The let-7f-5p-Nme4 pathway mediates tumor necrosis factor α-induced impairment in osteogenesis of bone marrow-derived mesenchymal stem cells.

Although tumor necrosis factor α (TNF-α)-mediated inflammation significantly impacts osteoporosis, the mechanisms underlying the osteogenic differentiation defects of bone marrow-derived mesenchymal stem cells (BM-MSCs) caused by TNF-α remain poorly understood. We found that TNF-α stimulation of murine BM-MSCs significantly upregulated the expression levels of several microRNAs (miRNAs), including let-7f-5p, but this increase was significantly reversed by treatment with the kinase inhibitor BAY 11-7082. To study gain- or loss of function, we transfected cells with an miRNA inhibitor or miRNA mimic. We then demonstrated that let-7f-5p impaired osteogenic differentiation of BM-MSCs in the absence and presence of TNF-α, as evidenced by alkaline phosphatase and alizarin red staining as well as quantitative assays of the mRNA levels of bone formation marker genes in differentiated BM-MSCs. Moreover, let-7f-5p targets the 3' untranslated region of Nucleoside diphosphate kinase 4 (Nme4) mRNA and negatively regulates Nme4 expression in mouse BM-MSCs. Ectopic expression of Nme4 completely reversed the inhibitory effects of the let-7f-5p mimic on osteogenic differentiation of mouse BM-MSCs. Furthermore, inhibition of let-7f-5p or overexpression of Nme4 in BM-MSCs restored in-vivo bone formation in an ovariectomized animal model. Collectively, our work indicates that let-7f-5p is involved in TNF-α-mediated reduction of BM-MSC osteogenesis via targeting Nme4.

Role of prostaglandin E2 in allogeneic mesenchymal stem cell therapy for cardiac repair.

Ischemic heart disease is among the primary causes of cardiovascular-related deaths worldwide. Conventional treatments including surgical interventions and medical therapies aid in preventing further damage to heart muscle but are unable to provide a permanent solution. In recent years, stem cell therapy has emerged as an attractive alternative to restore damaged myocardium after myocardial injury. Allogeneic (donor-derived) mesenchymal stem cells (MSCs) have shown great promise in preclinical and clinical studies, making them the most widely accepted candidates for cardiac cell therapy. MSCs promote cardiac repair by modulating host immune system and secreting various soluble factors, of which prostaglandin E2 (PGE2) is an important one. PGE2 plays a significant role in regulating cardiac remodeling following myocardial injury. In this review, we provide an overview of allogeneic MSCs as candidates for myocardial regeneration with a focus on the role of the PGE2/cyclooxygenase-2 (COX2) pathway in mediating these effects.

Homogeneity and heterogeneity of biological characteristics in mesenchymal stem cells from human umbilical cords and exfoliated deciduous teeth.

Mesenchymal stem cells (MSCs) have proven powerful potential for cell-based therapy both in regenerative medicine and disease treatment. Human umbilical cords and exfoliated deciduous teeth are the main sources of MSCs with no donor injury or ethical issues. The goal of this study was to investigate the differences in the biological characteristics of human umbilical cord mesenchymal stem cells (UCMSCs) and stem cells from human exfoliated deciduous teeth (SHEDs). UCMSCs and SHEDs were identified by flow cytometry. The proliferation, differentiation, migration, chemotaxis, paracrine, immunomodulatory, neurite growth-promoting capabilities, and acetaldehyde dehydrogenase (ALDH) activity were comparatively studied between these two MSCs in vitro. The results showed that both SHEDs and UCMSCs expressed cell surface markers characteristic of MSCs. Furthermore, SHEDs exhibited better capacity for proliferation, migration, promotion of neurite growth, and chondrogenic differentiation. Meanwhile, UCMSCs showed more outstanding adipogenic differentiation and chemotaxy. Additionally, there were no significant differences in osteogenic differentiation, immunomodulatory capacity, and the proportion of ALDHBright compartment. Our findings indicate that although both UCMSCs and SHEDs are mesenchymal stem cells and presented some similar biological characteristics, they also have differences in many aspects, which might be helpful for developing future clinical cellular therapies.

Cytoskeletal control of nuclear morphology and stiffness are required for OPN-induced bone-marrow-derived mesenchymal stem cell migration.

During cell migration, the movement of the nucleus must be coordinated with the cytoskeletal dynamics that influence the efficiency of cell migration. Our previous study demonstrated that osteopontin (OPN) significantly promotes the migration of bone-marrow-derived mesenchymal stem cells (BMSCs). However, the mechanism that regulates nuclear mechanics of the cytoskeleton during OPN-promoted BMSC migration remains unclear. In this study, we investigated how the actin cytoskeleton influences nuclear mechanics in BMSCs. We assessed the morphology and mechanics of the nuclei in the OPN-treated BMSCs subjected to disruption or polymerization of the actin cytoskeleton. We found that disruption of actin organization by cytochalasin D (Cyto D) resulted in a decrease in the nuclear projected area and nuclear stiffness. Stabilizing the actin assembly with jasplakinolide (JASP) resulted in an increase in the nuclear projected area and nuclear stiffness. SUN1 (Sad-1/UNC-84 1) is a component of the LINC (linker of nucleoskeleton and cytoskeleton) complex involved in the connections between the nucleus and the cytoskeleton. We found that SUN1 depletion by RNAi decreased the nuclear stiffness and OPN-promoted BMSC migration. Thus, the F-actin cytoskeleton plays an important role in determining the morphology and mechanical properties of the nucleus. We suggest that the cytoskeletal-nuclear interconnectivity through SUN1 proteins plays an important role in OPN-promoted BMSC migration.

High throughput screening reveals no significant changes in protein synthesis, processing, and degradation machinery during passaging of mesenchymal stem cells 1.

Increasing reports of successful and safe application of bone marrow derived mesenchymal stem cells (BM-MSCs) for cell therapy are pouring in from numerous studies. However poor survival of transplanted cells in the recipient has impaired the benefits of BM-MSCs based therapies. Therefore cell product preparation procedures pertaining to MSC therapy need to be optimized to improve the survival of transplanted cells. One of the important ex vivo procedures in the preparation of cells for therapy is passaging of BM-MSCs to ensure a suitable number of cells for transplantation, which may affect the turnover of proteins involved in regulation of cell survival and (or) death pathways. In the current study, we investigated the effect of an increase in passage number of BM-MSCs in cell culture on the intracellular protein turnover (protein synthesis, processing, and degradation machinery). We performed proteomic analysis of BM-MSCs at different passages. There was no significant difference observed in the ribosomal, protein processing, and proteasomal pathways related proteins in BM-MSCs with an increase in passage number from P3 to P7. Therefore, expansion of MSCs in the cell culture in clinically relevant passages (Passage 3-7) does not affect the quality of MSCs in terms of intracellular protein synthesis and turnover.

Cardiogenic effects of characterized Geum urbanum extracts on adipose-derived human mesenchymal stem cells.

Stem cell therapy is considered as a promising treatment for cardiovascular diseases. Adipose-derived mesenchymal stem cells (ADMSCs) have the ability to undergo cardiomyogenesis. Medicinal plants are effective and safe candidates for cell differentiation. Therefore, the aim of our study was to investigate cardiogenic effects of characterized (HPLC-UV) extracts of Geum urbanum on ADMSCs of adipose tissue. The methanolic extracts of the root and aerial parts of G. urbanum were obtained and MTT assay was used for studying their cytotoxic effects. Then, cells were treated with 50 or 100 µg/mL of the extracts from root and aerial parts of G. urbanum. MTT assay showed that the extracts of G. urbanum did not have any toxic effects on ADMSCs. Immunostaining results showed increase in the expression of α-actinin and cardiac troponin I (cTnI), and quantitative real-time reverse-transcription PCR data confirmed the upregulation of ACTN, ACTC1, and TNNI3 genes in ADMSCs after treatment. According to HPLC fingerprinting, some cardiogenic effects of G. urbanum extracts are probably due to ellagic and gallic acid derivatives. Our findings indicated that G. urbanum extracts effectively upregulated some essential cardiogenic markers, which confirmed the therapeutic role of this plant as a traditional cardiac medicine.

Protective mechanisms of melatonin against hydrogen-peroxide-induced toxicity in human bone-marrow-derived mesenchymal stem cells.

Many obstacles compromise the efficacy of bone marrow mesenchymal stem cells (BM-MSCs) by inducing apoptosis in the grafted BM-MSCs. The current study investigates the effect of melatonin on important mediators involved in survival of BM-MSCs in hydrogen peroxide (H2O2) apoptosis model. In brief, BM-MSCs were isolated, treated with melatonin, and then exposed to H2O2. Their viability was assessed by MTT assay and apoptotic fractions were evaluated through Annexin V, Hoechst staining, and ADP/ATP ratio. Oxidative stress biomarkers including ROS, total antioxidant power (TAP), superoxide dismutase (SOD) and catalase (CAT) activity, glutathione (GSH), thiol molecules, and lipid peroxidation (LPO) levels were determined. Secretion of inflammatory cytokines (TNF-α and IL-6) were measured by ELISA assay. The protein expression of caspase-3, Bax, and Bcl-2, was also evaluated by Western blotting. Melatonin pretreatment significantly increased viability and decreased apoptotic fraction of H2O2-exposed BM-MSCs. Melatonin also decreased ROS generation, as well as increasing the activity of SOD and CAT enzymes and GSH content. Secretion of inflammatory cytokines in H2O2-exposed cells was also reduced by melatonin. Expression of caspase-3 and Bax proteins in H2O2-exposed cells was diminished by melatonin pretreatment. The findings suggest that melatonin may be an effective protective agent against H2O2-induced oxidative stress and apoptosis in MSC.

Defining the identity of human adipose-derived mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (ADMSCs) are an ideal population for regenerative medical application. Both the isolation procedure and the culturing conditions are crucial steps, since low yield can limit further cell therapies, especially when minimal adipose tissue harvests are available for cell expansion. To date, a standardized procedure encompassing both isolation sites and expansion methods is missing, thus making the choice of the most appropriate conditions for the preparation of ADMSCs controversial, especially in view of the different applications needed. In this study, we compared the effects of three different commercial media (DMEM, aMEM, and EGM2), routinely used for ADMSCs expansion, and two supplements, FBS and human platelet lysate, recently proven to be an effective alternative to prevent xenogeneic antibody transfer and immune alloresponse in the host. Notably, all the conditions resulted in being safe for ADMSCs isolation and expansion with platelet lysate supplementation giving the highest isolation and proliferation rates, together with a commitment for osteogenic lineage. Then, we proved that the high ADMSC hematopoietic supportive potential is performed through a constant and abundant secretion of both GCSF and SCF. In conclusion, this study further expands the knowledge on ADMSCs, defining their identity definition and offers potential options for in vitro protocols for clinical production, especially related to HSC expansion without use of exogenous cytokines or genetic modifications.

Optimization of treatment with recombinant FGF-2 for proliferation and differentiation of human dental stem cells, mesenchymal stem cells, and osteoblasts.

Basic fibroblast growth factor (bFGF or FGF-2) is widely used to modulate the proliferation and differentiation of certain cell types. An expression and purification system for recombinant human FGF-2 in Escherichia coli was established for the purpose of securing a continuous supply of this protein. The purified recombinant FGF-2 significantly increased the population of human embryonic stem cells. The optimal concentrations of FGF-2 for cell proliferative induction in various adult stem cells including human dental pulp stem cells, full term human periodontal ligament stem cells, human gingival fibroblasts, mesenchymal stem cells, and osteogenic oseosarcoma were established in a dose-dependent manner. When cells were treated with recombinant FGF-2 for 6 days before osteogenic induction, the mRNA expression of the bone markers was upregulated in cells originated from human dental pulp tissue, indicating that pretreatment with FGF-2 during culture increase stem cell/progenitor population and osteogenic potential.

Impact of bone-marrow-derived mesenchymal stem cells on adriamycin-induced chronic nephropathy.

OBJECTIVES: To study the effects of bone-marrow-derived mesenchymal stem cells (BM-MSCs) on adriamycin (ADR)-induced chronic nephropathy in rats. METHODS: 60 male Sprague-Dawley rats were distributed among 3 groups (20 rats each): (i) the negative control group, which was normal rats that received saline (vehicle); (ii) the positive control (ADR) group, which was rats that received 2 intravenous injections of ADR into the penile vein at 14 day intervals without treatment, and (iii) the MSC group, which were rats treated as for the ADR group that were also given 2 intravenous injections of MSCs (5 days after each ADR injection). RESULTS: ADR caused a significant reduction in animal body mass, survival rate, hemoglobin (Hb) content, serum albumin, and renal GSH, and significantly increased serum levels of triglycerides, cholesterol, urinary protein excretion and kidney injury molecule-1 (KIM-1), renal MDA, as well as caspase-3 expression and glomerular and tubulointerstitial damage compared with the negative control group. MSC treatment failed to improve animal survival rate, body mass, Hb level, proteinuria, or hypoalbuminemia; however, it mildly improved the serum BUN, hyperlipidemia, caspase-3 expression, urinary levels of KIM-1, renal oxidative stress markers, and glomerular and tubulointerstitial damage score. CONCLUSION: administration of BM-MSCs during induction of ADR nephropathy provides partial protection, which could be due to improvements in the levels of of endogenous antioxidants, reduction of apoptosis, and maintenance of the integrity of the glomerular membrane.

GATA-4 protects against hypoxia-induced cardiomyocyte injury: effects on mitochondrial membrane potential.

Our previous studies have suggested that GATA-4 increases the differentiation of bone-marrow-derived mesenchymal stem cells (MSCs) into cardiac phenotypes. This study further investigated whether GATA-4 enhances MSC-mediated cardioprotection following hypoxia. MSCs were harvested from rat bone marrow and transduced with GATA-4 (MSC(GATA-4)). To mimic ischemic injury, cultured cardiomyocytes (CMs) isolated from neonatal rat ventricles were exposed to hypoxia or were pretreated with concentrated conditioned medium (CdM) from MSC(GATA-4) or transduced control MSC (MSC(Null)) for 16 h before exposure to hypoxic culture conditions (low glucose and low oxygen). Myocyte damage was estimated by annexin-V-PE and TUNEL technique and by lactate dehydrogenase (LDH) release. Cell survival was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium (MTT) uptake. Mitochondrial membrane potential was determined using confocal microscopy. ELISA studies indicated that insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) were significantly increased in MSC(GATA-4) compared with MSC(Null). Hypoxia-induced apoptosis/cell death was significantly reduced when CMs were co-cultured with MSC(GATA-4) in a dual-chamber system. Cell protection mediated by MSC(GATA-4) was mimicked by treating CMs with CdM from MSC(GATA-4) and abrogated with IGF-1- and VEGF-neutralizing antibodies. MSC(GATA-4) protects CMs under hypoxic conditions. The release of IGF-1 and VEGF from MSC(GATA-4) is likely to be responsible for protection of CMs.

Use of mesenchymal stem cells for cutaneous repair and skin substitute elaboration.

Human mesenchymal stem cells (MSCs) are a heterogeneous population of fibroblast-like cells, which are present in different locations, including bone marrow, adipose tissue, extra-foetal tissues, gingiva and dermis. MSCs, which present multipotency capacities, important expansive potential and immunotolerance properties, remain an attractive tool for tissue repair and regenerative medicine. Currently, several studies and clinical trials highlight the use of MSCs in cutaneous repair underlining that their effects are essentially due to the numerous factors that they release. MSCs are also used in skin substitute development. In this study, we will first discuss the different sources of MSCs actually available. We will then present results showing that bone marrow-derived MSCs prepared according to Good Manufacturing Practices and included in a dermal equivalent are able to promote appropriate epidermis growth and differentiation. These data demonstrate that bone marrow-derived MSCs represent a satisfactory alternative to dermal fibroblasts in order to develop skin substitute. In addition, MSCs could provide a useful alternative to sustain epidermis development and to promote wound healing.

High level of reactive oxygen species impaired mesenchymal stem cell migration via overpolymerization of F-actin cytoskeleton in systemic lupus erythematosus.

Some lines of evidence have demonstrated abnormalities of bone marrow mesenchymal stem cells (MSCs) in systemic lupus erythematosus (SLE) patients, characterized by defective phenotype of MSCs and slower growth with enhanced apoptosis and senescence. However, whether SLE MSCs demonstrate aberrant migration capacity or abnormalities in cytoskeleton are issues that remain poorly understood. In this study, we found that MSCs from SLE patients did show impairment in migration capacity as well as abnormalities in F-actin cytoskeleton, accompanied by a high level of intracellular reactive oxygen species (ROS). When normal MSCs were treated in vitro with H2O2, which increases intracellular ROS level as an oxidant, both reorganization of F-actin cytoskeleton and impairment of migration capability were observed. On the other hand, treatment with N-acetylcysteine (NAC), as an exogenous antioxidant, made F-actin more orderly and increased migration ratio in SLE MSCs. In addition, oral administration of NAC markedly reduced serum autoantibody levels and ameliorated lupus nephritis (LN) in MRL/lpr mice, partially reversing the abnormalities of MSCs. These results indicate that overpolymerization of F-actin cytoskeleton, which may be associated with high levels of ROS, causes impairment in the migration capacity of SLE MSCs and that oral administration of NAC may have potential therapeutic effects on MRL/lpr mice.

Intra-articular injection of autologous bone marrow aspirate concentrate in the treatment of osteoarthritis of the thumb first carpometacarpal joint: A pilot study.

This study aimed to evaluate the effect of bone marrow aspirate concentrate (BMAC) in the treatment of osteoarthritis of the thumb first carpometacarpal joint. Injections were carried out in 27 thumbs. According to the Dell classification, there were 2 stage I, 11 stage II, 13 stage III and 1 stage IV cases. The bone marrow was aspirated from the iliac crest, concentrated by centrifugation, and injected under fluoroscopic control into the pathological thumb. Results were assessed at a mean 16 months' follow-up (range, 8-26). Clinical evaluation comprised QuickDASH and PRWE scores, pain at rest on a numerical analog scale (NAS), and thumb column abduction on goniometry. QuickDASH and PRWE scores were 59 (range, 27-82) and 88 range, 37-125) preoperatively and 29 (range, 0-64) and 50 (range, 1-99) postoperatively, respectively. Mean pain at rest on NAS improved from 7 (range, 1-10) to 4 (range, 0-9). Thumb abduction improved by a mean 18° over preoperative data. No postoperative complications were found. Two patients had to be operated on for inefficacy of injection. This is the first article presenting the effect of an intra-articular injection of BMAC in the thumb first carpometacarpal joint and the results were encouraging. Many patients showed improved quality of life and pain relief. These injections appear to be an effective means of postponing surgery.

[Treatment of radiation-induced late effects: What's new?] / Traitement des effets tardifs après la radiothérapie : quoi de neuf ?

Mechanisms of late radio-induced lesions are the result of multiple and complex phenomena, with many entangled cellular and tissue factors. The biological continuum between acute and late radio-induced effects will be described, with firstly a break in homeostasis that leads to cellular redistributions. New insights into late toxicity will finally be addressed. Individual radiosensitivity is a primary factor for the development of late toxicity, and clinicians urgently need predictive tests to offer truly personalized radiation therapy. An update will be made on the various functional and genetic tests currently being validated. The management of radio-induced side effects remains a frequent issue for radiation oncologists, and an update will be made for certain specific clinical situations. Finally, an innovative management for patients with significant side effects after pelvic radiotherapy will be developed, involved mesenchymal stem cell transplantation, with the presentation of the "PRISME" protocol currently open to patients recruitment.

Gene therapy and cell therapy for the management of radiation damages to healthy tissues: Rationale and early results.

Nowadays, ionizing radiations have numerous applications, especially in medicine for diagnosis and therapy. Pharmacological radioprotection aims at increasing detoxification of free radicals. Radiomitigation aims at improving survival and proliferation of damaged cells. Both strategies are essential research area, as non-contained radiation can lead to harmful effects. Some advances allowing the comprehension of normal tissue injury mechanisms, and the discovery of related predictive biomarkers, have led to developing several highly promising radioprotector or radiomitigator drugs. Next to these drugs, a growing interest does exist for biotherapy in this field, including gene therapy and cell therapy through mesenchymal stem cells. In this review article, we provide an overview of the management of radiation damages to healthy tissues via gene or cell therapy in the context of radiotherapy. The early management aims at preventing the occurrence of these damages before exposure or just after exposure. The late management offers promises in the reversion of constituted late damages following irradiation.

Can Stem Cells Ameliorate the Pancreatic Damage Induced by Streptozotocin in Rats?

BACKGROUND: Stem cell therapy holds great promise for the repair of injured tissues and organs, and it is one of the most promising therapies for diabetes mellitus. Therefore, the present study was undertaken to elucidate the antidiabetic effect of both mesenchymal stem cells (MSCs) and insulin-producing cells (IPCs) on streptozotocin (STZ)-induced diabetes in rats. MATERIALS AND METHODS: MSCs were derived from bone marrow of male albino rats. MSCs were characterized morphologically and by Cluster of differentiation (CD-ve34) and (CD+ve105). They were then differentiated into IPCs, and both MSCs and IPCs were infused independently into tail veins of rats with STZ-induced diabetes. RESULTS: MSC and IPC therapy significantly improved the body weight and serum insulin, alpha-amylase, adiponectin, creatinine, total cholesterol, triacylglycerol, interleukin-6, tumour necrosis factor-alpha, liver L-malonaldehyde and glycogen levels in the STZ-induced diabetes model. CONCLUSIONS: Bone marrow-derived MSCs have the capacity to differentiate into IPCs capable of controlling the blood glucose level in rats with STZ-induced diabetes. Furthermore, treatment with MSCs and IPCs can improve aberrant biochemical parameters in an STZ-induced diabetes model.

Integrin-Linked Kinase Improves Functional Recovery of Diabetic Cystopathy and Mesenchymal Stem Cell Survival and Engraftment in Rats.

OBJECTIVES: Diabetic cystopathy (DCP), characterized by peripheral neuropathy-associated bladder dysfunction, is a common urinary complication in patients with diabetes (~80%). Bone marrow mesenchymal stem cell (BMMSC) transplantation, a new and emerging regenerative therapy, provides a curative option for DCP. However, the application of this therapy is limited by the low survival rate and engraftment of transplanted stem cells. This study was undertaken to determine whether integrin-linked kinase (ILK) overexpression would improve stem cell survival and engraftment after BMMSC transplantation. METHODS: Diabetes was induced in rats by injection of streptozotocin. ILK expression was detected by qRT-PCR and Western blot. Bladder function was measured by urodynamic analyses. Smooth-muscle regeneration and vascularization were evaluated by immunohistochemistry staining. RESULTS: ILK overexpression by adenovirus promotes proliferation of BMMSCs in vitro. ILK overexpression enhanced the ability of BMMSCs to decrease the volume threshold for micturition and residual urine in the rats with diabetes. The contractile response of bladder strips, tissue structure of bladder and smooth-muscle regeneration/vascularization were also improved in the rats receiving ILK-modified BMMSCs. CONCLUSIONS: Our data highlight the clinical potential of transplantation of gene-modified BMMSCs in the treatment of DCP, thereby serving as a rapid and effective first-line strategy to cure the bladder dysfunction resulting from long-term diabetes.

[Mesenchymal stem cell therapy, a new hope for eye disease]. / Thérapie cellulaire par cellules souches mésenchymateuses, un nouvel espoir pour les pathologies oculaires.

Mesenchymal stem cells (MSC) are adult stem cells, first identified in skeletal tissues and then found in the entire body. MSC are able to not only differentiate into specialized cells within skeletal tissue - chondrocytes, osteocytes, adipocytes and fibroblasts - but also secrete a large range of soluble mediators defining their secretome and allowing their interaction with a number of cell protagonists. Thus, in a general sense, MSC are involved in tissue homeostasis through their secretome and are specifically responsible for cell turn-over in skeletal tissues. For a decade and a half, safety and efficiency of MSC has led to the development of many clinical trials in various fields. However, results were often disappointing, probably because of difficulties in methods and evaluation. At a time when the first clinical trials using MSC are emerging in ophthalmology, the goal of this literature review is to gather and put into perspective preclinical and clinical results in order to better predict the future of this innovative therapeutic pathway.