Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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ROCK Inhibition extends passage of human embryonic stem cell derived retinal pigmented epithelium

ABSTRACT: Human embryonic stem cell derived retinal pigmented epithelial cells (hESC-RPE) are in clinical trials for the treatment of macular diseases. Currently, these cells take over three months to derive and subsequent months to mature and characterize. After only five to six passages the cells begin to undergo an epithelial-to-mesenchymal transition and are unsuitable for cellular therapies. We describe a novel passaging protocol and show that inhibition of Rho-associated, coiled coil containing protein kinases (ROCK1 and ROCK2) using Y-27632, allows extended passage of hESC-RPE in serum-free culture with maintenance of the RPE phenotype. After 30 population doublings, hESC-RPE at passage 13 maintain normal karyotype, display typical, polarized epithelial morphology, and continue to express RPE-specific genes. Passage 13 hESC-RPE show protein localization patterns similar to passage 2 cells, and display similar levels of growth factor secretion and phagocytosis of photoreceptor outer segments. Microarray analysis from day 2 cells shows several key pathways are altered by ROCK inhibition, including stimulation of the cell cycle and suppression of TGFM-NM-2 and Wnt signaling. These findings describe a means to greatly increase the yield of functional hESC-RPE for use in research and clinical trials. Two-condition experiment, Control vs Y-27632 treated hESC-RPE passage 5. Biological replicates: 4 control, 4 Y-27632. The experiments were technically carried out as dual channel (eg, Cy3 and Cy5-labeled samples hybridized to the same array) but processed as though they are single channel (Cy3 and Cy5 signals are calculated; Cy3/Cy5 ratios are not calculated). Therefore, there are 4 raw data files for total 8 samples.

ORGANISM(S): Homo sapiens

SUBMITTER: Roxanne Croze

PROVIDER: E-GEOD-56618 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Age-related macular degeneration (AMD) is a leading cause of blindness. Most vision loss occurs following the transition from a disease of deposit formation and inflammation to a disease of neovascular fibrosis and/or cell death. Here, we investigate how protracted wound stimulus leads to seminal changes in gene expression and the onset of a self-sustained state of wound response in retinal pigmented epithelial (RPE) cells. Using a human fetal RPE cell culture model and a systems level transcriptome analysis, we show that prolonged subconfluent culture resulting from repeated passage, leads to terminal acquisition of a mesenchymal-like phenotype post-confluence accompanied by altered expression of >40% of the transcriptome. In contrast, at subconfluence <5% of transcripts have >2-fold expression changes after repeated passage. Protein-protein interaction analysis reveals a core set of genes comprising two interconnected modules with functions pertaining to wound response and cell division. Among the wound response genes are the TGF-beta pathway activators: TGFB1, TGFG2, INHBA, INHBB, GDF6, CTGF, and THBS1. Small molecule inhibition of TGFBR1/ACVR1B mediated signaling both forestalls and reverses the passage-dependent loss of epithelial potential. Moreover, a disproportionate number of RPE wound response genes have altered expression in neovascular and geographic AMD; including key members of the TGF-beta pathway. In conclusiton, in RPE cells the switch to a terminal mesenchymal-like state following protracted or repeated wound stimulus is driven by activation of a self-perpetuating TGF-beta feedback loop. Targeted inhibition of TGF-beta signaling may be an effective approach towards retarding AMD progression and producing RPE cells in quantity for research and cell based therapies. Transcriptome profiles were determined from 44 human fetal RPE cultures of varying passage number, seeding density, culture maturity, and/or growth factor or small molecule treatment. Probes were labeled with Cy3 or Cy5 and Agilent whole genome microarrays were hybridized with a pair of Cy3 and Cy5 probes. After background subtraction and LOWESS correction to adjust for dye-dependent effects the net intensity values were determined as described in the Data Processing section and the entire data set was quantile normalized. The values reported in the Series Matrix are the quantile normalized net intensity values of the gene specific probes.

Project description:We have derived hESC from biopsied blastomeres of cleavage stage embryos under virtually the same conditions we used for the derivation of hESC lines from inner cell mass of blastocyst stage embryos. Blastomere-derived hESC lines exhibited all the standard characteristics of hESC including undifferentiated proliferation, genomic stability, expression of pluripotency markers and the ability to differentiate into the cells of all three germ layers both in vitro and in vivo. To examine whether hESC lines derived from two developmental stages of the embryo differ in gene expression, we have subjected three blastomere-derived hESC lines and two ICM-derived hESC lines grown under identical culture conditions to transcriptome analysis using gene expression arrays. Unlike previously reported comparisons of hESC lines which demonstrated, apart from core hESC-associated pluripotency signature, significant variations in gene expression profiles of different lines, our data show that hESC lines derived and grown under well-controlled defined culture conditions adopt nearly identical gene expression profiles. Moreover, blastomere-derived and ICM-derived hESC exhibited very similar transcriptional profiles independent of the developmental stage of the embryo from which they originated. Furthermore, this profile was evident in very early passages of the cells and did not appear to be affected by extensive passaging. These results suggest that during derivation process cells which give rise to hESC acquire virtually identical stable phenotype and are not affected by the developmental stage of the starting cell population. we investigated whether human stem cell lines derived from late totipotent blastomeres of the cleavage stage embryo develop a more “primitive” phenotype than ICM-derived lines by profiling their respective gene expression, molecular regulation and signaling pathways. In this study, we examined whether the inherent differences in the development stage of the starting embryonic cell population are maintained in the derived hESC lines by evaluating the stemness, differentiation capacity and gene expression profiles of blastomere- and ICM-derived hESC lines.

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ROCK Inhibition extends passage of human embryonic stem cell derived retinal pigmented epithelium

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