Comparing three methods of co-culture of retinal pigment epithelium with progenitor cells derived human embryonic stem cells

Close interaction between retinal pigment epithelium [RPE] and photoreceptors plays an essential role in visual function. The objective of this study is to determine the effects of RPE cells in the differentiation of progenitor derived human embryonic stem cells [hESC] into retinal cells; we developed in vitro co-culture models and compare these models to investigate in which model the expression of photoreceptor markers is superior. It seems the effects of RPE cells on differentiation of retinal progenitor cells [RPCs] through the cell-to-cell contact or with the use of insert and compare of these methods has not been reported yet. Initially, retinal progenitors [RPs] were differentiated from hESC. After isolation of RPE sheet from rabbit eyes, demonstrated these cells maintains the integrity and feature after 2 weeks. Next, we examined the induction of photoreceptors by the co-culture of RPE through insert in 1 week and 2 weeks [indirect] or without insert by the cell-to-cell contact [direct]. The differentiation of retinal cells was verified by protein and gene expression in these three methods. The adherent cells were morphologically examined using phase contrast microscopy and characterized by immunofluorescent staining and reverse transcription polymerase chain reaction [RT PCR]. Evaluation of immunostaining showed that hESC, highly [>80%] can be directed to the RPs fate. Upon co-culture of RPCs with RPE sheet using insert for 2 weeks or by the cell-to-cell contact, these cells differentiated to neural retina and expressed photoreceptor specific markers. However, in direct co-culture, some mature photoreceptor markers like arrestin expressed in compare with indirect co-culture. The expression of late photoreceptor marker could be improved when RPE cells seeded on RPCs in compare with the use of insert.

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

Characterization and functional assessment of mouse PPAR[gamma]1 promoter

Peroxisome Proliferator Activated Receptor gamma [PPAR[gamma]], a member of nuclear receptor superfamily, comprises two isoforms in mouse. These two isoforms are encoded by different mRNAs, which are arisen by alternative promoter usage. There are two promoter regions upstream of PPAR[gamma] gene. A 3 kb fragment, containing several transcription factor binding sites, acts as PPAR[gamma]1 promoter region. Thus, expression pattern of PPAR[gamma]1 isoform is due to the potential transcription factors that could influence its promoter activity. PPAR[gamma], Retinoid X Receptor [RXR] and Vitamin D Receptor [VDR], as nuclear receptors could influence PPAR[gamma] gene expression pattern during several differentiation processes. During neural differentiation, PPAR[gamma]1 isoform expression reaches to maximal level at neural precursor cell formation. A vast computational analysis was carried out to reveal the PPAR[gamma]1 promoter region. The putative promoter region was then subcloned upstream of an EGFP reporter gene. Then the functionality of PPAR[gamma]1 promoter was assessed in different cell lines. Results indicated that Rosiglitazone increased PPAR[gamma]1 promoter regulated EGFP expression of neural precursor cells during Embryoid Body [EB] formation. Furthermore vitamin D reduced PPAR[gamma]1 promoter regulated EGFP expression of neural precursor cells during EB formation through binding to its receptor. This study suggests that there are potential response elements for PPAR/RXR and VDR/RXR heterodimers in PPAR[gamma]1 isoform promoter. Also VDR/RXR heterodimers may decrease PPAR[gamma] expression through binding to its promoter

[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