml. Clinical signs were evaluated and scored before MSCs injection and weekly for 2 or 6 weeks. Animals were sacrificed at 2 or 6 weeks after injection. Eyes, liver, spleen, and gonads were assessed by histology and by fluorescent in situ hybridization to evaluate survival and extraocular migration of MSCs.[Results] There were no relevant clinical findings between control and MSC-injected rabbit eyes at any time point. There were also no relevant histological findings between control and MSC-injected rabbits related to ocular, liver, spleen, or gonad tissues modifications. MSCs survived intravitreally for at least 2 weeks after injection. Extraocular migration of MSCs was not detected.[Conclusions] MSCs are safe and well-tolerated when administered intravitreally at a dose of 15 × 106 cells/ml in pigmented rabbits. These findings enable future research to explore the intravitreal use of commercially prepared allogenic human MSCs in clinical trials of retinal diseases.This work was supported by grants from the Consejería de Educación de la Junta de Castilla y León (grant number VA118U14); and the Centro en Red de Medicina Regenerativa y Terapia Celular de la Junta de Castilla y León, Spain.Peer reviewe