ABSTRACT
Objective: Non-obstructive azoospermia is mostly irreversible. Efforts to cure this type of infertility have led to the application of stem cells in the reproduction field. In the present study, testicular cell-mediated differentiation of male germ-like cells from bone marrow-derived mesenchymal stem cells [BM-MSCs] in an in vitro indirect co-culture system is investigated
Materials and Methods: In this experimental study, mouse BM-MSCs were isolated and cultured up to passage three. Identification of the cells was evaluated using specific surface markers by flow-cytometry technique. Four experimental groups were investigated: control, treatment with retinoic acid [RA], indirect co-culture with testicular cells, and combination of RA and indirect co-culture with testicular cells. Finally, following differentiation, the quantitative expression of germ cell-specific markers including Dazl, Piwil2 and Stra8 were evaluated by real-time polymerase chain reaction [PCR]
Results: Molecular analysis revealed a significant increase in Dazl expression in the indirect co-culture with testicular cells group in comparison to the control group. Quantitative expression level of Piwil2 was not significantly changed in comparison to the control group. Stra8 expression was significantly higher in RA group in comparison to other groups
Conclusion: Indirect co-culture of BM-MSCs in the presence of testicular cells leads to expression of male germ cell-specific gene, Dazl, in the induced cells. Combination of co-culture with testicular cells and RA did not show any positive effect on the specific gene expressions
ABSTRACT
Objective: Ghrelin is a peptide which has a proliferative and antiapoptotic effect in many cells including bone marrow stromal cells [BMSCs]. Homeobox protein B4 [HOXB4] is a transcription factor involved in stem cell regeneration and survival. The aim of the study was to find out the effect of ghrelin on Hoxb4 expression in BMSCs
Materials and Methods: In this experimental study, rat BMSCs were cultivated in Dulbecco's Modified Eagle Medium [DMEM]. Passage three BMSCs were treated with ghrelin 100 micro M for 48 hours. Real-time polymerase chain reaction [PCR] was carried out from the untreated BMSCs [B], BMSCs treated with 125 micro M H2O2 [BH], BMSCs treated with 100 microM ghrelin then 125 microM H2O2[BGH] and BMSCs treated with 100 micro M ghrelin [BG] groups. For immunofluorescence, cells were incubated with an anti-HOXB4 monoclonal antibody. Primary antibodies were visualized using the Fluorescein isothiocyanate [FITC] method. All data are presented as mean +/- SEM and P<0.05 was considered as statistical significant
Results: Hoxb4 expression significantly increased in the BG compared with BH and BGH groups. Furthermore, 100 microM ghrelin, increased the mean of HOXB4 positive immunoreactive cells compared to the BH group
Conclusion: Ghrelin probably enhances proliferation and viability of BMSCs through Hoxb4 upregulation. However, the signaling pathway and other biological outcomes of this effect should be elucidated in different stem cells
ABSTRACT
Background and Aim: Recently, several studies have indicated that the central nervous system has the capacity for endogenous repair. But, the proliferation capacity of endogenous neural stem cells [NSCs] isn't sufficient for the treatment of neurodegenerative diseases. So, it sounds that stimulation of endogenous NSC proliferation is essential for neuroregeneration. The aim of this study was to examine the effects of Sambucus ebulus extract on the proliferation of neonatal rat hippocampus-derived neural stem cells [NSCs] under oxidative stress condition induced by H2O2
Material andmethods: The NSCs were isolated from neonatal rat hippocampus. To confirm neural characteristics of neural stem cells, the expression of neural-specific marker, Nestin was investigated by immunocytochemistry technique. 5×104 cells were cultured in every well of a 96 well plate and H2O2 was added to induce oxidative stress condition. Then NSCs were exposed to 50 microgram Sambucus ebulus extract for 24 hours, at various concentrations [25, 50, 100, 200, 400 and 500 microgram/ml]. The cell proliferation rate was assessed by MTT colorimetry assay before and after treatment with the extract
Results: Immunofluorescent studies showed that neural stem cells expressed specific neural marker; Nestin. The proliferation rate of NSCs increased in the treated groups in comparison to that in the control group. The highest rate of survival was observed when Sambucus ebulus was used at the concentration of 500 microgram/ml. [P<0.05]
ABSTRACT
The brain and spinal cord have a limited capacity for self-repair under damaged conditions. One of the best options to overcome these limitations involves the use of phytochemicals as potential therapeutic agents. In this study, we have aimed to investigate the effects of di-[2-ethylhexyl] phthalate [DEHP] on hippocampus-derived neural stem cells [NSCs] proliferation to search phytochemical candidates for possible treatment of neurological diseases using endogenous capacity. In this experimental study, neonatal rat hippocampus-derived NSCs were cultured and treated with various concentrations of DEHP [0, 100, 200, 400 and 600 micro M] and Cirsium vulgare [C. vulgare] hydroethanolic extract [0, 200, 400, 600, 800 and 1000 micro g/ml] for 48 hours under in vitro conditions. Cell proliferation rates and quantitative Sox2 gene expression were evaluated using MTT assay and real-time reverse transcription polymerase chain reaction [RT-PCR]. We observed the highest average growth rate in the 400 micro M DEHP and 800 micro g/ml C. vulgare extract treated groups. Sox2 expression in the DEHP-treated NSCs significantly increased compared to the control group. Gas chromatography/mass spectrometry [GC/ MS] results demonstrated that the active ingredients that naturally occurred in the C. vulgare hydroethanolic extract were 2-ethyl-1-hexanamine, n-heptacosane, 1-cyclopentanecarboxylic acid, 1-heptadecanamine, 2,6-octadien-1-ol,2,6,10,14,18,22-tetracosahexaene, and DEHP. DEHP profoundly stimulated NSCs proliferation through Sox2 gene overexpression. These results provide and opportunity for further use of the C. vulgure phytochemicals for prevention and/or treatment of neurological diseases via phytochemical mediated-proliferation of endogenous adult NSCs
Subject(s)
Animals, Laboratory , SOXB1 Transcription Factors , Cell Proliferation , Hippocampus/cytology , Neural Stem Cells/drug effectsABSTRACT
Objective: Cerebrospinal fluid [CSF] has a broad range of molecules and neurotrophic factors essential for neurogenesis. Bone marrow mesenchymal stem cells [BMSCs] are multipotent stem cells that can differentiate into the cells with neural-like phenotype under the induction of appropriate growth factors. According to the significant role of retinoic acid [RA] in neurogenesis, this study aims to differentiate BMSCs into neuronlike cells using CSF, RA, and the combination of CSF and RA
Methods: Rat BMSCs were isolated and characterized. The CSF was prepared from the cisterna magna of 19-day-old Wistar rat embryos. The BMSCs were induced by either 5% CSF [CSF group], 10-6 microM RA [RA group], or CSF plus RA [CSR group] for 12 days. Morphology of differentiated cells was examined by inverted microscope and axonal outgrowth measured using Image J software. In addition, the expression of neural-specific markers [Nestin and MAP-2] was examined by immunocytochemistry
Results: We observed specific-neuronal morphology in the differentiated cells. The maximum axon length was seen in the CSR group on the 12th day of induction. Immunocytochemistry results showed that the neural progenitor marker [Nestin] was expressed in all treated groups. However, MAP-2, as a mature neural marker, was only expressed in the CSR group
Conclusion: The findings suggest that CSF accompanied RA lead to differentiation of cells with neuronal and glial phenotypes from BMSCs in vitro
ABSTRACT
The present study was designed to evaluate the secondary microglial activation processes after spinal cord injury [SCI]. A quantitative histological study was performed to determine ED-1 positive cells, glial cell density, and cavitation size in untreated SCI rats at days 1, 2, and 4, and weeks 1, 2, 3, and 4. The results of glial cell quantification along the 4900- Micro m long injured spinal cord showed a significant increase in glial cell density percentage at day 2 as compared to other days. Whereas the highest increase in ED-1 immunoreactive cells [monocyte/phagocyte marker in rats] was observed at day 2 [23.15%] post-injury. Evaluation of cavity percentage showed a significant difference between weeks 3 and 4 post-injury groups. This study provides a new insight into the multiphase immune response to SCI, including cellular inflammation, macrophages/microglia activation, glial cell density, and cavitation. Better understanding of the inflammatory processes associated with acute SCI would permit the development of better therapeutic strategies
Subject(s)
Animals, Laboratory , Microglia , Macrophages , Inflammation , RatsABSTRACT
The primary phase of traumatic spinal cord injury [SCI] starts by a complex local inflammatory reaction such as secretion of pro-inflammatory cytokines from microglia and injured cells that substantially contribute to exacerbating pathogenic events in secondary phase. Valproic acid [VPA] is a histone deacetylase inhibitor. Acetylation of histones is critical to cellular inflammatory and repair processes. In this study, rats were randomly assigned to five experimental groups [laminectomy, untreated, and three VPA-treated groups]. For SCI, severe contusion was used. In treated groups, VPA was administered intraperitoneally at doses of 100, 200 and 400 mg/kg daily three hours after injury for 7 days. To compare locomotor improvement among experimental groups, behavioral assessments were performed by the Basso, Beattie and Bresnahan [BBB] rating scale. The expression of neurotrophins was evaluated by RT-PCR and real-time PCR. VPA administration increased regional brain-derived neurotrophic factor and glial cell-derived neurotrophic factor mRNA levels. Local inflammation and the expression of the lysosomal marker ED1 by activated macrophages/microglial cells were reduced by VPA and immunoreactivity of acetylated histone and microtubule-associated protein were increased. The results showed a reduction in the development of secondary damage in rat spinal cord trauma with an improvement in the open field test [BBB scale] with rapid recovery
Subject(s)
Animals, Laboratory , Spinal Cord Injuries/veterinary , Rats , Spinal Cord Injuries/drug therapy , Histone Deacetylase Inhibitors , Locomotion/drug effects , Epigenesis, GeneticABSTRACT
Adult stem cells [ASC] are undifferentiated cells found throughout the body. These cells are promising tools for cell replacement therapy in neurodegenerative disease. Adipose tissue is the most abundant and accessible source of ASC. This study was conducted to evaluate effect of selegiline on differentiation of adiposederived stem cells [ADSC] into functional neuron-like cells [NLC], and also level of the neurotrophin expression in differentiated cells. ADSC were transdifferentiated into NLC using selegiline where CD90, CD49d, CD31, CD106 and CD45 were used as markers for ADSC identification. Lipogenic and osteogenic differentiation of ADSC were used to characterize the ADSC. ADSC were treated with selegiline at different concentrations [from 10[-6] to 10[-11] mM] and time points [3, 6, 12, 24 and 48 h]. Percentage of viable cells, nestin and neurofilament 68 [NF-68] immunoreactive cells were used as markers for differentiation. The optimal dose for neurotrophin expressions in differentiating cells was evaluated using reverse transcriptase-PCR. NLC function was evaluated by loading and unloading with FM1-43 dye. ADSC were immunoreactive to CD90 [95.67 +/- 2.26], CD49d [71.52 +/- 6.64] and CD31 [0.6 +/- 0.86], but no immunoreactivity was detected for CD106 and CD45. The results of neural differentiation showed the highest percentage of nestin and NF-68 positive cells at 10[-9] mM concentration of selegiline [exposed for 24 h]. The differentiated cells expressed synapsin and neurotrophin genes except brainderived neurotrophic factor. ADSC can be an alternative source in cell-based therapy for neurodegenerative diseases using selegiline to induce ADSC differentiation to neuronal lineage