Recovering Spermatogenesis By Protected Cryopreservation Using Metformin and Transplanting Spermatogonial Stem Cells Into Testis in an Azoospermia Mouse Model.

Cryopreservation and transplantation of spermatogonial stem cells (SSCs) may serve as a new method to restore male fertility in patients undergoing chemotherapy or radiotherapy. However, SSCs may be damaged during cryopreservation due to the production of reactive oxygen species (ROS). Therefore, different antioxidants have been used as protective agents. Studies have shown that metformin (MET) has antioxidant activity. The aim of this study was to assess the antioxidant and antiapoptotic effects of MET in frozen-thawed SSCs. In addition, the effect of MET on the proliferation and differentiation of SSCs was evaluated. To this end, SSCs were isolated from mouse pups aged 3-6 days old, cultured, identified by flow cytometry (ID4, INTEGRIN ß1+), and finally evaluated for survival and ROS rate. SSCs were transplanted after busulfan and cadmium treatment. Cryopreserved SSCs with and without MET were transplanted after 1 month of cryopreservation. Eight weeks after transplantation, the recipient testes were evaluated for the expression of apoptosis (BAX, BCL2), proliferation (PLZF), and differentiation (SCP3, TP1, TP2, PRM1) markers using immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction. The findings revealed that the survival rate of SSCs was higher in the 500 µm/mL MET group compared to the other groups (50 and 5000 µm/mL). MET significantly decreased the intracellular ROS production. Transplantation of SSCs increased the expression level of proliferation (PLZF) and differentiation (SCP3, TP1, TP2, PRM1) markers compared to azoospermia group, and their levels were significantly higher in the MET group compared to the cryopreservation group containing basic freezing medium (p < 0.05). MET increased the survival rate of SSCs, proliferation, and differentiation and decreased the ROS production and the apoptosis rate. Cryopreservation by MET seems to be effective in treating infertility.

Antioxidant Effect of Melatonin on Proliferation, Apoptosis, and Oxidative Stress Variables in Frozen-Thawed Neonatal Mice Spermatogonial Stem Cells.

Cryopreservation of spermatogonial stem cells (SSCs) is an important method to restore and maintain fertility in preadolescent children suffering from cancer. For protection of SSCs from cryoinjury, various antioxidant agents have been used. The aim of this study was to assess the antiapoptotic and antioxidant effects of melatonin in frozen-thawed SSCs. SSCs were isolated from testes of neonatal mice (3-6 days old) and their purities were measured by flow cytometry with promyelocytic leukemia zinc finger protein. After culturing, the cells were frozen in two groups (1) control and (2) melatonin (100 µM) and stored for 1 month. Finally, the cell viability, colonization rate, expression of Bcl-2 and BAX gene, and intracellular reactive oxygen species (ROS) were evaluated after freezing-thawing. Melatonin increased the viability and colonization of SSCs and Bcl-2 gene expression. It also diminished BAX gene expression and intracellular ROS. The results of this study show that melatonin with antioxidant and antiapoptotic effects can be used as an additive for freezing and long-term storage of cells and infertility treatment in the clinic.

Transdifferentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Dopaminergic Neurons in a Three-Dimensional Culture.

Introduction: The induction of human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) toward dopaminergic neurons is a major challenge in tissue engineering and experimental and clinical treatments of various neurodegenerative diseases, including Parkinson disease. This study aims to differentiate HUC-MSCs into dopaminergic neuron-like cells. Methods: Following the isolation and characterization of HUC-MSCs, they were transferred to Matrigel-coated plates and incubated with a cocktail of dopaminergic neuronal differentiation factors. The capacity of differentiation into dopaminergic neuron-like cells in 2-dimensional culture and on Matrigel was assessed by real-time polymerase chain reaction, immunocytochemistry, and high-performance liquid chromatography. Results: Our results showed that dopaminergic neuronal markers' transcript and protein levels were significantly increased on the Matrigel differentiated cells compared to 2D culture plates. Conclusion: Overall, the results of this study suggest that HUC-MSCs can successfully differentiate toward dopaminergic neuron-like cells on Matrigel, having great potential for the treatment of dopaminergic neuron-related diseases.

Evaluating the Differential Expression of miR-146a, miR-222, and miR-9 in Matched Serum and Follicular Fluid of Polycystic Ovary Syndrome Patients: Profiling and Predictive Value.

Polycystic ovary syndrome (PCOS) is the most prevalent endocrine disorder of women in reproductive age with significant effects on reproductive and metabolic functions. Many molecular players may be involved in PCOS pathology; however, miRNAs possess great ability in gene expression control in normal ovarian function and folliculogenesis. We appraised the relative expression of miR-146a, miR-222, miR-9, and miR-224 in serum and follicular fluid (FF) of PCOS patients compared to control subjects. PCOS (n = 35) and control (n = 30) subjects were recruited in the study during their enrolment in IVF cycles. Serum and FF of human subjects were collected and stored. Total RNA was isolated from samples and cDNA was synthesized using miRNA-specific stem-loop RT primers. Quantitative real-time PCR was used to evaluate the expression of miRNAs relative to U6 expression. The predictive value of miRNAs' expression for discrimination of PCOS patients from control subjects was evaluated by receiver-operating characteristic (ROC) curve analysis. miR-224 was not detected in serum and FF samples. Significantly, higher levels of miR-146a and miR-9 in serum of PCOS group were detected. In contrast, relative expression of miR-146a and miR-9 significantly decreased in FF. In PCOS group, relative expression of all detected miRNAs was elevated in serum in comparison to FF, whereas in control group no change was noticed. Combination of FF miRNAs showed improved predictive value with area under the ROC curve (AUC) of 0.84, 93.8% sensitivity, and 83.3% specificity. Contradicting alternations of miRNAs in serum and FF are indicative of different sources of miRNAs in body fluids. Presumptive target genes of studied miRNAs in signalling pathways may show the potential role of these miRNA in folliculogenesis.

MicroRNA-30a-5p promotes differentiation in neonatal mouse spermatogonial stem cells (SSCs).

BACKGROUND: The importance of spermatogonial stem cells (SSCs) in spermatogenesis is crucial and intrinsic factors and extrinsic signals mediate fate decisions of SSCs. Among endogenous regulators, microRNAs (miRNAs) play critical role in spermatogenesis. However, the mechanisms which individual miRNAs regulate self- renewal and differentiation of SSCs are unknown. The aim of this study was to investigate effects of miRNA-30a-5p inhibitor on fate determinations of SSCs. METHODS: SSCs were isolated from testes of neonate mice (3-6 days old) and their purities were performed by flow cytometry with ID4 and Thy1 markers. Cultured cells were transfected with miRNA- 30a-5p inhibitor. Evaluation of the proliferation (GFRA1, PLZF and ID4) and differentiation (C-Kit & STRA8) markers of SSCs were accomplished by immunocytochemistry and western blot 48 h after transfection. RESULTS: Based on the results of flow cytometry with ID4 and Thy1 markers, percentage of purity of SSCs was about 84.3 and 97.4 % respectively. It was found that expression of differentiation markers after transfection was significantly higher in miRNA-30a- 5p inhibitor group compared to other groups. The results of proliferation markers evaluation also showed decrease of GFRA1, PLZF and ID4 protein in SSCs transfected with miRNA-30a-5p inhibitor compared to the other groups. CONCLUSIONS: It can be concluded that inhibition of miRNA-30a-5p by overexpression of differentiation markers promotes differentiation of Spermatogonial Stem Cells.

Effect of miR-30a-5p on Apoptosis, Colonization, and Oxidative Stress Variables in Frozen-Thawed Neonatal Mice Spermatogonial Stem Cells.

Cryopreservation of spermatogonial stem cells (SSCs) is a useful method for fertility preservation in preadolescent children suffering from cancer. However, SSCs may become damaged during cryopreservation due to the generation of reactive oxygen species (ROS). For this reason, various antioxidant agents have been used to protect SSCs from cryopreservation-induced damages. Recently, it has been reported that miR-30a-5p has antiapoptotic and antioxidant activity. The aim of this study was to assess the antiapoptotic and antioxidant effects of miR-30a-5p mimics in frozen-thawed SSCs. To this end, SSCs were isolated from male BALB/C mice (3-6 days old) and cultivated for 14 days. After the detection of optimum concentration, a miR-30a-5p mimic or miR-30a-5p inhibitor with Lipofectamine was transfected into SSCs and, finally, the cell groups were frozen for 1 week. After thawing, different properties, including cell viability (using MTT), colonization of SSCs (number and diameter of colonies), ROS generation (using DCFH-DA assay), levels of malondialdehyde (MDA) and superoxide dismutase (SOD), and gene expression of Bcl-2 and BAXBax (using quantitative real-time PCR), were investigated. The transfection of SSCs with miR-30a-5p mimics before the freezing-thawing process significantly increased the viability, number, and diameter of SSCs colonies. Also, the miR-30a-5p mimic decreased the levels of ROS production and MDA, but it increased the SOD levels. Moreover, the miR-30a-5p mimic decreased BAX and increased Bcl-2 expression in frozen-thawed SSCs. The transfection of SSCs with the miR-30a-5p mimic can increase cell viability and antioxidant defense, and it can decrease apoptosis during the freezing-thawing process. If SSC is able to produce spermatozoa after the transfection of miR-30a-5p and the freezing-thawing process, it can be suggested as a promising strategy for the cryopreservation of SSCs in prepubertal boys suffering from cancer.

Effect of a Freezing Medium Containing Melatonin on Markers of Pre-meiotic and Post-meiotic Spermatogonial Stem Cells (SSCs) After Transplantation in an Azoospermia Mouse Model Due to Testicular Torsion.

Spermatogonial stem cells (SSCs) are essential to the initiation of spermatogenesis. Cryopreservation, long-term maintenance, and auto-transplantation of SSCs could be a new treatment for infertility. The aim of this study was to add melatonin to the basic freezing medium and to evaluate its effect on the efficiency of the thawed SSCs after transplantation into the testicles of azoospermic mice. SSCs were isolated from newborn NMRI mice, and the cells were enriched to assess morphological features. The thawed SSCs were evaluated for survival, apoptosis, and ROS level before transplantation, and the proliferation (MVH and ID4) and differentiation (c-Kit, SCP3, TP1, TP2, and Prm1) markers of SSCs were examined using immunofluorescence, western blot, and quantitative real-time polymerase chain reaction (PCR) after transplantation. It was found that the survival rate of SSCs after thawing was significantly higher in the melatonin group compared with the cryopreservation group containing basic freezing medium, and the rate of apoptosis and level of ROS production also decreased significantly in the cryopreservation group with melatonin (p < 0.05). The expression of proliferation and differentiation markers after transplantation was significantly higher in the cryopreservation group with melatonin compared to the cryopreservation group (p < 0.05). The results suggest that adding melatonin to the basic freezing medium can effectively protect the SSCs by increasing the viability and reducing the ROS production and apoptosis and improve the transplantation efficiency of SSCs after cryopreservation, which will provide a significant suggestion for fertility protection in the clinic.

Role of cerebrospinal fluid in differentiation of human dental pulp stem cells into neuron-like cells.

Human dental pulp stem cells (hDPSCs) could be differentiated into neuron like-cells under particular microenvironments. It has been reported that a wide range of factors, presented in cerebrospinal fluid (CSF), playing part in neuronal differentiation during embryonic stages, we herein introduce a novel culture media complex to differentiate hDPSCs into neuron-like cells. The hDPSCs were initially isolated and characterized. The CSF was prepared from the Cisterna magna of 19-day-old Wistar rat embryos, embryonic cerebrospinal fluid (E-CSF). The hDPSCs were treated by 5% E-CSF for 2 days, then neurospheres were cultured in DMEM/F12 supplemented with 10-6 µm retinoic acid (RA), glial-derived neurotrophic factor and brain-derived neurotrophic factor for 6 days. The cells which were cultured in basic culture medium were considered as control group. Morphology of differentiated cells as well as process elongation were examined by an inverted microscope. In addition, the neural differentiation markers (Nestin and MAP2) were studied employing immunocytochemistry. Neuronal-like processes appeared 8 days after treatment. Neural progenitor marker (Nestin) and a mature neural marker (MAP2) were expressed in treated group. Moreover Nissl bodies were found in the cytoplasm of treated group. Taking these together, we have designed a simple protocol for generating neuron-like cells using CSF from the hDPSCs, applicable for cell therapy in several neurodegenerative disorders including Alzheimer's disease.

Aqueous Origanum vulgare Extract Improves the Quality of Cryopreserved Human Spermatozoa Through Its Antioxidant Effects.

Excessive production of reactive oxygen species (ROS) during semen cryopreservation can induce structural and functional changes in spermatozoa. It is well known that antioxidants can mitigate the effect of ROS. Moreover, the application of antioxidants in freezing media is an appropriate strategy for protecting spermatozoa against deleterious effects of ROS during the cryopreservation process. As an example, oregano is a medicinal plant with important activities, with antiseptic, antibacterial, antithrombotic, and antioxidant properties. This study aimed at evaluating the antioxidant effects of oregano extract on cryopreserved human spermatozoa. In the first phase, 13 semen samples with different concentrations of oregano extract (0.0, 50, 100, 150, 300, and 500 µg/mL) were cryopreserved to achieve an optimal dose of oregano extract. Then, motility, viability, and plasma membrane integrity were evaluated. In the second phase, 20 samples were cryopreserved in freezing media supplemented with or without the optimal concentration of oregano (100 µg/mL). After thawing, motility, the levels of ROS, lipid peroxidation, and translocation of phosphatidylserine (PS) were evaluated. The results showed that 100 µg/mL oregano extract significantly increased the total motility in frozen-thawed spermatozoa in comparison with the control group (28.2 ± 4.3 vs. 42.4 ± 1.6, p < 0.05). This concentration significantly decreased the percentage of 2',7'-dichlorofluorescein-positive cells (25.53 ± 1.2 vs. 21.48 ± 1.2) and the malondialdehyde level (4.25 ± 0.7 vs. 0.82 ± 0.4 µM) (p < 0.05). In the oregano group, the percentage of vital spermatozoa without PS externalization was significantly higher than that in the control group (25.88 ± 1.6 vs. 16.8 ± 1.9, p < 0.001), while the percentage of dead spermatozoa with PS externalization spermatozoa was significantly lower than that in the control group (51.65 ± 1.4 vs. 60.36 ± 1.9, p < 0.05). In general, the addition of oregano extract to sperm freezing extender has protective effects against oxidative stress and apoptosis.

Determination of the Excitatory Effects of MicroRNA-30 in the Self-Renewal and Differentiation Process of Neonatal Mouse Spermatogonial Stem Cells.

BACKGROUND: Spermatogonial stem cells (SSCs) are considered as special stem cells since they have the ability of self-renewal, differentiation, and transferring genetic information to the next generation. Also, they considered as vital players in initiating and preserving spermatogenesis. The fate decisions of SSCs are mediated by intrinsic and extrinsic factors, among which microRNAs (miRNAs) are one of the most essential factors in spermatogenesis among endogenous regulators. However, the mechanisms by which individual miRNAs regulate self-renewal and differentiation of SSCs are unclear. The present study aimed to evaluate the impact of miRNA-30 mimic on fate determinations of SSCs. MATERIALS AND METHODS: The obtained SSCs from neonatal mice (3-6 days old) were purified by MACS and flow cytometry with a promyelocytic leukemia zinc-finger marker. Then, the cultured cells were transfected with miRNA- 30 mimic, and finally, the changes in expressing ID4 and c-kit proteins were assessed by western blot analysis. RESULTS: According to flow cytometry findings, the percentage of SSC purity was about 98.32. The expression of ID4 protein and colonization increased significantly through the transfection of miRNA-30 mimic (P<0.05). CONCLUSION: The miRNA-30 controls spermatogonial stem cell self-renewal and differentiation, which may have significant implications for treating male infertility.

Schwann cell transplantation exerts neuroprotective roles in rat model of spinal cord injury by combating inflammasome activation and improving motor recovery and remyelination.

Inflammasome activation in the traumatic central nervous system (CNS) injuries is responsible for propagation of an inflammatory circuit and neuronal cell death resulting in sensory/motor deficiencies. NLRP1 and NLRP3 are known as activators of inflammasome complex in the spinal cord injury (SCI). In this study, cell therapy using Schwann cells (SCs) was applied for targeting NLRP inflammasome complexes outcomes in the motor recovery. These cells were chosen due to their regenerative roles for CNS injuries. SCs were isolated from sciatic nerves and transplanted to the contusive SCI-induced Wistar rats. NLRP1 and NLRP3 inflammasome complexes and their related pro-inflammatory cytokines were assayed in both mRNA and protein levels. Neuronal cell survival (Nissl staining), motor recovery and myelination (Luxol fast blue/LFB) were also evaluated. The groups were laminectomy, SCI, vehicle and treatment. The treatment group received Schwann cells, and the vehicle group received solvent for the cells. SCI caused increased expressions for both NLRP1 and NLRP3 inflammasome complexes along with their related pro-inflammatory cytokines, all of which were abrogated after administration of SCs (except for IL-18 protein showing no change to the cell therapy). Motor deficits in the hind limb, neuronal cell death and demyelination were also found in the SCI group, which were counteracted in the treatment group. From our findings we conclude promising role for cell therapy with SCs for targeting axonal demyelination and degeneration possibly through attenuation of the activity for inflammasome complexes and related inflammatory circuit.

Co-Administration of Progesterone and Melatonin Attenuates Ischemia-Induced Hippocampal Damage in Rats.

Stroke is the second leading reason for death worldwide and is one of the fundamental causes of long-term disabilities. The aim of this investigation was to assess the impact of combined administration progesterone (PROG) and melatonin (MEL) on stroke complications. Male Wistar rats (9-10 weeks) weighing 250-300 g were used as a part of this examination. They were randomly separated into eight groups (nine rats for every group). Common carotid arteries on the two sides clamped (BCCAO model) with non-traumatic clips for 20 min. At that point, the rats were treated with 8 mg/kg PROG, 10 mg/kg MEL, and vehicles (sesamoid and normal saline). Morris water maze testing was performed following 2 weeks. At that point, the rats were euthanized, and histological examination was directed. The outcome demonstrated that utilization of PROG and MEL in treatment groups essentially increases the quantity of pyramidal cells and enhances spatial memory compared to non-treatment groups (p < 0.05). Moreover, the neuroleptic factor gene expression and protein concentration were significantly enhanced in the treated groups (p < 0.05). As indicated by the outcomes, co-administration of PROG and MEL can enhance learning and memory by surviving the pyramidal neurons and diminishing neural death by means of increasing neuroleptic factors in the hippocampal CA1 zone.

The Effect of Rosa Damascena Extract on Expression of Neurotrophic Factors in the CA1 Neurons of Adult Rat Hippocampus Following Ischemia.

Ischemic stroke is an important cause of death and disability in the world. Brain ischemia causes damage to brain cell, and among brain neurons, pyramidal neurons of the hippocampal CA1 region are more susceptive to ischemic injury. Recent findings suggest that neurotrophic factors protect against ischemic cell death. A dietary component of Rosa damascene extract possibly is associated with expression of neurotrophic factors mRNA following ischemia, so it can have therapeutic effect on cerebral ischemia. The present study attempts to evaluate the neuroprotective effect of Rosa damascene extract on adult rat hippocampal neurons following ischemic brain injury. Forty-eight adult male Wistar rats (weighing 250±20 gr and ages 10-12 weeks) used in this study, animals randomly were divided into 6 groups including Control, ischemia/ reperfusion (IR), vehicle and three treated groups (IR+0.5, 1, 2 mg/ml extract). Global ischemia was induced by bilateral common carotid arteries occlusion for 20 minutes. The treatment was done by different doses of Rosa damascena extract for 30 days. After 30 days cell death and gene expression in neurons of the CA1 region of the hippocampus were evaluated by Nissl staining and real time PCR assay. We found a significant decrease in NGF, BDNF and NT3 mRNA expression in neurons of CA1 region of the hippocampus in ischemia group compared to control group (P<0.0001). Our results also revealed that the number of dark neurons significantly increases in ischemia group compared to control group (P<0.0001). Following treatment with Rosa damascene extract reduced the number of dark neurons that was associated with NGF, NT3, and BDNF mRNA expression. All doses level had positive effects, but the most effective dose of Rosa damascena extract was 1 mg/ml. Our results suggest that neuroprotective activity of Rosa damascena can enhance hippocampal CA1 neuronal survival after global ischemia.

Progesterone Enhanced Remyelination in the Mouse Corpus Callosum after Cuprizone Induced Demyelination.

BACKGROUND: Progesterone as a sex steroid hormone is thought to affect and prevent demyelination, but its role in promoting myelin repair is far less investigated. In this study, remyelinating potential of progesterone in corpus callosum was evaluated on an experimental model of MS. METHODS: In this experimental study, adult male C57BL/6 mice were fed with 0.2% (w/w) cuprizone in ground breeder chow ad libitum for 6 weeks. At day zero, after cuprizone removal, mice were divided randomly into two groups: (a) placebo group, which received saline pellet implant, (b) progesterone group, which received progesterone pellet implant. Some mice of the same age were fed with their normal diet to serve as the healthy control group. Two weeks after progesterone administration, Myelin content was assessed by Luxol-fast blue staining. The myelin basic protein (MBP) and proteolipid protein (PLP) expression were assessed using Western blot analysis and the changes in the number of oligodendrocytes and oligodendroglial progenitor cells were assessed by immunohistochemistry (IHC) and flow cytometry. RESULTS: Luxol-fast blue staining revealed enhanced remyelination in the progesterone group when compared with the placebo group. Densitometry measurements of immunoblots demonstrated that MBP and PLP proteins contents were significantly increased in the progesterone group compared with the placebo group. Flow cytometry and IHC analysis showed increases in Olig2 and O4 cells in the progesterone group compared with the placebo group. CONCLUSION: Overall, our results indicate that progesterone treatment can stimulate myelin production and that it may provide a feasible and practical way for remyelination in diseases such as multiple sclerosis.

Remyelination of the Corpus Callosum by Olfactory Ensheathing Cell in an Experimental Model of Multiple Sclerosis.

Multiple Sclerosis (MS) causes loss of the myelin sheath, which leads to loss of neurons. Regeneration of myelin sheath stimulates axon regeneration and neurons' survival. In this study, olfactory ensheathing cell (OEC) transplantation is investigated to restore myelin sheath in an experimental model of MS in male mice.OECs were isolated from the olfactory mucosa of seven-day-old infant rats and cultured. Then, cells were evaluated and approved by flow cytometry by p75 and GFAP markers. A total of 32 mice (C57BL /6) were studied in four groups; 1) without any treatment (control), 2) Sham (receiving PBS), 3) MS model and 4) MS and OEC transplantation. MS was induced by adding Cuprizon in the diet of animals for six weeks. After the expiration of 20 days, histologic analysis was performed with approval of the presence of cells in the graft area and the removal of myelin and myelin regeneration with two types of luxal fast blue (LFB) staining and immunohistochemistry. The purity of the cells ensheathing the olfactory was 90%. There was a significant difference in Myelin percentage of PBS and OEC recipient groups (P≤0.05). MBP and PLP of the myelin sheath in the group receiving OECs were more than MS group.According to the findings, in MS model MBP and PLP of the myelin sheath is reduced. In the group receiving OECs, it was returned to a normal level significantly compared to the sham group received only PBS significant differences were observed. The OECs transplantation can improve myelin restoration.

Do Pilea Microphylla Improve Sperm DNA Fragmentation and Sperm Parameters in Varicocelized Rats?

Varicocele is one of the most common causes of primary male infertility. Pilea microphylla (PM) is being used as folk medicine. This study was aimed to investigate the effects of PM in a rat model of varicocele. A total of 30 male Wistar rats were divided into control, sham, varicocele, accessory varicocele and PM-treated groups. After 10 weeks of varicocele induction, sperm parameters and chromatin (Aniline blue, acridine orange and toluidine blue) were evaluated, except for the treated and accessory groups that received 50 mg/kg PM orally daily for 10 weeks and then were sacrificed. Sperm parameters significantly decreased in varicocele groups (P < 0.01). Moreover, there was a negative correlation between the DNA fragmentation and sperm parameters in varicocelized rats. Administration of PM led to significantly increased sperm parameters and AO staining (P < 0.05). These findings suggest that PM improves sperm parameters and DNA fragmentation in varicocelized rats. PM can reduce the damage to sperm DNA but not chromatin condensation.

Promotion of remyelination by adipose mesenchymal stem cell transplantation in a cuprizone model of multiple sclerosis.

OBJECTIVE: Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). Stem cell transplantation is a new therapeutic approach for demyelinating diseases such as MS which may promote remyelination. In this study, we evaluate the remyelinating potential of adipose mesenchymal stem cells (ADSCs) and their effect on neural cell composition in the corpus callosum in an experimental model of MS. MATERIALS AND METHODS: This experimental study used adult male C57BL/6 mice. Cultured ADSCs were confirmed to be CD73(+),CD90(+), CD31(-),CD45(-), and labeled by PKH26. Animals were fed with 0.2% w/w cuprizone added to ground breeder chow ad libitum for six weeks. At day 0 after cuprizone removal, mice were randomly divided into two groups: the ADSCs-transplanted group and the control vehicle group (received medium alone). Some mice of the same age were fed with their normal diet to serve as healthy control group. Homing of ADSCs in demyelinated lesions was examined by fluorescent microscope. At ten days after transplantation, the mice were euthanized and their cells analyzed by luxol fast blue staining (LFB), transmission electron microscopy and flow cytometry. Results were analyzed by one-way analysis of variance (ANOVA). RESULTS: According to fluorescent cell labeling, transplanted ADSCs appeared to survive and exhibited homing specificity. LFB staining and transmission electron microscope evaluation revealed enhanced remyelination in the transplanted group compared to the control vehicle group. Flow cytometry analysis showedan increase in Olig2 and O4 cells and a decrease in GFAP and Iba-1 cells in the transplanted group. CONCLUSION: Our results indicate that ADSCs may provide a feasible, practical way for remyelination in diseases such as MS.

17ß-Estradiol enhances the efficacy of adipose-derived mesenchymal stem cells on remyelination in mouse model of multiple sclerosis.

Previous studies have demonstrated the potential of monotherapy with either mesenchymal stem cells (MSCs) or estrogen in autoimmune and cuprizone models of multiple sclerosis (MS). The aim of this study was to examine the effects of co-administration of 17ß-estradiol (E2) and adipose-derived mesenchymal stem cells (ADSCs) on remyelination of corpus callosum axons in a cuprizone model of MS. Forty eight male C57BL/6 mice were fed cuprizone (0.2%) for 6 weeks. At day 0 after cuprizone removal, animals were randomly divided into four groups. The E2 monotherapy, ADSCs monotherapy, E2/ADSCs combined therapy and vehicle control. Some mice of the same age were fed with their normal diet to serve as healthy control group. E2 pellets, designed to release 5.0 mg E2 over 10 days, were implanted subcutaneously. 10(6) PKH26 labeled ADSCs were transplanted into lateral tail. The extent of demyelination, remyelination, and cell type's composition of host brain were examined at 10 days post-transplantation in the body of the corpus callosum. Transplanted cells migrated to the corpus callosum injury. Histological examination revealed efficacy of intravenous ADSCs transplantation in remyelination of mouse cuprizone model of MS can be significantly enhanced by E2 administration. Flow cytometry showed that the mean percentages of expression of Iba-1, Olig2 and O4 were significantly increased in E2/ADSCs combined therapy in comparison with ADSCs monotherapy. In conclusion, the findings of this study revealed that E2 administration enhanced efficacy of intravenous ADSCs transplantation in remyelination of corpus callosum axons in mouse cuprizone model of MS.

Effect of Trolox addition to cryopreservation media on human sperm motility.

BACKGROUND: Sperm parameters and motion kinetics are affected by cryopreservation. OBJECTIVE: The main purpose of the current study was to determine the effect of different concentrations of Trolox as an antioxidant to freezing-thawing procedure on human sperm kinematic parameter. MATERIALS AND METHODS: Semen was collected from 20 normal donors and divided into five aliquots prior to cryopreservation. The first aliquot was analyzed by computer-assisted sperm analysis (CASA). Other aliquots were mixed with cryo-protective agent containing 0, 20, 40, and 80 µmol Trolox and treated samples were cryopreserved in liquid nitrogen. After two weeks samples were thawed and sperm motion kinematics was measured by CASA. Percent motility (Mot), curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and amplitude of lateral head displacement (ALH) were compared before and after freeze. RESULTS: Addition of 40µmol Trolox resulted in significantly higher (p<0.05) post thaw VCL, VSL and VAP compared to other groups. Therefore the percentage of post thaw motile spermatozoa were significantly higher (p<0.01). CONCLUSION: The supplementation of Trolox significantly improved the post-thawed human semen quality, especially progressive motility and average path velocity.

Osteogenic differentiation of rat mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells: melatonin as a differentiation factor.

BACKGROUND: Adipose-derived stem cells (ADSC) could be an appealing alternative to bone marrow stem cells (BMSC) for engineering cell-based osteoinductive grafts. Meanwhile, prior studies have demonstrated that melatonin can stimulate osteogenic differentiation. Therefore, we assayed and compared the melatonin effect on osteogenic differentiation of BMSC with that of ADSC. METHODS: Mesenchymal stem cells (MSC) were isolated from the bone marrow and fat of adult rats. Both cell types were cultured in osteogenic medium in the absence and presence of melatonin at physiological concentrations (20-200 pg/ml). After 4 weeks, the expression of osteocalcin gene was analyzed by reverse transcription-PCR, alkaline phosphatase (ALP) activity was assayed and alizarin red S and von Kossa staining were done. Cell viability and apoptosis were also assayed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a tetrazole (MTT) and flow cytometry, respectively. RESULTS: The osteoblastic differentiation of ADSC as demonstrated by ALP activity was less than that of BMSC. The amount of matrix mineralization has shown by alizarin red S and von Kossa staining also showed statistical differences between the two MSC. The incidence of apoptotic cells was higher among ADSC than BMSC. The flow cytometry proves that cell growth reduction is due to a decrease in the number of the cells entering the S phase of the cell cycle. MTT assay indicated that viable cells were fewer among ADSC than BMSC in control groups. CONCLUSION: The results of the study suggest that BMSC have greater osteogenic potential than ADSC and that melatonin promotes osteogenic differentiation to BMSC, but has a negative effect on ADSC osteogenic differentiation.