Trehalose and carnosic acid induced LC3I, LC3II ratio, P62 down-regulation and cleaved caspase 3 expression in neural stem cells.

BACKGROUND: Using neural stem cells (NSCs) in cell therapy and regenerative medicine is a growing knowledge. In this study, the protective role of carnosic acid and trehalose against H2O2-induced oxidative stress in autophagy induction and apoptosis inhibition in NSCs was investigated. MATERIAL AND METHODS: The bone marrow stromal cells (BMSCs) were isolated from the femur of the rat and differentiated into NSCs using basic fibroblast and epidermal growth factors (bFGF and EGF), and B27 serum free media. To evaluate the autophagy, the P62 protein was assessed by immunocytochemistry and LC3II / LC3I ratio by Western blotting. Further, we used 3-Methyladenine (3-MA), a widely used autophagy inhibitor to study whether combined treatment of 3-MA with carnosic acid and trehalose modulates autophagy in NSCs. For studying apoptosis, the cleaved caspase-3 protein was evaluated. Carnosic acid and trehalose increased the survival of the NSCs. RESULTS: The H2O2 decreased the autophagy and induced apoptosis with increasing time during 24 hours, however, a pre-treatment with 2 µM carnosic acid and trehalose 3 % induced the autophagy proteins (while increasing the LC3II / LC3I ratio and decreasing the P62) and decreased the apoptosis (while decreasing the expression of the cleaved caspase-3). The results showed that the carnosic acid and trehalose increased the survival of NSCs against the oxidative stress caused by H2O2, decreased apoptosis, and induced autophagy. CONCLUSION: Due to the carnosic acid and trehalose unique properties and its low toxicity, it can be used as an agent in cellular transplantation for reducing oxidative stress and inducing autophagy (Fig. 4, Ref. 37).

Optimization of nanofibrous silk fibroin scaffold as a delivery system for bone marrow adherent cells: in vitro and in vivo studies.

Electrospun silk fibroin nanofibrous scaffolds (ESFNSs) were successfully prepared by electrospinning of various Bombyx mori silk fibroin concentrations (10, 12, and 14% in formic acid). After characterizing the purified silk fibroin, the morphology, porosity, fibers' diameter, and uniformity of the prepared scaffolds were examined in detail. In addition, biological responses such as effects on bone marrow cell viability, cytotoxicity, and cell adhesion were evaluated in vitro. Biocompatibility and bioactivity properties of the ESFNSs were evaluated in vitro and in vivo by cell culturing and subcutaneous implantation in rat models for 7 and 28 days, respectively. According to the obtained results, no beaded fibers were seen in any of the prepared scaffolds, whereas ESFNS-10% provided more uniformity and porosity with nanoscaled fibers (90 ± 0.021 nm). Furthermore, the scaffolds also showed good cell adhesion and spreading (68.7 ± 11.8 and 7.6 ± 3.3 total length and width, respectively) with no detectable effect on cell viability and cytotoxicity. The in vivo biocompatibility evaluation indicated that the scaffolds did not stimulate detectable cellular inflammatory response (lymphocytes) and increased the total cell number (cellularity) in the implantation area. Furthermore, the results suggest the potential use of the prepared ESFNS-10% bone marrow cell constructs in direct implantation for tissue engineering applications.

Combined treatment of high-intensity interval training with neural stem cell generation on contusive model of spinal cord injury in rats.

INTRODUCTION: Spinal cord injury (SCI) leads to inflammation, axonal degeneration, and gliosis. A combined treatment of exercise and neural stem cells (NSC) has been proposed to improve neural repair. This study evaluated a combined treatment of high-intensity interval training (HIIT) with NSC generation from adipose-derived stem cells (ADSCs) on a contusive model of SCI in rats. MATERIALS AND METHODS: In vitro, rat ADSCs were isolated from the perinephric regions of Sprague-Dawley rats using enzymatic digestion. The ADSCs were transdifferentiated into neurospheres using B27, EGF, and bFGF. After production of NSC, they were labeled using green fluorescent protein (GFP). For the in vivo study, rats were divided into eight groups: control group, sham operation group, sham operation + HIIT group, sham operation + NSC group, SCI group, SCI + HIIT group, SCI + NSC group, and SCI/HIIT/NSC group. Laminectomy was carried out at the T12 level using the impactor system. HIIT was performed three times per week. To assess behavioral function, the Basso-Beattie-Bresnahan (BBB) locomotor test and H-reflex was carried out once a week for 12 weeks. We examined glial fibrillary acidic protein (GFAP), S100ß, and NF200 expression. RESULTS: NSC transplantation, HIIT and combined therapy with NSC transplantation, and the HIIT protocol improved locomotor function with decreased maximum H to maximum M reflexes (H/M ratio) and increased the Basso-Beattie-Bresnahan score. CONCLUSION: Combined therapy in contused rats using the HIIT protocol and neurosphere-derived NSC transplantation improves functional and histological outcomes.

Quercetin as a possible complementary therapy in multiple sclerosis: Anti-oxidative, anti-inflammatory and remyelination potential properties.

Multiple sclerosis (MS) is a complex autoimmune disorder of the central nervous system (CNS) which causes various symptoms such as fatigue, dyscoordination weakness and visual weakness. The intricacy of the immune system and obscure etiology are the main reasons for the lack of a definite treatment for MS. Oxidative stress is one of the most important key factors in MS pathogenesis. It can enhance inflammation, neurodegeneration and autoimmune-mediated processes, which can lead to excessive demyelination and axonal disruption. Recently, promising effects of Quercetin as a non-pharmacological anti-oxidant therapy have been reported in preclinical studies of MS disease. In this review, we provide a compendium of preclinical and clinical studies that have investigated the effects of Quercetin on MS disease to evaluate its potential utility as a complementary therapy in MS. Quercetin treatment in MS disease not only protects the CNS against oxidative stress and neuroinflammation, but it also declines the demyelination process and promotes remyelination potential. The present study clarifies the reported knowledge on the beneficial effects of Quercetin against MS, with future implication as a neuroprotective complementary therapy.

Potential role of human chorionic gonadotropin supplementation in spermatogenesis in rats subjected to forced swimming exercise.

AIM: The aim of this study was to evaluate the supportive effect of human chorionic gonadotropin (hCG) on the quality of spermatogenesis, including count, motility, morphology, viability and apoptosis of sperm following forced swimming exercise. MATERIALS AND METHODS: Twenty-four adult male Sprague-Dawley rats were used in this study. All rats were divided into four groups: control group; swimming exercise group (S); hCG administration group and swimming (SG) with hCG administration group (G). The experimental group was trained to force swimming stress for 10 min for 6 days. Then the sperm quality parameters were measured after dissection and epididymis removal. Spermatogenesis and germ cell apoptosis were evaluated by using Miller & Johnsen's score and TUNEL staining respectively. RESULTS: Results showed the count (control: 113±3.1, S: 74±1.9, G: 111±3, and SG: 103±2.4), motility (control:  93±2, S: 67±2.8,G: 90±2.7, and SG: 78±1), morphology (control: 89±3%, S: 47±2.4%, G: 90±3.1%, and SG: 67±1.1%), and viability of sperm (control: 91±2.9, S: 50±2, G: 91±1.9, and SG: 70±1.3) in swimming exercised-hCG administered group, significantly enhanced by hCG treatment compared to the swimming exercise group (p≤0.01). Also the number of apoptotic germ cells significantly decreased in swimming exercised-hCG administered group compared to the swimming exercised group. CONCLUSIONS: These results suggest that administration of hCG can protect the testes against the detrimental effect of forced swimming exercise in adult male rats.

Differentiation of human dental pulp stem cells into functional motor neuron: In vitro and ex vivo study.

There are several therapeutic options for spinal cord injury (SCI), among these strategies stem cell therapy is a potential treatment. The stem cells based therapies have been investigating in acute phase of clinical trials for promoting spinal repair in humans through replacement of functional neuronal and glial cells. The aim of this study was to evaluate the differentiation of Human Dental Pulp Stem Cells (hDPSCs) into functional motor neuron like cells (MNLCs) and promote neuroregeneration by stimulating local neurogenesis in the adult spinal cord slice culture. The immunocytochemistry analysis demonstrated that hDPSCs were positive for mesenchymal stem cell markers (CD73, CD90 and CD105) and negative for the hematopoietic markers (CD34 and CD45). hDPSCs were induced to neurospheres (via implementing B27, EGF, and bFGF) and then neural stem cells (NSC). The NSC differentiated into MNLCs in two steps: first by Shh and RA and ; then with GDNF and BDNF administration. The NS and the NSC were assessed for Oct4, nestin, Nanog, Sox2 expression while the MNLCs were evaluated by ISLET1, Olig2, and HB9 genes. Our results showed that hDPSC can be differentiated into motor neuron phenotype with expression of the motor neuron genes. The functionality of MNLCs was demonstrated by FM1-43, intracellular calcium ion shift and co- culture with C2C12. We co-cultivated hDPSCs with adult rat spinal slices in vitro. Immunostaining and hoechst assay showed that hDPSCs were able to migrate, proliferate and integrate in both the anterolateral zone and the edges of the spinal slices.

Promoting motor functions in a spinal cord injury model of rats using transplantation of differentiated human olfactory stem cells: A step towards future therapy.

Human olfactory ecto-mesenchymal stem cells (hOE-MSCs) derived from the human olfactory mucosa (OM) can be easily isolated and expanded in cultures while their immense plasticity is maintained. To mitigate ethical concerns, the hOE-MSCs can be also transplanted across allogeneic barriers, making them desirable cells for clinical applications. The main purpose of this study was to evaluate the effects of administering the hOE-MSCs on a spinal cord injury (SCI) model of rats. These cells were accordingly isolated and cultured, and then treated in the neurobasal medium containing serum-free Dulbecco's Modified Essential Medium (DMEM) and Ham's F-12 Medium (DMEM/F12) with 2% B27 for two days. Afterwards, the pre-induced cells were incubated in N2B27 with basic fibroblast growth factor (bFGF), fibroblast growth factor 8b (FGF8b), sonic hedgehog (SHH), and ascorbic acid (vitamin C) for six days. The efficacy of the induced cells was additionally evaluated using immunocytochemistry (ICC) and real-time polymerase chain reaction (RT-PCR). The differentiated cells were similarly transplanted into the SC contusions. Functional recovery was further conducted on a weekly basis for eight consecutive weeks. Moreover, cell integration was assessed via conventional histology and ICC, whose results revealed the expression of choline acetyltransferase (ChAT) marker at the induction stage. According to the RT-PCR findings, the highest expression level of insulin gene-enhancer protein (islet-1), oligodendrocyte transcription factor (Olig2), and homeobox protein HB9 was observed at the induction stage. The number of engraftment cells also rose (approximately by 2.5 % ± 0.1) in the motor neuron-like cells derived from the hOE-MSCs-grafted group compared with the OE-MSCs-grafted one. The functional analysis correspondingly revealed that locomotor and sensory scores considerably improved in the rats in the treatment group. These findings suggested that motor neuron-like cells derived from the hOE-MSCs could be utilized as an alternative cell-based therapeutic strategy for SCI.

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.

Neurogenic differentiation of human dental pulp stem cells by optogenetics stimulation.

INTRODUCTION: Human dental pulp stem cells (hDPSCs), a promising source for autologous transplantation in regenerative medicine, have been shown to be able to differentiate into neural precursors. Optogenetics is considered as an advanced biological technique in neuroscience which is able to control the activity of genetically modified stem cells by light. The purpose of this study is to investigate the neurogenic differentiation of hDPSCs following optogenetic stimulation. METHODS: The hDPSCs were isolated by mechanical enzymatic digestion from an impacted third molar and cultured in DMEM/F12. The cells were infected with lentiviruses carrying CaMKIIa-hChR2 (H134R). Opsin-expressing hDPSCs were plated at the density of 5 × 104 cells/well in 6-well plates and optical stimulation was conducted with blue light (470 nm) pulsing at 15 Hz, 90 % Duty Cycle and 10 mW power for 10 s every 90 minutes, 6 times a day for 5 days. Two control groups including non-opsin-expressing hDPSCs and opsin-expressing hDPSCs with no optical stimulation were also included in the study. A day after last light stimulation, the viability of cells was analyzed by the MTT assay and the morphological changes were examined by phase contrast microscopy. The expression of Nestin, Microtubule-Associated protein 2 (MAP2) and Doublecortin (DCX) were examined by immunocytochemistry. RESULTS: Human DPSCs expressed the reporter gene, mCherry, 72 hours after lentiviral infection. The result of MTT assay revealed a significant more viability in optical stimulated opsin-expressing hDPSCs as compared with two control groups. Moreover, optical stimulation increased the expression of Nestin, Doublecortin and MAP2 along with morphological changes from spindle shape to neuron-like shape. CONCLUSION: Optogenetics stimulation through depolarizing the hDPSCs can increase the cells viability and/or proliferation and also promote the differentiation toward neuron-like cells.

Antimicrobial peptides-loaded smart chitosan hydrogel: Release behavior and antibacterial potential against antibiotic resistant clinical isolates.

In this study, we synthesized thermo-responsive chitosan (TCTS) hydrogels, and loaded with different concentrations of antimicrobial peptide (AMP) (0, 4, 8 and 16 µg·ml-1) to fabricate an antibacterial wound dressing against resistant clinical isolates. Physico-chemical properties, release behavior, cytobiocompatibility and antibacterial activity of the AMP-TCTS hydrogels against standard strain and resistant Acinetobacter baumannii were fully determined in vitro. The TCTS-40% ß-glycerolphosphate hydrogels showed a gelation time of 15 min at 37 °C. 80% weight loss at day 35 with no changes in pH value was observed. AMP-TCTS hydrogels showed a burst release of AMP (around 40%) at day 1, and a controlled release up to day 7. A dramatic water uptake was observed at first 4 h, and then continued for 10 h in a steady manner. All the AMP-TCTS hydrogels showed excellent cytobiocompatibility for human fibroblasts. The TCTS showed no antibacterial activity against both standard strain and clinical isolates. All the AMP-TCTS hydrogels had strong antibacterial activity against standard strains, but only 16 µg·ml-1 showed antibacterial behavior against resistant A. baumannii. Our results strongly suggest the 16 µg·ml-1 AMP-TCTS hydrogel as an excellent antibacterial wound dressing against resistant A. baumannii, and now promises to proceed with pre-clinical investigations.

Silk nanofibrous electrospun scaffold enhances differentiation of embryonic stem like cells derived from testis in to mature neuron.

The scaffolds accompanied with stem cells have great potential for applications in neural tissue engineering. Fabrication of nanofibrous scaffold similar to extracellular matrix is one of the applicable methods in neural tissue regeneration. The aim of this study was the fabrication of a silk nanofibrous scaffold as a microenvironment for neural guiding differentiation of embryonic stem like cells (ES Like cells) derived from testis toward neuron-like cells. ES Like derived from culturing of testicular cells in vitro, were seeded on silk scaffolds and induced to neuronal phenotype using 4-/4± RA technique following culturing the cells in the neurobasal medium supplemented with 20 ng/mL bFGF,10 ng/mL EGF, B27, and N2 for 8-12 days. The neural differentiation was confirmed via the evaluation of specific neural markers; Nestin, NF68, MAP2 and ß tubulin using immunocytochemistry and real-time polymerase chain reaction. Our results showed that silk scaffold support the attachment and proliferation of ES Like cells. The expression of Nestin, NF68, Map2, and ß tubulin markers were higher in cells grown on silk scaffold in compare to monolayer group. This study suggests electrospun silk nanofibrous scaffold as an appropriate substrate for neural induction of stem cells that is applicable for repairmen of damaged neural tissues. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2662-2669, 2018.

A Review of Herbal Therapy in Multiple Sclerosis.

Multiple sclerosis is a complex autoimmune disorder which characterized by demyelination and axonal loss in the central nervous system (CNS). Several evidences indicate that some new drugs and stem cell therapy have opened a new horizon for multiple sclerosis treatment, but current therapies are partially effective or not safe in the long term. Recently, herbal therapies represent a promising therapeutic approach for multiple sclerosis disease. Here, we consider the potential benefits of some herbal compounds on different aspects of multiple sclerosis disease. The medicinal plants and their derivatives; Ginkgo biloba, Zingiber officinale, Curcuma longa, Hypericum perforatum, Valeriana officinalis, Vaccinium macrocarpon, Nigella sativa,Piper methysticum, Crocus sativus, Panax ginseng, Boswellia papyrifera, Vitis vinifera, Gastrodia elata, Camellia sinensis, Oenothera biennis, MS14 and Cannabis sativa have been informed to have several therapeutic effects in MS patients.

Repair of Critical-Sized Rat Calvarial Defects With Three-Dimensional Hydroxyapatite-Gelatin Scaffolds and Bone Marrow Stromal Stem Cells.

INTRODUCTION: The repair of critical-sized defects (CSDs) are one of the most challenging orthopedic problems and the attempts for development of an ideal scaffold for treatment of large bone defect are ongoing. AIM: The aim of this study was the effectiveness of hydroxyapatite-gelatin seeded with bone marrow stromal cells construct for healing of critical-sized bone defect in vivo. MATERIAL AND METHODS: In this experimental study, the bone marrow stromal cells (BMSCs) were isolated by flushing method. For in vitro study, the cells were seeded on the scaffold and the cell viability as well as cytotoxicity were tested by MTT and LDH specific activity. The scaffold-cell construct was implanted into the critical-sized bone defect created in calvaria of Wistar male rats.15 rats were randomly divided into 3 groups (n=5), group 1 (control group): Injury without transplantation, group 2: implanted with hydroxyapatite-gelatin scaffold, group 3: hydroxyapatite-gelatin scaffold seeded with BMSCs. At different days post-implantation, the implanted site was collected and the bone healing was evaluated through H&E and Masson's Trichrome staining. ANOVA and paired t-test were used for data comparison and P<0.05 was considered significant. RESULTS: The results of MTT showed that the scaffold has no toxic effects on stromal cells. The first signs of ossification in hydroxyapatite-gelatin with BMSCs cells group appeared in the first week. However, in the fourth week, ossification was completed and the scaffold remaining was found as embedded islands in the spongy bone tissue. The greatest number of lymphocytes in the experimental group was observed after one week of planting scaffold. CONCLUSION: Hydroxyapatite-gelatin scaffold coated with BMSCs cells has a potential role in the healing process of bone and would be a possible new therapeutic strategy to repair extensive bone lesions.

Differentiation of Spermatogonia Stem Cells into Functional Mature Neurons Characterized with Differential Gene Expression.

Transplantation of embryonic stem cells (ESCs) is a promising therapeutic approach for the treatment of neurodegenerative diseases. However, ESCs are not usable clinically due to immunological and ethical limitations. The identification of an alternative safe cell source opens novel options via autologous transplantation in neuro-regeneration circumventing these problems. Here, we examined the neurogenic capacity of embryonic stem-like cells (ES-like cells) derived from the testis using neural growth factor inducers and utilized them to generate functional mature neurons. The neuronal differentiation of ES-like cells is induced in three stages. Stage 1 is related to embryoid body (EB) formation. To induce neuroprogenitor cells, EBs were cultured in the presence of retinoic acid, N2 supplement and fibroblast growth factor followed by culturing in a neurobasal medium containing B27, N2 supplements for additional 10 days, to allow the maturation and development of neuronal progenitor cells. The neurogenic differentiation was confirmed by immunostaining for markers of mature neurons. The differentiated neurons were positive for Tuj1 and Tau1. Real-time PCR dates indicated the expression of Nestin and Neuro D (neuroprogenitor markers) in induced cells at the second stage of the differentiation protocol. The differentiated mature neurons exhibited the specific neuron markers Map2 and ß-tubulin. The functional maturity of neurons was confirmed by an electrophysiological analysis of passive and active neural membrane properties. These findings indicated a differentiation capacity of ES-like cells derived from the testis to functionally mature neurons, which proposes them as a novel cell source for neuroregenerative medicine.

Functional Concentrations of BMP4 on Differentiation of Mouse Embryonic Stem Cells to Primordial Germ Cells.

BACKGROUND: Bone morphogenetic protein 4 (BMP4) has a significant role in primordial germ cells (PGCs) differentiation from mouse embryonic stem cell (mESC). The aim of this study is to determine the best concentration of BMP4 at a time of two days on differentiation PGCs from mESC. MATERIALS AND METHODS: To differentiate PGCs, embryoid bodies (EBs) from mESCs were cultured in concentrations of 0, 5 and 10 ng/ml BMP4 for two days. Germ cell markers Oct4 (Pou5f1), Stella (Dppa3) and Mvh (Ddx4) were analyzed by flow cytometry, immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Flow cytometry data demonstrated most Mvh-positive cells were observed only in the treated groups. Immunocytochemistry of EBs in the treated groups identified cells positive for Mvh. PCR results showed expression of Oct4 in the control group and treated groups. Stella and Mvh were expressed only in the treated groups. CONCLUSION: Low concentrations of BMP4 during two days had an optimal effect on differentiation of PGCs from mESC.