[Studying the morphology and growth of human retinal pigment epithelial cells on a thin layer of alginate and alginate/gelatin as a culture substrate]

Human retinal pigment epithelium [hRPE] is a cell monolayer located in the outer part of the retina that is in contact with photoreceptors. In many diseases RPE cells damage. One way for treating this disease is the implantation of intact instead of damaged cells. For this reason different types of substrates have been used for cell cultivation. This study has used alginate and a blend of alginate/gelatin [A/G] to study RPE cell growth. We prepared alginate solutions in concentrations of 1% and 2% [w/v] in water and DMEM/F12. The solutions were infused into each well of 6-well micro plates until a uniform culture substrate that had a 1 mm thickness was generated. Passage-4 hRPE cells were cultivated on the substrate and the cell characteristics studied. hRPE cells did not adhere to alginate in DMEM/F12 and did not exhibit interaction with alginate substrate. For this reason A/G solutions at concentrations of 1% and 2% [w/v] in water were prepared. We prepared A/G blends at weight ratios of: 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, and 80:20. These blends were infused into each well of 6-well plates until the appropriate 1 mm thickness of A/G was achieved. Isolated hRPE cells were cultured on synthetic substrate after which we studied the cells' characteristics. hRPE cell generated adhesive colonies on the A/G substrate. In all studied combinations of A/G, the diffused hRPE cells formed a monolayer under the substrate sheets. However the A/G 20:80 ratio had cell growth in the upper face of the substrate. hRPE survived indefinitely on A/G substrate. After the cells were re-cultured on polystyrene, they showed general morphological features of normal hRPE cells. The A/G blend at a 20:80 ratio was chosen to be used for future studies

Investigation of the mitochondrial ATPase 6/8 and tRNALys genes mutations in Autism

Autism results from developmental factors that affect many or all functional brain systems. Brain is one of tissues which are crucially in need of adenosine triphos-phate [ATP]. Autism is noticeably affected by mitochondrial dysfunction which impairs energy metabolism. Considering mutations within ATPase 6, ATPase 8 and tRNALys genes, associated with different neural diseases, and the main role of ATPase 6/8 in energy generation, we decided to investigate mutations on these mtDNA-encoded genes to reveal their roles in autism pathogenesis. In this experimental study, mutation analysis for the mentioned genes were performed in a cohort of 24 unrelated patients with idiopathic autism by employing amplicon sequencing of mtDNA fragments. In this study, 12 patients [50%] showed point mutations that represent a significant correlation between autism and mtDNA variations. Most of the identified substitutions [55.55%] were observed on MT-ATP6, altering some conserved amino acids to other ones which could potentially affect ATPase 6 function. Mutations causing amino acid replacement denote involvement of mtDNA genes, especially ATPase 6 in autism pathogenesis. MtDNA mutations in relation with autism could be remarkable to realize an understandable mechanism of pathogenesis in order to achieve therapeutic solutions

Retinal pigment epithelium culture; a potential source of retinal stem cells

To establish human retinal pigment epithelial [RPE] cell culture as a source for cell replacement therapy in ocular diseases Human cadaver globes were used to isolate RPE cells. Each globe was cut into several pieces of a few millimeters in size. After removing the sclera and choroid, remaining tissues were washed in phosphate buffer saline and RPE cells were isolated using dispase enzyme solution and cultured in Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 supplemented with 10% fetal calf serum. Primary cultures of RPE cells were established and spheroid colonies related to progenitor/stem cells developed in a number of cultures. The colonies included purely pigmented or mixed pigmented and non-pigmented cells. After multiple cellular passages, several types of photoreceptors and neural-like cells were detected morphologically. Cellular plasticity in RPE cell cultures revealed promising results in terms of generation of stem/progenitor cells from human RPE cells. Whether the spheroids and neural-like retinal cells were directly derived from retinal stem cells or offspring of trans-differentiating or de-differentiating RPE cells remains to be answered

efficiency of CD40 down regulation by siRNA and antisense ODN: comparison of lipofectamine and FuGENE6

Dendritic cells [DCs] are ideal accessory cells in the field of gene therapy. Delivery of DNA and siRNA into mammalian cells is a useful technique in treating various diseases caused by single gene defects. Selective gene silencing by small interfering RNAs [siRNAs] and antisense oligodeoxynucleotides [ODN]s is an efficient method for the manipulation of cellular functions. An efficient, functional delivery system with no toxicity problems would be attractive. We compared two commercially available cationic lipids, Lipofectamine and FuGENE6, in the delivery of both siRNA and antisense ODNs into mice spleen-derived DCs. Cellular uptake was measured by the means of fluorescein-labelled non-silencing siRNA and antisense ODNs as a model system using flow cytometry. Cytotoxicity of the two delivery systems was compared with propidium iodide and annexin-V staining, and quantified with flow cytometry. The efficiency of our oligonucleotide delivery systems was compared by measuring CD40 expression by flow cytometry. CD40 expression in DCs was 38%. After siRNA transfection by Lipofectamine, CD40 expression decreased to 13%, and after transfection by FuGENE6, it decreased to 18%. The difference was statistically significant. CD40 down regulation in DCs transfected with the two different antisense sequences by Lipofectamine was 21% and 23%, and down regulation after transfection by FuGENE6 was 19% and 18%, respectively. The differences were not statistically significant. The effects of siRNA and antisense ODNs were specific. Lipofectamine was a more potent delivery system in siRNA effect, followed by FuGENE6. There was no significant difference between Lipofectamine and FuGENE6 as a delivery system of antisense ODNs

Designing of relative quantitative Real-Time PCR for detection of CD40 gene expression in dendritic cell after suppression with antisense

Dendritic cells have a critical role in control and regulation of immune responses. It is believed that these cells can be used for the treatment of many diseases. One of the methods used in immunotherapy is based on generating of tolerogenic dendritic cells through inhibition of expression costimulatory molecules. CD40 is one of the costimulatory molecules, and inhibition of expression by antisense or siRNA techniques, can generate tolerogenic dendritic cells. Generation of tolerogenic dendritic cells will be useful in the treatment of many diseases. By developing a quantitive RT-PCR for evaluation of gene expression, generation of these cells could be possible. Using proper software we designed an Antisense and transfection of dendritic cells by lipofectamine 2000 [Invitrogen] could lead us to generate tolerogenic dendritic cells. In this study dendritic cells were extracted from of Balb/c mice Spleen and the purity of this extraction was determined by flow cytometry. BCL 1 cell line as a CD40 expressing control group and Wehi-164 cell line were cultured in RPMI-1640+10%FCS. Primer design for CD40 gene and house keeping gene [GADPH] was done by bioinformatic soft wares such as Beacon designer, mfold and Blast. RNasy plus mini kit [Qiagen] was used for RNA extraction and the Purity and Integrity were determined by O.D at 260/280 and agarose gel electrophoresis. In the next step cDNA synthesized and quantitative RT-PCR for CD 40 using IQ sybergreen [Biorad] were setup. Finally, standard curve for CD40 and internal control in different RNA concentrations were performed. After transfection with lipofectamin 2000 the amount of gene suppression were quantified by qualitative RT-PCR. Using gradient real time PCR, optimum annealing temperature, C[t] and delta Rn for CD40 and GADPH were determined, annealing temperature was 59.5 degree sign c and melting temperature was 84 degree sign c. Slope of the curve and the efficacy of PCR for CD40 and GADPH genes were quantified by serial dilution method. CD 40 Standard curve efficiency is 96.5 and the slope is -3.408 and the efficacy of GADPH standard curve is 94.1 and its slope is -3.471. The amount of CD40 gene suppression by antisense in dendritic cells was 1/32 and in BCL1 cell line was 1/64. Also the transfection reagent had no effect on the gene expression. The best time for CD40 gene expression is 48h after transfection. Semi quantitative PCR, absolute quantitative PCR and relative quantitative PCR are used to evaluative the expression of different genes such as CD40. In this study we found that for making CD40 and GADPH standard curves, Biorad two steps PCR kit [IQ -sybergreen] and Oligo dt for cDNA synthesis were very suitable. In this case, GADPH is a good internal control. Also for evaluation of gene suppression relative quantitative RT-PCR was more efficient compare to other methods such as northern blotting. The CD40 gene Suppression after 48 hours in dendritic cells was 1/32 and in BCL1 cells was 1/64