Objective: The neuronal repressor REST (RE1-silencing transcription factor) is expressed at high levels in embryonic stem cells (ESC) with a critical role in self-renewal and pluripotency signaling network of these cells. REST is an essential element for brain development and also neuronal differentiation of ESC in vitro. Although REST is a well-known regulatory element in embryonic stem cells, but according to our knowledge, it has not been evaluated in induced pluripotent stem cell (iPSC) till now.Materials and Methods: The quantitative expression of REST in each step was evaluated by real-time PCR and the presence of REST was showed by immunofluorescent assay.Results: Q-PCR analysis showed that the expression level of REST decreased significantly during differentiation of hiPSCs to neural precursor cells, and remained stable until neuronal differentiation. immunofluorescent data revealed the both nuclear as cytoplasmic presence of REST in hiPSCs as well as NSCs, while in the maturated neurons it was only detectable in the cytoplasm.Conclusion: Our findings show a distinct expression pattern for REST during HESCS and hiPSCs neural differentiation. This study opens a new window for further experiments in this field to receive efficient differentiation of neuronal cells.

Aiming to develop a stem cell based therapy for deafness; we have pursued the isolation and characterization of human auditory stem cells from different sources. Initially, we identified a population of auditory stem cells from the developing cochlea (human Foetal Auditory Stem Cells, hFASCs) that could be used as an in vitro model of differentiation. Using optimized culture conditions and growth factors we have selectively expanded a population of cells that express several stem cell markers such as NESTIN, SOX2 and OCT4. Cells proliferate for several months and remain undifferentiated. We have defined culture conditions that induce differentiation into neurons and hair cells. When transferred to neutralizing conditions, cells extend processes and readily differentiate into bipolar auditory neurons that express neuronal markers. They also display typical neuronal potassium and sodium currents. When exposed to hair cell conditions, several hair cell markers as well as potassium and calcium currents are induced. Second, we have induced differentiation of inner ear phenotypes from human Embryonic Stem Cells (HESCS). To achieve this we manipulated the culture conditions using factors that mimic the normal development of ear placodes in vivo. By this technique we have isolated cells expressing early placodal markers such as PAX8, PAX2 and GATA3. These cells can be induced to differentiate further into sensory neurons and hair cell-like phenotypes. These systems provide ideal models to study inner ear differentiation in humans and could prove highly useful for drug development and more importantly, for the generation of stem cell-based therapies for deafness.

The isolation and culture of human embryonic stem cells (HESCS) has opened up new opportunities not only for regenerative medicine and other biomedical applications but also for the study of early human development, signaling pathway networks and mechanisms of human diseases. However, it is remain unclear about the factors important for the maintenance of undifferentiated HESCS in vitro and factors /signaling pathways leading HESCS differentiation to specific lineages. Genetic modification of HESCS will facilitate our investigation to achieve thesegoals. For example, incorporation of fluorescent reporter genes in a lineage specific expression pattern will enable more effective screening to identify molecules regulating specific cell fate commitment. Ectopic expression, or silencing, of key developmental genes/ will enhance our understanding their function in HESCS self-renewal or differentiation. We have generated HESCS reporter cell lines expressing green fluorescent protein (GFP) under the control of ES cell (e.g. Oct4) or lineage specific (e.g. a-fetoprotein, AFP for endodermal lineage) promoters using either chemical transfection or lentiviral transduction techniques. Using these reporter cell lines, we have compared effect of bFGF in regulation of human ES cell self-renewal, studied Oct4 expression in regulation of HESCS differentiation and development of better system to differentiate HESCS to hepatic cell fate.

Optimization and development of more defined culture methods for human embryonic and induced pluripotent stem cells (HESCS and hiPSCs) will provide an invaluable contribution to the field of regenerative medicine. However, one problem is that HESCS and hiPSCs are vulnerable to apoptosis upon cellular detachment and dissociation and therefore have a low plating efficiency upon passaging. Here, we report the effective freezing/thawing of single dissociated HESCS and hiPSCs in a feeder-free culture in the presence of ROCK inhibitor Y-27632. Our results show that the addition of Y-27632 to medium of HESCS and hiPSCs increases the survival of single dissociated HESCS and hiPSCs in feeder-free culture. The cloning efficiency of hiPSCs and HESCS improves when ROCK inhibitor is added both in Matrigel and in medium in comparison with conventional addition to medium alone. Under these treatments HESCS and hiPSCs retain typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture. Therefore, we believe this method will be useful for current and future applications of these pluripotent stem cells.

Objective: Human embryonic stem cells (HESCS) are pluripotent cells with ability to differentiate to three germ layers. However, dissimilar to mouse embryonic stem cells (mESCs), HESCS are vulnerable to be cultured in dissociated state due to induced apoptosis that has been reflected as an important obstacle to extend genetically manipulation of them. This phenomenon is arisen by activation of Rho\Rock signaling pathway is adversely affecting dissociated HESCS proliferation and survival through E-Cadherin dependent cell-cell adhesion disruption. E-cadherin is one of cytoskeleton elements that will be decreased after Rho/Rock pathway activation. Thus attenuation of Rho/Rock pathway may inhibit apoptosis of dissociated HESCS. One of the proposed components, which have been shown to be potential attenuator of this signaling pathway, is Pioglitzone. Pioglitzone, a specific of agonist of proxisome proliferative activated receptor γ (PPARy), decreases the MYPT phosphorylation level a key mediator of Rho/Rock signaling pathway. Proxisome proliferative activated receptor γ (PPARy) is a member of the PPAR superfamily ligand dependent nuclear receptors (a, b/δ, y) that has numerous roles in variety of cells. PPARy could be activated by both natural and synthetic ligands. The aim of this study is implementation of Rock inhibitor (Y-27632) with Pioglitzone to increase HESCS colonies formation rate through induction of E-Cadherin and b-Catenin expression independent of cells survival. Furthremore, we have evaluated the effects of bFGF supplementation on essential transcriptional factors involving in pluripotency of dissociated HESCS and combinational effects of Pioglitazone and bFGF on proliferation state of dissociated HESCS have been examined.Materials and Methods: To study effects of Pioglitazone on b-Catenin and E-Cadherin along with Rock inhibitor (Y-27632), HESCS were treated with Pioglitazone and Y-27632. Four and 24 hours and 1 week post treating, cells were harvested and quantitative real time PCR was applied to demonstrate effects of using Pioglitazone and Y-27632 on E-cadherin and b-catenin expression. To analyze colony formation rate and cells survival, alkaline phosphatase (AP) and annexinV/ PI tests were performed respectively. Furthermore, HESCS were grown in serum-free N2B27-based media and treated with Pioglitazone in presence or absence of bFGF for 4 days and expression level of pluripotent markers was assessed by real time PCR. Localization and intensity of these markers were also carried out by immunostaining.Results: Data showed that treating with Rock inhibitor along with Pioglitzone increased HESCS colonies formation rate significantly without influencing cell survival as compared with Rock inhibitor implementation. Moreover, treatment of dissociated HESCS with Y-27632 and Pioglitazone increased E-cadherin and b -catenin expression. Co-treating with bFGF and Pioglitazone increased Nanog expression, whereas did not any significantly effect on OCT4 or SOX2 expression as compared with bFGF supplementation. Moreover, pioglitazone decreased Pax6 and Sox1 as neural precursor cell markers.Conclusion: The present study suggests that implementation of Rock inhibitor (Y-27632) with Pioglitazone increases HESCS colonies efficiency formation rate through an induction of E-Cadherin and b-catenin expression in an independent pathway of cells survival. FGF/ERK signaling plays a positively acting role in HESCS by supporting Nanog expression. Thus, Pioglitazone could positively effect on NANOG, downstream of FGF2 and decrease PAX6 and SOX1 neural precursor genes, which lead to improvement in self-renewal of HESCS.

Human embryonic stem cells (HESCS) represent a population of undifferentiated cells with both self renewal and multilineage differentiation characteristics representing all three embryonic germ layes thus revealing their pluriptent potential. Human ESCs originate from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. The pluripoent nature of HESCS has attracted great interest in using them as an important source for functional genomics, drug screening, and, perhaps eventually, cell and gene therapy. The derivation of the first HESCS was reported in 1998. Since then we have learnt a great deal about how to isolate and culture these cells to use them for the aforementioned objectives. This review gives an verview of the different methods of establishment, characterization, and maintenance of HESCS.

Age-related Macular Degeneration (AMD) is one of the retinal degenerative diseases associated with some degree of dysfunction and loss of Retinal Pigmented Epithelium (RPE) cells and leads to permanent sight loss. Available treatments only slow down its progression. Applying a scaffold to help RPE cells proliferation and make layers has been proposed as a promising approach to treat this group of diseases. In this study, a fuzzy system was used to optimize the situation of making a scaffold. For better adhesion and proliferation of cells, the polycaprolactone scaffold's surface was modified by alkaline hydrolysis and plasma. Some analyses, such as water uptake and biodegradation rate, were done. Then, differentiated human embryonic stem cells (HESCS) were cultured on several groups of scaffolds. Finally, the viability, proliferation, and morphology of differentiated hESC-RPE cells on all groups of the scaffolds were investigated. The nanofibers' diameter was minimized by optimizing voltage and solution concentration with a fuzzy model for the first time, which obtained 110. 5 nm, 18. 9 kV, and 0. 065 g/mL (w/v), respectively. The immersion time of the scaffold in alkaline solution and solution concentration during surface modification were achieved 4. 3 M and 104 minutes, respectively, by response surface methodology. Results of the MTT assay showed that the hydrolyzed group had a high proliferation of cells. Scanning electron microscopy observation of cell morphology after 60 days confirmed this result. In conclusion, our results demonstrate that the hydrolyzed scaffold is a suitable bed for cell proliferation, a good option for AMD treatment.

Background: HESCS-MSCs open a new insight into future cell therapy applications, due to their unique characteristics, including immunomodulatory features, proliferation, and differentiation. Methods: Herein, HESCSMSCs were characterized by IF technique with CD105 and FIBRONECTIN as markers and FIBRONECTIN, VIMENTIN, CD10, CD105, and CD14 genes using RT-PCR technique. FACS was performed for CD44, CD73, CD90, and CD105 markers. Moreover, these fibroblast-like cells, due to multipotent characteristics, differentiated to the osteoblast. Results: MSCs were derived from diploid and triploid hESC lines using sequential 3D and 2D cultures and characterized with the specific markers. IF showed the expression of FIBRONECTIN and CD105 in HESCS-MSCs. Flow cytometry data indicated no significant difference in the expression of MSC markers after 6 and 13 passages. Interestingly, gene expression profiles revealed slight differences between MSCs from diploid and triploid HESCS. The HESCS-MSCs displayed osteogenic differentiation capacity, which was confirmed by Alizarin red staining. Conclusion: Our findings reveal that both diploid and triploid hESC lines are capable of forming MSCs; however, there are some differences in their gene expression profiles. Generation of MSCs from HESCS, as a non-invasive procedure in large scale, will lend itself for the future cell-based therapeutic applications.