Fndc5 knockdown significantly decreased neural differentiation rate of mouse embryonic stem cells.

Fibronectin type III domain-containing 5 protein (Fndc5) or peroxisomal protein, is a type I membrane protein that has 209 amino acid residues. Previous studies by our group have shown an increase in its expression after retinoic acid treatment of mouse embryonic stem cells (mESCs) during the process of neural differentiation, leading us to conclude that it might be involved in neurogenesis. In the present study, we have constructed an inducible short hairpin RNA (shRNA) vector that is expressed under induction by doxycycline. Next, we generated a stably transformed mESCs line that expressed shRNA against the Fndc5 gene. The knockdown of Fndc5 was performed in two stages of mESC neural differentiation during and post-neural progenitor (NP) formation. Our results indicated that in the process of NPs formation, decreased Fndc5 expression significantly reduced expression of NPs and mature neuronal markers which modulated neuronal differentiation. Decreased Fndc5 expression during the post-NPs formation stage also caused significant reduction in the levels of mature neuronal markers. Fndc5 knockdown during both stages significantly affected both neuronal and astrocytes maturation. We have concluded that Fndc5 expression is required for the appropriate neural differentiation of mESCs. These data confirm the importance of Fndc5 in the generation and development of the nervous system.

Functional expression of potassium channels in cardiomyocytes derived from embryonic stem cells.

Royan B(1) stem cell can be differentiated to specialized cell types including cardiomyocytes. This developmental change is accompanied with expression of various K(+) channel types. The aim of this study was to detect functional expression of K(+) currents from stem cell stage and one week and two weeks after differentiation into cardiomyocyte. Mouse stem cell derived cardiomyocytes (ES-cardiomyocytes) were isolated to single cell suspension for K(+) current recording using whole cell patch-clamp technique. The predominant depolarizing current in ES-cardiomyocytes was a tetraethylammonium (TEA) (10 mM) sensitive current which was partially blocked by nifedipine (1 µM) and attenuated by increasing concentration of EGTA (10 mM) in the pipette solution. Pharmacology and electrophysiological properties of this oscillatory sustained current very well matched with characteristics of Ca(2+) activated K(+) current. In addition there was another kind of sustained outward K(+) current which was resistance to TEA but was inhibited by 3,4-diaminopyridine. The characteristic features of this current indicate that this current was due to activation of delayed rectifier K(+) channels. RT-PCR study also confirmed expression of these two types of K(+) channels in ES-cardiomyocytes. Therefore, present study shows functional expression of two types of K(+) ionic current in ES-cardiomyocytes.

Quantitative expression of phospholipase C zeta, as an index to assess fertilization potential of a semen sample.

BACKGROUND: Failed fertilization post-ICSI has been mainly attributed to the sperm's inability to induce oocyte activation. Phospholipase C zeta (PLCζ) is considered to be one of the factors for the induction of oocyte activation. The aim of this study was to quantitatively assess the expression of PLCζ in globozoospermic men or those with previously low or failed fertilization in comparison with fertile men or those with high fertilization potential. In addition, the relationship between expression of PLCζ and that of other sperm markers was evaluated. METHODS: Real-time PCR was carried out to evaluate relative expression of PLCζ mRNA. Chromatin maturity and acrosin activity were assessed by CMA3 staining and a colorimetric method. RESULTS: The expression of PLCζ was significantly lower in globozoospermic men (P< 0.01, n= 8) or individuals with previously low or failed fertilization (P< 0.01, n= 36) in comparison to fertile men (n= 24). In addition, a significant difference was observed between globozoospermic (P< 0.01) and individuals with previously low or failed fertilization (P= 0.003) in comparison to high fertilization individuals (n= 17). Expression of PLCζ was not correlated with either chromatin maturity or acrosin activity. However, a significant correlation was observed between the percentage of fertilization and relative expression of PLCζ (r= 0.4, P< 0.01). CONCLUSION: In this study, for the first time, we have shown that assessment of relative expression of PLCζ may provide a useful marker for the ability of sperm to induce oocyte activation after ICSI.