In Vitro Differentiation of Neural-Like Cells from Human Embryonic Stem Cells by A Combination of Dorsomorphin, XAV939, and A8301

Objective: Motor neuron differentiation from human embryonic stem cells (hESCs) is a goal of regenerative medicine to provide cell therapy as treatments for diseases that damage motor neurons. Most protocols lack adequate efficiency in generating functional motor neurons. However, small molecules present a new approach to overcome this challenge. The aim of this research is to replace morphogen factors with a cocktail of efficient, affordable small molecules for effective, low cost motor neuron differentiation. Materials and Methods: In this experimental study, hESCs were differentiated into motor neuron by the application of a small molecule cocktail that consisted of dorsomorphin, A8301, and XAV939. During the differentiation protocol, we selected five stages and assessed expressions of neural markers by real-time polymerase chain reaction (PCR), immunofluorescence staining, and flow cytometry. Motor neuron ion currents were determined by whole cell patch clamp recording. Results: Immunofluorescence staining and flow cytometry analysis of hESC-derived neural ectoderm (NE) indicated that they were positive for NESTIN (92.68%), PAX6 (64.40%), and SOX1 (82.11%) in a chemically defined adherent culture. The replated (hESC)-derived NE differentiated cells were positive for TUJ1, MAP2, HB9 and ISL1. We evaluated the gene expression levels with real-time reverse transcriptase-PCR at different stages of the differentiation protocol. Voltage gated channel currents of differentiated cells were examined by the whole-cell patch clamp technique. The hESC-derived motor neurons showed voltage gated delay rectifier K+, Na+ and Ca2+ inward currents. Conclusion: Our results indicated that hESC-derived neurons expressed the specific motor neuron markers specially HB9 and ISL1 but voltage clamp recording showed small ionic currents therefore it seems that voltage gated channel population were inadequate for firing action potentials

The effect of varicocelectomy on serum testosterone level in infertile men with varicocele: an interventional study

Background: Varicocele is recognized as the most common cause of male factor infertility and is found in 15% of the general population. This prevalence increases to 35% in men presenting with primary infertility and between 70 to 80% in men presenting with secondary infertility. The effect of varicocele on Leydig cell function and testosterone production has been always a question. In this study we examined the effect of varicocelectomy on serum testosterone. Methods: This research protocol was approved by the institutional review board at Royan Institute in infertility department and also this study has been done in Royan Institute (Tehran, Iran) during one year since September 2012 till October 2013. In this cross-sectional study, Serum levels of total testosterone in 79 men with clinical varicocele and in 70 fertile men who served as a control group were compared. Men aged 23–46 years with clinically palpable varicoceles as determined by physical examination were studied. Three to 6 months, testosterone levels were measured again after varicocelectomy, then testosterone levels were compared before and after varicocelectomy. Results: The mean of serum testosterone levels before surgery in infertile men with varicocele and fertile men were 590(230) vs. 583(237) ng/dl respectively. No statistically significant changes were noted in serum testosterone levels for any groups. Three month after varicocelectomy mean serum testosterone levels were significantly increased in infertile men with varicocele compared with preoperative levels from 590 (230) to 663 (242) ng/dl (P=0.009). Also the testis volume of patients were examined, which were divided into two groups included the men with testis volume less than 16 ml (<16) and more than 16 ml (≥16). Conclusion: In infertile men affected with clinical varicocele, varicocelectomy seems to have caused positive impact on the level of serum testosterone increase. It is thought that positive effect is probably caused by improvement of the Leydig cell functions which induce the increase of serum testosterone level