Eva Villaron

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.

Both expanded and uncultured mesenchymal stem cells from MDS patients are genomically abnormal, showing a specific genetic profile for the 5q-syndrome

The presence of cytogenetic aberrations on mesenchymal stem cells (MSC) from myelodysplastic syndrome (MDS) patients is controversial. The aim of the study is to characterize bone marrow (BM) derived MSC from patients with MDS using: kinetic studies, immunophenotyping, fluorescent in situ hybridization (FISH) and genetic changes by array-based comparative genomic hybridization (array-CGH). In all 36 cases of untreated MDS were studied. MDS–MSC achieved confluence at a significantly slower rate than donor-MSC, and the antigenic expression of CD105 and CD104 was lower. Array-CGH studies showed DNA genomic changes that were proved not to be somatic. These results were confirmed by FISH. To confirm that genomic changes were also present in freshly obtained MSCs they were enriched by sorting BM cells with the following phenotype: CD45−/CD73++/CD34−/CD271++. They also showed genomic changes that were confirmed by FISH. To analyze the relationship of these aberrations with clinical–biological data an unsupervized hierarchical cluster analysis was performed, two clusters were identified: the first one included the 5q− syndrome patients, whereas the other incorporated other MDS. Our results show, for the first time that MSC from MDS display genomic aberrations, assessed by array-CGH and FISH, some of them specially linked to a particular MDS subtype, the 5q− syndrome.

Allogeneic mesenchymal stem cell therapy for refractory cytopenias after hematopoietic stem cell transplantation.

BACKGROUND: Posttransplant cytopenias are a severe complication after allogeneic stem cell transplantation (allo‐SCT) and their origin is often multifactorial or unknown. They are frequently refractory to standard therapy, which may include steroids and/or immunoglobulins. Mesenchymal stem cells (MSCs) are an attractive therapeutic tool in the allo‐SCT setting for the ability to enhance engraftment as well as acting as immunosuppressants for graft‐versus‐host disease. There is no prior experience in the literature of the use of MSCs to treat cytopenias after allo‐SCT.

Optimisation of mesenchymal stromal cells karyotyping analysis: implications for clinical use.

Purpose: The aim of this study was to optimise the yield of metaphases in mesenchymal stromal cells (MSC) in vitro cultures and to study the karyotype of MSC expanded in good manufacturing practice (GMP) conditions for clinical use.

Prospective comparative analysis of the angiogenic capacity of monocytes and CD133+ cells in a murine model of hind limb ischemia

BACKGROUND AIMS The aim of this study was to compare prospectively the vasculogenic capacity of two cell sources, monocytes and CD133+ cells. METHODS Cells were obtained from healthy donors by adherence or magnetic selection. Animals studies were performed in a model of hind limb ischemia and different groups were established according to type and number of cells infused. Revascularization was measured by sequential blood flow analysis using a laser Doppler device and by assessing capillary density in the ischemic muscles. In order to locate the infused cells, immunofluorescence and immunocytochemistry techniques were performed and analyzed by light and confocal microscopy. RESULTS During the study period there was a significant improvement in both limb perfusion and capillary density in mice treated with either human monocytes or CD133+ cells (P<0.05) compared with non-treated mice. No cells were detected as incorporated into the vessels when 1 x 10(5) cells were used but with higher doses (1 x 10(6)) a few human cells were observed integrated into the vessels in both groups of treated mice. Supernatants of both cell types showed vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and platelet-derived growth factor- AB (PDGF-AB) expression. CONCLUSIONS Treatment with human monocytes or CD133+ cells improves blood perfusion and capillary density in a murine model and both cell types seem to stimulate vasculogenesis in a fairly similar way.

Sequential intravenous allogeneic mesenchymal stromal cells as a potential treatment for thromboangiitis obliterans (Buerger’s disease)

Thromboangiitis obliterans (TAO), also known as Buerger’s Disease, is an occlusive vasculitis linked with high morbidity and amputation risk. To date, TAO is deemed incurable due to the lack of a definitive treatment. The immune system and inflammation are proposed to play a central role in TAO pathogenesis. Due to their immunomodulatory effects, mesenchymal stromal cells (MSCs) are the subject of intense research for the treatment of a wide range of immune-mediated diseases. Thus far, local intramuscular injections of autologous or allogeneic MSCs have shown promising results in TAO. However, sequential intravenous allogeneic MSC administration has not yet been explored, which we hypothesized could exert a systemic anti-inflammatory effect in the vasculature and modulate the immune response. Here, we report the first case of a TAO patient at amputation risk treated with four sequential intravenous infusions of bone marrow-derived allogeneic MSCs from a healthy donor. Following administration, there was significant regression of foot skin ulcers and improvements in rest pain, Walking Impairment Questionnaire scores, and quality of life. Sixteen months after the infusion, the patient had not required any further amputations. This report highlights the potential of sequential allogeneic MSC infusions as an effective treatment for TAO, warranting further studies to compare this approach with the more conventionally used intramuscular MSC administration and other cell-based therapies.