Xeno-free culture of human spermatogonial stem cells supported by human embryonic stem cell-derived fibroblast-like cells

Spermatogonial stem cells （SSCs） divide continuously to support spermatogenesis throughout postnatal life and transmit genetic information to the next generation. Here, we report the successful establishment of the method for the isolation and identification of human SSCs from testicular tissue, and to determine the culture conditions required to expand SSCs on human embryonic stem cell-derived fibroblast-like cells （hdFs）. Large-scale cultures of SSCs were maintained on hdF feeder layers and expanded in the presence of a combination of cytokines and glial cell line-derived neurotrophic factor for at least 2 months. Cell surface marker analysis showed that SSCs retained high levels of alkaline phosphatase activity and stained strongly for anti-stage-specific embryonic antigen （SSEA）-1, OCT4 and CD49f. They also expressed the genes OCT4, SOX3 and STRA8 as detected by reverse transcription polymerase chain reaction （RT-PCR） analysis. These data clearly illustrate a novel approach for the growth of human SSCs using hdFs as feeder cells, potentially eliminating xenogeneic contaminants. This system provides a new opportunity for the study of the regulatory mechanism of the ‘niche＇ that governs SSC self-renewal, and will be a valuable source of SSCs for potential clinical applications.

Postreperfusion hyperkalemia in liver trans-plantation using donation after cardiac death grafts with pathological changes

BACKGROUND: With the increasing use of donation after cardiac death (DCD), especially of the graft liver with steatosis or other pathological changes, the frequency of postreperfu-sion hyperkalemia in liver transplantation has increased sig-niifcantly. The present study aimed to determine the factors associated with developing postreperfusion hyperkalemia in liver transplantation from DCD. METHODS: One hundred thirty-one consecutive adult pa-tients who underwent orthotopic liver transplantation from DCD were retrospectively studied. Based on serum potassium within 5 minutes after reperfusion, recipients were divided into two groups: hyperkalemia and normokalemia. According to preoperative biopsy results, the DCD graft livers were clas-siifed into ifve categories. Univariate analysis was performed using Chi-square test to identify variables that were signiif-cantly different between two groups. Multivariate logistic regression was used to conifrm the risk factors of developing hyperkalemia and postreperfusion syndrome. Correlation analysis was used to identify the relationship between the serum concentration of potassium within 5 minutes after re-perfusion and the difference in mean arterial pressure values before and within 5 minutes after reperfusion. RESULTS: Twenty-two of 131 liver recipients had hyperkale-mia episodes within 5 minutes after reperfusion. The rate of hyperkalemia was signiifcantly higher in recipients of macro-steatotic DCD graft liver (78.6%,P<0.001) than that in recipi-ents of non-macrosteatotic DCD graft liver. The odds ratio of developing postreperfusion hyperkalemia in recipients of&nbsp;macrosteatotic DCD graft liver was 51.3 (P<0.001). Macroste-atosis in the DCD graft liver was an independent risk factor of developing hyperkalemia within 5 minutes after reperfusion. The highest rate of postreperfusion syndrome also occurred in the recipients with macrosteatotic DCD graft liver (71.4%, P<0.001). A strong relationship existed between the serum po-tassium within 5 minutes after reperfusion and the difference in mean arterial pressure values before and within 5 minutes after reperfusion in macrosteatotic DCD graft liver recipients. CONCLUSION: Macrosteatosis in the DCD graft liver was an independent risk factor of developing hyperkalemia and postreperfusion syndrome in the recipients.

GIT1 enhances neurite outgrowth by stimulating microtubule assembly

GIT1,a G-protein-coupled receptor kinase interacting protein,has been reported to be involved in neurite outgrowth.However,the neurobiological functions of the protein remain unclear.In this study,we found that GIT1 was highly expressed in the nervous system,and its expression was maintained throughout all stages of neuritogenesis in the brain.In primary cultured mouse hippocampal neurons from GIT1 knockout mice,there was a significant reduction in total neurite length per neuron,as well as in the average length of axon-like structures,which could not be prevented by nerve growth factor treatment.Overexpression of GIT1 significantly promoted axon growth and fully rescued the axon outgrowth defect in the primary hippocampal neuron cultures from GIT1 knockout mice.The GIT1 N terminal region,including the ADP ribosylation factor-GTPase activating protein domain,the ankyrin domains and the Spa2 homology domain,were sufficient to enhance axonal extension.Importantly,GIT1 bound to many tubulin proteins and microtubule-associated proteins,and it accelerated microtubule assembly in vitro.Collectively,our findings suggest that GIT1 promotes neurite outgrowth,at least partially by stimulating microtubule assembly.This study provides new insight into the cellular and molecular pathogenesis of GIT1-associated neurological diseases.

Novel double-layer Silastic testicular prosthesis with controlled release of testosterone in vitro, and its effects on castrated rats

Testicular prostheses have been used to deal with anorchia for nearly 80 years. Here, we evaluated a novel testicular prosthesis that can controllably release hormones to maintain physiological levels of testosterone in vivo for a long time. Silastic testicular prostheses with controlled release of testosterone （STPT） with different dosages of testosterone undecanoate （TU） were prepared and implanted into castrated Sprague-Dawley rats. TU oil was applied by oral administration to a separate group of castrated rats. Castrated untreated and sham-operated groups were used as controls. Serum samples from every group were collected to measure the levels of testosterone （T）, follicle-stimulating hormone and luteinizing hormone （LH）. Maximum intracavernous penile pressure （ICPmax） was recorded. The prostates and seminal vesicles were weighed and subjected to histology, and a terminal dexynucleotidyl transferase-mediated UTP nick end labeling （TUNEL） assay was used to evaluate apoptosis. Our results revealed that the weights of these tissues and the levels of T and LH showed significant statistical differences in the oral administration and TU replacement groups compared with the castrated group （P 〈 0.05）. Compared with the sham-operated group, the ICPmax, histology and TUNEL staining for apoptosis, showed no significant differences in the hormone replacement groups implanted with medium and high doses of STPT. Our results suggested that this new STPT could release TU stably through its double semi-permeable membranes with excellent biocompatibility. The study provides a new approach for testosterone replacement therapy.