Stem cells possess significant age-dependent differences in their immune-response profile. These differences were analysed by Next-Generation Sequencing of six age groups from bone marrow mesenchymal stem cells. A total of 9,628 genes presenting differential expression between age groups were grouped into metabolic pathways. We focused our research on young, pre-pubertal and adult groups, which presented the highest amount of differentially expressed genes related to inflammation mediated by chemokine and cytokine signalling pathways compared with the newborn group, which was used as a control. Extracellular vesicles extracted from each group were characterized by nanoparticle tracking and flow cytometry analysis, and several micro-RNAs were verified by quantitative real-time polymerase chain reaction because of their relationship with the pathway of interest. Since miR-21-5p showed the highest statistically significant expression in extracellular vesicles from mesenchymal stem cells of the pre-pubertal group, we conducted a functional experiment inhibiting its expression and investigating the modulation of Toll-Like Receptor 4 and their link to damage-associated molecular patterns. Together, these results indicate for the first time that mesenchymal stem cell-derived extracellular vesicles have significant age-dependent differences in their immune profiles.

Mesenchymal stem cells promising role in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age. Six age groups from bone marrow mesenchymal stem cells of Wistar rats were studied (newborn, infant, young, pre-pubertal, pubertal and adult). Quantitative proteomic assay was performance by iTRAQ using an 8-plex iTRAQ labeling and the proteins differentially expressed were grouped in pluripotency, proliferative and metabolism processes. Proliferation makers, CD117 and Ki67 were measure by flow cytometry assay. Real time polymerase chain reaction analysis of pluripotency markers Rex1, Oct4, Sox2 and Nanog were done. Biological differentiation was realized using specific mediums for 14 days to induce osteogenesis, adipogenesis or chondrogenesis and immunostain analysis of differentiated cell resulting were done. Enzimoimmunoassay analysis of several enzymes as L-lactate dehydrogenase and glucose-6-phosphate isomerase were also done to validate iTRAQ data. Taking together these results indicate for the first time that mesenchymal stem cells have significant differences in their proliferative, pluripotency and metabolism profiles and those differences are age depending.

In the present study, we examined the effect of the over-expression of LMNA, or its mutant form progerin (PG), on the mesoderm differentiation potential of mesenchymal stem cells (MSCs) from human umbilical cord (UC) stroma using a recently described differentiation model employing spheroid formation. Accumulation of lamin A (LMNA) was previously associated with the osteoarthritis (OA) chondrocyte phenotype. Mutations of this protein are linked to laminopathies and specifically to Hutchinson-Gilford Progeria Syndrome (HGPS), an accelerated aging disease. Some authors have proposed that a deregulation of LMNA affects the differentiation potential of stem cells. The chondrogenic potential is defective in PG-MSCs, although both PG and LMNA transduced MSCs, have an increase in hypertrophy markers during chondrogenic differentiation. Furthermore, both PG and LMNA-MSCs showed a decrease in manganese superoxide dismutase (MnSODM), an increase of mitochondrial MnSODM-dependent reactive oxygen species (ROS) and alterations in their migration capacity. Finally, defects in chondrogenesis are partially reversed by periodic incubation with ROS-scavenger agent that mimics MnSODM effect. Our results indicate that over-expression of LMNA or PG by lentiviral gene delivery leads to defects in chondrogenic differentiation potential partially due to an imbalance in oxidative stress.

IntroductionNuclear accumulation of a mutant form of the nuclear protein Lamin-A, called Progerin (PG) or Lamin AΔ50, occurs in Hutchinson-Gilford Progeria Syndrome (HGPS) or Progeria, an accelerated aging disease. One of the main symptoms of this genetic disorder is a loss of sub-cutaneous fat due to a dramatic lipodystrophy.MethodsWe stably induced the expression of human PG and GFP -Green Fluorescent Protein- as control in 3T3L1 cells using a lentiviral system to study the effect of PG expression in the differentiation capacity of this cell line, one of the most used adipogenic models. Quantitative proteomics (iTRAQ) was done to study the effect of the PG accumulation. Several of the modulated proteins were validated by immunoblotting and real-time PCR. Mitochondrial function was analyzed by measurement of a) the mitochondrial basal activity, b) the superoxide anion production and c) the individual efficiency of the different complex of the respiratory chain.ResultsWe found that over-expression PG by lentiviral gene delivery leads to a decrease in the proliferation rate and to defects in adipogenic capacity when compared to the control. Quantitative proteomics analysis showed 181 proteins significantly (p < 0.05) modulated in PG-expressing preadipocytes. Mitochondrial function is impaired in PG-expressing cells. Specifically, we have detected an increase in the activity of the complex I and an overproduction of Superoxide anion. Incubation with Reactive Oxygen Species (ROS) scavenger agents drives to a decrease in autophagic proteolysis as revealed by LC3-II/LC3-I ratio.ConclusionPG expression in 3T3L1 cells promotes changes in several Biological Processes, including structure of cytoskeleton, lipid metabolism, calcium regulation, translation, protein folding and energy generation by the mitochondria. Our data strengthen the contribution of ROS accumulation to the premature aging phenotype and establish a link between mitochondrial dysfunction and loss of proteostasis in HGPS.

Mesenchymal stem cells are being the focus of connective tissue technology and regenerative medicine, presenting a good choice cell source for improving old and well recognized techniques of cartilage defect repair. For instance, the autologous chondrocyte transplantation using new concepts of regenerative medicine. The present study investigated the risk of xenogenicity of human synovial membrane-derived MSCs, injected into the monkeys using intravenous and intra-articular administration.The animal models used were adult monkeys Rhesus which had been injured into the left knee to create an Osteoarthritis (OA) animal model. CD105+-MSCs were injected twice into the OA monkeys with an interval of one week between them. The animals were euthanized one month after treatment. Immunohistochemistry analysis of different organs: spleen, heart, fat, liver, gut, pancreas, lung, skeletal muscle and kidney from the animals revealed that CD105+-MSCs migrated towards the injured knee joint. MSCs naive were found statistically significant increased in the injured knee in front of healthy one. CD105+-MSCs were negatives for CD68 and the area where CD105+-MSCs were found presented SDF-1 increased levels in front of healthy knee. We concluded that a characterized MSCs subset could be a safe alternative for cell therapy in clearly localized pathologies.

Xenogeneic chondrocytes and allogeneic mesenchymal stem cells (MSC) are considered a potential source of cells for articular cartilage repair. We here assessed the immune response triggered by xenogeneic chondrocytes when injected intraarticularly, as well as the immunoregulatory effect of allogeneic bone marrow-derived MSC after systemic administration. To this end, a discordant xenotransplantation model was established by injecting three million porcine articular chondrocytes (PAC) into the femorotibial joint of Lewis rats and monitoring the immune response. First, the fate of MSC injected using various routes was monitored in an in vivo imaging system. The biodistribution revealed a dependency on the injection route with MSC injected intravenously (i.v.) succumbing early after 24 h and MSC injected intraperitoneally (i.p.) lasting locally for at least 5 days. Importantly, no migration of MSC to the joint was detected in rats previously injected with PAC. MSC were then administered either i.v. 1 week before PAC injection or i.p. 3 weeks after to assess their immunomodulatory function on humoral and adaptive immune parameters. Anti-PAC IgM and IgG responses were detected in all PAC-injected rats with a peak at week 2 postinjection and reactivity remaining above baseline levels by week 18. IgG2a and IgG2b were the predominant and long-lasting IgG subtypes. By contrast, no anti-MSC antibody response was detected in the cohort injected with MSC only, but infusion of MSC before PAC injection temporarily augmented the anti-PAC antibody response. Consistent with a cellular immune response to PAC in PAC-injected rats, cytokine/chemokine profiling in serum by antibody array revealed a distinct pattern relative to controls characterized by elevation of multiple markers at week 2, as well as increases in proliferation in draining lymph nodes. Notably, systemic administration of allogeneic MSC under the described conditions did not diminish the immune response. IL-2 measurements in cocultures of rat peripheral blood lymphocytes with PAC indicated that PAC injection induced some T-cell hyporesponsiveness that was not enhanced in the cohorts additionally receiving MSC. Thus, PAC injected intraarticularly in Lewis rats induced a cellular and humoral immune response that was not counteracted by the systemic administration of allogeneic MSC under the described conditions.

Rheumatic diseases such as osteoarthritis (OA) are a major social and economic burden because of the population aging and the lack of curative solutions. An effective cell therapy may be the best treatment option for OA and other cartilage diseases. However, the main cellular strategy used to repair articular cartilage, the transplantation of autologous chondrocytes, is limited to a small number of patients with traumatic lesions. The use of joint replacement after years of disease progression proves the great medical need in current practice. Mesenchymal stromal/stem cells (MSCs) provide an alternative cell source for cartilage regeneration due to numerous advantages, comprising relative ease to isolate and culture, chondrogenic capacity, and antiinflammatory effects. Initial clinical trials with MSCs have led to encouraging results, but many variables have to be considered to attain true amelioration of disease or repair (type and status of cartilage disease, source and conditions of cells, administration regime, combinatorial approaches). Particularly, allogeneic MSCs are an advantageous cellular product. The animal models chosen for preclinical evaluation are also relevant for successful translation into clinical practice. Considering the limitations in the field, rigorous comparative and validating studies in well-established animal models (including large animals) are still needed to set up the bases for additional clinical trials. The present review of studies performed in small and large animal models should help clarify the applicability of MSC-based therapies for articular cartilage repair.

Our group focuses on the study of mesenchymal stem cells (MSCs) from human umbilical cord stroma or Warthońs jelly and their directed differentiation toward chondrocyte-like cells capable of regenerating damaged cartilage when transplanted into an injured joint. This study aimed to determine whether lactogenic hormone prolactin (PRL) or 3, 3', 5-triiodo-L-thyronine (T3), the active thyroid hormone, modulates chondrogenesis in our in vitro model of directed chondrogenic differentiation, and whether Wnt signalling is involved in this modulation. MSCs from human umbilical cord stroma underwent directed differentiation toward chondrocyte-like cells by spheroid formation. The addition of T3 to the chondrogenic medium increased the expression of genes linked to chondrogenesis like collagen type 2, integrin alpha 10 beta 1, and Sox9 measured by quantitative real time polymerase chain reaction (qRT-PCR) analysis. Levels of collagen type 2 and aggrecane analyzed by immunohistochemistry, and staining by Safranin O were increased after 14 days in spheroid culture with T3 compared to those without T3 or only with PRL. B-catenin, Frizzled, and GSK-3β gene expressions were significantly higher in spheroids cultured with chondrogenic medium (CM) plus T3 compared to CM alone after 14 days in culture. The increase of chondrogenic differentiation was inhibited when the cells were treated with T3 plus ML151, an inhibitor of the T3 steroid receptor. This work demonstrates, for first time, that T3 promotes differentiation towards chondrocytes-like cells in our in vitro model, that this differentiation is mediated by steroid receptor co-activator 2 (SRC2) and does not induce hypertrophy. J. Cell. Biochem. 117: 2097-2108, 2016. © 2016 Wiley Periodicals, Inc.