Novel method to obtain highly enriched cultures of adult rat Schwann cells.

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.

Evaluation of Fas positive sperm and complement mediated lysis in subfertile individuals.

PURPOSE: Evaluation of Fas receptor on surface of sperm, as an apoptotic marker, using flow cytometry and confirming the results using an antibody-antigen complex through the classic complement pathway. MATERIALS AND METHODS: Semen samples were obtained from 10 fertile and 73 infertile individuals with diagnoses of male factor infertility. Expressions of Fas receptor and phosphatidyl serine on sperm were assessed by flow cytometry. Fas expression was further assessed by antibody-antigen complex through the complement pathway. Lysis was detected via PI (propidium iodide) staining. RESULTS: The mean Fas expression was considerably lower than previously reported values. No significant differences in the percentage of PI were detected before and after activation of the classic complement pathway. Annexin V positive samples showed low Fas expression. CONCLUSION: Our results have confirmed the presence of selected apoptotic markers such as Fas or phosphatidyl serine on ejaculate sperm, but suggest that Fas expression is low. Further studies are required to investigate the "abortive apoptosis" mechanism through Fas/Fas L.

Transplantation of undifferentiated and induced human exfoliated deciduous teeth-derived stem cells promote functional recovery of rat spinal cord contusion injury model.

Regarding both the neural crest origin and neuronal potential of stem cells from human exfoliated deciduous teeth (SHED), here, we assessed their potential in addition to neural induced SHED (iSHED) for functional recovery when transplanted in a rat model for acute contused spinal cord injury (SCI). Following transplantation, a significant functional recovery was observed in both groups relative to the vehicle and control groups as determined by the open field locomotor functional test. We also observed that animals that received iSHED were in a better state as compared with the SHED group. Immunohistofluorescence evaluation 5 weeks after transplantation showed neuronal and glial differentiation and limited proliferation in both groups. However, myelin basic protein and chondroitin sulfate proteoglycan NG2-oligodendrocyte markers-were increased and glial fibrillary acidic protein-astrocyte marker-was decreased in the iSHED group in comparison with the SHED group. These findings have demonstrated that transplantation of SHED or its derivatives could be a suitable candidate for the treatment of SCI as well as other neuronal degenerative diseases.

Differentiation of human ES cell-derived neural progenitors to neuronal cells with regional specific identity by co-culturing of notochord and somite.

Limitations to the in vivo study of human nervous system development make it necessary to design an in vitro model to evaluate the in vivo effects of surrounding tissues on neurogenesis and regional identity of the human neural plate. Rostral neural progenitors (NPs) were initially generated from adherent human embryonic stem cells (hESCs) in a defined condition and characterized. Then, to find the role of somites (S) and notochords (N) in rostro-caudal (RC) and dorso-ventral (DV) patterning of neuronal cells, NPs were co-cultured with microencapsulated chicken S or N in alginate beads. In this study, N induced more neurogenesis as evaluated by expression of TUJ1 and MAP2-positive cells. Additionally, N induced spinal cord ventral brachiothoracic identity as well as NPs proliferation. We observed a synergic effect on motoneuron induction when S and N were used together. Moreover, S induced hindbrain identity in differentiated neuronal cells from NPs. These results indicate that highly enriched NPs can be generated in an adherent and defined system from hESCs. Moreover, S and N tissues highly influenced neuronal differentiation in point of proliferation, neurogenesis, and RC and DV regional identity. These results indicate a very simple and efficient protocol to mimic in vivo events of human neural development in vitro which is important in the context of developmental neuroscience and cellular replacement therapies.

Flow Cytometry: A Novel Approach for Indirect Assessment of Protamine Deficiency by CMA3 Staining, Taking into Account the Presence of M540 or Apoptotic Bodies.

BACKGROUND: Chromomycin A3 (CMA3) staining, either by the slide method or fluorescence microscopy, is widely used for indirect assessment of protamine deficiency in a semen sample. Flow cytometry is the most suitable tool to improve assessment accuracy, both in terms of statistical analysis and for prevention of observer variation. This study provides a simple procedure to account for merocyanine 540 (M540) or apoptotic bodies, which result in underestimation of the percentage of CMA3 positivity, by using propidium iodide (PI) staining. Therefore, this study aims to evaluate the percentage of CMA3 by PI staining to exclude M540 bodies that prevent underestimation of CMA3 staining. MATERIALS AND METHODS: This study is an experimental study. Semen samples collected from 104 infertile men who referred to the Andrology Unit of the Isfahan Fertility and Infertility Center were initially assessed according to World Health Organization (WHO) criteria. Samples were washed twice with Ham's. Each sample was divided into two portions, a control and the other processed for density gradient centrifugation (DGC). Each portion was assessed for CMA3 staining by both the slide and flow cytometry methods. Coefficients of correlation and student t-test were carried out using the Statistical Package for the Social Studies (SPSS 11.5). RESULTS: Detection of CMA3 staining was more appropriate with fluorescence detector 3 (FL-3) rather than fluorescence detector 2 (FL-2) in the evaluation of protamine deficiency to exclude M540 bodies. CONCLUSION: This study, for the first time, provides the basis for assessment of CMA3 staining for flow cytometry. However, since the maximum excitation for CMA3 is not covered by the 488 nm laser, we recommend further experimentation using a flow cytometer with optimal excitation.