M.C. Arufe

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. J. Cell. Biochem. 108: 145–155, 2009.

iTRAQ-based analysis of progerin expression reveals mitochondrial dysfunction, reactive oxygen species accumulation and altered proteostasis

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.

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.

3, 3′, 5-triiodo-L-thyronine Increases In Vitro Chondrogenesis of Mesenchymal Stem Cells From Human Umbilical Cord Stroma Through SRC2

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.

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.

172 CHARACTERIZATION OF microRNA EXPRESSION PROFILES IN NORMAL AND OSTEOARTHRITIC HUMAN CHONDROCYTES

entiation of both of them confirmed by staining with Oil Red O and Alizarin Red respectively. We carried out RT-PCR and immunohistochemistry analysis to test expression of SOX9, type II collagen, type I collagen and C-20 aggrecan which are proteoglycan compounds of extra-cellular matrix and all of them were positive as soon as after 4 days in culture. Expressions of type X collagen and MMP-13 proteins also were measured raising their expression as well as increase the time in culture.

225 DIFFERENTIATION OF CD-105+ MSC FROM SYNOVIAL MEMBRANE TOWARDS CHONDROCYTE-LIKE CELLS THROUGH SPHEROID FORMATION

genetic processes. Functional studies in primary human chondrocytes have long been hampered by the low transfection efficiencies of non-viral gene delivery methods in these cells. Only with the development of specific electroporation methods like nucleofection and radiofrequency (RF) electroporation highly efficient non-viral transfection protocols for human chondrocytes became available, but these methods require relatively high cell numbers and cannot be easily adjusted to the analysis of a larger number of samples. The purpose of our work was therefore the establishment of a high-throughput transfection protocol for primary human articular chondrocytes (PHC) that allows the simultaneous transfection of multiple cell samples while reducing the number of cells necessary per experiment. In parallel analogous protocols should be established for the human chondrocytic cell lines T/C-28a2 and HCS-2/8. Methods: Primary human chondrocytes were isolated from adult knee cartilage by an optimized enzymatic digestion protocol and cultivated in high-density monolayer culture for three to five days. T/C-28a2 and HCS2/8 cells were cultivated under standard conditions. Chondrocytes and chondrocytic cell lines were transfected with an GFP expressing reporter construct applying amaxa’s nucleofector and 96-well shuttle devices with experimental variation of transfection parameters, pulse solutions and cell numbers. Transfection efficiencies were measured by FACS and cell viability was determined by an ATP assay. SiRNA constructs targeting GAPDH expression were transfected into the cells using the established nucleofection protocols and mRNA knockdown values were determined by a branched-DNA assay or by quantitative real-time PCR. Results: For high-throughput transfection of primary human chondrocytes transfection efficiencies of more than 70% of surviving cells could be achieved routinely with the presented nucleofection protocol. Cell viability after transfection was around 80%. Transfection efficiency and survival rate were highly dependent on the proper preparation of the PHCs prior to transfection. The cell number used for transfection could be reduced to 2×105 per sample in comparison to the established single cuvette protocol. The protocol proved also well suited for the transfer of siRNA molecules to primary human chondrocytes with suppression rates of more than 95% for GAPDH mRNA levels. Transfection efficiencies for T/C-28a2 cells surpassed 90% for both single cuvette and high-throughput formats with survival rates of more than 80%. Single cuvette nucleofection of HCS-2/8 cells reached transfection efficiencies of around 40%. Conclusions: We present the successful adaptation of the nucleofection procedure for primary human chondrocytes to a high-throughput format. The protocol allows the efficient transfection of up to 96 cell samples in parallel and is not limited to the transfection of DNA but can be easily adapted to the transfection of siRNA constructs. The protocol requires substantially lower cell numbers than single cuvette protocols and is therefore very well suited for applications requiring multiple experimental replicates. Likewise, the established protocols for T/C-28a2 cells allow the highly efficient transfection in single cuvette and 96-well formats opening novel application areas. In summary, we present efficient high-throughput transfection protocols for primary human chondrocytes and chondrocytederived cell lines with widespread application potential in osteoarthritis research (siRNA screening assays etc.).