Role of cerebrospinal fluid in differentiation of human dental pulp stem cells into neuron-like cells / 대한해부학회지

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), glialderived 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.

Role of cerebrospinal fluid in differentiation of human dental pulp stem cells into neuron-like cells / 대한해부학회지

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), glialderived 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.

Effects of mono-[2-ethylhexyl] phthalate and DI-[2-ethylhexyl] phthalate administrations on oocyte meiotic maturation, apoptosis and gene quantification in mouse model

Objective: Phthalates, which are commonly used to render plastics into soft and flexible materials, have also been determined as developmental and reproductive toxicants in human and animals. The purpose of this study was to evaluate the effect of mono-[2-ethylhexyl] phthalate [MEHP] and di-[2-ethylhexyl] phthalate [DEHP] oral administrations on maturation of mouse oocytes, apoptosis and gene transcription levels

Materials and Methods: In this experimental study, immature oocytes recovered from Naval Medical Research Institute [NMRI] mouse strain [6-8 weeks], were divided into seven different experimental and control groups. Control group oocytes were retrieved from mice that received only normal saline. The experimental groups I, II or III oocytes were retrieved from mice treated with 50, 100 or 200 micro l DEHP [2.56 micro M] solution, respectively. The experimental groups IV, V or VI oocytes were retrieved from mouse exposed to 50, 100 or 200 micro l MEHP [2.56 micro M] solution, respectively. Fertilization and embryonic development were carried out in OMM and T6 medium. Apoptosis was assessed by annexin V-FITC/Dead Cell Apoptosis Kit, with PI staining. In addition, the mRNA levels of Pou5f1, Ccna1 and Asah1 were examined in oocytes. Finally, mouse embryo at early blastocyst stage was stained with acridine-orange [AO] and ethidium-bromide [EB], in order to access their viability

Results: The proportion of oocytes that progressed up to metaphase II [MII] and 2-cells embryo formation stage was significantly decreased by exposure to MEHP or DEHP, in a dose-dependent manner. Annexin V and PI positive oocytes showed greater quantity in the treated mice than control. Quantitative reverse transcriptase-polymerase chain reaction [qRT-PCR] revealed that expression levels of Pou5f1, Asah1 and Ccna1 were significantly lower in the treated mouse oocytes than control. The total cell count for blastocyst developed from the treated mouse oocytes was lower than the controls

Conclusion: These results indicate that oral administration of MEHP and DEHP could negatively affect mouse oocyte meiotic maturation and development in vivo, suggesting that phthalates could be risk factors for mammalians' reproductive health. Additionally, phthalate-induced changes in Pou5f1, Asah1 and Ccna1 transcription level could explain in part, the reduced developmental ability of mouse-treated oocytes

Impact of Cell Density on Differentiation Efficiency of Rat Adipose-derived Stem Cells into Schwann-like Cells

BACKGROUND AND OBJECTIVES: Schwann-like (SC-like) cells induced from adipose-derived stem cells (ASCs) may be one of the ideal alternative cell sources for obtaining Schwann cells (SCs). They can be used for treating peripheral nerve injuries. Co-culture with SCs or exposure to glial growth factors are commonly used for differentiation of ASCs to SC-like cells. However, the effect of initial cell density as an inductive factor on the differentiation potential of ASCs into the SC-like cells has not been yet investigated. METHODS AND RESULTS: ASCs were harvested from rat and characterized. The cells were seeded into the culture flasks at three different initial cell densities i.e. 2×10³, 4×10³ and 8×10³ cells/cm² an overnight and differentiated toward SC-like cells using glial growth factors. After two weeks, the differentiation rate of ASCs to SC-like cells at different densities was assessed by immunofluorescence, fluorescence-activated cell sorting analysis and real time RT-PCR. Expression of the typical SCs markers, S-100 proteins and glial fibrillary acidic protein (GFAP) protein, was observed in all cell densities groups although the number of S100-positive and GFAP-positive cells, and the expression of p75(NTR) mRNA, another SC marker, were significantly higher at the density of 8×10³ cells/cm² when compared with the other cell densities groups (p<0.001). CONCLUSIONS: The results suggest that the higher differentiation rate of ASCs to SC-like cells can be obtained at initial cell density of 8×10³ cells/cm², possibly via increased cell-cell interaction and cell density-dependent influence of glial growth factors.

effects of different doses of ketamine on quality of normal ejaculated sperm

Background: Ketamine, an injectable anesthetic in human and animal medicine, is also a recreational drug used by young adults. The aim of this study is to evaluate the effects of ketamine on membrane integrity, DNA fragmentation and sperm parameters in humans

Materials and Methods: This prospective study was conducted on 40 males with normal semen samples over one month [August 2012]. Subjects were randomly allocated to four groups [Control and case I, II and III] whose semen samples were adjusted to different concentrations of ketamine [1, 3, 5 microL] for one hour. Sperm analysis was performed for routine parameters, motility and morphology. Evaluation of membrane integrity and DNA fragmentation was done by eosin-Y staining and the sperm chromatin dispersion [SCD] test, respectively. The results were analyzed by ANOVA and Tukey's tests. P

Results: Total sperm motility in all case groups were significantly lower compared with the control group. In case group III, progressive motility showed significant difference with case group II. After addition of ketamine, sperm had evidence of coiled tails in all case groups compared to the control group however this observation was not significant. Evaluation of membrane integrity showed the rate of necrospermia increased in all case groups. However, ketamine only significantly affected membrane integrity in case group III. SCD staining showed that in the control group nucleoids with medium halos [63.44 +/- 1.2] were significantly different compared to the case groups I [15.44 +/- 0.45], II [9.05+/-1.16] and III [10.55 +/- 1.14], respectively. Between case groups, nucleoids with large and medium halos showed significant differences in case groups II and III compared with case group I. Nucleoids with medium halos were significantly different between case groups II and III

Conclusion: Ketamine, through its effect on membrane integrity and DNA fragmentation, decreased sperm viability and caused abnormal sperm parameters in progressive motility

Reproductive outcome following thawed embryo transfer in management of ovarian hyperstimulation syndrome

The purpose of the study was to compare clinical pregnancy and delivery rates with fresh and frozen embryo transfer in patients admitted to Shiraz- Human Assisted Reproductive Center with ovarian hyperstimulation syndrome [OHSS]. OHSS patients randomly divided in two groups, group A [n=50] with fresh embryo transfer and group B [n=50] with frozen embryo transfer. We used vitrification method for freezing the embryos. Patient age, combination of female and male factors, total number of retrieved oocytes, number of cryopreserved embryo, number of transferred embryos, clinical pregnancy and delivery rates were recorded for all patients. All statistical calculations were done using SPSS software. Generalized linear model was used to adjust the confounding factors to compare the clinical pregnancy and delivery rates between two groups. The p<0.05 was considered statistically significant. Mean [ +/- SD] ages of these patients were 26.78 +/- 3.5 and 28.42 +/- 4.2 yrs in fresh [A] and frozen [B] embryo transfer groups respectively. Combinations of male and female factors were 28.3% and 32.1% respectively. Average numbers of oocytes retrieved in two groups were 22.14 +/- 4.3 and 21.02 +/- 4.9, and after fertilization, embryos cryopreserved per patient yielded averages of 13.82 +/- 3.5 and 12.5 +/- 4.3. Thaw and ET were performed and the means for transferred embryos were 3.22 +/- 0.6 and 4.1 +/- 0.7. We didn't find any significant differences in implicit parameters between the two groups. The pregnancy and delivery rates in OHSS patients were significantly higher in frozen embryo transfer, 63.1% and 45.6%, compared with fresh embryo transfer, 55.1% and 35.4%, respectively. The pregnancy and delivery rates in OHSS cases, both fresh and subsequently with frozen embryo transfer, were exceptionally high. There was statistically significant difference of pregnancy and delivery rates between fresh and frozen embryo transfer. As a result, an elective embryo freezing policy to moderate the severity and duration of OHSS has compromising outcomes for women at risk of OHSS

Evaluation of apoptotic genes expression and its protein after treatment of cryptorchid mice

Cryptorchidism has been proved to cause apoptosis in germ cells in respond to changes in the stimulation levels of specific physiological events. The purpose of this study was to determine whether treatment of bilateral cryptorchidism was associated with alterations in testicular gene expression. To induce bilateral cryptorchid model, immature mice were anesthetized and a small incision was made in the abdominal skin and peritoneum, then fat pad at the upper end of testis was sutured to the peritoneum. Transcript level of Bax, Bcl-2 proper, p53 and survivin mRNA and protein were determined after performing the two treatment methods: surgical return of testis into scrotum [Exp1] and transplantation of spermatogonial stem cells with later orchidopexy [Exp2], performed 2 and 3 months after heat exposure, respectively. RT-PCR data showed decreased levels of p53 and Bax expression as well as decreased levels of Bcl-2 mRNA in treatment groups especially after transplantation compared with control group. The expression of survivin 140 was increased significantly after treatment, whereas that of survivin 40 was lower especially in the orchidopexy group. Immunohistochemistry staining showed that the intensity of Bax expression mainly was decreased in treated cryptorchid testis and rates of Bcl-2 were increased significantly, but expression of p53 and survivin proteins did not changed significantly after treatment. These observations suggest that cell-type-specific and many apoptotic systems control germ cell apoptosis after treatment of cryptorchidism

Spermatogonial stem cell transplantation and subsequent orchidopexy in the bilateral cryptorchid mouse model

Testicular cell transplantation has been widely used to investigate the restoration of fertility in rodent models. In this research we apply transplantation as a treatment method in the cryptorchid model and compare this method with orchidopexy, which is the routine treatment for this problem. We studied the controversial effects of treatment on the number of germ cells and other morphometrical characteristics of testicular and epididymal parameters in cryptorchid mice. Bilateral cryptorchidism was induced in immature mice by returning two testes to the abdominal cavity via a surgical procedure. Respectively orchidopexy and transplantation of spermatogonial stem cells [were isolated from bilateral cryptorchid testes] with later orchidopexy was performed two and three months after heat exposure in separate cryptorchid mice. The weight of testes, spermatogenic cell numbers, as well as epididymal sperm parameters were measured at two and eight weeks after treatment. The results were analyzed by performing ANOVA and Tukey's tests. Our results showed that after orchidopexy, the testis remained atrophied and the number of spermatogonia returned to the near normal range, but spermatogenesis was recovered only partially at the stage of differentiated germ cells. After transplantation we observed significant changes in the stage of sperm formation compared to orchidopexy. We demonstrated that the spermatogonia isolated from bilateral cryptorchid mice have the ability to regenerate spermatogenesis. Also, while orchidopexy is a routine treatment for cryptorchidism, transplantation may thus prove to be a promising technique for the preservation of fertility for severely damaged cryptorchid testes that have scarce spermatogonia

effects of laser assisted hatching on pregnancy rates

For infertile women aged over 35 years, failure of the ZP [zona pellucida] to rupture is believed to be associated with a decreased implantation rate in vitro fertilization [IVF] or intra cytoplasmic sperm injection [ICSI]. In this research, laser assisted hatching [LAH] was offered to patients with advanced maternal age to evaluate a possible benefit. Nine hundred thirty two cycles of IVF/ICSI in females were analyzed. Women included in this study were allocated in 4 groups. In group I and II, embryos were cultured and transferred with and without LAH in women aged /= 35. Laser manipulations were performed using a suturn-Tm3 system using 2-3 pulses of 0.8 millisecond with 400 voltage duration. The size of the hole made in the zona was measured to be 5-10 micro m, depending on the zona thickness of each individual embryo. The performance of LAH significantly increased clinical pregnancy rates in all patients. In group I and II, the chemical [50.99% and 31.61% respectively], clinical [50% and 30.69% respectively] and multiple pregnancies [22.27% and 5.94% respectively] significantly differ between these groups. In the patients with advanced female age >/= 35 the performance of LAH significantly increased chemical [30.12%] and clinical pregnancy [27.71%] rates compared to whom without LAH [18.96% and 16.37% respectively]. Our data demonstrate in the patients who were less than 35 years old, multiple pregnancy rates were significantly increased compared to other groups who aged over 35 years old. In addition benefit of LAH in improving pregnancy rates after IVF or ICSI in women of advanced age [>/= 35] was shown