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Background: Type 1 diabetes mellitus (T1D) is characterized by autoimmune responses resulting in destruction of insulin-producing pancreatic beta cells. Multipotent mesenchymal stromal cells (MSCs) exhibit immunomodulatory potential, migratory capacity to injured areas and may contribute to tissue regeneration by the secretion of bioactive factors. Therefore, MSCs are considered as a promising approach to treat patients with different autoimmune diseases (AID), including T1D patients. Phenotypical and functional alterations have been reported in MSCs derived from patients with different AID. However, little is known about the properties of MSCs derived from patients with T1D. Since autoimmunity and the diabetic microenvironment may affect the biology of MSCs, it becomes important to investigate whether these cells are suitable for autologous transplantation. Thus, the aim of the present study was to evaluate the in vitro properties and the in vivo therapeutic efficacy of MSCs isolated from bone marrow of newly diagnosed T1D patients (T1D-MSCs) and to compare them with MSCs from healthy individuals (C-MSCs)., Methods: T1D-MSCs and C-MSCs were isolated and cultured until third passage. Then, morphology, cell diameter, expression of surface markers, differentiation potential, global microarray analyses and immunosuppressive capacity were in vitro analyzed. T1D-MSCs and C-MSCs therapeutic potential were evaluated using a murine experimental model of streptozotocin (STZ)-induced diabetes., Results: T1D-MSCs and C-MSCs presented similar morphology, immunophenotype, differentiation potential, gene expression of immunomodulatory molecules and in vitro immunosuppressive capacity. When administered into diabetic mice, both T1D-MSCs and C-MSCs were able to reverse hyperglycemia, improve beta cell function and modulate pancreatic cytokine levels., Conclusions: Thus, bone marrow MSCs isolated from T1D patients recently after diagnosis are not phenotypically or functionally impaired by harmful inflammatory and metabolic diabetic conditions. Our results provide support for the use of autologous MSCs for treatment of newly diagnosed T1D patients.

Major skin burns are difficult to treat. Patients often require special care and long-term hospitalization. Besides specific complications associated with the wounds themselves, there may be impairment of the immune system and of other organs. Mesenchymal stromal cells (MSCs) are a recent therapeutic alternative to treat burns, mainly aiming to accelerate the healing process. Several MSC properties favor their use as therapeutic approach, as they promote angiogenesis, stimulate regeneration, and enhance the immunoregulatory function. Moreover, since patients with extensive burns require urgent treatment and because the expansion of autologous MSCs is a time-consuming process, in this present study we chose to evaluate the therapeutic potential of xenogeneic MSCs in the treatment of severe burns in rats. MSCs were isolated from mouse bone marrow, expanded in vitro, and intradermally injected in the periphery of burn wounds. MSC-treated rats presented higher survival rates (76.19%) than control animals treated with PBS (60.86%, p < 0.05). In addition, 60 days after the thermal injury, the MSC-treated group showed larger proportion of healed areas within the burn wounds (90.81 ± 5.05%) than the PBS-treated group (76.11 ± 3.46%, p = 0.03). We also observed that CD4(+) and CD8(+) T cells in spleens and in damaged skin, as well as the percentage of neutrophils in the burned area, were modulated by MSC treatment. Plasma cytokine (TGF-β, IL-10, IL-6, and CINC-1) levels were also altered in the MSC-treated rats, when compared to controls. Number of injected GFP(+) MSCs progressively decreased over time, and 60 days after injection, few MSCs were still detected in the skin of treated animals. This study demonstrates the therapeutic effectiveness of intradermal application of MSCs in a rat model of deep burns, providing basis for future regenerative therapies in patients suffering from deep burn injuries.

Mesenchymal stromal cells (MSCs) are cultured cells that can give rise to mature mesenchymal cells under appropriate conditions and secrete a number of biologically relevant molecules that may play an important role in regenerative medicine. Evidence indicates that pericytes (PCs) correspond to mesenchymal stem cells in vivo and can give rise to MSCs when cultured, but a comparison between the gene expression profiles of cultured PCs (cPCs) and MSCs is lacking. We have devised a novel methodology to isolate PCs from human adipose tissue and compared cPCs to MSCs obtained through traditional methods. Freshly isolated PCs expressed CD34, CD140b, and CD271 on their surface, but not CD146. Both MSCs and cPCs were able to differentiate along mesenchymal pathways in vitro, displayed an essentially identical surface immunophenotype, and exhibited the ability to suppress CD3(+) lymphocyte proliferation in vitro. Microarray expression data of cPCs and MSCs formed a single cluster among other cell types. Further analyses showed that the gene expression profiles of cPCs and MSCs are extremely similar, although MSCs differentially expressed endothelial cell (EC)-specific transcripts. These results confirm, using the power of transcriptomic analysis, that PCs give rise to MSCs and suggest that low levels of ECs may persist in MSC cultures established using traditional protocols.

Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway, responsible for the biosysnthesis of cholesterol. However, statins also have pleiotropic effects that interfere with several signaling pathways. Mesenchymal stromal cells (MSC) are a heterogeneous mixture of cells that can be isolated from a variety of tissues and are identified by the expression of a panel of surface markers and by their ability to differentiate in vitro into osteocytes, adipocytes and chondrocytes. MSC were isolated from amniotic membranes and bone marrows and characterized based on ISCT (International Society for Cell Therapy) minimal criteria. Simvastatin-treated cells and controls were directly assayed by CFSE (Carboxyfluorescein diacetate succinimidyl ester) staining to assess their cell proliferation and their RNA was used for microarray analyses and quantitative PCR (qPCR). These MSC were also evaluated for their ability to inhibit PBMC (peripheral blood mononuclear cells) proliferation. We show here that simvastatin negatively modulates MSC proliferation in a dose-dependent way and regulates the expression of proliferation-related genes. Importantly, we observed that simvastatin increased the percentage of a subset of smaller MSC, which also were actively proliferating. The association of MSC decreased size with increased pluripotency and the accumulating evidence that statins may prevent cellular senescence led us to hypothesize that simvastatin induces a smaller subpopulation that may have increased ability to maintain the entire pool of MSC and also to protect them from cellular senescence induced by long-term cultures/passages in vitro. These results may be important to better understand the pleiotropic effects of statins and its effects on the biology of cells with regenerative potential.

It has been demonstrated that genomic alterations of cells in the hematopoietic microenvironment could induce myelodysplastic syndromes (MDS) with ineffective hematopoiesis and dysmorphic hematopoietic cells, and subsequent transformation to acute myeloid leukemia. This investigation is the first attempt to correlate the gene expression profile of AURKA and AURKB in a cytogenetically stratified population of mesenchymal stem cells (MSCs) from MDS patients. We found that AURKA messenger RNA was expressed at significantly higher levels in MSCs even with normal/altered karyotype when compared with hematopoietic cells and healthy donors. In addition, we found that the presence of chromosomal abnormalities (mainly aneuploidy) in hematopoietic cells/MSCs was also associated with higher levels of AURKA. Different from previous investigations, our findings, regarding AURKA expression support the hypothesis that the presence of chromosomal abnormalities in MSCs from MDS is not a consequence of the method used for chromosome preparation. They may reflect the genomic instability present in the bone marrow microenvironment of MDS patients. This information is also supported by differences observed in the growth kinetics between MSCs from healthy donors (normal karyotype) and from MDS patients with abnormal karyotype. In summary, our results may not be considered evidence that MDS and MSCs are originated from a single neoplastic clone. In fact, both cells (hematopoietic and MSCs) may probably be altered in response to damage-inducing factors, and the presence of genomic abnormalities in MSCs suggests that an unstable bone marrow microenvironment may facilitate the expansion of MDS/leukemic cells., (Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background Aims: Mesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues., Methods: MSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays., Results: Post-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure., Conclusions: A method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.

Objective: High-dose chemotherapy (HDCT) followed by autologous stem cell transplantation is a widely applied treatment for hematological and autoimmune diseases. Little is known about the effects of this therapy on multipotent mesenchymal stromal cells (MSCs). We aimed to characterize, morphologically and functionally, MSCs isolated from bone marrow aspirates of patients after HDCT., Materials and Methods: We studied 12 consecutive lymphoma patients submitted to BEAM conditioning regimen followed by autologous stem cell transplantation 28 to 1836 days before the sample collection. Thirteen normal donors were used as control. MSCs were isolated by adherence to plastic and expanded ex vivo by culture in flasks containing alpha-minimum essential medium plus 15% fetal bovine serum., Results: The cell population isolated showed a typical MSC morphology, immunophenotype, and differentiation capacity into adipogenic, osteogenic, and chondrogenic lineages. The MSCs obtained from patients with Hodgkin's disease and non-Hodgkin's lymphoma showed decreased fibroblastoid colony-forming unit count (p = 0.023) and increased doubling time (p = 0.031) related to the control group. The total cell expansion of MSCs from normal subjects was marginally superior to the patient group (p = 0.064). There were no differences in gene expression profile, MSCs plasticity, or hematopoiesis support capability between control and patient group., Conclusions: Results suggest that HDCT applied to lymphoma patients damaged MSCs, which was demonstrated by their reduced clonogenic potential, doubling time, and cell expansion rates when compared to controls.