ABSTRACT
Objective: The incidence rate of testicular cancer among young males is high. Co-administration of bleomycin, etoposide and cisplatin [BEP] has increased survival rate of patients with testicular cancer. Although BEP is one of the most effective treatment for testicular cancer, but it severely affects the reproductive system that ultimately leads to infertility. In addition to its antioxidant activity, zinc has an important role in progression of spermiogenesis. This study aimed to evaluate the effect of zinc on sperm parameters, chromatin condensation and testicular structure after BEP treatment
Materials and Methods: In this experimental study, 40 male rats were divided into 4 groups [control, BEP, BEP+ zinc and zinc] and examined for 2 spermatogenesis periods [i.e. 18 weeks]. The rats in BEP and BEP+ zinc group were treated with BEP at appropriate doses [0.75, 7.5, and 1.5 mg/kg] for three cycles of three weeks. Zinc at a dose of 10 mg/kg/day was administered to BEP+ zinc and zinc groups. After 18 weeks, we assessed sperm parameters, and excessive histone in sperm chromatin using aniline blue staining, as well as testicular structure and germ line cells using periodic acid-Schiff staining
Results: After BEP treatment, significant decreases were observed in normal sperm morphology, motility, and concentration, as well as alterations in rat sperm chromatin condensation and testicular tissue [P<0.001]. Furthermore, after zinc consumption for 9 weeks, we observed significant improvements of sperm parameters and chromatin condensation as well as a significant retrieval of spermatogonia, leydig cells and tubular architecture [P<0.05]
Conclusion: Zinc administration after chemotherapy with BEP in testicular cancer might be potentially useful in declining the off target consequence associated with oxidative stress
ABSTRACT
Objective: The presence of neurotrophic factors is critical for regeneration of neural lesions. Here, we transplanted combination of neurotrophic factor secreting cells [NTF-SCs] and human adipose derived stem cells [hADSCs] into a lysolecithin model of multiple sclerosis [MS] and determined the myelinization efficiency of these cells
Materials and Methods: In this experimental study, 50 adult rats were randomly divided into five groups: control, lysolecithin, vehicle, hADSCs transplantation and NTF-SCs/ hADSCs co-transplantation group. Focal demyelization was induced by lysolecithin injection into the spinal cord. In order to assess motor functions, all rats were scored weekly with a standard experimental autoimmune encephalomyelitis scoring scale before and after cell transplantation. Four weeks after cell transplantation, the extent of demyelination and remyelination were examined with Luxol Fast Blue [LFB] staining. Also, immunofluorescence method was used for evaluation of oligodendrocyte differentiation markers including; myelin basic protein [MBP] and Olig2 in the lesion area
Results: Histological study show somewhat remyelinzation in cell transplantation groups related to others. In addition, the immunofluorescence results indicated that the MBP and Olig2 positive labeled cells were significantly higher in co-cell transplantation group than hADSCs group [P<0.05]. Also, outcome of motor functional test showed significant improvement function in cell transplantation groups, as compared to the others [P<0.01]
Conclusion: Our results indicated that the remyelinization process in co-cell transplantation group was better than other groups. Thus, NTF-SCs/ hADSCs transplantation can be proper candidate for cell based therapy in neurodegenerative diseases, such as MS
ABSTRACT
Objective: This study aimed to isolate and culture SADS cells, investigate their neurogenic capacity and evaluate their application for nerve tissue engineering
Materials and Methods: In this experimental study, SADS cells were isolated from human adipose tissue. After 7-day treatment of SADS cells with insulin, indomethacin and isobutylmethylxanthine, neurogenic differentiation of SADS cells was investigated. During this study, Poly [?-caprolactone] [PCL] and PCL/gelatin nanofibrous scaffolds were fabricated using electrospinning and subsequently nanofibrous scaffolds were coated with platelet-rich plasma [PRP]. SADS cells were also seeded on nanofibrous scaffolds and neurogentic differentiation of these cells on nanofibers was also evaluated. Effect of PRP on proliferation and differentiation of SADS cells on scaffolds was also studied
Results: Our results showed that after 7-day treatment of SADS cells with insulin, indomethacin and isobutylmethylxanthine, SADS cells expressed markers characteristic of neural cells such as nestin and neuron specific nuclear protein [NEUN] [as early neuronal markers] as well as microtubule-associated protein 2 [MAP2] and neuronal microtubule-associated [TAU] [as mature neuronal markers] while mature astrocyte maker [GFAP] was not expressed. MTT assay and SEM results showed that incorporation of gelatin and PRP into the structure of nanofibrous scaffolds has a significant positive influence on the bioactivity of scaffolds. Our results also showed neurogentic differentiation of SADS cells on scaffolds
Conclusion: Our results demonstrated that SADS cells have potential to differentiate into early and mature progenitor neurons, in vitro. PCL/gelatin/PRP was found to be a promising substrate for proliferation of SADS cells and differentiation of these cells into neural cells which make these scaffolds a candidate for further in vivo experiments and suggest their application for nerve tissue engineering
ABSTRACT
Multiple sclerosis [MS] is a chronic inflammatory disease characterized by central nervous system [CNS] lesions that can lead to severe physical or cognitive disability as well as neurological defects. Although the etiology and pathogenesis of MS remains unclear, the present documents illustrate that the cause of MS is multifactorial and include genetic predisposition together with environmental factors such as exposure to infectious agents, vitamin deficiencies, and smoking. These agents are able to trigger a cascade of events in the immune system which lead to neuronal cell death accompanied by nerve demyelination and neuronal dysfunction. Conventional therapies for MS are based on the use of anti-inflammatory and immunomodulatory drugs, but these treatments are not able to stop the destruction of nerve tissue. Thus, other strategies such as stem cell transplantation have been proposed for the treatment of MS. Overall, it is important that neurologists be aware of current information regarding the pathogenesis, etiology, diagnostic criteria, and treatment of MS. Thus, this issue has been discussed according to recent available information
Subject(s)
Humans , Male , Women , Multiple Sclerosis/pathology , Cell- and Tissue-Based Therapy , Genetic Predisposition to Disease , Anti-Inflammatory Agents , Stem Cell TransplantationABSTRACT
Background: Some antidepressant drugs can promote neuronal cell proliferation in vitro as well as hippocampal neurogenesis in human and animal models. Furthermore, adipose tissue is an available source of adult stem cells with the ability to differentiate in to multiple lineages. Therefore, human Adipose-Derived Stem Cells [hADSCs] may be a suitable source for regenerative medical applications. Since there is no evidence for the effect of Paroxetine as the most commonly prescribed antidepressant drug for neurogenic potential of hADSCs, an attempt was made to determine the effect of Paroxetine on proliferation and neural differentiation of hADSCs
Methods: ADSCs were isolated from human abdominal fat. These cells differentiated to neuron-like cells and were treated with Paroxetine. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide [MTT] assay and immunofluorescence technique were used for assessment of cell proliferation and neurogenic differentiation potential of induced cells, respectively
Results: MTT assay analysis showed that Paroxetine significantly increased the proliferation rate of induced hADSCs [p<0.05], while immunofluorescent staining indicated that Paroxetine treatment during neurogenic differentiation could enhance the mean percentage of Nestin and MAP2 [Microtubule-associated protein-2] positive cells but the mean percentage of GFAP [Glial acidic fibrillary protein] positive cells significantly decreased relative to control group [p<0.05]
Conclusion: Our results provide evidence that Paroxetine can promote proliferation and differentiation rate during neurogenic differentiation of hADSCs. Moreover, Paroxetine can reduce gliogenesis of induced hADSCs during neurogenic differentiation
ABSTRACT
Objective: In this study, nano-biocomposite composed of poly [lactide-co-glycolide] [PLGA] and chitosan [CS] were electrospun through a single nozzle by dispersing the CS nano-powders in PLGA solution. The cellular behavior of human adipose derived stem cells [h-ADSCs] on random and aligned scaffolds was then evaluated
Materials and Methods: In this experimental study, the PLGA/CS scaffolds were prepared at the different ratios of 90/10, 80/20, and 70/30 [w/w]%. Morphology, cell adhesion and proliferation rate of h-ADSCs on the scaffolds were assessed using scanning electron microscope [SEM], 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide [MTT] assay and trypan blue staining respectively
Results: H-ADSCs seeded on the matrices indicated that the PLGA/CS composite matrix with aligned nanofibres and higher content of CS nano-powders gave significantly better performance than others in terms of cell adhesion and proliferation rate [P<0.05]
Conclusion: We found that CS enhanced cell adhesion and proliferation rate, and aligned nanofibers guided cell growth along the longitudinal axis of the nanofibers, which would provide a beneficial approach for tissue engineering
ABSTRACT
Due to the restricted potential of neural stem cells for regeneration of central nervous system [CNS] after injury, providing an alternative source for neural stem cells is essential. Adipose derived stem cells [ADSCs] are multipotent cells with properties suitable for tissue engineering. In addition, alginate hydrogel is a biocompatible polysaccharide polymer that has been used to encapsulate many types of cells. The aim of this study was to assess the proliferation rate and level of expression of neural markers; NESTIN, glial fibrillary acidic protein [GFAP] and microtubule-associated protein 2 [MAP2] in encapsulated human ADSCs [hADSCs] 10 and14 days after neural induction. In this experimental study, ADSCs isolated from human were cultured in neural induction media and seeded into alginate hydrogel. The rate of proliferation and differentiation of encapsulated cells were evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide [MTT] assay, immunocytoflourescent and real-time reverse transcriptase polymerase chain reaction [RT-PCR] analyzes 10 and 14 days after induction. The rate of proliferation of encapsulated cells was not significantly changed with time passage. The expression of NESTIN and GFAP significantly decreased on day 14 relative to day 10 [P<0.001] but MAP2 expression was increased. Alginate hydrogel can promote the neural differentiation of encapsulated hADSCs with time passage
ABSTRACT
In recent years, adipose tissue, due to the stem cells contained within, has found a new special place in laboratory and clinical applications. These adipose-derived stem cells [ADSCs] have the same characteristics of bone marrow mesenchymal stem cells [BMSCs]. Although bone marrow [BM] is not easily accessible and its procurements may be painful, most patients possess excess fat which can be obtained by less invasive methods; this makes adipose tissue ubiquitous, available and an ideal large-scale source for research on clinical applications. BMSCs and ADSCs were harvested from three healthy human and were characterized using flow-cytometry. After they were treated for neurosphere formation using basic fibroblast growth factor, epidermal growth factor, B27; terminal differentiation was performed. In this study, we used immunocytochemistry, real time-polymerase chain reaction and western blotting techniques for detection and comparison of Nestin, microtubule-associated protein-2 [MAP-2] and glial fibrillary acidic protein [GFAP] markers in human ADSCs and BMSCs. Under appropriate conditions ADSCs can differentiate into neuron-like cells and express neural markers the same as BMSCs, also the expression of GFAP marker in differentiated cells derived from ADSCs was significantly lower than the cells derived from BMSCs [P < 0.05]. While the expression of MAP-2 marker in both groups was the same. However, due to its advantages and according to our results based on the expression levels of GFAP and MAP-2, adipose tissue rather than BM could represent a more appropriate stem cell source for investigating the application of these cells in understanding the pathophysiology and in treatment of neurodegenerative disorders
ABSTRACT
Currently, relation between reactive oxygen species [ROS] ROS concentration and semen quality was indicated. Saffron has traditionally been not only considered as a food additive but also as a medicinal herb, which has a good antioxidant properties. The aim of this study was to evaluate the protection potency of saffron and vitamin E on sperm chromatin integrity. Thirty adult male Wistar rats divided equally into saffron [100 mg/kg], vitamin E [100 mg/kg] and control [0.5cc distilled water /day] groups. After 60 days, cauda epididymis dissected and sperm cells were used for analysis of sperm chromatin packaging by chromomycin A3 [CMA3] staining, and sperm chromatin susceptibility to acid denaturation by acridine orange [AO] staining. The mean percentage of CMA3 positive sperm was significantly decreased in saffron and vitamin E groups relative to control group [p<0.001]. Moreover, the AO staining results showed that the mean percentage of sperm with DNA damage was significantly decreased in saffron and vitamin E groups as compared with control group [p<0.001]. Our results purposed that saffron can protect sperm against DNA damage and chromatin anomalies
ABSTRACT
Close interaction between retinal pigment epithelium [RPE] and photoreceptors plays an essential role in visual function. The objective of this study is to determine the effects of RPE cells in the differentiation of progenitor derived human embryonic stem cells [hESC] into retinal cells; we developed in vitro co-culture models and compare these models to investigate in which model the expression of photoreceptor markers is superior. It seems the effects of RPE cells on differentiation of retinal progenitor cells [RPCs] through the cell-to-cell contact or with the use of insert and compare of these methods has not been reported yet. Initially, retinal progenitors [RPs] were differentiated from hESC. After isolation of RPE sheet from rabbit eyes, demonstrated these cells maintains the integrity and feature after 2 weeks. Next, we examined the induction of photoreceptors by the co-culture of RPE through insert in 1 week and 2 weeks [indirect] or without insert by the cell-to-cell contact [direct]. The differentiation of retinal cells was verified by protein and gene expression in these three methods. The adherent cells were morphologically examined using phase contrast microscopy and characterized by immunofluorescent staining and reverse transcription polymerase chain reaction [RT PCR]. Evaluation of immunostaining showed that hESC, highly [>80%] can be directed to the RPs fate. Upon co-culture of RPCs with RPE sheet using insert for 2 weeks or by the cell-to-cell contact, these cells differentiated to neural retina and expressed photoreceptor specific markers. However, in direct co-culture, some mature photoreceptor markers like arrestin expressed in compare with indirect co-culture. The expression of late photoreceptor marker could be improved when RPE cells seeded on RPCs in compare with the use of insert.
Subject(s)
Cell Differentiation , Photoreceptor Cells, Vertebrate , Gene Expression , Embryonic Stem Cells , Evaluation Studies as Topic , Reverse Transcriptase Polymerase Chain Reaction , Microscopy, Electron, Scanning Transmission , Coculture TechniquesABSTRACT
Peroxisomes are ubiquitous organelles in nearly all eukaryotes that participate in the metabolism of fatty acids and other metabolites. In order to investigate peroxisome biogenesis and gene profile expression analysis during neurogenesis, we have performed to set a semi-quantitative analysis for two peroxisomal genes expression [Catalase and PEX3] during neurogenesis comparing with stem cell marker genes [Oct4 and Nanog] in P19 cells. In this project we have used P19 cells which are suitable progenitor cells for the study of neurogenesis. Moreover, two different peroxisomal genes were selected to chase both kinetics of peroxisomal matrix protein synthesis [Catalase] and peroxisomal membrane protein biogenesis [Pex3p]. Total RNA extracted from P19 cells, and was used for cDNA synthesis at the next step. Specific primers of Oct4, Nanog, PEX3 and Catalase cDNAs were amplified by RT-PCR for quantitative analysis. Various parameters were changed to optimize the PCR profiles for each gene for further assessments. Here we are going to describe the conditions which we used, e.g.: PEX3 band was detected sharply after 35 cycles and at annealing temperature of 52.5-55.1°C. For PCR of Catalase we observed a sharp band after 35 cycles and annealing temperature of 52.7-55°C. Nanog cDNA was amplified after 36 cycles and between 60.1-64.1°C. Oct4 band was observed sharply after 28 cycles of amplification and annealing temperature in range of 53.4-57°C
ABSTRACT
Chromomycin A3 [CMA3] staining has been used to assess protamine deficiency. The aim of this study was to determine credibility of CMA3 along with semen parameters for assessment of fertility potential. Semen analysis and CMA3 staining were carried out on 234 fertile and 178 subfertile individuals. Semen analysis was assessed according to WHO criteria. Protamine deficiency was assessed by CMA3 staining. Means, range of variables, coefficients of correlation and receiver operating characteristic [ROC] analyses of semen parameters and protamine deficiency were determined. Mean values of three main sperm parameters and the percentage of sperm with negative CM A3 were significantly different between fertile and sub fertile groups. The results of CMA3 assessment showed significant correlation with sperm density, percentage of motility and normal morphology in the total population, while in the subfertile group the results of CMA3 showed significant correlation with sperm density and normal morphology. However in fertile men, the only significant correlation was observed between sperm with negative CMA3 and normal morphology. ROC analyses revealed that CMA3 staining has a higher potential to predict fertility status, compared to semen parameters. Assessment of protamine deficiency could be considered as one of the complementary tests along with semen analysis for assessment of fertility
Subject(s)
Humans , Male , Fertility , Semen , Staining and Labeling , Protamines , Sperm Motility , SpermatozoaABSTRACT
Single nucleotide polymorphism [SNPs] are considered as one of the underlying causes of male infertility. Proper sperm chromatin packaging which involves replacement of histones with protamines has profound effect on male fertility. Over 20 SNPs have been reported for the protamine 1 and 2. The aim of this study was to evaluate the frequency of two previously reported SNPs using polymerase chain reaction [PCR]-restriction fragment length polymorphism [RFLP] approach in 35, 96 and 177 normal, oligozoospermic and azoospermic individuals. These SNPs are: 1. A base pair substitution [G] at position 197 instead of T in protamine type 1 Open reading frame [ORF] including untranslated region, which causes an Arg residue change to Ser residue in a highly conserved region. 2. cytidine nucleotide change to thymidine in position of 248 of protamine type 2 ORF which caused a nonsense point mutation. The two mentioned SNPs were not present in the studied population, thus concluding that these SNPs can not serves as molecular markers for male infertility diagnosis. The results of our study reveal that in a selected Iranian population, the SNP G197T and C248T are completely absent and are not associated with male infertility and therefore these SNPs may not represent a molecular marker for genetic diagnosis of male infertility
Subject(s)
Humans , Male , Infertility, Male , Polymorphism, Single Nucleotide , Mutation/genetics , Polymerase Chain ReactionABSTRACT
Adult stem cells which are derived from different tissues, with their unique abilities to self-renew and differentiate into various phenotypes have the potential for cell therapy and tissue engineering. Human adipose tissue is an appropriate source of mesenchymal stem cells with wide differentiation potential for tissue engineering research. In this study isolated stem cells from human subcutaneous adipose tissue were investigated for chondrogenic potential of adipose-derived stem cells [ADSCs] in pellet culture system treated with transforming growth factor- beta3 [TGF-beta3]. Human ADSCs were isolated from subcutaneous adipose tissue and digested with collagenase type I. Immunocytochemical method for cell surface antigens was done in order to characterize the cells. The isolated cells were treated with chondrogenic medium, supplemented with TGF-beta3 in pellet culture system and harvested after 21 days. Histological staining was used to evaluate the presence of proteoglycan, with alcian blue. Immunohistochemical method performed for the assessment of cartilage'specific type II collagen and aggrecan. Also, in order to confirm our results, we managed RT-PCR technique. Chondrogenesis of ADSCs in pellet culture, induced by TGF-beta3 growth factor. Histological and immunohistochemical methods showed deposition of typical cartilage extracellular matrix components in pellets. RT-PCR analysis of cartilage matrix genes, such as type II collagen and aggrecan, also, confirmed the induction of the chondrocytic phenotype in high-density culture upon stimulation with TGF-beta3. TGF-beta3 promoted chondrogenesis of ADSC in pellet culture system. We suggest that human subcutaneous adipose stem cells could be excellent candidates for the cartilage tissue engineering
Subject(s)
Humans , Adipose Tissue , Subcutaneous Fat , Transforming Growth Factor beta3 , Chondrogenesis , Cell Culture Techniques , Tissue Engineering , Immunohistochemistry , Polymerase Chain Reaction , RNA-Directed DNA Polymerase , Cell DifferentiationABSTRACT
Articular cartilage tissue defects cannot be repaired by the proliferation of resident chondrocytes. Autologous chondrocyte transplantation [ACT] is a relatively new therapeutic approach to cover full thickness articular cartilage defects by in vitro grown chondrocytes from the joint of a patient. Therefore, we investigated the differentiation capability of human chondrocytes maintained in alginate culture. The cartilage specimens obtained from 50 patients who underwent total knee and hip operations at the teaching hospital of Isfahan University of Medical Sciences, Isfahan Iran. Isolated primary chondrocytes were first grown in monolayer cultures for 1 to 6 passages [each passage lasting about 3 days]. At each passage, monolayer cells seeded in alginate culture and investigated morphologically and immuno-cytologically for expression of cartilage-specific markers [collagen type II and cartilage-specific proteoglycans]. The chondrocytes from monolayer passages PI to P4 introduced in alginate cultures regained a chondrocyte phenotype. Cells were interconnected by typical gap junctions and after few days, they produced a cartilage-specific extracellular matrix [collagen type II and cartilage-specific proteoglycans]. In contrast, cells from monolayer passages P5 and P6 did not redifferentiate to chondrocytes in the alginate cultures. Chondrocyte culture was established for the first time in Iran. The alginate culture conditions promote the redifferentiation of dedifferentiated chondrocytes that have still a chondrogenic potential. This procedure opens up a promising approach to produce sufficient numbers of differentiated chondrocytes for ACT. Indeed, in some patients the harvested cells were used immediately and successfully for transplantation
Subject(s)
Humans , Cartilage, Articular/injuries , Cell Differentiation , Cartilage Diseases/therapy , Transplantation, Autologous , Tissue Engineering , Microscopy, Electron, Scanning , Alginates , Collagen Type II , ProteoglycansABSTRACT
In this study, chondrocyte culture was established for the first time in Iran, and calcium alginate was used for longer culture of chondrocyte in vitro. The study was programmed in order to be used for future human chondrocyte transplantation. The cartilage specimen obtained from 50 patients who underwent total knee and hip operations in Isfahan University of Medical Sciences. Cartilage specimens were used for monolayer as well as suspension culture in alginate beads. Approximately 12 +/- 1 millions cells were harvested from the 3[RD] passage. The cells were round with large euchromatic nucleus and several nucleoli and small vacuoles. The cells derived from passages 1 to 4, which were grown up then, in alginate beads, showed higher staining with alcian blue. The harvested cells in some patients were immediately and successfully used for autologus transplantation. This later work will be reported separately
Subject(s)
Humans , Alginates/metabolism , Transplantation, Autologous , Extracellular Matrix Proteins , Cartilage , Cell Culture Techniques , ProteoglycansABSTRACT
According to the recent biological research spectra, tissue engineering is an issue which has become the focus of practical medicine attention for repairing skin, skeletal and cartilage damages. As it is obvious any damage to these tissues neither repairs in a spontaneously manner nor implanting is a reliable proposal idea due to the immunogenicity response. Regarding to what have been mentioned, it is essential to apply basic tools such as scaffold, active biological molecules and cells. One of the most important steps in tissue engineering is finding appropriate cells without immunogenicity and tumorogenicity which can be divided, and able to differentiate to other tissues. Among all kind of stem cells, mesenchymal stem cells, seems to have all of these features. In this review we will introduce with the basis of tissue engineering and its profits
ABSTRACT
The aim of this report has been evaluating protamine content and DNA integrity of two patients with globozoospermia undergoing ICSI. Semen analysis was carried out according to WHO criteria. Protamine deficiency and DNA fragmentation was assessed using Chromomycin A3 and sperm chromatin dispersion assay respectively. ICSI and chemical activation were carried out on inseminated oocyte. Both cases demonstrated high degrees of protamine deficiency, while one of the cases indicated high level of DNA fragmentation, too. High fertilization rates were achieved in both cases. However, embryo transfer did not lead to implantation or pregnancy. Artificial oocyte activation overcomes low fertilization rate reported in cases with high level of protamine deficiency. In the present study, failed implantation in one of the cases may be caused by high DNA fragmentation
Subject(s)
Humans , Male , Sperm Injections, Intracytoplasmic , DNA FragmentationABSTRACT
Sperm DNA is known to contribute one half of the genomic material to the offspring. The integrity of sperm DNA is important in fertilization, embryonic and fetal development, and postnatal child well being. The nature has created multiple barriers that allow only the fittest sperm to reach and fertilize an oocyte. However, assisted reproductive techniques [ART], like IVF and ICSI, may allow sperms with abnormal genomic material to enter the oocyte with minimal effort. This article describes structure of sperm DNA and different mechanism involved in sperm chromatin anomalies and DNA damage. Furthermore, this study elaborates possible sperm selection methods that may improve the outcome of ART