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ABSTRACT: Cotransplantation of neural progenitors (NPs) with Schwann cells (SCs) might be a way to overcome low rate of neuronal differentiation of NPs following transplantation in spinal cord injury (SCI) and the improvement of locomotor recovery. In this study, we initially generated NPs from human embryonic stem cells (hESCs) and investigated their potential for neuronal differentiation and functional recovery when cocultured with SCs in vitro and cotransplanted in a rat acute model of contused SCI. Cocultivation results revealed that the presence of SCs provided a consistent status for hESC-NPs and recharged their neural differentiation toward a predominantly neuronal fate. Following transplantation, a significant functional recovery was observed in all engrafted groups (NPs, SCs, NPs + SCs) relative to the vehicle and control groups. We also observed that animals receiving cotransplants established a better state as assessed with the BBB functional test. Immunohistofluorescence evaluation 5 weeks after transplantation showed invigorated neuronal differentiation and limited proliferation in the cotransplanted group when compared to the individual hESC-NP-grafted group. These findings have demonstrated that the cotransplantation of SCs with hESC-NPs could offer a synergistic effect, promoting neuronal differentiation and functional recovery.
Cell Transplantation 09/2011; 21(5):827-43. · 5.13 Impact Factor
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Nosrat Nourbakhsh,
Mitra Soleimani,
Zahra Taghipour,
Khadijeh Karbalaie,
Seeid-Behrouz Mousavi,
Ardeshir Talebi,
Fatemeh Nadali,
Somayeh Tanhaei,
Gholam-Abbas Kiyani,
Marziyeh Nematollahi,
Farzaneh Rabiei, Mohammad Mardani,
Hamid Bahramiyan,
Mahmood Torabinejad,
Mohammad-Hossein Nasr-Esfahani,
Hossein Baharvand
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ABSTRACT: Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative, clonogenic and multipotent stem cells with a neural crest cell origin. Additionally, they can be collected with minimal invasiveness in comparison with other sources of mesenchymal stem cells (MSCs). Therefore, SHED could be a desirable option for potential therapeutic applications. In this study, SHEDs were established from enzyme-disaggregated deciduous dental pulp obtained from 6 to 9 year-old children. The cells had typical fibroblastoid morphology and expressed antigens characteristic of MSCs, STRO1, CD146, CD45, CD90, CD106 and CD166, but not the hematopoietic and endothelial markers, CD34 and CD31, as assessed by FACS analysis. Differentiation assessment revealed a strong osteogenic and adipogenic potential of SHEDs. In order to further evaluate the in vitro differentiation potential of SHED into neural cells, a simple short time growth factor-mediated induction was used. Immunofluorescence staining and flow cytometric analysis revealed that SHED rapidly expressed nestin and b-III tubulin, and later expressed intermediate neural markers. In addition, the intensity and percentages of nestin and b-III tubulin and mature neural markers (PSA-NCAM, NeuN, Tau, TH, or GFAP) increased significantly following treatment. Moreover, RT-PCR and Western blot analyses showed that the neural markers were strongly up-regulated after induction. In conclusion, these results provide evidence that SHED can differentiate into neural cells by the expression of a comprehensive set of genes and proteins that define neural-like cells in vitro. SHED cells might be considered as new candidates for the autologous transplantation of a wide variety of neurological diseases and neurotraumatic injuries.
The International journal of developmental biology 01/2011; 55(2):189-95. · 2.16 Impact Factor
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ABSTRACT: Schwann cells (SCs) can be used to repair both the peripheral and central nervous systems. Therefore, establishment of a procedure to obtain activated, highly proliferative SCs, in an appropriate time for clinical applications, is a prerequisite. Purification is complicated by contamination with fibroblasts which often become the predominant cell type in an in vitro SC culture. This study describes a novel and efficient method to enrich SCs by utilizing the differential detachment properties of the two cell types. In culture, cells were treated with two different media and the chelator, EGTA, which detached SCs faster than fibroblasts and allowed for easy isolation of SCs. Within seven days, high yields of SCs with a purity of greater than 99% were achieved. This was confirmed by immunostaining characterization and flow-cytometric analyses using an antibody against the p75 low affinity nerve growth factor receptor (p75LNGFR). The entire procedure was completed in approximately 21 days. This method has the advantage of being technically easier, faster, and more efficient than other previously described methods. An SC culture that was about 99% homogenous was achieved.
Biotechnology Letters 03/2010; 32(6):781-6. · 1.68 Impact Factor
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ABSTRACT: Understanding neuroectoderm formation and its subsequent diversification to functional neural subtypes remains elusive. We have shown here for the first time that embryonic stem cells (ESCs) can differentiate into neurons and motor neurons (MNs) by using a coculture embryonic notochord model in vitro. Mouse ESCs were induced to form neural precursors via timed exposure to retinoic acid (RA) using the 4-/4+ RA protocol. These cells were then cocultured with alginate bead-encapsulated notochords isolated from Hamburger and Hamilton stage 6-10 chick embryos. The use of notochord alone was not able to induce neural differentiation from ESCs, and, therefore, notochord does not possess neural inducing activity. Hence, the most successful neuronal cells and MN differentiation was only observed following the coculture of RA-pretreated ESCs with notochord. This resulted in a significantly greater number of cells expressing microtubule-associated protein-2 (MAP2), HB9, choline acetyltransferase (ChAT) and MN-specific genes. While further characterization of these differentiated cells will be essential before transplantation studies commence, these data illustrate the effectiveness of embryonic notochord coculture in providing valuable molecular cues for directed differentiation of ESCs toward an MN lineage.
Stem cells and development 05/2008; 18(2):259-67. · 4.15 Impact Factor
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ABSTRACT: Sperm premature chromosomal condensation (PCC) has been associated with failed fertilization. Previous studies suggest that protamine deficiency or failed oocyte activation may make spermatozoa prone to PCC. However, it is not clear which of these two factors has a more profound effect on fertilization failure. In order to distinguish between these two phenomena, oocytes that failed to fertilize after intracytoplasmic sperm injection (ICSI) were artificially activated and the association between protamine deficiency and PCC was evaluated in the remaining oocytes that failed to fertilize. The results of this study reveal that after artificial activation, fertilization rate post-ICSI increased from 59.95 to 87.7% and PCC spermatozoa appeared to be present in over 50% of the remaining oocytes that failed to fertilize. The percentage of sperm PCC was significantly higher in protamine deficient samples, thus suggesting that after failed oocyte activation, sperm PCC induced by protamine deficiency may be considered as an alternative cause of failed fertilization post-ICSI. Furthermore, the results of this study did not show any correlation between pronuclei size asynchrony and protamine deficiency.
Reproductive biomedicine online 04/2007; 14(4):422-9. · 2.04 Impact Factor
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ABSTRACT: The aim of this study was to evaluate the effect of sperm DNA damage and protamine deficiency on fertilization and embryo development post-intracytoplasmic sperm injection (ICSI), and also to assess the effect of protamine deficiency on DNA damage. Semen samples were collected from 28 patients participating in the ICSI programme. Following sperm preparation and ICSI, the remaining processed semen samples were used to assess protamine deficiency and DNA damage employing chromomycin A3 (CMA3) staining and comet assay, respectively. Comet parameters, CMA3 percentage positivity, fertilization rate, embryo cleavage score and embryo quality score were assessed. Except for CMA3, none of the comet parameters showed significant correlation with fertilization rate. However, among comet parameters, head area and head intensity showed positive correlation with the embryo cleavage score, while comet mean intensity and head mean intensity showed a significant negative correlation with CMA3 positivity. Results of this study demonstrate that DNA fragmentation is more frequent in protamine-deficient spermatozoa. Unlike protamine deficiency, sperm DNA fragmentation does not preclude fertilization. Nonetheless, embryos derived from spermatozoa with high DNA damage have a lower potential to reach blastocyst stage.
Reproductive biomedicine online 09/2005; 11(2):198-205. · 2.04 Impact Factor
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ABSTRACT: MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is commonly used as a cell proliferation assay. The objective of this study was to evaluate the capability of MTT assay to discriminate between viable and nonviable sperms and compare it sefficiency with E&N (eosin and nigrosin) and HOST (hypo-osmotic swelling test).
MTT assay was modified to obtain optimal result for assessment of sperm viability. After standardization of method, MTT, E&N, and HOST were carried out simultaneously on 57 semen samples from patients referring to Isfahan Fertility and Infertility Center. The correlation coefficient between these tests and sperm motility was calculated using the SPSS statistical program. Specificity and sensitivity of each test was also obtained.
The optimal conditions for sperm MTT viability assay were 2 h after addition of sperm to MTT in HAM'S F10 + 25 mM HEPES + 10% HSA at 37 degrees C and pH 7.4-7.45. Inter- and intra-assay coefficients of variations were 9 and 7%, respectively. The sensitivity and specificity for sperm MTT viability assay, E&N, and HOST were 97,98, and 99%, and 100, 100, and 83% respectively. High significant correlations were obtained between sperm MTT viability assay, E&N, HOST and motility.
Sperm MTT viability assay can be used as a diagnostic test for discrimination of viable sperms from sperm population.
Journal of Assisted Reproduction and Genetics 10/2002; 19(10):477-82. · 1.84 Impact Factor
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ABSTRACT: Background: Sperm selection for ICSI based on morphology and motility might not be relevant to chromatin integrity. Thus sperm selection based on sperm characteristics has been suggested. Objective: The aim of this study was to compare the efficiency of Zeta method with routine Density Gradient Centrifugation method (DGC) for the selection of sperm with higher DNA integrity. Materials and Methods: Semen samples were obtained from 63 individuals referring to Andrology Unit of Isfahan Fertility and Infertility Center. Semen analysis was carried out according to WHO criteria. Each semen sample was divided into three equal portions. One portion was used as control, the second portion was used for Zeta method and the third portion underwent DGC method. Each portion was evaluated to DNA integrity by TUNEL assay. Student t-test was carried out using SPSS and p-value lower than 0.05 was considered significant. Results: The mean number of sperm DNA fragmentation in Zeta and DGC methods were significantly decreased compare to the control group (p<0.001). In addition, Zeta method was more efficient than the DGC method in the selection of sperm with intact DNA (p<0.001). Conclusion: The Zeta method appears to be a suitable procedure to recover sperm with normal DNA integrity.
Iranian Journal of Reproductive Medicine (ISSN: 1680-6433) Vol 7 Num 2.
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ABSTRACT: Background: The aim of present study is to evaluate the effect of PolyVinyl Pyrrolidone (PVP) routinly used during ICSI procedure on sperm membrane integrity, and sperm chromatin status. Materials and Methods: This study was carried out on 21 semen samples from the infertile men referring to Isfahan Fertility and Infertility Center. The processed semen samples were divided into two portions. One portion was added to Ham's F10+ 10% PVP, and the other portion was added to Ham's F10 as a control group. Hypo-osmotic swelling test (HOST), SDS, and SDS+EDTA tests were carried out on the control and PVP groups at 15, 30, and 60min. Results: The results show that sperm membrane integrity measured by HOST, and sperm chromatin stability measured by SDS test, reduces by increasing the exposure time to PVP. However, the ability of sperm chromatin undergoing decondensation (that has been assessed by SDS+EDTA), does not show any changes by increasing the exposure time to PVP. Conclusion: The results of current study shows that reducing the exposure time of sperm to PVP may protect sperm membrane, and chromatin integrity.
