Mesenchymal stromal cells for articular cartilage repair: preclinical studies.

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 anti-inflammatory 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.

Chondrogenic potential of subpopulations of cells expressing mesenchymal stem cell markers derived from human synovial membranes.

In this study we analyzed the chondrogenic potential of subpopulations of mesenchymal stem cells (MSCs) derived from human synovial membranes enriched for CD73, CD106, and CD271 markers. Subpopulations of human synovial membrane MSCs enriched for CD73, CD106, and CD271 markers were isolated using a cytometry sorter and characterized by flow cytometry for MSC markers. The expression of Sox9, Nanog, and Runx2 genes by these cells was measured by reverse transcriptase-polymerase chain reaction. The chondrogenesis of each subpopulation was assessed by culturing the cells in a defined medium to produce spontaneous spheroid formation and differentiation towards chondrocyte-like cells. The examination of the spheroids by histological and immunohistochemical analyses for collagen type II (COL2), aggrecan, collagen type I (COL1), metalloprotease 13 (MMP13), and collagen type X (COLX) levels were performed to assess their chondrogenesis capacity. The adipogenesis and osteogenesis potential of each subpopulation was determined using commercial media; the resulting cells were stained with oil red O or red alizarin to test the degree of differentiation. The subpopulations had different profiles of cells positive for the MSC markers CD44, CD69, CD73, CD90, and CD105 and showed different expression levels of the genes Sox9, Nanog, and Runx2 involved in chondrogenesis, undifferentiation, and osteoblastogenesis, respectively. Immunohistochemical analysis demonstrated that COL1, COL2, COLX, MMP13, and aggrecan were expressed in the spheroids as soon as 14 days of culture. The CD271(+) subpopulation expressed the highest levels of COL2 staining compared to the other subpopulations. CD105 and Runx2 were shown by immunohistochemistry and genetic analysis to have significantly higher expression CD271(+) subpopulation than the other subpopulations. Spheroids formed from CD271-enriched and CD73-enriched MSCs from normal human synovial membranes mimic the native cartilage extracellular matrix more closely than CD106(+) MSCs and are possible candidates for use in cartilage tissue engineering. Both cell types have potential for promoting the differentiation of MSCs into chondrocytes, presenting new possibilities for achieving intrinsic cartilage repair.

Differentiation of synovial CD-105(+) human mesenchymal stem cells into chondrocyte-like cells through spheroid formation.

Mesenchymal stem cells (MSCs) have the capacity to differentiate into several cell lineages, some of which can generate bone, cartilage, or adipose tissue. The presence of MSCs in the synovial membrane was recently reported. Data from comparative studies of MSCs derived from various mesenchymal tissues suggest that MSCs from synovial membranes have a superior chondrogenesis capacity. Previous chondrogenic differentiation studies have used the total population of MSCs, including cells with several MSC markers, such as CD44, CD90, CD105, or CD73. However the chondrogenic capacity of an individual population of MSCs has not been examined. Our aim was to study the chondrogenic capacity of the cellular MSC subset, CD105(+), derived from synovial membrane tissues of patients with osteoarthritis (OA) and normal donors. The tissues were digested with a cocktail of collagenase/dispase and the isolated MSCs were seeded into plates. The subpopulation of CD105(+)-MSCs was separated using a magnetic separator. The MSCs were then differentiated towards chondrocyte-like cells using a specific medium to promote spheroid formation. Spheroids were collected after 14, 28, and 46 days in chondrogenic medium and stained with hematoxylin, eosin, Safranin O or Alcian blue to evaluate the extracellular matrix. Immunohistochemistry was performed to study collagen types I (COLI) and II (COLII) and aggrecan expression. Phenotypic characterization of the isolated CD105(+)-MSCs shows that these cells are also positive for CD90 and CD44, but negatives for CD34 and CD45. In addition, this cellular subset expressed Sox-9. Spheroids appeared after 7 days in culture in the presence of chondrogenic medium. Our studies show no differences between MSCs obtained from OA and normal synovial membranes during chondrogenesis. The morphological analysis of spheroids revealed characteristics typical of chondrocyte cells. The intensity of Safranin O, Alcian blue and aggrecan staining was positive and constant throughout the culture period. However, the intensity of COL2 staining was higher at 28 days (84.29 +/- 0.1 U) than at 46 days (61.28 +/- 01 U), while COL1 staining was not detected in any samples analyzed. These results were confirmed by reverse transcriptase-polymerase chain reaction assays. We conclude that the cellular subset of CD105(+)-MSCs has chondrogenic capacity. The study also show the similar chondrogenic capacity of CD105(+)-MSCs cultured from normal and OA synovial membranes.

Corrigendum: Effect of age on pro-inflammatory miRNAs contained in mesenchymal stem cell-derived extracellular vesicles.

This corrects the article DOI: 10.1038/srep43923.

Effect of age on pro-inflammatory miRNAs contained in mesenchymal stem cell-derived extracellular vesicles.

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.

CD105+-mesenchymal stem cells migrate into osteoarthritis joint: An animal model.

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.

Influence of age on rat bone-marrow mesenchymal stem cells potential.

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.

Validation of an EIA kit for determination of total thyroid hormones in rat serum. Effects of different anaesthetics.

Two enzyme immunoassays for the quantitative determination of total thyroxine (T4) and total triiodothyronine (T3) in human serum were validated to measure total T4 and T3 in rat serum. The specificity, sensitivity, detectability and reproducibility have been tested as well as the effects of different anaesthetics (pentobarbital and a mixture of ketamine and xylacine) on serum thyroid hormone levels. Hormones were quantified, by means of a previously validated technique, 18 hours after surgery for the placement of a stem for blood extraction in conscious and freely moving animals. Thyroid serum levels, especially T3, were slightly lower in xylacine plus ketamine treated animals than in those treated with pentobarbital. The administration of glutamic acid, stimulated the secretion of thyroid hormones, this effect appearing 30 minutes after its administration and it was independent of the anaesthetic used during the surgery for the cannula placement, although in pentobarbital treated rats, the serum T3 level increase induced by glutamate appears 60 minutes after the treatment.

Effect of excitatory amino acids on serum TSH and thyroid hormone levels in freely moving rats.

The actions of glutamate (L-Glu), and glutamate receptor agonists on serum thyroid hormones (T4 and T3) and TSH levels have been studied in conscious and freely moving adult male rats. The excitatory amino acids (EAA), L-Glu, N-methyl-D-aspartate (NMDA), kainic acid (KA) and domoic acid (Dom) were administered intraperitoneally. Blood samples were collected through a cannula implanted in the rats jugular 0--60 min after injection. Thyroid hormone concentrations were measured by enzyme immunoassay, and thyrotrophin (TSH) concentrations were determined by radioimmunoassay. The results showed that L-Glu (20 and 25 mg/kg) and NMDA (25 mg/kg) increased serum thyroxine (T4), triiodothyronine (T3) and TSH concentrations. Serum thyroid hormone levels increased 30 min after treatment, while serum TSH levels increased 5 min after i.p. administration, in both cases serum levels remained elevated during one hour. Injection of the non-NMDA glutamatergic agonists KA (30 mg/kg) and Dom (1 mg/kg) produced an increase in serum thyroid hormones and TSH levels. These results suggest the importance of EAAs in the regulation of hormone secretion from the pituitary-thyroid axis, as well as the importance of the NMDA and non-NMDA receptors in this stimulatory effect.

Effects of domoic acid on serum levels of TSH and thyroid hormones.

The actions of Domoic Acid (Dom), a marine toxin, on the levels of serum TSH and thyroid hormones (T4 and T3) has been studied to determine if these actions could be mediated by the serotoninergic system. In all the experiments, adult male Wistar rats were used. The Dom dissolved in saline was administered via i.p. in doses of 0.5 and 1 mg/kg. The T4 and T3 concentrations were determined by enzimoinmunoassay and TSH concentration was determined by radioinmunoassay. The results show that Dom 1 mg/kg increases the serum T4 levels one hour after treatment and decreases these levels 2 and 3 hr after treatment. Dom 0.5 mg/kg decreased the serum T4 levels 2 and 3 hr after treatment. The concentrations of T3 in serum were unchanged by both doses of Dom. The concentration of TSH was increased by Dom. In order to study the possible mediation of the serotoninergic system in the effect of Dom on the hormone levels, PCPA, a tryptophan hydroxylase inhibitor, was administered i.p. 90 min before blood sampling. In this case, with both doses of Dom a decrease in the levels of both hormones occurred with respect to the PCPA group. These results indicate that the serotoninergic system could affect the actions of Dom on TSH and thyroid hormone secretion.

Effect of domoic acid on brain amino acid levels.

The administration of Domoic Acid (Dom) in a 0.2 mg/kg i.p. dose induces changes in the levels of amino acids of neurochemical interest (Asp, Glu, Gly, Tau, Ala, GABA) in different rat brain regions (hypothalamus, hippocampus, amygdala, striatum, cortex and midbrain). The most affected amino acid is the GABA, the main inhibitory amino acid neurotransmitter, whereas glutamate, the main excitatory amino acid, is not affected. The rat brain regions that seem to be the main target of the Dom action belong to the limbic system (hippocampus, amygdala). The possible implication of the amino acids in the actions of Dom is also discussed.

Effect of okadaic acid and calyculin-A, two protein phosphatase inhibitors, on thyrotropin-stimulated triiodothyronine secretion in cultured sheep thyroid cells.

We have studied the effect of two protein phosphatase inhibitors on thyrotropin (TSH)-stimulated triiodothyronine (T3) production by sheep thyroid cells grown in primary culture. Incubation of sheep thyrocytes with okadaic acid (OA) and calyculin-A (CL-A), two potent inhibitors of type 1 (PP1) and type 2A (PP2A) protein phosphatases, resulted in an increase of TSH-stimulated T3 production. This effect was detected using concentrations as low as 0.1 pM with OA and 1 fM with CL-A. An inhibitory effect on T3 production, due to cellular death, was observed with 6 nM OA and 1 nM CL-A. In the absence of TSH, OA or CL-A had no effect on T3 production by thyrocytes. Forskoline (10 microM), an activator of adenylate cyclase, increased the basal and TSH-stimulated T3 release by sheep thyroid cells; this effect was increased by OA in cells grown in the basal state but not in the presence of TSH. These results suggest that the marine toxins OA and CL-A, two potent inhibitors of PP-1 and PP-2A, have significant stimulatory effects on T3 secretion promoted by TSH and FK. These observations indicate that these proteins could be important mediators of thyroid hormone production.

Endogenous excitatory amino acid neurotransmission regulates thyroid-stimulating hormone and thyroid hormone secretion in conscious freely moving male rats.

The role of neurotransmission of endogenous excitatory amino acid (EAA) on serum thyroid hormones and thyroid-stimulating hormone (TSH) levels was examined in conscious and freely moving adult male Sprague-Dawley rats. The rats were cannulated at the third ventricle 2 d before the experiments. Several glutamate receptor agonists, such as kainic acid and domoic acid, and antagonists, such as 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and dizocilpine (MK-801) were administered into the third ventricle. Serum TSH levels were assesed by radioimmunoassay, and serum thyroid hormone levels were assessed by enzyme immunoassay. The results showed that the administration of CNQX and MK-801 produced a decrease in serum levels of TSH and thyroid hormones. The administration of kainic acid and domoic acid increased TSH concentrations, whereas CNQX completely blocked the release of TSH induced by kainic acid and domoic acid. These results suggest the importance of endogenous EAA in the regulation of hormone secretion from the pituitary-thyroid axis, as well as the role of the N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the stimulatory effect of EAAs on the pituitary-thyroid axis.