Pre-Implantation Genetic Testing for Monogenic Disorders (PGT-M) in A Family with A Novel Mutation in DPAGT1 Gene.

Congenital disorders of glycosylation (CDG) are a heterogeneous group of systemic disorders characterized by defects in glycosylation of lipids and proteins. One of the rare subtypes of CDG is CDG-Ij (MIM # 608093), which is caused by pathogenic mutations in DPAGT1, a gene encoding UDP-N-acetylglucosaminedolichyl-phosphate N-acetylglucosaminephosphotransferase enzyme. This enzyme catalyzes the first step of oligosaccharide synthesis in glycoprotein biosynthesis pathway. Preimplantation genetic testing for monogenic disorders (PGT-M) is a diagnostic technique that can reveal the genetic profile of embryos before implantation phase of in vitro fertilization (IVF). Currently, this approach is performed using next generation sequencing (NGS) technology. Herein, with the help of whole-exome and Sanger sequencing, we detected a novel missense mutation (NM_001382, c.1217 A>G) in DPAGT1 gene in two families with consanguineous marriage. Using different online bioinformatics tools including MutationTaster, I-Mutant v2.0, T- Coffee, and CADD v1.0, this mutation was predicted pathogen. Finally, after performing PGT-M followed by successful pregnancy, a normal child was born in one of these families. In conclusion, we identified a novel pathogenic mutation in DPAGT1 in a family with multiple members affected by CDG, which extends the range of pathogenic variants associated with CDG and therefore facilitates early detection of the disease.

Stem Cells from Human Exfoliated Deciduous Tooth Exhibit Stromal-Derived Inducing Activity and Lead to Generation of Neural Crest Cells from Human Embryonic Stem Cells.

In this article which was published in Cell J, Vol 17, No 1, Spring 2015, on pages 37-48, we found that Figure 1H, Figure 2 (OTX2, row 3), and Figure 3 (row 4) had been published incorrectly. The following figures are corrected. The authors would like to apologies for any inconvenience caused.

ERK1/2 is a key regulator of Fndc5 and PGC1α expression during neural differentiation of mESCs.

Fibronectin type III domain containing 5 (Fndc5) has already been distinguished to be involved in neural differentiation. However, cellular events of Fndc5 function are still ambiguous in the nervous system. One approach to shed light on duty of this protein in the nervous system is to find its cross-talks with various signaling pathways with defined characteristics and roles. Identification of the underlying molecular mechanism which controls Fndc5 expression and switches its activity up and down enables us to find out the Fndc5 functional map in the nervous system and other human body systems. Retinoic acid (RA) is a bio-small molecule which exerts its role as a neural inducer in the neurodevelopmental process of neural tube. RA up-regulates the expression of various genes involved in neural differentiation process via two distinct pathways: the genomic and the non-genomic. Our previous study has revealed that RA induces Fndc5 expression during neural differentiation process. In this study we have evaluated our hypothesis about the non-genomic up regulation of Fndc5 expression by RA. Interestingly we have identified that there is an association between ERK signaling pathway and Fndc5 expression. Furthermore, inhibition of this pathway by PD0325901 dramatically reduced Fndc5 mRNA level, while activating the pathway up-regulated Fndc5 transcription. In addition, it has been proven that ERK1/2 modulation via RA has more significant controlling effect on Fndc5 promoter rather than bFGF. This led us to conclude that RA enhances Fndc5 expression through a non-genomic pathway via the ERK signaling pathway.

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.

Assessment of potassium current in Royan B(1) stem cell derived cardiomyocytes by patch-clamp technique.

Embryonic stem cells are capable of differentiating to variety of cell tissues including cardiomyocytes. This developmental change is accompanied with a great deal of ion channel expression and functions. Mouse stem cell derived cardiomyocytes were prepared and separated to yield isolated single cell suspension for cell current recording. In the present study some properties of the K(+)-current in Royan B(1) stem cell derived cardiomyocytes were investigated using whole cell patch-clamp technique. When the holding potential was - 60 mV, in some cells a major outward current was elicited by square depolarizing pulses from -60 mV to +50 mV. This outward current was sustained for the duration of 300 ms test pulse. The sustained outward K(+) current was inhibited by tetraethylammonium (10 mM) indicating the activity of Ca(2+) activated K(+) channel in these cells. In some of the cells with 0.2 mM 3,ethylene glycol-bis (ß-aminoethyl ether) N,N,N(`),N(`)-tetraacetic acid in the pipette, only a very small outward current was recorded which suggests that in these cells the voltage activated K(+) channels is either absent or if existed it is not fully functional. Other cells were in far between, indicating that voltage activated K(+) channels are developing in these cells but it is not yet fully functional. In conclusion, we have identified functional large conductance Ca(2+) activated K(+) channel in Royan B(1) stem cell derived cardiomyocytes.

Embryonic stem cell sphere: a controlled method for production of mouse embryonic stem cell aggregates for differentiation.

OBJECTIVES: Embryonic stem cells (ESCs) are of significant interest as a renewable source of nonproliferating cells. Differentiation of ESCs is initiated by the formation of embryoid bodies (EBs). Standard methods of EB formation are limited in their production capacity, in any variations in EB size and formation of EBs through frequent passages. Here we have reported the utility of a microencapsulation technique for overcoming these limitations by mass production of mouse ESCs in alginate beads called ESC spheres. METHODS: The mouse ESCs were encapsulated in 1.2% alginate solution and cocultured on a feeder layer. The cells were evaluated by flow cytometry, in vitro differentiation, immunofluorescence, and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Analysis of encapsulated ESC spheres by flow cytometry showed similar percentages of Oct-4 and stage-specific embryonic antigen-1 (SSEA-1) expression in comparison with routine culture of ESCs. Moreover, the ESC spheres maintained a pluripotency potential which was comparable with ESCs cultured on feeder cells directly, as demonstrated by immunofluorescence and RT-PCR. CONCLUSIONS: The results demonstrated that alginate encapsulation as a simple bioreactor, provides a scalable system for mass undifferentiated ESC sphere production with similar sizes and without the need for frequent passages for differentiation and clinical and pharmaceutical applications.