Project description:I. Exp Design 1. Type of experiment: Comparison of native versus cultured RPE cells 2. Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices 3. How many hybridizations in exp: 21 4. If a common reference used for all the hybs: no 5. Quality control steps: three independent arrays for each condition 6. Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors. II. Samples used, extract prep, and labeling 1. Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells. 2. Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin). 3. Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturerâs instructions. 4. Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP. 5. No external controls were added. III. Hybridization procedures and parameters 1. Sample, array type, batch and serial # used 2. Hybridization protocol: Hybridization was carried out using the manufacturerâs recommendations. Arrays were prehybridized with Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS. IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis. 3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

Project description:Description I. Exp Design 1. Type of experiment: Comparison of native versus cultured RPE cells 2. Experimental factors: Native RPE versus ARPE-19 cells grown in different culture conditions 3. How many hybridizations in exp: 20 4. If a common reference used for all the hybs: no 5. Quality control steps: three independent arrays for each condition, and five different donor globes for native RPE 6. Description: The expression profile of ARPE-19 cells grown under different culture conditions were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 5 donors. II. Samples used, extract prep, and labeling 1. Biosource: Human donor globes from NDRI and Sierra Eye and Tissue Bank, and ARPE-19 cells. 2. Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (subconfluent culture density in presence of serum, confluent culture density in presence of serum, confluent culture density with serum withdrawn medium, differentiated (grown for 2.5 months) in serum, and differentiated (grown for 2.5 months) and then serum withdrawn medium). 3. Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer's instructions. 4. Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP. 5. No external controls were added. III. Hybridization procedures and parameters 1. Sample, array type, batch and serial # used 2. Hybridization protocol: Hybridization was carried out using the manufacturer's recommendations. Arrays were prehybridized with Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS. IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis. 3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis. Keywords: other

Project description:Description I. Exp Design 1. Type of experiment: Comparison of native versus cultured RPE cells 2. Experimental factors: Native RPE versus ARPE-19 cells grown in different culture conditions 3. How many hybridizations in exp: 20 4. If a common reference used for all the hybs: no 5. Quality control steps: three independent arrays for each condition, and five different donor globes for native RPE 6. Description: The expression profile of ARPE-19 cells grown under different culture conditions were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 5 donors. II. Samples used, extract prep, and labeling 1. Biosource: Human donor globes from NDRI and Sierra Eye and Tissue Bank, and ARPE-19 cells. 2. Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (subconfluent culture density in presence of serum, confluent culture density in presence of serum, confluent culture density with serum withdrawn medium, differentiated (grown for 2.5 months) in serum, and differentiated (grown for 2.5 months) and then serum withdrawn medium). 3. Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer's instructions. 4. Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP. 5. No external controls were added. III. Hybridization procedures and parameters 1. Sample, array type, batch and serial # used 2. Hybridization protocol: Hybridization was carried out using the manufacturer's recommendations. Arrays were prehybridized with Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS. IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis. 3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

Project description:The therapeutic potential of pluripotent stem cells is great as they promise to Q7 usher in a new era of medicine where cells or organs may be prescribed to replace dysfunctional tissue. At the forefront are efforts in the eye to develop Q8 this technology as it lends itself to in vivo monitoring and sophisticated imaging modalities. In the retina, retinal pigment epithelium (RPE) is the most promising replacement cell as it has a single layer, is relatively simple to transplant, and is associated with dozens of eye diseases. However, after transplantation, they may transform and cause complications. This transformation may be partially due to incomplete maturation. With the goal of learning how to mature RPE, we compared iPSC-RPE with adult primary RPE and the immortalized ARPE-19 line. We cultured ARPE-19, iPSC-RPE, and adult human RPE for one month, evaluating morphology, RPE marker staining, and transepithelial electrical resistance as quality control indicators. We then isolated RNA for bulk RNAsequencing and DNA for genotyping. We genotyped adult RPE lines for the top age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR) risk allele polymorphisms. Transcriptome data verified that both adult and iPSC-RPE exhibit similar RPE gene expression signatures, significantly more than ARPE-19. In addition, in iPSC-RPE, gene networks relating to epithelial to mesenchymal transition (EMT), stem cell maintenance, retina development, and muscle contraction were significantly elevated compared to those of adult RPE. We compared adult human RPE to iPSC-RPE in a model of EMT and observed an increased sensitivity of iPSC-RPE to producing contractile aggregates in vitro which resembles incident reports upon transplantation. P38 inhibition was capable of inhibiting iPSC-RPE–derived aggregates. In summary, we find that the transcriptomic signature of iPSC-RPE conveys an immature RPE state which may be ameliorated by targeting “immature” gene regulatory networks.

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Native versus cultured retinal pigment epithelium cells

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