Methylation status of H19/IGF2 differentially methylated region in in vitro human blastocysts donated by healthy couples

Backgrund: Imprinted genes are a unique subset of few genes, which have been differentially methylated region [DMR] in a parental origin-dependent manner during gametogenesis, and these genes are highly protected during pre-implantation epigenetic reprogramming. Several studies have shown that the particular vulnerability of imprinting genes during suboptimal pre- and peri-conception micro-environments often is occurred by assisted reproduction techniques [ART]. This study investigated the methylation status of H19/IGF2 DMR at high-quality expanding/expanded human blastocysts donated by healthy individuals to evaluate the risks linked to ART

Method: Methylation levels of H19/IGF2 DMR were analyzed by bisulfite conversion and sequencing at 18 CpG sites [CpGs] located in this region

Result: The overall percentage of methylated CpGs and the proportion of hyper-methylated clones of H19/IGF2 DMR in analyzed blastocysts were 37.85 +/- 4.87% and 43.75 +/- 5.1%, respectively. For validation of our technique, the corresponding methylation levels of peripheral human lymphocytes were defined [49.52 +/- 1.86% and 50%, respectively]

Conclusion: Considering the absence of in vivoproduced human embryos, it is not possible to conclude that the methylation found in H19/IGF2 DMR is actually normal or abnormal. Regarding the possible risks associated with ART, the procedures should be optimized in order to at least reduce some of the epigenetic risks

Analysis of promyelocytic leukemia in human embryonic carcinoma stem cells during retinoic acid-induced neural differentiation

Background: Promyelocytic leukemia protein [PML] is a tumor suppressor protein that is involved in myeloid cell differentiation in response to retinoic acid [RA]. In addition, RA acts as a natural morphogen in neural development

Objectives: This study aimed to examine PML gene expression in different stages of in vitro neural differentiation of NT2 cells, and to investigate the possible role of PML in pluripotency and/or neural development

Materials and Methods: RA was used as a neural inducer for in vitro neural differentiation of NT2 cells. During this process PML mRNA and protein levels were assessed by quantitative real time RT-PCR [QRT-PCR] and Immunoblotting, respectively. Furthermore bisulfite sequencing PCR [BSP] was used to assess PML promoter methylation in NT2 cells and NT2 derived neuronal precursor cells [NT2.NPCs]

Results: QRT-PCR results showed that, PML had maximum expression with significant differences in NT2 derived neuronal precursor cells relative to NT2 cells and NT2 derived neural cells [NT2.NCs]. Numerous isoforms of PML with different intensities appeared in immunoblots of pluripotent NT2 cells, NT2.NPCs, and NT2.NCs. Furthermore, the methylation of the PML promoter in NT2.NCs was 2.6 percent lower than NT2 cell

Conclusions: The observed differences in PML expression in different cellular stages possibly could be attributed to the fact that PML in each developmental state might be involved in different cell signaling machinery and different functions. The appearance of different PML isoforms with more intensity in neural progenitor cells; may suggest apossible role for this protein in neural development

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

The neural crest is a transient structure of early vertebrate embryos that generates neural crest cells [NCCs]. These cells can migrate throughout the body and produce a diverse array of mature tissue types. Due to the ethical and technical problems surrounding the isolation of these early human embryo cells, researchers have focused on in vitro studies to produce NCCs and increase their knowledge of neural crest development. In this experimental study, we cultured human embryonic stem cells [hESCs] on stromal stem cells from human exfoliated deciduous teeth [SHED] for a two-week period. We used different approaches to characterize these differentiated cells as neural precursor cells [NPCs] and NCCs. In the first co-culture week, hESCs appeared as crater-like structures with marginal rosettes. NPCs derived from these structures expressed the early neural crest marker p75 in addition to numerous other genes associated with neural crest induction such as SNAIL, SLUG, PTX3 and SOX9. Flow cytometry analysis showed 70% of the cells were AP2/P75 positive. Moreover, the cells were able to self-renew, sustain multipotent differentiation potential, and readily form neurospheres in suspension culture. SHED, as an adult stem cell with a neural crest origin, has stromal-derived inducing activity [SDIA] and can be used as an NCC inducer from hESCs. These cells provide an invaluable resource to study neural crest differentiation in both normal and disordered human neural crest development

in vitro study on chick somite ability to express cerberus, chordin, FGF8, follistatin, and noggin transcripts

In vitro simulation of developmental processes is an invaluable tool to shed light on the intrinsic mechanism of developmental biosystems such as central nervous system in mammals. Chick somites have been used to simulate the neural differentiation of human neural progenitor cells. In the present study, we aimed to indicate whether somites have the ability to express required neural differentiation factors at mRNA level. Chick embryos were isolated from the yolk surface of the fertilized eggs and somites were subsequently isolated from embryos under a dissecting microscope. Total RNA of the somites was extracted and RT-PCR carried out with specific primers of cerberus, chordin, FGF8, follistatin and noggin. Data showed that five aforementioned factors were co-expressed after 7 days in vitro by somites. We concluded that neural induction property of somites appeared by production of required neural differentiation factors including cerberus, chordin, FGF8, follistatin and noggin

Constructing a mouse Oct4 promoter/EGFP vector, as a whole-cellular reporter to monitor the pluripotent state of cells

The transcription factor Oct-4, is an important marker of undifferentiating level and a key regulating factor for maintenance of pluripotency in cells. Establishment of an Oct-4 promoter-based reporter ystem is an appropriate tool for monitoring the differentiation of embryonic stem cells both in vivo and in vitro. In the present study, we report construction of a recombinant vector, pDB2 Oct4 promoter/EGFP, in which expression of Enhanced Green Fluorescent Protein [EGFP] was controlled by the mouse Oct-4 promoter. In transfected mouse embryonic stem cells with this vector, EGFP was predicted to be specifically expressed in pluripotency state. After transfection, high-level expression of EGFP under the control of Oct-4 promoter was observed in manipulated embryonic stem cells. Thus, our new cellular reporter showed that both the properties of embryonic cells and expression the EGFP could be of great help in studying the differentiating and reprogramming mechanisms of mESCs

Bone morphogenetic protein-4 influences neural differentiation of induced mouse mesenchymal stem cells

Members of the transforming growth factor-beta superfamily, including bone morphogenetic protein 4 [BMP4], have been implicated as regulators of neural differentiation. The aim of this study was to establish whether BMP4 could influence neuronal differentiation of mesenchymal stem cells [MSCs]. Therefore, neuronal differentiation of MSCs was induced by basic fibroblast growth factor [bFGF] and epidermal growth factor [EOF] and treatment. The expression of neuronal specific markers such as Nestin, MAP2, p-Tubulin III and NKX6.1 were detected by RT-PCR, flow cytometery and/or immunostaining. While the percentage of Nestin positive cells was increased significantly during treatment, the addition of BMP4 during the first 4 days of treatment with bFGF and EGF reduced Nestin expression as showed by flow cytometry. This observation was further confirmed by relative gene expression which showed the reduction in expression of neural markers such as Nestin, MAP2 and NKX6.1 following treatment with BMP4. The results of this study suggest that BMP4 downregulates the neural fate of induced mouse MSCs

[Creation of tenecteplase-producing CHO cell line using site-specific integrase from the phage phi C31]

The aim of this study was to produce a stable CHO cell line expressing tenecteplase. In the first step, the tenecteplase coding sequence was cloned in a pDB2 vector containing attB recognition sites for the phage phi C31 integrase. Then, using lipofection, the CHO cells were co-transfected with constructed recombinant plasmid encoding tenecteplase and attB recognition sites and the integrase coding sequence containing pCMV-Int plasmid. As the recombinant plasmid contained the neomycin resistance gene [neo], stable cells were then selected using G418 as an antibiotic. Stable transformed cells were assessed using genomic PCR and RT-PCR. Finally, the functionality of tenecteplase was evaluated on the cell culture media. our results indicated that tenecteplase coding sequence was inserted into the CHO cell genome and was successfully expressed. Moreover, tenecteplase activity assessment indicated the presence of our functional tenecteplase in the cell culture medium. Considering the data obtained from this study, phi C31 integrase can be used for the production of a stable cell line and it be used to introduce ectopic genes into mammalian cells