Mesenchymal Stem Cells Ameliorate Cuprizone-Induced Demyelination by Targeting Oxidative Stress and Mitochondrial Dysfunction.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. The main causes of MS disease progression, demyelination, and tissue damage are oxidative stress and mitochondrial dysfunction. Hence, the latter are considered as important therapeutic targets. Recent studies have demonstrated that mesenchymal stem cells (MSCs) possess antioxidative properties and are able to target mitochondrial dysfunction. Therefore, we investigated the effect of transplanting Wharton's jelly-derived MSCs in a demyelination mouse model of MS in which mice were fed cuprizone (CPZ) for 12 weeks. CPZ is a copper chelator that impairs the activity of cytochrome oxidase, decreases oxidative phosphorylation, and produces degenerative changes in oligodendrocytes, leading to toxic demyelination similar to those found in MS patients. Results showed that MSCs caused a significant increase in the percentage of myelinated areas and in the number of myelinated fibers in the corpus callosum of the CPZ + MSC group, compared to the CPZ group, as assessed by Luxol fast blue staining and transmission electron microscopy. In addition, transplantation of MSCs significantly increased the number of oligodendrocytes while decreasing astrogliosis and microgliosis in the corpus callosum of the CPZ + MSC group, evaluated by immunofluorescence. Moreover, the mechanism by which MSCs exert these physiological effects was found to be through abolishing the effect of CPZ on oxidative stress markers and mitochondrial dysfunction. Indeed, malondialdehyde significantly decreased while glutathione and superoxide dismutase significantly increased in CPZ + MSC mice group, in comparison witth the CPZ group alone. Furthermore, cell therapy with MSC transplantation increased the expression levels of mitochondrial biogenesis transcripts PGC1α, NRF1, MFN2, and TFAM. In summary, these results demonstrate that MSCs may attenuate MS by promoting an antioxidant response, reducing oxidative stress, and improving mitochondrial homeostasis.

Incorporation of SPION-casein core-shells into silk-fibroin nanofibers for cardiac tissue engineering.

Mimicking the structure of extracellular matrix (ECM) of myocardium is necessary for fabrication of functional cardiac tissue. The superparamagnetic iron oxide nanoparticles (SPIONs, Fe3 O4 ), as new generation of magnetic nanoparticles (NPs), are highly intended in biomedical studies. Here, SPION NPs (1 wt%) were synthesized and incorporated into silk-fibroin (SF) electrospun nanofibers to enhance mechanical properties and topography of the scaffolds. Then, the mouse embryonic cardiac cells (ECCs) were seeded on the scaffolds for in vitro studies. The SPION NPs were studied by scanning electron microscope (SEM), X-ray diffraction (XRD), and transmission electron microscope (TEM). SF nanofibers were characterized after incorporation of SPIONs by SEM, TEM, water contact angle measurement, and tensile test. Furthermore, cytocompatibility of scaffolds was confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. SEM images showed that ECCs attached to the scaffolds with elongated morphologies. Also, the real-time PCR and immunostaining studies approved upregulation of cardiac functional genes in ECCs seeded on the SF/SPION-casein scaffolds including GATA-4, cardiac troponin T, Nkx 2.5, and alpha-myosin heavy chain, compared with the ones in SF. In conclusion, incorporation of core-shells in SF supports cardiac differentiation, while has no negative impact on ECCs' proliferation and self-renewal capacity.

Inhibition of H1N1 influenza virus infection by zinc oxide nanoparticles: another emerging application of nanomedicine.

BACKGROUND: Currently available anti-influenza drugs are often associated with limitations such as toxicity and the appearance of drug-resistant strains. Therefore, there is a pressing need for the development of novel, safe and more efficient antiviral agents. In this study, we evaluated the antiviral activity of zinc oxide nanoparticles (ZnO-NPs) and PEGylated zinc oxide nanoparticles against H1N1 influenza virus. METHODS: The nanoparticles were characterized using the inductively coupled plasma mass spectrometry, x-ray diffraction analysis, and electron microscopy. MTT assay was applied to assess the cytotoxicity of the nanoparticles, and anti-influenza activity was determined by TCID50 and quantitative Real-Time PCR assays. To study the inhibitory impact of nanoparticles on the expression of viral antigens, an indirect immunofluorescence assay was also performed. RESULTS: Post-exposure of influenza virus with PEGylated ZnO-NPs and bare ZnO-NPs at the highest non-toxic concentrations could be led to 2.8 and 1.2 log10 TCID50 reduction in virus titer when compared to the virus control, respectively (P < 0.0001). At the highest non-toxic concentrations, the PEGylated and unPEGylated ZnO-NPs led to inhibition rates of 94.6 and 52.2%, respectively, which were calculated based on the viral loads. There was a substantial decrease in fluorescence emission intensity in viral-infected cell treated with PEGylated ZnO-NPs compared to the positive control. CONCLUSIONS: Taken together, our study indicated that PEGylated ZnO-NPs could be a novel, effective, and promising antiviral agent against H1N1 influenza virus infection, and future studies can be designed to explore the exact antiviral mechanism of these nanoparticles.

In vitro differentiation of neural stem cells derived from human olfactory bulb into dopaminergic-like neurons.

This study describes a new accessible source of neuronal stem cells that can be used in Parkinson's disease cell transplant. The human olfactory bulb contains neural stem cells (NSCs) that are responsible for neurogenesis in the brain and the replacement of damaged cellular components throughout life. NSCs are capable of differentiating into neuronal and glial cells. We isolated NSCs from the olfactory bulb of brain-death donors and differentiated them into dopaminergic neurons. The olfactory bulb tissues obtained were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F12, B27 supplemented with basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor. The NSCs and proliferation markers were assessed. The multipotentiality of olfactory bulb NSCs was demonstrated by their capacity to differentiate into neurons, oligodendrocytes and astrocytes. To generate dopaminergic neurons, olfactory bulb NSCs were differentiated in neurobasal medium, supplemented with B27, and treated with sonic hedgehog, fibroblast growth factor 8 and glial cell-derived neurotrophic factor from the 7th to the 21st day, followed by detection of dopaminergic neuronal markers including tyrosine hydroxylase and aromatic l-amino acid decarboxylase. The cells were expanded, established in continuous cell lines and differentiated into the two classical neuronal phenotypes. The percentage of co-positive cells (microtubule-associated protein 2 and tyrosine hydroxylase; aromatic l-amino acid decarboxylase and tyrosine hydroxylase) in the treated cells was significantly higher than in the untreated cells. These results illustrate the existence of multipotent NSCs in the adult human olfactory bulb that are capable of differentiating toward putative dopaminergic neurons in the presence of trophic factors. Taken together, our data encourage further investigations of the possible use of olfactory bulb NSCs as a promising cell-based therapeutic strategy for Parkinson's disease.

In vitro proliferation and differentiation of mouse spermatogonial stem cells in decellularized human placenta matrix.

Utilizing natural scaffold production derived from extracellular matrix components presents a promising strategy for advancing in vitro spermatogenesis. In this study, we employed decellularized human placental tissue as a scaffold, upon which neonatal mouse spermatogonial cells (SCs) were cultured three-dimensional (3D) configuration. To assess cellular proliferation, we examined the expression of key markers (Id4 and Gfrα1) at both 1 and 14 days into the culture. Our quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed a notable increase in Gfrα1 gene expression, with the 3D culture group exhibiting the highest levels. Furthermore, the relative frequency of Gfrα1-positive cells significantly rose from 38.1% in isolated SCs to 46.13% and 76.93% in the two-dimensional (2D) and 3D culture systems, respectively. Moving forward to days 14 and 35 of the culture period, we evaluated the expression of differentiating markers (Sycp3, acrosin, and Protamine 1). Sycp3 and Prm1 gene expression levels were upregulated in both 2D and 3D cultures, with the 3D group displaying the highest expression. Additionally, acrosin gene expression increased notably within the 3D culture. Notably, at the 35-day mark, the percentage of Prm1-positive cells in the 3D group (36.4%) significantly surpassed that in the 2D group (10.96%). This study suggests that the utilization of placental scaffolds holds significant promise as a bio-scaffold for enhancing mouse in vitro spermatogenesis.

Enhanced anti-tumor activity of transferrin/folate dual-targeting magnetic nanoparticles using chemo-thermo therapy on retinoblastoma cancer cells Y79.

Malignant neoplasms are one of the main causes of death, especially in children, on a global scale, despite strenuous efforts made at advancing both diagnostic and therapeutic modalities. In this regard, a new nanocarrier Vincristine (VCR)-loaded Pluronic f127 polymer-coated magnetic nanoparticles conjugated with folic acid and transferrin (PMNP-VCR-FA-TF) were synthesized and characterized by various methods. The cytotoxicity of these nanoparticles was evaluated in vitro and ex vivo conditions. The in vitro anti-tumor effect of the nanoparticles was evaluated by colony formation assay (CFA) and reactive oxygen species (ROS) in Y79 cell line. The results showed that nanoparticles with two ligands conferred greater toxicity toward Y79 cancer cells than ARPE19 normal cells. Under an alternating magnetic field (AMF), these nanoparticles demonstrated a high specific absorption rate. The CFA and ROS results indicated that the AMF in combination with PMNP-VCR-FA-TF conferred the highest cytotoxicity toward Y79 cells compared with other groups (P < 0.05). PMNP-VCR-FA-TF could play an important role in converting externally applied radiofrequency energy into heat in cancer cells. The present study confirmed that dual targeting chemo-hyperthermia using PMNP-VCR-FA-TF was significantly more effective than hyperthermia or chemotherapy alone, providing a promising platform for precision drug delivery as an essential component in the chemotherapy of retinoblastoma.

Anti-Leishmanial Effects of a Novel Biocompatible Non-Invasive Nanofibers Containing Royal Jelly and Propolis against Iranian Strain of Leishmania major (MRHO/IR/75/ER): an In-Vitro Study.

Background: Current medications especially the pentavalent antimonial compounds have been used as the first line treatment of cutaneous leishmaniasis (CL), but they have limitations due to serious side effects such as drug resistance, cardio and nephrotoxicity, and high costs. Hence, the demand to find more usable drugs is evident. Synthesis and development of natural, effective, biocompatible, and harmless compounds against Leishmania major is the principal priority of this study. Methods: By electrospinning method, a new type of nanofiber were synthesized from royal jelly and propolis with different ratios. Nanofibers were characterized by Scanning Electron Microscope (SEM), Transmission Electron Microscopy (TEM), Thermogravimetric Analysis (TGA), Contact angle, and Fourier-transform infrared spectroscopy (FTIR). The Half-maximal inhibitory concentration (IC50), Half-maximal effective concentration (EC50) and the 50% cytotoxic concentration (CC50) for different concentrations of nanofibers were determined using quantitative calorimetric methods. Inductively coupled plasma-optical emission spectrometry (ICP-OES) and flow cytometry were performed as complementary tests. Results: The results showed that the proposed formulas provide a new achievement that, despite the significant killing activity on L. major, has negligible cytotoxicity on the host cells. Royal jelly nanofibers have significantly shown the best 72 hours results (IC50= 35 µg/ml and EC50=16.4 µg/ml) and the least cytotoxicity. Conclusion: This study presents a great challenge to introduce a new low-cost treatment method for CL, accelerate wound healing, and reduce scarring with minimal side effects and biocompatible materials. Royal jelly and propolis nanofibers significantly inhibit the growth of L. major in-vitro.

Antifungal activity of green-synthesized curcumin-coated silver nanoparticles alone and in combination with fluconazole and itraconazole against Candida and Aspergillus species.

Background and Purpose: Regarding the wide-spectrum antimicrobial effects of curcumin and silver, this study aimed to evaluate the antifungal activity of green-synthesized curcumin-coated silver nanoparticles (Cur-Ag NPs) against a set of Candida and Aspergillus species. Materials and Methods: Cur-Ag NPs were synthesized by mixing 200 µL of curcumin solution (40 mM) and 15 mL of deionized water. The mixture was stirred for 3-5 min, followed by the addition of 2.5 mL of silver nitrate solution (2.5 mM). The resulting solution was incubated for 3 days. Antifungal susceptibility of 30 fungal isolates of Aspergillus and Candida to fluconazole and itraconazole, as well as the activity of Cur-Ag NPs against the isolates, were determined, both alone and in combination, using broth microdilution according to the Clinical and Laboratory Standards Institute guidelines. Results: Cur-Ag NPs demonstrated promising antifungal activity, particularly against Candida species. The geometric mean value of the minimum inhibitory concentration of Cur-Ag NPs was significantly lower than that of fluconazole for all the studied fungi. Similarly, it was lower than those of itraconazole in C. albicans and A. fumigatus. The minimum fungicidal concentrations of Cur-Ag NPs were markedly better than those of fluconazole but still inferior to those of itraconazole. Conclusion: Cur-Ag NPs demonstrated indisputable antifungal activity and great potential that can be harnessed to combat fungal infections, particularly those caused by azole-resistant strains of Aspergillus and Candida.

Metabotropic glutamate receptor: A new possible therapeutic target for cochlear synaptopathy.

Objectives: Cochlear synaptopathy is a common cause of auditory disorders in which glutamate over-activation occurs. Modulating glutamatergic pathways has been proposed to down-regulate post-synaptic excitation. Materials and Methods: 12-guinea pigs as sham and test groups were exposed to a 4-kHz noise at 104 dB SPL, for 2 hr. Pre-exposure intra-tympanic injection with LY354740 and normal saline 9% was applied in the test and sham groups. The amplitude growth of ABR-wave-I and wave-III latency shift with noise were considered in pre- and post-exposure times. The synapses were observed by transmission electron-microscopy. Results: ABR thresholds recovered 1-week post-exposure in both groups. The reduction of wave-I amplitude at 4, 6, and 8 kHz were statistically different between pre- and 1- day post-exposure and recovered mostly in the sham group. The amount of latency shift in masked ABR was different between pre- and all post-exposure, and the response could not be detected at higher than 50 dB SL noise. However, the response detectability increased to 60 dB SL noise, and the significance of differences between pre- and post-exposure persisted only at the high level of noise in the test group. In electron-microscopy of sham samples, the size of the ribbon was larger, spherical with an irregularity, and hollow. The post-synaptic density was thicker and missed its flat orientation. Conclusion: The higher slope of the ABR-wave I amplitude, the more tolerance of noise in masked ABR, concomitant with the histological finding that revealed less synaptic damage, confirmed the therapeutic effect of LY354740 in cochlear synaptopathy.

Effect of PLGA Nanoparticle-Mediated Delivery of miRNA 503 on The Apoptosis of Ovarian Endometriosis Cells.

OBJECTIVE: One of the challenges in gene therapy is the transfer of the gene to the target cell. MicroRNAs (miRNAs) regulate gene expression after transcription by binding directly to the messenger and play a vital role in cell behaviors and the pathogenesis of some diseases. This study was aimed at developing poly (lactic-co-glycolic acid) (PLGA)- based nanoparticles (NPs) for gene delivery to endometriotic cyst stromal cells (ECSCs). MATERIALS AND METHODS: In this experimental study, endometriosis cells were isolated from women with severe endometriosis (DIE) and digested by the enzymatic method (40 µg/ml DNAase I and 300 µg/ml collagenase type 3). PLGA-based NPs were synthesized and characterized. The size of sole PLGA NPs and PLGA/miRNA were 60 ± 4 nm and 70 ± 5.1 nm respectively. Poly lactic-co-glycolic-based NPs were used as vector carriers for miRNA 503 transfection in endometriosis cells. The cells were divided into the five groups of control and four doses (25, 50, 75, and 100 µm) of miRNA 503/PLGA at 12, 24, 48, and 72 hours. Viability and apoptosis were evaluated by the MTT assay and Annexin Kits. Data were analyzed by one-way analysis of variance. RESULTS: The results show that the size of PLGA/miRNA complex with dynamic light scattering (DLS) was 70 ± 5.1 nm and zeta potential values of the PLGA/PEI/miRNA complexes were 27.9 mV. Based on the MTT assay results, the optimal dose of miRNA 503/PLGA was 75 µm, at which the viability of ECSCs was 52.6% ± 1.2 (P≤0.001), and the optimal time was 48 hours. The apoptotic rates of ECSCs treated with PLGA/miRNA503 (34.75 ± 4.9%) were significantly higher than those of ECSCs treated with PLGA alone (3.35 ± 2.58%, P≤0.01). CONCLUSION: Cell death increased with increasing the concentration of miRNA; thus, it can be suggested as a treatment for endometriosis.

Anti-biofilm Activity of a Lytic Phage Against Vancomycin-Resistant Enterococcus faecalis.

Background & Objective: This study aims to isolate a lytic bacteriophage against planktonic Enterococcus faecalis V583 culture and evaluate its ability to disrupt and inhibit biofilm. Methods: An anti-E. faecalis phage was isolated from sewage and visualized by electron microscopy, the vB_EfsS_V583 (V583) host range was determined by spot test on 13 E. faecalis clinical strains. Inhibition and degradation experiments were designed to investigate the effect of phage on biofilm. In the inhibition and degradation assay, biofilms were formed in the presence and absence of phage, respectively. Finally, crystal violet method tested the effect of phage on biofilm. Results: Phage V583 belongs to the Siphoviridae family and can infect all E. faecalis strains. Antibacterial activity has been shown to degrade and inhibit biofilm produced by V583. The study results showed that phage v583 is more efficient in biofilm inhibition than biofilm degradation. In both assays, phage-treated wells' absorption is less than untreated wells. These results were confirmed by Colony-forming unit reduction in the treated biofilm. Conclusion: The anti-biofilm activity showed that phage therapy using phage V583 might be an alternative tool to remove E. faecalis biofilms.

Radio-sensitivity enhancement in HT29 cells through magnetic hyperthermia in combination with targeted nano-carrier of 5-Flourouracil.

Normal tissue complication and development of radioresistance in cancer cells are known as the main challenges of ionizing radiation treatment. In the current study, we intended to induce selective radiosensitization in HT29 cancer cells by developing folic acid modified magnetic triblock copolymer nanoparticles as carrier of 5-Flourouracil (5-FU) which was further used in combination with hyperthermia. The aforementioned nanoparticles were synthesized and characterized by differential scanning calorimetric analysis (DSC), UV-visible spectroscopy, dynamic light scattering (DLS), zeta sizer, and transmission electron microscopy (TEM). These nanoparticles were also assessed to determine drug loading capacity (DLC %) and drug release profile. The cytotoxicity of nanoparticles was evaluated on two different cell lines: HUVEC and HT29. Furthermore, radiosensitivity induction of the nanoparticles with and without exposure of alternative magnetic field was investigated. MTT-based cytotoxicity assay demonstrated that the therapeutic ratio was enhanced in response to using 5-FU-loaded nanoparticles as compared to 5-FU. Various characterizations including gene expression study, measurement of reactive oxygen species (ROS) generation, Annexin V/PI staining, and clonogenic assay revealed that ionizing radiation in combination with hyperthermia in the presence of the synthesized nanoparticles led to maximal anti-cancer effects as compared to other single (P < 0.001) and combined treatments (P < 0.01). Our results suggested that combined treatment based on using folic acid modified magnetic copolymer nanoparticle as carrier of 5-FU accompanied with hyperthermia could be proposed as an efficient approach to enhance radiation effects in cancer cells.

Anti-inflammatory and anti-apoptotic effects of hyperbaric oxygen preconditioning in a rat model of cisplatin-induced peripheral neuropathy.

OBJECTIVES: Cisplatin-induced peripheral neuropathy is a debilitating side effect in patients receiving this drug. Recent studies suggest hyperbaric oxygen (HBO) therapy as a new treatment approach for models of neural injury. The aim of the current study was to determine the protective effects of HBO preconditioning against peripheral neuropathy induced by Cisplatin (CDDP). MATERIALS AND METHODS: The present study was conducted on 4 groups of rats: Sham group; HBO group (60 min/d); Control group (CDDP 2 mg/kg/d); Precondition group (HBO+CDDP). Mechanical threshold testing was weekly carried out using von Frey filament. Sciatic nerve and associated ganglia were removed five weeks after the first CDDP injection for biochemical evaluation of malondialdehyde (MDA) content and myeloperoxidase (MPO) activity, immunohistochemistry of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), TNF-α, caspase-3 and iNOS, and transmission electron microscopic (TEM) assessments. RESULTS: MDA levels and MPO activities were significantly decreased in preconditioned rats. Attenuated TUNEL reaction along with attenuated caspase-3, TNF-α, and iNOS expression could be significantly detected in preconditioned rats. Also, HBO preconditioning improved the nociceptive threshold. CONCLUSION: The results suggest that HBO preconditioning can attenuate peripheral neuropathy caused by cisplatin in rats.

DNA methyltransferase inhibitor 5-azacytidine in high dose promotes ultrastructural maturation of cardiomyocyte.

BACKGROUND: The adult human heart muscle cells, cardiomyocytes are not capable of regenerate after injury. Stem cells are a powerful means for future regenerative medicine because of their capacity for self-renewal and multipotency. Several studies have reported the cardiogenic potential in human adipose tissue-derived stem cells (ADSCs) differentiation, but there is still no efficient protocol for the induction of cardiac differentiation by 5-azacytidine (5-Aza). The present study involves characterization and mainly, the ultrastructure of ADSCs derived cardiomyocyte-like cells. METHODS: The cultured ADSCs were treated with 50 µM 5-Aza for 24 hours, followed by a 10-week extension. At different time points, cardiomyocyte-like cells were assessed by qRT-PCR and were evaluated by transmission electron microscopy at 10th week. RESULTS: The expression of cardiac-specific markers entailing cardiac troponin I (cTnI), connexin 43, myosin light chain-2v (Mlc-2v), increased over 10 weeks and the highest expression was at 10th week. The expression of the ß-myosin heavy chain (ß-MHC) increased significantly over 5 weeks and then decreased. At the ultrastructural level myofibrils, transverse tubules (T-tubules), sarcoplasmic reticular membrane, and intercalated discs were present. CONCLUSIONS: These data suggest that treatment with 5-Aza in high dose could promote differentiation of ADSCs into cardiomyocyte-like cells. These differentiated cells could be used for regeneration of damaged cardiomyocytes with the 3D scaffold for delivery of the cells.

Ultrastructural and optical characteristics of cancer cells treated by a nanotechnology based chemo-photothermal therapy method.

The current chemotherapy method demonstrates the need for improvement in terms of efficacy and safety. Given the beneficiary effect of heat in combination with chemotherapy, the purpose of this study is to develop a multifunctional nanoplatform by co-incorporating gold nanoparticles (AuNPs) as photothermal agent and cisplatin as anticancer drug into alginate hydrogel (named as ACA) to enable concurrent thermo-chemotherapy. The in vitro cytotoxicity experiment showed that the as-developed nanocomplex was able to induce greater cytotoxicity in KB human nasopharyngeal cancer cells compared to free cisplatin at the same concentration. Moreover, the interaction of ACA and laser irradiation acted synergistically and resulted in higher cell death rate compared to separate application of photothermal therapy and chemotherapy. The micrograph of KB cells also revealed that ACA was able to selectively accumulate into the mitochondria, so that laser irradiation of KB cells pre-treated with ACA resulted in intensive morphological damages such as plasma membrane disruption, chromatin condensation, autophagic vacuoles formation and organelle degeneration. Moreover, the sign and magnitude of optical nonlinear refractive index measured by Z-scan technique was shown to be significantly altered in cells exposed to ACA with and without laser irradiation. Consequently, the nanocomplex developed herein could be a promising platform to combine photothermal therapy and chemotherapy effectively, thereby achieving synergistic therapeutic outcome.

In Vitro Cytotoxicity of Folate-Silica-Gold Nanorods on Mouse Acute Lymphoblastic Leukemia and Spermatogonial Cells.

OBJECTIVE: The purpose of this study was to evaluate in vitro cytotoxicity of gold nanorods (GNRs) on the viability of spermatogonial cells (SSCs) and mouse acute lymphoblastic leukemia cells (EL4s). MATERIALS AND METHODS: In this experimental study, SSCs were isolated from the neonate mice, following enzymatic digestion and differential plating. GNRs were synthesized, then modified by silica and finally conjugated with folic acid to form F-Si-GNRs. Different doses of F-Si-GNRs (25, 50, 75, 100, 125 and 140 µM) were used on SSCs and EL4s. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation assay was performed to examine the GNRs toxicity. Flow cytometry was used to confirm the identity of the EL4s and SSCs. Also, the identity and functionality of SSCs were determined by the expression of specific spermatogonial genes and transplantation into recipient testes. Apoptosis was determined by flow cytometry using an annexin V/propidium iodide (PI) kit. RESULTS: Flow cytometry showed that SSCs and EL4s were positive for Plzf and H-2kb, respectively. The viability percentage of SSCs and EL4s that were treated with 25, 50, 75, 100, 125 and 140 µM of F-Si-GNRs was 65.33 ± 3.51%, 60 ± 3.6%, 51.33 ± 3.51%, 49 ± 3%, 30.66 ± 2.08% and 16.33 ± 2.51% for SSCs and 57.66 ± 0.57%, 54.66 ± 1.5%, 39.66 ± 1.52%, 12.33 ± 2.51%, 10 ± 1% and 5.66 ± 1.15% for EL4s respectively. The results of the MTT assay indicated that 100 µM is the optimal dose to reach the highest and lowest level of cell death in EL4s and in SSCs, respectively. CONCLUSION: Cell death increased with increasing concentrations of F-Si-GNRs. Following utilization of F-Si-GNRs, there was a significant difference in the extent of apoptosis between cancer cells and SSCs.

In vitro differentiation of neural stem cells into noradrenergic-like cells.

Neural stem cells (NSCs) as a heterogeneous multipotent and self- renewal population are found in different areas in the developing mammalian nervous system, as well as the sub-ventricular zone (SVZ) and the hippocampus of the adult brain. NSCs can give rise to neurons, astrocytes and oligodendrocytes. The aim of this study was to differentiate neural stem cells into noradrenergic-like cells in vitro. Neural stem cells were harvested from SVZ of newborn rat brains. The cells were cultured in DMEM12, B-27 supplemented with 20 ng/ ml (hFGF) and 20 ng/ ml (EGF) for 2 weeks. Neurospheres were differentiated in neurobasal medium, B-27 supplemented with BDNF (50 ng/ ml) and GDNF (30 ng/ ml) for 3 and 5 days. Cell culture techniques and immunocytochemistry were applied to examine neurospheres and tyrosine hydroxylase positive cells. The number of neurites was counted 3 and 5 days after the induction of differentiation. Nestin and Sox2 were expressed in NSCs and neurospheres. NSCs were differentiated into noradrenergic- like cells (NACs). Tyrosine hydroxylase was detected in these cells. The results of NSCs differentiation for 5 days culture had a significant decrease (P≤0.05) in the number of TH positive cells with one or two neurite per cell, and a significant increase (P≤0.05) in the number of TH positive cells with three, four or more neurites per cell, compared with 3 days culture. Based on these results, NSCs have the ability to differentiate into noradrenergic cells in the presence of BDNF and GDNF growth factors.

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.

Comparison of transplantation of bone marrow stromal cells (BMSC) and stem cell mobilization by granulocyte colony stimulating factor after traumatic brain injury in rat.

BACKGROUND: Recent clinical studies of treating traumatic brain injury (TBI) with autologous adult stem cells led us to compare effect of intravenous injection of bone marrow mesenchymal stem cells (BMSC) and bone marrow hematopoietic stem cell mobilization, induced by granulocyte colony stimulating factor (G-CSF), in rats with a cortical compact device. METHODS: Forty adult male Wistar rats were injured with controlled cortical impact device and divided randomly into four groups. The treatment groups were injected with 2 × 106 intravenous bone marrow stromal stem cell (n = 10) and also with subcutaneous G-CSF (n = 10) and sham-operation group (n = 10) received PBS and "bromodeoxyuridine (Brdu)" alone, i.p. All injections were performed 1 day after injury into the tail veins of rats. All cells were labeled with Brdu before injection into the tail veins of rats. Functional neurological evaluation of animals was performed before and after injury using modified neurological severity scores (mNSS). Animals were sacrificed 42 days after TBI and brain sections were stained by Brdu immunohistochemistry. RESULTS: Statistically, significant improvement in functional outcome was observed in treatment groups compared with control group (P<0.01). mNSS showed no significant difference between the BMSC and G-CSF-treated groups during the study period (end of the trial). Histological analyses showed that Brdu-labeled (MSC) were present in the lesion boundary zone at 42nd day in all injected animals. CONCLUSION: In our study, we found that administration of a bone marrow-stimulating factor (G-CSF) and BMSC in a TBI model provides functional benefits.