Cell Journal (Yakhteh)
Volume:15 Issue: 1, Spring 2013

Management of mesenchymal stem cells (MSCs) capabilities to differentiate into osteogenic and chondrogenic lineages would be of utmost importance for their future use in difficult to treat cases of destroyed bone and cartilage. Thus, an understanding of the epigenetic mechanisms as important modulators of stem cell differentiation might be useful. Epigenetic mechanism refers to a process that regulates heritable and long-lasting alterations in gene expression without changing the DNA sequence. Such stable changes would be mediated by several mechanisms including DNA methylation and histone modifications. The involvement of epigenetic mechanisms during MSC bone and cartilage differentiation has been investigated during the past decade. The purpose of this review is to cover outstanding research works that have attempted to ascertain the underlying epigenetic changes of the nuclear genome during in vitro differentiation of MSCs into bone and cartilage cell lineages. Understanding such genomic alterations may assist scientists to develop and recognize reagents that are able to efficiently promote this cellular differentiation. Before summarizing the progress on epigenetic regulation of MSC bone and cartilage differentiation, a brief description will be given regarding in vitro conditions that favor MSC osteocytic and chondrocytic differentiation and the main mechanisms responsible for epigenetic regulation of differentiation.

Vasectomy, though in some cases are being confronted with irreversibility, has been accepted as an effective contraceptive method. It is estimated that near 2-6% of vasectomised men ultimately show a tendency to restore their fertility. In some cases, vasectomy has been considered as an irreversible procedure due to many post-vasectomy complications causing this debate. The aim of present study was to investigate the pattern of expression of galectin-3, an inflammatory factor secreted by macrophages and immune cells, following the vasectomy in mice testis tissue. In this experimental study, twenty mature male Balb/c mice, aged two months, were divided into two equal groups: sham and vasectomised groups (n=10). They were sacrificed four months after vasectomy, while the pattern of galectin-3 expression was investigated using a standard immunohistochemistry technique on testicular tissues. Stereological analyses of testes parameters in vasectomised and sham-operated groups were compared by mixed model analysis. Based on observations, although galectin-3 was not expressed in sham-operated group, it was expressed in 40% of testicular tissues of vasectomised mice, like: seminiferous tubules, interstitial tissues and tunica albugina. Also, our result showed a significant alteration in number of germ and sertoli cells of testicular tissue in vasectomised group in comparison to sham-operated group. In addition, the result of mixed model method confirmed a significant reduction in germ and sertoli cells of vasectomised group. The expression of galectin-3 at different parts of testicular tissue in vasectomised group is higher than sham group. This express illustrates the increase of degenerative changes and inflammation reactions in testicular tissue, leading to chronic complications and infertility, after the vasovasostomy.

A study of the histological events under interleukin-1α (IL-lα) induction of bovine nasal cartilage (BNC) could result in useful data to better understand the mechanisms involved in tissue breakdown in joint diseases. The aim of this study was to investigate the effects of IL-lα on chondrocyte phenotype and extracellular matrix (ECM) changes in BNC explants. In this experimental study, samples were divided into two groups. Group I (control group) BNC explants were cultured only in Dulbecco’s modified Eagle’s medium (DMEM). In group II, BNC explants were treated with IL-lα (10 ng/ml) for 28 days. Then, samples were harvested on culture days 3, 7, 14, 21 and 28 and chondrocyte morphology and ECM alterations were assessed by invert microscopy and histology by hematoxylin and eosin (H&E) and Alcian blue. Cell viability was evaluated by the lactate dehydrogenase (LDH) assay test. Data were analyzed by the t test and p<0.05 was considered significant. IL-lα induced significant morphological changes in cartilage. In the presence of IL-lα, most chondrocytes transformed into a fibroblast-like morphology with a granular black point appearance. An increase in the cell: matrix ratio was observed and there were decreased numbers of chondrocytes.IL-lα induced breakdown of ECM. We observed partial degradation of ECM between days 7-14 and complete degradation occurred between days 21-28 of culture. The LDH levels increased. IL-1α induced morphological changes in chondrocytes and increased destruction of cartilage ECM. There was a parallel correlation between proteoglycan degradation and changes in chondrocyte morpholgy.

Embryonic cerebrospinal fluid (e-CSF) has an important role in development of embryonic and adult brain. Proteomic analysis suggests that this fluid has many morphogenes and cytokines that alter in time and space throughout embryonic life. The aim of this study was to evaluate the developmental effect of embryonic CSF on proliferation and differentiation of neuroprogenitor cells in different gestational age. In this In this experimental study, we examined the role of e-CSF on proliferation and differentiation of neuroprogenitor cells using neurosphere culture method. Neurospheres size analysis and MTT assay were used to assess cell proliferation after four days in vitro. Glial differentiation study was carried out by immunocytochemistry. Neurospheres size and percentage of glial fibrialy acidic protein (GFAP) positive cells were measured by image analyzer (image J). The data were analyzed by one-way ANOVA, followed by the Tukey’s post hoc test. Data were expressed as mean ± SEM, and differences were considered significant when p<0.05, 0.01 and 0.001. Viability and proliferation of neuro progenitor cells in cultures conditioned with E16 CSF and E18 CSF were significantly increased compare to control group. A dramatic decrease in percentage of GFAP-positive cells was found following the application of CSF from E16 and E18 embryos, but not E20 CSF. Our data suggest that, e-CSF altered proliferation and differentiation of neuro progenitor cells in age dependent manner. E16 and E18 CSF enhanced proliferation and viability of neuro progenitor cells, and inhibited differentiation to glial fate in comparison with control group.

(AFB1) suppresses the immune system. To decrease such suppressive effects on the immune system, a wide range of herbal medicines like garlic are utilized. Biological activities of garlic in vitro and in ous studies demonstrated that aged garlic (dry garlic bulbs preserved in the freezer for suppressor fractions. This study focuses on the immunosuppressor activity of AFB1 and immunostimulator activity of aged garlic extract (AGE) through the evaluation of CD4+ CD25+ FoxP+ regulator cell (Treg) counts and the pattern of cytokine production in Balb/c normal mice. In this experimental research, AFB1 was separated from As- Delayed-Type Hypersensitivity (DTH) test was carried out to determinate the effectiveness of different doses of AGE and AFB1, which can both have an effect on the immune system. Subsequent experiments were carried out on 20 Balb/c mice to estimate the effects of AGE and AFB1 on the number of Treg cell in 4 groups: 10 μl/kg/day of AFB1 and AGE diluents were administered for 4 consecutive days to group 1. AFB1, 2. control, 3. AGE + splenocytes harvested and the percentage of splenic cytometry analysis. The ELISA method was utilized to measure Cytokine production.and IL-4 cytokines produced by splenocytes stimulated by and decreased the number of Treg cells in the spleen (p<0.05). AFB1 increased the number Treg cells in the spleen and decreased cytokine production (p<0.05). In groups 2 (control) and 4 (AGE) the number of Treg cells decreased (p value<0.05) whereas in groups 1 and 3 the number of Treg cells increased (p<0.05). This study indicated that AGE is able to alter the cytokine production in normal mice into a Th1 and anti-tumor immunity in particular. AFB1 is able to alter the cytokine production into a Th2 protective pattern. Therefore, AGE might be used as herbal medicine with few side effects as compared to chemotherapy in treating cancers caused by substances like AFB1

Macrophages influence their environment and surrounding immune cells as soon as stimulators affect them. Different sources of macrophages induce different reactions in their neighboring immune cells,which result in non-uniform immunologic outcomes. In this experimental research, we compare the behavior of peritoneal macrophages to lipopolysaccharide (LPS) stimulation from BALB/cmice as an indicator of a type 2 immune response and from C57BL/6 mice as an indicator of a type 1 immune response. In this experimental study, peritoneal macrophages prepared from thioglycolate stimulated BALB/c and C57BL/6 micewere treated with 1μg/ml LPS. At different time points after LPS treatment, nitric oxide (NO), interferon gamma (IFN-γ), interleukin 4 (IL-4),transforming growth factor β1(TGF-β1), interleukin 17 (IL-17), and interleukin 10(IL-10) production were measured in the supernatants of all macrophage cultures.Indoleamine 2, 3 dioxygenase (IDO) and phagocytic activitywere analyzed in the different experimental groups. The supernatant effects of LPS-treated macrophages on splenocyte proliferation was assessed by the colorimetric method using a 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reagent. According to cytokine analysis, different mouse strains show different cytokine patterns in response to LPS. C57BL/6 macrophages produced more IL-17, IL-10, and IFN-γ, while BALB/c macrophages produced more TGF-β1 and IL-4. There was no significant difference in IDO activity between strains (p≤0.05). BALB/c mice produced more NO inthe first 24 hours after LPS treatment,but C57BL/6 produced more NO at 72 hours post-LPS treatment. Macrophages from both strains hada suppressor effect on splenocyte proliferation, but this effect was stronger in BALB/c mice. The results show that macrophages from different genetic backgrounds respond differently to the same stimulus in aspects of type, intensity, and time of response. The consideration of these aspects will enableresearchers to use correct treatment programs for immune-regulation or immunotherapy.

There is longstanding experimental and clinical evidence that supports the idea that replacement of dopaminergic (DAergic) neurons can ameliorate functional disabilities of Parkinson’s disease (PD). The purpose of the present study is to examine the efficacy of transplantation of rat bone marrow stromal cell (BMSCs)-derived tyrosine hydroxylase-positive (TH+) cells induced by deprenyl into 6-hydroxydopamine (6-OHDA)-lesioned rat models, using behavioral tests and immunohistochemical evaluations. In this experimental study, undifferentiated BrdU-labeled BMSCs were incubated in serum-free medium that contained 10-8 M deprenyl for 24 hours. Afterwards, BMSCs were cultured for 48 hours in α-minimal essential medium (α-MEM) supplemented with 10% FBS, then differentiated into TH+ neurons. We randomly divided 24 hemiparkinsonian rats as follows: group 1 (control) received only medium, while groups 2 and 3 were injected with 2×105 BMSCs and deprenyl-treated cells in 4 μl medium. Injections were made into the injured strata of the rats. Rotational behavior in response to apomorphine was tested before transplantation and at 2, 4, and 6 weeks post-graft. Animals were then sacrificed, and the brains were extracted for immunohistochemical and electron microscopic studies. Apomorphine-induced rotation analysis indicated that animals with grafted cells in groups 2 and 3 exhibited significantly less rotational behavior than those in the control group at 2, 4, and 6 weeks after transplantation. Immunohistochemical analysis demonstrated that BrdU-labeled cells expressed specific neuronal markers, such as NF 200 and TH, at the implantation site. The presence of TH+ cells in conjunction with the reduction in rotation might show the capacity of grafted cells to release dopamine. Ultrastructural analysis revealed the presence of immature neurons and astrocyte-like cells at the graft site. TH+ neurons induced by deprenyl can be considered as a cell source for PD autograft therapy.

Garlic (Allium sativum) has anti-inflammatory, anti-mutagenesis, and immunomodulatory properties that modulate anti-tumor immunity and inhibit tumor growth. In this study we have examined the effect of a protein fraction isolated from fresh garlic on anti-tumor response and intra-tumor lymphocyte infiltration. In this experimental study a protein fraction was purified from fresh garlic bulbs using ultra-filtration, followed by chromatofocusing, and SDS-PAGE analysis. Anti-tumor activity was assessed by intra-tumor injection of the protein fraction and garlic extract, itself, into groups of 5 mice each. The percentage of peripheral blood and intra-tumor CD4+ and CD8+ cells were assessed by flow cytometry. Unpaired student’s t test using the SPSS program was applied for all statistical analyses. Garlic extract included different type of proteins with different molecular weight. One of protein’s fraction was immunomodeulator and was composed of three single polypeptides, with molecular masses of ~10-13 kDa and different isoelectric points (pI). These molecules augmented the delayed type hypersensitivity (DTH) response compared to the control group. Intra-tumor injection of the fraction provoked a significant increase in the CD8+ subpopulation of T-lymphocytes, as well as a decrease in tumor size. The fraction increased peripheral blood CD8+ T-lymphocytes in treated animals. The data confirms that protein fractions purified from fresh garlic bulbs augment CD8+ T-cell infiltration into the tumor site, inhibiting tumor growth more efficiently than garlic extract. These fi ndings provide a basis for further investigations on the purified polypeptide as a useful candidate for immunomodulation and tumor treatment.

Osteoporosis is a bone disorder that reduces bone mineral density (BMD) and leads to bone fracture. In addition to different factors, gene polymorphisms have been revealed to be associated with osteoporosis. In this study, we investigated the association between the BsmI polymorphism of vitamin D receptor (VDR) gene (rs1544410) and BMD in a population of Iranian women. In this case control study, clinical risk factors for osteoporosis were obtained from the participants through a questionnaire for a case-control study. The World Health Organisation (WHO) criteria were applied for the diagnosis of the disease. Peripheral blood samples were obtained from 146 pre- and or postmenopausal Iranian women aged between 35 and 71 years (53.53 ± 9.8). The study population was classified for BMD into normal and osteoporotic groups, who matched for age, pregnancy status, menstrual condition, and body mass index (BMI). The BMD of the lumbar spine (L1-4) and femoral neck was measured. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to detect and analyze the genotype. The frequencies of AA and GG were significantly different between the two groups (p value<0.05), with the first genotype being higher in the patients and the second being higher in the normal group. The GG genotype was significantly associated with increased BMD in the lumbar spine (p value<0.05) but non-significant in the femoral neck (p value>0.05). BsmI polymorphism of VDR gene has a significant association with BMD in the lumbar spine and may have a minor effect on the proximal femur BMD in Iranian women.

The bioscaffold can be used in tissue engineering and regenerative medicine. The scaffolds used in tissue engineering must have high porosity to facilitate accelerated angiogenesis for feeding cells and repelling cell waste outside the scaffold. In this experimental study, we attempted to produce lung three-dimensional scaffold and assay its effect on cell penetration and migration. In an experimental study, rabbit lung tissue was decellularized and used as a scaffold for rabbit blastema cells. The scaffolds were studied on the 15th day after culturing. Microscopic features revealed high porosity in the lung tissue scaffold. Electron microscopic imaging also showed collagen and elastin were intact, which are important properties in scaffolds designed for tissue engineering. Migration and permeation of blastema cells into the lung tissue scaffold was also observed. Rabbit lung tissue scaffolds have high porosity. Blastema cells successfully migrated toward and permeated the scaffold inside.