Effect of miR-21 in mesenchymal stem cells-derived extracellular vesicles behavior.

BACKGROUND: A challenging new branch of research related to aging-associated diseases is the identification of miRNAs capable of modulating the senescence-associated secretory phenotype (SASP) which characterizes senescent cells and contributes to driving inflammation. METHODS: Mesenchymal stem cells (MSC) from human umbilical cord stroma were stable modified using lentivirus transduction to inhibit miR-21-5p and shotgun proteomic analysis was performed in the MSC-derived extracellular vesicles (EV) to check the effect of miR-21 inhibition in their protein cargo. Besides, we studied the paracrine effect of those modified extracellular vesicles and also their effect on SASP. RESULTS: Syndecan-1 (SDC1) was the most decreased protein in MSC-miR21--derived EV, and it was involved in inflammation and EV production. MSC-miR21--derived EV were found to produce a statistically significant inhibitory effect on SASP and inflammaging markers expression in receptor cells, and in the opposite way, these receptor cells increased their SASP and inflammaging expression statistically significantly when treated with MSC-miR-21+-derived EV. CONCLUSION: This work demonstrates the importance of miR-21 in inflammaging and its role in SASP through SDC1.

Development of new non-viral systems for genetic modification of senescent cells.

Senescence is a process characterized by a prolonged irreversible cell-cycle arrest. The accumulation of senescent cells in tissues is related to aging and to the development of age-related diseases. Recently, gene therapy has emerged as a powerful tool for treating age-associated diseases by the transference of specific genes into the target cell population. However, the high sensitivity of senescent cells significantly precludes their genetic modification via classical viral and non-viral systems. Niosomes are self-assembled non-viral nanocarriers that exhibit important advantages due to their elevated cytocompatibility, versatility, and cost-efficiency, arising as a new alternative for genetic modification of senescent cells. In this work, we explore for the first time the use of niosomes for genetic modification of senescent umbilical cord-derived mesenchymal stem cells. We report that niosome composition greatly affected transfection efficiency; those formulations prepared in medium with sucrose and containing cholesterol as helper lipid being the most suitable to transfect senescent cells. Moreover, resulting niosome formulations exhibited a superior transfection efficiency with a markedly less cytotoxicity than the commercial reagent Lipofectamine. These findings highlight the potentiality of niosomes as effective vectors for genetic modification of senescent cells, providing new tools for the prevention and/or treatment of age-related diseases.

Study of Ferroptosis Transmission by Small Extracellular Vesicles in Epithelial Ovarian Cancer Cells.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. The current treatment for EOC involves surgical debulking of the tumors followed by a combination of chemotherapy. While most patients achieve complete remission, many EOCs will recur and develop chemo-resistance. The cancer cells can adapt to several stress stimuli, becoming resistant. Because of this, new ways to fight resistant cells during the disease are being studied. However, the clinical outcomes remain unsatisfactory. Recently, ferroptosis, a novel form of regulated cell death trigged by the accumulation of iron and toxic species of lipid metabolism in cells, has emerged as a promising anti-tumor strategy for EOC treatment. This process has a high potential to become a complementary treatment to the current anti-tumor strategies to eliminate resistant cells and to avoid relapse. Cancer cells, like other cells in the body, release small extracellular vesicles (sEV) that allow the transport of substances from the cells themselves to communicate with their environment. To achieve this, we analyzed the capacity of epithelial ovarian cancer cells (OVCA), treated with ferroptosis inducers, to generate sEV, assessing their size and number, and study the transmission of ferroptosis by sEV. Our results reveal that OVCA cells treated with ferroptotic inducers can modify intercellular communication by sEV, inducing cell death in recipient cells. Furthermore, these receptor cells are able to generate a greater amount of sEV, contributing to a much higher ferroptosis paracrine transmission. Thus, we discovered the importance of the sEV in the communication between cells in OVCA, focusing on the ferroptosis process. These findings could be the beginning form to study the molecular mechanism ferroptosis transmission through sEV.

Therapy free of cells vs human mesenchymal stem cells from umbilical cord stroma to treat the inflammation in OA.

Osteoarthritis (OA) is closely linked to the increase in the number of senescent cells in joint tissues, and the senescence-associated secretory phenotype (SASP) is implicated in cartilage degradation. In the last decade, extracellular vesicles (EV) in combination with the use of miRNAs to modify post-transcriptional expressions of multiple genes have shown their utility in new therapies to treat inflammatory diseases. This work delves into the anti-inflammatory effect of extracellular vesicles derived from mesenchymal stem cells (MSC) previously modified to inhibit the expression of miR-21. We compare the efficacy of two treatments, MSC with their miR-21 inhibited through lentiviral transfection and their EV, against inflammation in a new OA animal model. The modified MSC and their EV were intraperitoneally injected in an OA animal model twice. One month after treatment, we checked which therapy was the most effective to reduce inflammation compared with animals untreated. Treated OA model sera were analyzed for cytokines and chemokines. Subsequently, different organs were analyzed to validate the results obtained. EV were the most effective treatment to reduce chemokines and cytokines in serum of OA animals as well as SASP, in their organs checked by proteomic and genomic techniques, compared with MSC alone in a statistically significant way. In conclusion, MSC-miR-21--derived EV showed a higher therapeutic potential in comparison with MSCs-miR-21-. They ameliorate the systemic inflammation through inactivation of ERK1/2 pathway in OA in vivo model. Workflow of the realization of the animal model of OA by injecting cells into the joint cavity of the left knee of the animals, which produces an increase in serum cytokines and chemokines in the animals in addition to the increase in SASP and markers of inflammation. Inhibition of miR-21 in MSCs, from the stroma of the human umbilical cord, by lentivirus and extraction of their EVs by ultracentrifugation. Finally, application of MSC therapy with its miR-21 inhibited or its EVs produces a decrease in serum cytokines and chemokines in the treated animals, in addition to an increase in SASP and markers of inflammation. The cell-free therapy being the one that produces a greater decrease in the parameters studied.

Action Mechanisms of Small Extracellular Vesicles in Inflammaging.

The accumulation process of proinflammatory components in the body due to aging influences intercellular communication and is known as inflammaging. This biological mechanism relates the development of inflammation to the aging process. Recently, it has been reported that small extracellular vesicles (sEVs) are mediators in the transmission of paracrine senescence involved in inflammatory aging. For this reason, their components, as well as mechanisms of action of sEVs, are relevant to develop a new therapy called senodrugs (senolytics and senomorphic) that regulates the intercellular communication of inflammaging. In this review, we include the most recent and relevant studies on the role of sEVs in the inflammatory aging process and in age-related diseases such as cancer and type 2 diabetes.

Acellular nerve graft enriched with mesenchymal stem cells in the transfer of the phrenic nerve to the musculocutaneous nerve in a C5-C6 brachial plexus avulsion in a rat model.

INTRODUCTION: Phrenic nerve transfer has been shown to achieve good nerve regeneration in brachial plexus avulsion. Acellular nerve allografts (ANAs) showed inferior results to autografts, which is why its use with mesenchymal stem cells (MSCs) is currently being studied. The aim is to study the effect of BM-MSCs associated with ANAs in a rat model of phrenic nerve transfer to the musculocutaneous nerve in a C5-C6 avulsion. MATERIAL AND METHODS: 42 Wistar-Lewis rats underwent a C5-C6 lesion in the right forelimb by excising a 3 mm segment from both roots, followed by a phrenic nerve transfer to the musculocutaneous nerve associated with the interposition of a three types of nerve graft (randomly distributed): control (autograft) group (n = 12), ANAs group (n = 12), and ANAs + BM-MSCs group (n = 18) After 12 weeks, amplitude and latency of the NAP and the compound motor action potential (CMAP) were measured. Biceps muscles were studied by histological analysis and nerve grafts by electron microscopy and fluorescence analysis. RESULTS: Statistically significant reductions were found in latency of the CMAP between groups control (2.48 ± 0.47 ms) and experimental (ANAs: 4.38 ± 0.78 ms, ANAs + BM-MSCs: 4.08 ± 0.85 ms) and increases in the amplitude of the CMAP between groups control (0.04388 ± 0.02 V) and ANAs + BM-MSCs (0.02275 ± 0.02 V), as well as in the thickness of the myelin sheath between groups control (0.81 ± 0.07 µm) and experimental (ANAs: 0.72 ± 0.08 µm, ANAs + BM-MSCs: 0.72 ± 0.07 µm) and in the area of the myelin sheath between groups control (13.09 ± 2.67 µm2 ) and ANAs (10.01 ± 2.97 µm2 ) (p < .05). No statistically significant differences have been found between groups ANAs and ANAs + BM-MSCs. CONCLUSIONS: This study presents a model for the study of lesions of the upper trunk and validates the autologous graft as the gold standard.

High-Throughput Screen Detects Calcium Signaling Dysfunction in Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a deadly childhood disorder, which is considered a very rare disease. It is caused by an autosomal dominant mutation on the LMNA gene, and it is characterized by accelerated aging. Human cell lines from HGPS patients and healthy parental controls were studied in parallel using next-generation sequencing (NGS) to unravel new non-previously altered molecular pathways. Nine hundred and eleven transcripts were differentially expressed when comparing healthy versus HGPS cell lines from a total of 21,872 transcripts; ITPR1, ITPR3, CACNA2D1, and CAMK2N1 stood out among them due to their links with calcium signaling, and these were validated by Western blot analysis. It was observed that the basal concentration of intracellular Ca2+ was statistically higher in HGPS cell lines compared to healthy ones. The relationship between genes involved in Ca2+ signaling and mitochondria-associated membranes (MAM) was demonstrated through cytosolic calcium handling by means of an automated fluorescent plate reading system (FlexStation 3, Molecular Devices), and apoptosis and mitochondrial ROS production were examined by means of flow cytometry analysis. Altogether, our data suggest that the Ca2+ signaling pathway is altered in HGPS at least in part due to the overproduction of reactive oxygen species (ROS). Our results unravel a new therapeutic window for the treatment of this rare disease and open new strategies to study pathologies involving both accelerated and healthy aging.

Therapeutic Potential for Regulation of the Nuclear Factor Kappa-B Transcription Factor p65 to Prevent Cellular Senescence and Activation of Pro-Inflammatory in Mesenchymal Stem Cells.

Mesenchymal stem cells have an important potential in the treatment of age-related diseases. In the last years, small extracellular vesicles derived from these stem cells have been proposed as cell-free therapies. Cellular senescence and proinflammatory activation are involved in the loss of therapeutic capacity and in the phenomenon called inflamm-aging. The regulators of these two biological processes in mesenchymal stem cells are not well-known. In this study, we found that p65 is activated during cellular senescence and inflammatory activation in human umbilical cord-derived mesenchymal stem cell. To demonstrate the central role of p65 in these two processes, we used small-molecular inhibitors of p65, such as JSH-23, MG-132 and curcumin. We found that the inhibition of p65 prevents the cellular senescence phenotype in human umbilical cord-derived mesenchymal stem cells. Besides, p65 inhibition produced the inactivation of proinflammatory molecules as components of a senescence-associated secretory phenotype (SASP) (interleukin-6 and interleukin-8 (IL-6 and IL-8)). Additionally, we found that the inhibition of p65 prevents the transmission of paracrine senescence between mesenchymal stem cells and the proinflammatory message through small extracellular vesicles. Our work highlights the important role of p65 and its inhibition to restore the loss of functionality of small extracellular vesicles from senescent mesenchymal stem cells and their inflamm-aging signature.

Mesenchymal Stem Cell-Derived Extracellular Vesicle Isolation and Their Protein Cargo Characterization.

In the present protocol, extracellular vesicles (EVs) released from a primary culture of human umbilical cord mesenchymal stem cells (MSCs) were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (TEM) and measured by nanoparticle tracking analysis (NTA). Protein was extracted from EVs using RIPA buffer and then was assessed for integrity. The proteomic content of the total EV protein samples was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after labeling by tandem mass tag (TMT). This combined approach allowed the development of an effective strategy to study the protein cargo from MSC-derived EVs.

Influence of mesenchymal stem cell-derived extracellular vesicles in vitro and their role in ageing.

INTRODUCTION: This study assessed whether mesenchymal stem cell (MSC)-derived extracellular vesicles influenced ageing and pluripotency markers in cell cultures where they are added. METHODS: MSC-derived extracellular vesicles from old and young rat bone marrows were isolated by ultracentrifugation and were characterised by western blotting, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). They were added to young and old MSC cultures. Real-time quantitative reverse transcription polymerase chain reactions and western blot analysis were performed to check the markers of ageing (vinculin and lamin A), pluripotency markers (Nanog and Oct4) and components of the mTOR signalling pathway (Rictor, Raptor, AKT and mTOR) in these cell populations. Subsequently, microRNA (miR)-188-3p expression was transiently inhibited in young MSCs to demonstrate the influence of mTOR2 on MSC ageing. RESULTS: Incubation with young MSC-derived extracellular vesicles decreased the levels of ageing markers and components of the mTOR pathway and increased the pluripotency markers from old MSC populations. By contrast, incubation of young MSCs with old MSC-derived extracellular vesicles generated the reverse effects. Inhibition of miR-188-3p expression in young MSCs produced extracellular vesicles that when incubated with old MSCs produced an increase in the levels of Rictor, as well as a decrease of phosphor-AKT, as indicated by a significant decrease in beta-galactosidase staining. CONCLUSIONS: MSC-derived extracellular vesicles affected the behaviour of MSC cultures, based on their composition, which could be modified in vitro. These experiments represented the basis for the development of new therapies against ageing-associated diseases using MSC-derived extracellular vesicles.

Effect of aging on behaviour of mesenchymal stem cells.

Organs whose source is the mesoderm lineage contain a subpopulation of stem cells that are able to differentiate among mesodermal derivatives (chondrocytes, osteocytes, adipocytes). This subpopulation of adult stem cells, called "mesenchymal stem cells" or "mesenchymal stromal cells (MSCs)", contributes directly to the homeostatic maintenance of their organs; hence, their senescence could be very deleterious for human bodily functions. MSCs are easily isolated and amenable their expansion in vitro because of the research demanding to test them in many diverse clinical indications. All of these works are shown by the rapidly expanding literature that includes many in vivo animal models. We do not have an in-depth understanding of mechanisms that induce cellular senescence, and to further clarify the consequences of the senescence process in MSCs, some hints may be derived from the study of cellular behaviour in vivo and in vitro, autophagy, mitochondrial stress and exosomal activity. In this particular work, we decided to review these biological features in the literature on MSC senescence over the last three years.

Next-Generation Sequencing and Quantitative Proteomics of Hutchinson-Gilford progeria syndrome-derived cells point to a role of nucleotide metabolism in premature aging.

Hutchinson-Gilford progeria syndrome (HGPS) is a very rare fatal disease characterized for accelerated aging. Although the causal agent, a point mutation in LMNA gene, was identified more than a decade ago, the molecular mechanisms underlying HGPS are still not fully understood and, currently, there is no cure for the patients, which die at a mean age of thirteen. With the aim of unraveling non-previously altered molecular pathways in the premature aging process, human cell lines from HGPS patients and from healthy parental controls were studied in parallel using Next-Generation Sequencing (RNAseq) and High-Resolution Quantitative Proteomics (iTRAQ) techniques. After selection of significant proteins and transcripts and crosschecking of the results a small set of protein/transcript pairs were chosen for validation. One of those proteins, ribose-phosphate pyrophosphokinase 1 (PRPS1), is essential for nucleotide synthesis. PRPS1 loss-of-function mutants present lower levels of purine. PRPS1 protein and transcript levels are detected as significantly decreased in HGPS cell lines vs. healthy parental controls. This modulation was orthogonally confirmed by targeted techniques in cell lines and also in an animal model of Progeria, the ZMPSTE24 knock-out mouse. In addition, functional experiments through supplementation with S-adenosyl-methionine (SAMe), a metabolite that is an alternative source of purine, were done. Results indicate that SAMe has a positive effect in the proliferative capacity and reduces senescence-associated Beta-galactosidase staining of the HPGS cell lines. Altogether, our data suggests that nucleotide and, specifically, purine-metabolism, are altered in premature aging, opening a new window for the therapeutic treatment of the disease.

Biodistribution and Immunogenicity of Allogeneic Mesenchymal Stem Cells in a Rat Model of Intraarticular Chondrocyte Xenotransplantation.

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.

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 Warthons 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-3ß 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.

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

INTRODUCTION: Nuclear 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. METHODS: We 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. RESULTS: We 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. CONCLUSION: PG 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.

Influence of flap prefabrication on seeding of subcutaneously injected mesenchymal stem cells in microvascular beds in rats.

BACKGROUND: In this article, the authors investigated whether the prefabrication of an autologous pedicled flap by isolation from the surrounding with artificial skin substitutes would increase mesenchymal stem cell (MSC) seeding. METHODS: Mesenchymal stem cells were isolated from human umbilical cords and were cultured and characterized by fluorescence-activated cell sorting. Oxacarbocyanine and its green fluorescence emission were used to label the MSCs population.Sixteen adult Wistar rats were randomized in 4 groups (n = 4 animals per group). In group 1, a prefabricated groin flap (GF) with skin substitutes was harvested without cell injection; in group 2, 1 million MSCs were injected subcutaneously in the area corresponding to the GF without flap harvesting; in Group 3, a prefabricated GF with skin substitutes was harvested and 1 million MSCs were injected subcutaneously; and in Group 4, a prefabricated GF with skin substitutes was harvested and 2 million MSCs were injected subcutaneously. All procedures were performed bilaterally in each animal. Animals were sacrificed 2 weeks after the surgery. Flap viability was then assessed by clinical inspection and histology, and seeding of MSCs was observed. RESULTS: All flaps survived 2 weeks after the surgery. Oxacarbocyanine-labeled cells were found in all prefabricated flaps injected (Groups 3 and 4) in higher number in comparison with the group where subcutaneous injection without flap harvesting was performed (Group 2). This difference was statistically significant (P < 0.05). CONCLUSIONS: Prefabricated skin flaps with skin substitutes may provide a useful vehicle for the implantation of MSCs to serve as an autologous microvascular bioscaffold.

Lamin A deregulation in human mesenchymal stem cells promotes an impairment in their chondrogenic potential and imbalance in their response to oxidative stress.

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.

Proteome analysis during chondrocyte differentiation in a new chondrogenesis model using human umbilical cord stroma mesenchymal stem cells.

Umbilical cord stroma mesenchymal stem cells were differentiated toward chondrocyte-like cells using a new in vitro model that consists of the random formation of spheroids in a medium supplemented with fetal bovine serum on a nonadherent surface. The medium was changed after 2 days to one specific for the induction of chondrocyte differentiation. We assessed this model using reverse transcriptase-polymerase chain reaction, flow cytometry, immunohistochemistry, and secretome analyses. The purpose of this study was to determine which proteins were differentially expressed during chondrogenesis. Differential gel electrophoresis analysis was performed, followed by matrix-assisted laser desorption/ionization mass spectrometry protein identification. A total of 97 spots were modulated during the chondrogenesis process, 54 of these spots were identified as 39 different proteins and 15 were isoforms. Of the 39 different proteins identified 15 were down-regulated, 21 were up-regulated, and 3 were up- and down-regulated during the chondrogenesis process. Using Pathway Studio 7.0 software, our results showed that the major cell functions modulated during chondrogenesis were cellular differentiation, proliferation, and migration. Five proteins involved in cartilage extracellular matrix metabolism found during the differential gel electrophoresis study were confirmed using Western blot. The results indicate that our in vitro chondrogenesis model is an efficient and rapid technique for obtaining cells similar to chondrocytes that express proteins characteristic of the cartilage extracellular matrix. These chondrocyte-like cells could prove useful for future cell therapy treatment of cartilage pathologies.

Analysis of the chondrogenic potential and secretome of mesenchymal stem cells derived from human umbilical cord stroma.

Mesenchymal stem cells (MSCs) from umbilical cord stroma were isolated by plastic adherence and characterized by flow cytometry, looking for cells positive for OCT3/4 and SSEA-4 as well as the classic MSC markers CD44, CD73, CD90, Ki67, CD105, and CD106 and negative for CD34 and CD45. Quantitative reverse transcriptase-polymerase chain reaction analysis of the genes ALP, MEF2C, MyoD, LPL, FAB4, and AMP, characteristic for the differentiated lineages, were used to evaluate early and late differentiation of 3 germ lines. Direct chondrogenic differentiation was achieved through spheroid formation by MSCs in a chondrogenic medium and the presence of chondrogenic markers at 4, 7, 14, 28, and 46 days of culture was tested. Immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction analyses were utilized to assess the expression of collagen type I, collagen type II, and collagen type X throughout the time studied. We found expression of all the markers as early as 4 days of chondrogenic differentiation culture, with their expression increasing with time, except for collagen type I, which decreased in expression in the formed spheroids after 4 days of differentiation. The signaling role of Wnt during chondrogenic differentiation was studied by western blot. We observed that ß-catenin expression decreased during the chondrogenic process. Further, a secretome study to validate our model of differentiation in vitro was performed on spheroids formed during the chondrogenesis process. Our results indicate the multipotential capacity of this source of human cells; their chondrogenic capacity could be useful for future cell therapy in articular diseases.

Differentiation of murine embryonic stem cells to thyrocytes requires insulin and insulin-like growth factor-1.

The mechanisms controlling thyrocyte development during embryonic stem (ES) cell differentiation have only been partially elucidated, although previous studies have suggested the participation of thyroid stimulating hormone (TSH) in these processes. To further define the role of TSH in this context, we have studied a murine ES cell line in which green fluorescent protein (GFP) cDNA is targeted to the TSH receptor (TSHR) gene, linking the expression of GFP to the transcription of the endogenous TSHR gene. We demonstrate that, in the initial stages of embryoid body formation, activin A and TSH induce the differentiation of definitive endoderm and thyrocyte progenitors expressing Sox17, Foxa2, and TSHR. These thyrocyte progenitors are then converted into cellular aggregates that, in the presence of insulin and IGF-1, further differentiate into mature thyroglobulin-expressing thyrocytes. Our data suggest that, despite the fact that TSH is important for the induction and specification of thyrocytes from ES cells, insulin and IGF-1 are crucial for thyrocyte maturation. Our method provides a powerful in vitro differentiation model for studying the mechanisms of early thyrocyte lineage development.