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Objective: implantation failure is an obstacle in assisted reproduction techniques [ART]. Calcitonin is a molecules involved in uterine receptivity and embryo implantation. Melatonin can promote embryo quality and improve implantation. This study examines the effect of pretreatment of blastocysts with melatonin and calcitonin on heparin binding-epidermal growth factor [HB-EGF] expression in murine endometrium
Materials and Methods: in this experimental study, we collected 2-cell embryos from the oviducts of 1.5 day pregnant NMRI mice. Embryos were cultured to the blastocyst in G[TM] medium with or without 10[-9] M melatonin. Pregnant and pseudo-pregnant mice received intraperitoneal [IP] injections of 2 IU calcitonin. After 24 hours, we transferred the cultured blastocysts into the uteri of pseudo-pregnant mice. Two days later, implantation sites were counted and we assessed the levels of HB-EGF mRNA and protein in the uteri of naturally pregnant and pseudo-pregnant mice by quantitative real-time polymerase chain reaction [qRT-PCR] and Western blot. Statistical analysis was performed with one-way ANOVA followed by the Tukey post hoc test. P<0.05 was considered statistically significant
Results: melatonin pretreatment of blastocysts along with calcitonin administration significantly increased HB-EGF mRNA and protein [P<0.001] in the endometrium of pseudo-pregnant mice. Administration of calcitonin in naturally pregnant mice significantly increased HB-EGF mRNA and protein levels [P<0.001]. Compared with the control group [2.6 +/- 0.5], the average number of implantation sites in the melatonin group [4.6 +/- 0.5, P<0.05] and calcitonin group [7 +/- 1, P<0.001] significantly increased. There was a significant increase in implantation sites in the combined melatonin and calcitonin group [8.6 +/- 0.5, P<0.001]. Calcitonin significantly enhanced calcitonin receptor mRNA [P<0.001] and protein [P<0.05] in the uteri of naturally pregnant and pseudo-pregnant mice
Conclusion: melatonin pretreated blastocysts along with calcitonin increased HB-EGF expression in the uteri of pseudopregnant mice. Calcitonin administration upregulated HB-EGF in uteri of naturally pregnant mice
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Objective: Bone marrow has recently been recognized as a novel source of stem cells for the treatment of wide range of diseases. A number of studies on murine bone mar-row have shown a homogenous population of rare stage-specific embryonic antigen 1 [SSEA-1] positive cells that express markers of pluripotent stem cells. This study focuses on SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiate them into insulin-secreting cells [ISCs] in order to investigate their differentiation potential for future use in cell therapy
Materials and Methods: This study is an experimental research. Mouse SSEA-1 positive cells were isolated by Magnetic-activated cell sorting [MACS] followed by characterization with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs with specific differentiation media. In order to evaluate differentiation quality and analysis, dithizone [DTZ] staining was use, followed by reverse transcription polymerase chain reaction [RT-PCR], immunocytochemistry and insulin secretion assay. Statistical results were analyzed by one-way ANOVA
Results: The results achieved in this study reveal that mouse bone marrow contains a population of SSEA-1 positive cells that expresses pluripotent stem cells markers such as SSEA-1, octamer-binding transcription factor 4 [OCT-4] detected by immunocytochemistry and C-X-C chemokine receptor type 4 [CXCR4] and stem cell antigen-1 [SCA-1] detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCs cell clusters as evidenced by their DTZ positive staining and expression of genes such as Pdx1 [pancreatic transcription factors], Ngn3 [endocrine progenitor marker], Insulin1 and Insulin2 [pancreaticbeta-cell markers]. Additionally, our results demonstrate expression of PDX1 and GLUT2 protein and insulin secretion in response to a glucose challenge in the differentiated cells
Conclusion: Our study clearly demonstrates the potential of SSEA-1 positive cells to differentiate into insulin secreting cells in defined culture conditions for clinical applications
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Background: Bone marrow-derived mesenchymal stem cells [BMMSCs] transplantation has been considered as a promising milestone in liver fibrosis treatment. However, low amounts of homing are a major obstacle. We aimed to investigate the role of melatonin pretreatment in BMMSC homing into experimental liver fibrosis
Methods: BMMSCs were obtained, grown, propagated and preconditioned with 5 µM melatonin and analyzed for multipotency and immunophenotypic features at passage three. The cells were labelled with CM-Dil and infused into the rats received the i.p. injection of carbon tetrachloride [CCl4] for five weeks to induce liver fibrosis. Animals were divided into two groups: One group received BMMSCs, whereas the other group received melatonin-pretreated BMMSCs [MT-BMMSCs]. After cell injection at 72 h, animals were sacrificed, and the liver tissues were assessed for further evaluations: fibrosis using Masson's trichrome and hematoxylin and eosin staining and homing using fluorescent microscopy and flow cytometry
Results: BMMSCs and MT-BMMSCs expressed a high level of CD44 but low levels of CD11b, CD45 and CD34 [for all P
Conclusion: This study indicates the improved homing potential of BMMSCs in pretreatment with melatonin. Therefore, this strategy may represent an applied approach for improving the stem cell therapy of liver fibrosis
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Wharton's Jelly-Mesenchymal Stem Cells [WJ-MSCs] are pluripotent cells with differentiation capability into most cell lineages. The aim of the current work was to examine the role of Retinoic Acid [RA] in differentiation process of these cells into hepatocyte-like cells and determine the morphological and functional patterns. Human WJ-MSCs were extracted, cultured and expanded; after approximately 95% of confluence, the cells were treated with hepatogenic media containing RA. The cells were subsequently analyzed for morphological changes, glycogen storage, albumin production, and specific gene expression. WJ-MSCs expressed high levels of CD90 [93.6%] and CD105 [90.7%], but low levels of CD34 [0.3%] and CD45 [0.8%]. Albumin production had significant difference in the two groups [p
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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
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Diabetes Mellitus [DM], simply known as diabetes, refers to a group of metabolic diseases in which there are high blood sugar levels over a prolonged period. In this study, the feasibility and safety of intravenous transplantation of Very Small Embryonic Like stem cells [VSELs] were investigated for diabetes repair, and finally the migration and distribution of these cells in hosts were observed. Mouse bone marrow VSELs were isolated by Fluorescent Activating Cell Sorting [FACS] method by using fluorescent antibodies against CD45, CXCR4 and Sca1 markers. Sorted cells were analyzed for expression of oct4 and SSEA1 markers with immunocytochemistry staining method. To determine multilineage differentiation, sorted cells were differentiated to Schwann, osteocyte and beta cells. Ten days after the establishment of a mouse model of pancreas necrosis, Dil-labeled VSELs were injected into these mice via tail vein. Pancreases were harvested 4 weeks after transplantation and the sections of these tissues were observed under fluorescent microscope. It was proved that CD45-, CXCR4+, and Sca1+ sorted cells express oct4 and SSEA1. Our results revealed that intravenously implanted VSELs could migrate into the pancreas of hosts and survive in the diabetic pancreas. In treated groups, blood glucose decreased significantly for at least two month and the weights of mice increased gradually. This study provides a strategy for using VSELs for curing diabetes and other regenerative diseases, and the strategy is considered an alternative for other stem cell types
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Animales de Laboratorio , Diabetes Mellitus/terapia , Ratones , Antígenos Comunes de Leucocito , Receptores CXCR4 , Páncreas , Inyecciones IntravenosasRESUMEN
Numerous in vitro reports suggest that Low Level Laser Therapy [LLLT] affects cellular processes by biostimulation, however most of them emphasize on using visible light lasers which have low penetration. The aim of this study was to determine the effect of infrared laser light [which is more useful in clinic because of its higher penetration] on secretion of Fibroblast Growth Factor [FGF], Platelet Derived Growth Factor [PDGF] and Vascular Endothelial Growth Factor [VEGF], as important growth factors in wound healing. Fibroblasts were extracted from the skin of 7 diabetic and 7 nondiabetic mice and cultured. Cell cultures of experimental group were irradiated with single dose of LLLT [energy density of 1 J/ cm[2]] using an 810 nm continuous wave laser and the control group was not irradiated. Secretion of growth factors by skin fibroblasts were quantified through real time polymerase chain reaction. Diabetic irradiated group showed significant increase in FGF [p=0.017] expression, although PDGF increased and VEGF decreased in both diabetic and nondiabetic irradiated groups, but these variations were not statistically significant. These results suggest that LLLT may play an important role in wound healing by stimulating the fibroblasts
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A number of studies on reproduction have mentioned Origanum Vulgare extract's ability to reduce mortality rates and improve fertility rates. However, other studies have suggested that it is possible to use Origanum Vulgare extract to induce abortion. The aim of this study was to investigate the effect of different doses of Origanum Vulgare on embryo survival and macroscopic abnormalities in mice. In this study, 24 mice Balb/c female weighting approximately 25-30 g were divided into 4 groups. Origanum Vulgare extract was prepared; different concentrations [2.5, 12.5, and 25 mg in 0.25 ml distilled water] were administered, by oral gavage, to three experimental groups of mice between day 6 [starting gastrulation] until day 15 of pregnancy [end of organogenesis]. The control group consisted of six mice that received 0.25 ml of distilled water daily. On day 16 of study, pregnant mice were anesthetized by chloroform and fetuses were removed and stained with Alcian Blue, Alizarin Red s and microwave irradiation. Morphological and skeletal abnormalities were investigated by light and stereomicroscopes. The results of this study showed that high doses of the Origanum Vulgare extract significantly decreased the mean number of embryos [10 +/- 0.5, P>0.05], mean number of live embryos [7 +/- 0.5, P>0.05] in each mouse and resulted in significant reduction in mean weight[1184 +/- 8 mg, P>0.05] and crown-rump length[11.9 +/- 0.23 mm, P>0.05] and the overall size of fetuses compared to control group, whereas there was no significant difference between the groups receiving low dose of Origanum Vulgare extract with control group. In addition, under the effect of the Origanum Vulgare extract the subcutaneous bleeding seemed [2 +/- 0.1, P>0.05] significantly more frequent compared to the control group. Origanum Vulgare extract did not have any positive effect on fetal development; and high dosages led to an increased incidence rate of abortion and fetal malformations in the fetuses of women who received it.
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Animales , Femenino , Aborto Veterinario/inducido químicamente , Desarrollo Embrionario y Fetal/efectos de los fármacos , Teratogénesis , Estructuras Embrionarias/efectos de los fármacos , Ratones Endogámicos BALB CRESUMEN
Spermatogonial stem cells [SSCs] maintain spermatogenesis throughout life in the male. Maintenance of SSCs and induction of spermiogenesis in vitro may provide a therapeutic strategy to treat male infertility. This study investigated in vitro differentiation of mouse SSCs in presence or absence of Sertoli cells, hormones and vitamins. Spermatogonial populations were enriched from testes of 4-6 week old males by magnetic activated cell sorting and anti-Thy-1 antibody. Sertoli cells isolated from 6-8 week old testes were enriched using lectin-DSA-coated plates. Isolated SSCs were cultured in the presence of Leukemia inhibitory factor [LIF] for 7 days in gelatin-coated dishes, then dissociated and cultured for 7 days in media lacking LIF in the presence or absence of Sertoli cells, with or without FSH, testosterone and vitamins. After one week, the effects of Sertoli cells +/- supplementary media on SSC differentiation was evaluated by microscopy and expression of meiotic and postmeiotic transcripts using RT-PCR. SSC colonies had limited development after LIF removal alone, exhibiting low expression of meiotic [Scp3, Th2b] but not postmeiotic transcript, and loss of Stra8 and Dazl expression. SSCs co-cultured with Sertoli cells, hormones and vitamins developed spermatid-like cells expressing postmeiotic markers [TP1, TP2, Prm1] at levels over 2-fold higher than Sertoli cells or hormone/vitamins alone. Our present SSC-Sertoli co-culture provides conditions that may allow efficient in vitro differentiation of SSCs for the treatment of male infertility
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Wound healing of burned skin remains a major goal in public health. Previous reports showed that the bone marrow stem cells were potent in keratinization and vascularization of full thickness skin wounds. In this study, mesenchymal stem cells were derived from rat adipose tissues and characterized by flowcytometry. Staining methods were used to evaluate their differentiation ability. A collagen-chitosan scaffold was prepared by freeze-drying method and crosslinked by carbodiimide-based crosslinker. The results of immunecytochemistry and PCR experiments confirmed the adipose-derived stem cells [ASC] in differentiation to the keratinocytes under the treatment of keratinocyte growth factor. The isolated ASC were seeded on the scaffolds and implanted at the prepared wounds. The scaffolds without cells were considered as a control and implanted on the other side of the rat. Histopathological analyses confirmed the formation of new tissue on the scaffold-cell side after 14 days with the formation of dermis and epidermis. These results indicated the capacity of ASC in differentiation to keratinocytes and also wound healing in vivo
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Animales , Animales de Laboratorio , Cicatrización de Heridas , Piel , Regeneración , Colágeno , Andamios del Tejido , Ratas , Ingeniería de Tejidos , QueratinocitosRESUMEN
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 17beta-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
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Bone marrow is the traditional source of human multipotent mesenchymal stem cells [MSCs], but adipose tissue appears to be an alternative and more readily available source. In this study, rat adipose-derived stem cells [ADSCs] were induced to differentiate into Schwann-like cells and compared with rat bone marrow stem cells [BMSCs] for their Schwann-like cells differentiation potential. BMSCs and ADSCs were characterized for expression of MSCs-specific markers, osteogenic and adipogenic differentiation. They were induced to differentiate into Schwann-like cells and analyzed for expression of the Schwann specific markers. The immunocytochemical differentiation markers were S-100 and real time quantitative Real-time polymerase chain reaction [RT-PCR] markers were S100, P75 and glial fibrillary acidic protein [GFAP]. 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide [MTT] assay and Annexin V-Fluorescein isothiocyanate [FITC]/ Propidium iodide [PI] double labeling method were employed to detect early stage cell apoptosis. BMSCs and ADSCs showed similarities in expression of the MSC-specific markers, osteogenic and adipogenic differentiation. Both quantitative RT-PCR and immunocytochemical analysis demonstrated that BMSCs and ADSCs had equal expression of the Schwann-specific markers following Schwann-like cells differentiation. However, gene expression of P75 was higher in BMSCs compared with ADSCs. MTT assay and flow cytometry found that of the total BMSCs and ADSCs in the culture medium, 20% to 30% of the cells died, but the remaining cell population remained strongly attached to the substrate and differentiated. Comparative analysis showed that Schwann-like cell differentiation potential of ADSCs was slightly decreased in comparison with BMSCs. Therefore, BMSCs are more favorable choice than ADSCs for tissue engineering
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Masculino , Animales de Laboratorio , Tejido Adiposo , Células Madre Mesenquimatosas , Células Madre Multipotentes , Células de Schwann , Médula Ósea , Células de la Médula Ósea , Fenotipo , Ratas WistarRESUMEN
Free radical formation and oxidative stress might play an important role in the pathogenesis of Parkinson's disease [PD]. In vitro data indicate that neuromelanin [NM] pigment is formed the excess cytosolic catecholamine that is not accumulated into synaptic vesicles via the vesicular monoamine transporter 2 [VMAT2]. We designed this study to investigate the neuroprotective effects of vitamin E in the early model of PD. Male rats [n = 40] with unbiased rotational behavior were randomly divided into five groups: sham operated group [SH, n = 8], vehicle-treated SH group [SH + V, n = 8], vitamin E-treated SH group [SH + E, n = 8], vehicle-treated lesion group [L + V, n = 8] and vitamin E-treated lesion group [L + E, n = 8]. Unilateral intrastriatal 6-hydroxydopamine [12.5 micro l] lesioned rats were treated intramuscularly with alpha-tocopherol acid succinate [24 I.U/kg, intramuscular [i.m.]] 1 h before surgery and three times per week for 2 month post-surgery. To evaluate the vitamin E pretreatment efficacy, tyrosine hydroxylase [TH] immunoreactivity and immunostaining intensity [ISI] for monoamine transporter 2 were used. TH immunohistochemical analyses showed a reduction of 20% in locus coeruleus [LC] cell number of vitamin E pretreated lesioned group but the cell number dropped to 60% in the lesioned group. The ISI of the cells was measured for VMAT2 in LC. Lesioned groups: 1] had the lowest VMAT2 ISI of all neurons; 2] There was an inverse relationship between VMAT2 ISI and NM pigment in the locus and 3] Neurons with the highest VMAT2 ISI also had high TH ISI. The data support the hypothesis that repeated i.m. administration of vitamin E exerts a protective effect on the LC neurons in the early model of PD
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Masculino , Animales de Laboratorio , Locus Coeruleus/efectos de los fármacos , Ratas Sprague-Dawley , Melaninas , Antihipertensivos , Modelos AnimalesRESUMEN
This study was performed to determine whether melatonin at physiological concentrations [0.01-10nM] could affect the proliferation and osteogenic differentiation of Rat ADSCs in vitro. ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced on a monolayer culture with osteogenic medium with or without melatonin at physiological concentrations [0.01-10nM]. After 4 weeks cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase [ALP] activity by ALP kit. Cell viability and apoptosis were also assayed by 3-[4, 5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenlyl]-2-[4-ulfophenyl]-2H-tetrazolium assay and flowcytometry, respectively. All assays were performed in triplicate. The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to the osteogenic medium alone. Similarly, the mineral deposition [calcium level] also decreased. The flow cytometry proved that the cell growth decreased and the apoptotic cells increased. These results suggest that physiological concentration of melatonin has a negative effect on ADSCs osteogenesis
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Masculino , Animales de Laboratorio , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Tejido Adiposo/citología , Osteogénesis , Ratas Sprague-Dawley , Células MadreRESUMEN
Norepinephrine plays a trophic role in the control of cell replication and differentiation in target cells that express adrenergic receptors. In this study, we have tested the influence of infraphysiological, physiological and supraphysiological concentrations [0.0001 nM, 1 nM, 10000 nM] of human norepinephrine on the proliferation of breast cancer cells [human breast adenocarcinoma [MCF-7]] in co-culture with human adipocytes in three-dimensional collagen gel matrix culture. Cell proliferation and lipolysis rate were measured by 3-[4, 5-dimethylthiazolyl]-2, 5-diphenyl-tetrazolium bromide [MTT] and Oil red O colorimetric assay in second, 7[th] and 14[th] days of culture experiments. Our results showed a direct correlation between lipolysis rate of adipocytes and proliferation rate of MCF-7 cells. Both physiological and supraphysiological concentrations of human norepinephrine significantly [P<0.05] increased the proliferation of MCF-7 cells synchronously with progress of adipocyte lipolysis. The proliferations of MCF-7 cells were significantly decreased after conversion of adipocytes to fibroblast-like cells by supraphysiological concentration of norepinephrine. There was no statistical difference in lipolysis of adipocytes and proliferation of MCF-7 cells in response to infraphysiological concentration of norepinephrine. These findings indicated that norepinephrine stimulated the proliferation of MCF-7 cells in co-culture with human adipocytes as a lipolytic factor and that norepinephrine effect was suppressed by conversion of adipocytes to fibroblast-like cells, suggesting adipocytes as another target for prevention and therapy of breast cancer