ABSTRACT
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
ABSTRACT
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
ABSTRACT
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
ABSTRACT
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
ABSTRACT
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
ABSTRACT
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
Subject(s)
Animals, Laboratory , Diabetes Mellitus/therapy , Mice , Leukocyte Common Antigens , Receptors, CXCR4 , Pancreas , Injections, IntravenousABSTRACT
Acquaintance with the different anatomical variations of the arterial supply of the gallbladder is of great importance in hepatobiliary surgical procedures. A rare variation of the hepatobiliary arterial system was found during anatomical dissection of a female Iranian cadaver. Two cystic arteries were present, the first arising directly from the right hepatic artery and the second from the gastroduodenal artery. Knowledge of the different anatomical variations of the arterial supply of the gallbladder is of great importance in hepatobiliary surgical procedures
ABSTRACT
Recent publications about differentiation of stem cells to germ cells have motivated researchers to make new approaches to infertility. In vitro production of germ cells improves understanding differentiation process of male and female germ cells. Due to the problem of using embryonic stem cells [ESC], it's necessary the mentioned cells be replaced with some adult multi-potent stem cells in laboratories. The aim of this study was to obtain germ cells from appropriate source beyond ESC and compare differential potentials of adipocytes derived stem cells [ADMSCs] with bone marrow derived stem cells [BMMSCs]. To find multi-potential entity, after providing purified ADMSCs and BMMSCs, differentiation to osteoblast and adipocyte was confirmed by using appropriate culture medium. To confirm mesenchymal lineage production superficial markers [expression of CD90 and CD44 and non-expression of CD45 and CD31] were investigated by flowcytometry. Then the cells were differentiated to germ cells in inductive medium containing retinoic acid for 7days. To evaluate germ cells characteristic markers [Dazl [Deleted in azoospermia-like], Mvh [Mouse vasa homolog gene], Stra8 [Stimulated by retinoic acid] and Scp3 [Synaptonemal complex protein 3]] flowcytometry, imunoflorescence and real time PCR were used. Both types of cells were able to differentiate into osteoblast and adipocyte cells and presentation of stem cell superficial markers [CD90, CD44] and absence of endothelial and blood cell markers [CD31, CD45] were confirmative The flowcytometry, imunoflorescence and real time PCR results showed remarkable expression of germ cells characteristic markers [Mvh, Dazl, Stra8, and Scp3]. It was found that although ADMSCs were attained easier and also cultured and differentiated rapidly, germ cell markers were expressed in BMMSCs significantly more than ADMSCs
Subject(s)
Animals, Laboratory , Germ Cells , Infertility , Tretinoin , Flow Cytometry , Real-Time Polymerase Chain Reaction , MiceABSTRACT
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. 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]. 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 showed an increase in Olig2 and O4 cells and a decrease in GFAP and Iba-1 cells in the transplanted group. Our results indicate that ADSCs may provide a feasible, practical way for remyelination in diseases such as MS
Subject(s)
Male , Animals, Laboratory , Mesenchymal Stem Cell Transplantation , Adipose Tissue , Cuprizone , MiceABSTRACT
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
ABSTRACT
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. 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]. 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. Low concentrations of BMP4 during two days had an optimal effect on differentiation of PGCs from mESC
ABSTRACT
The aim of the present study was to investigate the neuroprotective effects of Melissa officinalis, a major antioxidant plant, against neuron toxicity in hippocampal primary culture induced by 3,4-methylenedioxymethamphetamine [MDMA] or ecstasy, one of the most abused drugs, which causes neurotoxicity. 3-[4,5-dimethyl2 thiazoyl]2,5-diphenyketrazolium bromide [MTT] assay was used to assess mitochondrial activity, reflecting cell survival. Caspase-3 activity assay and Hoechst / propiedium iodide [PI] staining were done to show apoptotic cell death. A high dose of ecstasy caused profound mitochondrial dysfunction, around 40% less than the control value, and increased apoptotic neuronal death to around 35% more than the control value in hippocampal neuronal culture. Co-treatment with Melissa officinalis significantly reversed these damages to around 15% and 20% respectively of the MDMA alone group, and provided protection against MDMA-induced mitochondrial dysfunction and apoptosis in neurons. Melissa officinalis has revealed neuroprotective effects against apoptosis induced by MDMA in the primary neurons of hippocampal culture, which could be due to its free radical scavenging properties and monoamine oxidase [MAO] inhibitory effects
Subject(s)
Animals, Laboratory , Female , Neuroprotective Agents , N-Methyl-3,4-methylenedioxyamphetamine/toxicity , Plant Extracts , Rats, WistarABSTRACT
The failure of regeneration after spinal cord injury [SCI] has been attributed to axonal demyelination and neuronal death. Cellular replacement and white matter regeneration are both necessary for SCI repair. In this study, we evaluated the co-transplantation of olfactory ensheathing cells [OEC] and embryonic stem [ES] cell-derived motor neurons [ESMN] on contused SCI. OEC cultured from olfactory nerve rootlets and olfactory bulbs. ESMN was generated by exposing mouse ES cells to retinoic acid and sonic hedgehog. Thirty female rats were used to prepare SCI models in five groups. Control and medium-injected groups was subjected to induce lesion without cell transplantation. OEC or ESMN or both were transplanted into the site of the lesion in other groups. The purity of OEC culture was 95%. Motor neuron progenitor markers [Olig2, Nkx6.1 and Pax6] and motor neuron markers [Is11, Is12 and Hb9] were expressed. Histological analysis showed that significantly more [P<0.001] spinal tissue was spared in OEC, ESMN and OEC+ ESMN groups but the OEC+ ESMN group had a significantly greater percentage of spared tissue and myelination than other groups [P< 0.05]. The numbers of ESMN in co-transplanted group were significantly higher than ESMN group [P<0.05]. A significant [P<0.05] recovery of hindlimb function was observed in rats in the transplanted groups. We found that the co-transplantation of ESMN and OEC into an injured spinal cord has a synergistic effect, promoting neural regeneration, ESMN survival and partial functional recovery
Subject(s)
Female , Animals, Laboratory , Embryonic Stem Cells/transplantation , Motor Neurons , Olfactory Bulb , Olfactory Nerve , Rats, Wistar , Cell Culture Techniques , Reverse Transcriptase Polymerase Chain Reaction , RNA , ImmunohistochemistryABSTRACT
Mediodorsal [MD] thalamic nucleus, which is considered to take place between extra pyramidal and limbic feedback circuit, receives projective fibers from ventrolateral neurons of reticular part of substantia nigra [SNr]. In order to better understand the influence and chemical reaction of these fibers upon MD nucleus, the morphology and synaptology of them were examined in the present study. Phaseolous vulgaris-leucoagglutin [PHA-L] was injected into substantia nigra pars reticulate. After 3-4 days, the sections of SNr injection site and MD nucleus were prepared. Then, we examined organization, morphology and, synaptology of PHA-L labeled SNr fibers that go to caudal and lateral part of MD thalamic nucleus. At the electron microscopic level, the SNr terminals made synapses predominantly with the medium to small dendrites and far less frequently with soma and large dendrites. These terminals were packed with polymorphic synaptic vesicles and formed symmetrical synapses; furthermore, it has been already recognized that cortico straital fibers from sensory-motor cortex go to region of the SNr that give rise to the nigrothalamic fibers. This data suggest that upon the synaptic organization, morphology and chemical nature of GABAergic, SNr fibers may have different inhibitory influence on MD neurons regulating the thalamic output from MD to cerebral cortex in the control of limbic and extra pyramidal feedback system
Subject(s)
Male , Animals, Laboratory , Synapses , Substantia Nigra/anatomy & histology , Phytohemagglutinins , Rats, Sprague-DawleyABSTRACT
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
Subject(s)
Male , Animals, Laboratory , Locus Coeruleus/drug effects , Rats, Sprague-Dawley , Melanins , Antihypertensive Agents , Models, AnimalABSTRACT
In vitro maturation [IVM] of oocytes is a promising technique to reduce the costs and avert the side-effects of gonadotropin stimulation for in vitro fertilization [IVF]. The pregnancy rates from oocytes matured in vitro are much lower than those of in vivo stimulation cycles, indicating that optimization of IVM remains a challenge. In this study, we investigated the effect of cumulus cells on maturation and fertilization rate of immature oocytes [Germinal vesicle]. Germinal vesicle [GV] oocytes were recovered from 6-8 weeks old Balb C female mice 48hr after injection of 10 IU pregnant mare serum gonadotropin [PMSG]. Collected oocytes were divided into two groups. Group A: GV oocytes without cumulus [denuded oocyte]. Group B: GV oocytes with cumulus cells [cumulus-oocyte complex]. The oocytes in both groups were cultured in TCM-199 medium in a humidified atmosphere of 5% CO2 in air at 37?C. The maturation, fertilization and developmental rates were recorded after 24hr. Maturation, fertilization and developmental rates in denuded oocytes [DO] were 65.1%, 68.02%, 78.63% respectively, and in cumulus-oocyte complex [COC] were 78.20%, 85.57% and 85.05%, respectively. The maturation, fertilization and developmental rates of COC were significantly higher than those of DO [p<0.05]. The results show that cumulus cells have beneficial effects on maturation, fertilization and cleavage rates of mice oocytes
ABSTRACT
Early in the development of many animals, before transcription begins, any change in the pattern of protein synthesis is attributed to a change in the translational activity or stability of mRNA in the egg and early embryo. As a result, translational control is critical for a variety of developmental decisions, including oocyte maturation and initiation of preimplantation development. In this study, using real-time RT-PCR method, we defined the time course of degradation and deadenylation of an oocyte specific gene [c-mos] more precisely and a gene that is re-synthesized after ZGA [cyclin A2]. Our data indicate that oocyte-specific transcript, c-mos, degrades rapidly while cyclin A2 mRNA does not and the deadenylation of c-mos mRNA precedes the process of degradation. Our findings suggest that time-dependent elimination of different maternal mRNA is a way for regulation of translation in early development of mouse embryos