Effects of EdU labeling on mesenchymal stem cells

BackgroundThymidine analog 5-ethynyl-2-deoxyuridine (EdU) has recently been used for tracking mesenchymal stem cells (MSCs). In the present study, we tested whether EdU was cytotoxic and whether it interfered with differentiation, cytokine secretion and migration of MSCs.MethodsEdU labeling was performed by incubating adipose-derived stem cells (ADSCs) with 10^?8 mol/L of EdU for 48 h. Incorporation of EdU was detected by reaction with azide-conjugated Alexa594. The labeled and unlabeled ADSCs were compared for proliferation and apoptosis as determined by CellTiter and comet assays, respectively. They were also compared for neuron-like and endothelial differentiation as determined by morphology, marker expression and function. Comparison of their secreted cytokine profile was performed by cytokine antibody array. Comparison of their response to homing factor SDF-1 was performed by migration assay.ResultsEdU was incorporated into the nucleus in approximately 70% of ADSCs. No significant differences in proliferation and apoptosis rates were observed between EdU-labeled and unlabeled ADSCs. Isobutylmethylxanthine induced both EdU-labeled and unlabeled ADSCs to assume a neuron-like morphology and to express β-III tubulin. Endothelial growth medium-2 (EGM2) induced endothelial differentiation in both EdU-labeled and unlabeled ADSCs, including the ability to uptake low-density lipoprotein and to form capillary-like structures as well as the expression of vWF, eNOS and CD31. EdU-labeled and unlabeled ADSCs exhibited identical secreted cytokine profile and identical migratory response to SDF-1.DiscussionAt the recommended dosage of 10^?8 mol/L, EdU is non-toxic to ADSCs. EdU label did not interfere with differentiation, cytokine secretion or migratory response to SDF-1 by ADSCs.     

Treatment of graft-versus-host-disease with mesenchymal stromal cells

Mesenchymal stromal cells (MSC) are a population of phenotypically heterogeneous cells that can be isolated from many readily accessible tissues, including bone marrow, umbilical cord, placenta and adipose tissue, where they form part of the supportive, stromal micro-environment. Extensive ex vivo and pre-clinical data suggest that subpopulations within MSC contribute to immunomodulation of the host, without provoking immunologic responses from alloreactive T cells or other effector cells, as well as contributing to tissue repair. These unique properties make MSC an ideal investigational agent for treating graft-versus-host disease (GvHD). Therapeutic trials with varied MSC dosing schedules and clinical end-points have shown mixed results. We have reviewed the biology of MSC gleaned from pre-clinical models, and summarized the results of clinical trials utilizing MSC for the treatment of acute and chronic GvHD.     

Labeling of cynomolgus monkey bone marrow-derived mesenchymal stem cells for cell tracking by multimodality imaging

Recently, transplantation of allogeneic and autologous cells has been used for regenerative medicine. A critical issue is monitoring migration and homing of transplanted cells, as well as engraftment efficiency and functional capability in vivo. Monitoring of superparamagnetic iron oxide (SPIO) particles by magnetic resonance imaging (MRI) has been used in animal models and clinical settings to track labeled cells. A major limitation of MRI is that the signals do not show biological characteristics of transplanted cells in vivo. Bone marrow mesenchymal stem cells (MSCs) have been extensively investigated for their various therapeutic properties, and exhibit the potential to differentiate into cells of diverse lineages. In this study, cynomolgus monkey MSCs (cMSCs) were labeled with Molday ION Rhodamine-B™ (MIRB), a new SPIO agent, to investigate and characterize the biophysical and MRI properties of labeled cMSCs in vitro and in vivo. The results indicate that MIRB is biocompatible and useful for cMSCs labeling and cell tracking by multimodality imaging. Our method is helpful for detection of transplanted stem cells in vivo, which is required for understanding mechanisms of cell therapy.     

Comparison of treatments of peripheral arterial disease with mesenchymal stromal cells and mesenchymal stromal cells modified with granulocyte and macrophage colony-stimulating factor

Background aimsGranulocyte macrophage-colony stimulating factor (GM-CSF) promotes vessel formation through several molecular signaling pathways. Mesenchymal stromal cells (MSCs) have an important role in neovasculogenesis during ischemia because they release pro-angiogenic paracrine factors, pro-survival and immunomodulatory substances and can differentiate into endothelial cells. The objective of this study was to evaluate whether there is synergy between GM-CSF and MSCs in recovering ischemic limbs.MethodsMSCs from mouse bone marrow were transduced with a lentiviral vector expressing GM-CSF and injected into animals with surgically induced limb ischemia, with unmodified MCSs used as control. The evolution of limb necrosis was evaluated for 1 month. Muscle strength was assessed on the 30th day, and the animals were euthanized to determine the muscle mass and to perform histological analyses to determine the degree of cellular infiltration, capillary and microvessel densities, fibrosis, necrosis and tissue regeneration.ResultsBoth treatments were able to ameliorate ischemia, decrease the areas of fibrosis, necrosis, adipocytes and leukocyte infiltrates and increase the number of capillaries. The addition of GM-CSF promoted the formation of larger vessels, but it also resulted in more fibrosis and less muscle mass without affecting muscle force.ConclusionsBoth treatments resulted in a remarkable amelioration of ischemia. More fibrosis and less muscle mass produced by the overexpression of GM-CSF did not affect muscle functionality significantly. Importantly, MSCs overexpressing GM-CSF produced larger vessels, which is an important long-term advantage because larger vessels are more efficient in the reperfusion of ischemic tissues physiologically.     

Therapeutic applications of mesenchymal stromal cells

Mesenchymal stromal cells (MSC) are multipotent cells that can be derived from many different organs and tissues. They have been demonstrated to play a role in tissue repair and regeneration in both preclinical and clinical studies. They also have remarkable immunosuppressive properties. We describe their application in settings that include the cardiovascular, central nervous, gastrointestinal, renal, orthopaedic and haematopoietic systems. Manufacturing of MSC for clinical trials is also discussed. Since tissue matching between MSC donor and recipient does not appear to be required, MSC may be the first cell type able to be used as an "off-the-shelf" therapeutic product.     

Identity and ranking of colonic mesenchymal stromal cells

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.     

Co-culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N -succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34⁺CD38⁻ fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34⁺, CD133⁺ and CD38⁻) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC–MSC interaction might further enhance cord blood expansion on MSC.     

Immunophenotype of human lymphocytes after interaction with mesenchymal stromal cells

Human multipotent mesenchymal stromal cells (MMSCs) were cocultured with allogenic blood-born mononuclear cells (MNCs). The MNCs consisted of cells that differed in their maturity or functional state, such as lymphocytes from adult peripheral blood vs. umbilical cord blood (cb) or nonstimulated vs. phytohemagglutinin (PHA)-activated lymphocytes from peripheral blood, respectively. The share of T, B, and natural killer (NK) cells or T cell subsets within the initial MNCs or cbMNCs were within physiological reference range for adult peripheral blood. After coculturing with the MMSCs, the populations of B cells decreased in both MNCs and cbMNCs, whereas the populations of the T and NK cells decreased among cbMNC only (p < 0.05). A decrease in the subset of T-NK cells was observed in the T cells of both MNCs and cbMNCs. In the coculture of MMSCs and PHA-MNCs, we found decrease in the number of CD8⁺ and HLA-DR⁺ cells and an increase in the number of CD25⁺ lymphocytes compared to monocultured PHA-MNCs. Our data show that the interaction with MMSCs did not substantially modify the composition of allogenic lymphocytes independent of their maturation (MNCs vs. cbMNCs) or activation (MNCs vs. PHA-MNCs), and the means were within the physiological limits. Moreover, exposure to the MMSCs did not reduce the viability of lymphocytes and even promoted the survival of cells in case of cbMNCs.     

Intracellular immunoglobulins in Namalva and U266 cells cocultivated with mesenchymal stromal cells

The data concerning the influence of mesenchymal stromal cells (MSCs) on immunoglobulin (Ig) production are contradictory. Most results were obtained using MSC derived from bone marrow. The properties of MSCs obtained from other tissues are not well studied. In the present work, MSC cultures have been established from umbilical cord, adipose tissue, and bone marrow of healthy donors, as well as from bone marrow of patients with autoimmune diseases. MSCs from all these sources exhibited similar surface markers. We assayed the influence of MSC cocultivation at exponential or stationary growth phases on IgM content in Namalva and IgE content in U266 cells. Bone marrow MSCs from healthy donors did not affect IgM and IgE production. Proliferating MSCs from patients with Crohn’s disease and multiple sclerosis stimulated Ig production. Exponentially growing MSCs derived from umbilical cord and adipose tissue also stimulated Ig synthesis. MSCs at stationary cultures enhanced IgM production in Namalva (cells) and suppressed IgE synthesis in U266 cells. Thus, MSCs from various tissues with common phenotypes differed in their capacity to modulate Ig production by B-lymphoid cells. The effect of MSCs depends on their growth stage and may be different for lymphoblastoid and myeloma cells.     

Cell therapy with autologous mesenchymal stromal cells in post-traumatic syringomyelia

Background aims

Recently, clinical studies show that cell therapy with mesenchymal stromal cells (MSCs) improves the sequelae chronically established in paraplegic patients, being necessary to know which of them can obtain better benefit.

Uptake and delivery of antigens by mesenchymal stromal cells

Background aimsMesenchymal stromal cells (MSCs) are multipotent stem cells with immunosuppressive properties. Nevertheless, it has been previously reported that MSCs might also trigger the immune response. We studied whether MSCs may act as carriers, capturing antigens that can be endocytosed by antigen-presenting cells later on.MethodsWe measured the cellular uptake of mannose receptor-mediated fluid phase macropinocytosis, assessed as cellular uptake of fluorescein isothiocyanate-dextran, and PKH-67-labeled cell lysates as a surrogate marker for antigen capture among dendritic cells (DCs, positive control), T lymphocytes (negative control) and MSCs.ResultsAll experiments confirmed that MCSs displayed pinocytic and endocytic capacities, which were lower than those observed for DCs but significantly higher than those observed for T cells. We also demonstrated that MSCs release previously endocytosed antigens, which subsequently can be captured by DCs.ConclusionsMSCs have the ability to capture and release antigens.     

Interaction of human multipotential mesenchymal stromal and immune cells

Multipotential mesenchymal stromal cells (MMSCs) are the subject of increasing scientific interest due to their key role in physiological renewal and repair. Allogeneic MMSCs interaction with other components of tissue environment, in particular with immune cells, represent one of the most intriguing question of modern cell physiology. MMSCs possess pronounced immunomodulatory capabilities based on their "immmunopriveledge" properties and the ability to suppress immune response. This review is highlighted the current state of art in the field of MMSCs immunomodulatory effects realization and mechanisms. MMSCs and immune cells interaction represents complex multidirectional process governed by both direct cell-to-cell interactions and soluble factors (interferon-gamma, tumor necrosis factor, prostaglandin E2, hepatocyte growth factor, interleukins ets.). The importance of physical environmental factors, primarily oxygen tension, on peculiarities of MMSCs and immune cells interaction is discussed.