ABSTRACT
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.
ABSTRACT
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.
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
The anatomy of the palmar vascular arches and their variations, being one of the most challenging anatomical regions for reconstructive surgeon. During a routine dissection of a male adult cadaver in dissection hall of zahedan university of medical sciences, a complex, unilateral and rare variation in the pattern of blood supply to the palm of the right hand was observed. The history of the individual and cause of the death is not known. In this cadaver there was an incomplete superficial palmar arterial arch had no contribution from the radial artery. The superficial palmar arch giving only one common palmar digital artery, that supply second interdigital space and then it terminated by giving rise to a common trunk for princeps pollicis and radialis indicis arteries. Absence of the second and third common palmar digital artery with the contiguous sides of the third and forth interdigital spaces supply by the second and third palmar metacarpal arteries from the deep palmar arch respectively. The third palmar metacarpal artery giving rise to a branch which supplies the medial side of the little finger. Having knowledge of the variations of vascular patterns resulting from a number of developmental errors could provide an important source of information for Anatomists, Radiologist, reconstructive and vascular surgeons
ABSTRACT
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
Sperm parameters and motion kinetics are affected by cryopreservation. The main purpose of the current study was to determine the effect of different concentrations of Trolox as an antioxidant to freezing-thawing procedure on human sperm kinematic parameter. Semen was collected from 20 normal donors and divided into five aliquots prior to cryopreservation. The first aliquot was analyzed by computer-assisted sperm analysis [CASA]. Other aliquots were mixed with cryo-protective agent containing 0, 20, 40, and 80 micro mol Trolox and treated samples were cryopreserved in liquid nitrogen. After two weeks samples were thawed and sperm motion kinematics was measured by CASA. Percent motility [Mot], curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP], linearity [LIN], and amplitude of lateral head displacement [ALH] were compared before and after freeze. Addition of 40 micro mol Trolox resulted in significantly higher [p<0.05] post thaw VCL, VSL and VAP compared to other groups. Therefore the percentage of post thaw motile spermatozoa were significantly higher [p<0.01]. The supplementation of Trolox significantly improved the post-thawed human semen quality, especially progressive motility and average path velocity
Subject(s)
Humans , Male , Chromans/pharmacology , Cryopreservation , Antioxidants , Freezing , SpermatozoaABSTRACT
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
Subject(s)
Male , Animals, Laboratory , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Adipose Tissue/cytology , Osteogenesis , Rats, Sprague-Dawley , Stem CellsABSTRACT
In human fertilization, the sperm centrosome nucleates a radial array of microtubules called the sperm aster. The sperm aster is responsible for apposition of male and female pronuclei, and later gives rise to the first meiotic spindle. The objective of this study was to determine microtubule assembly and chromatin configuration in rabbit oocytes following intracytoplasmic injection with human sperm by piezo-driven pipette. Oocytes were collected from superovulated dose 14-15 h after hCG injection and were fertilized by injection of a single human sperm into the ooplasm of each oocyte without additional activation treatment. Four hours post heterologous intracytoplamic sperm injection [ICSI], rabbit eggs were fixed and microtubule organization and chromatin configuration were examined by immunofluorescence microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle. Following human sperm injection, an aster of microtubules formed adjacent to the sperm head, around mid-piece, and sperm aster was enlarged and assembled around male and female pronuclei. During pronuclear centration, male and female pronuclei were surrounded by a microtubule array without nucleation sites. With fertile human sperm, the sperm aster formation rate was 54.6%. From our data we concluded that human spermatozoa can be injected successfully into rabbit oocytes, resulting in a reasonable survival rate, and that rabbit oocytes provide a reliable tools for assessing human sperm centrosomal function using the Piezo-ICSI system