Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Conversion of Human Somatic Cells to Multipotent Endoderm Stem Cells by Small Molecules [array]

ABSTRACT: Forced expression of transcription factors for lineage reprogramming brings hope to cell-based therapy. However, its application is hampered by risks of potential genetic aberrations and tumorigenicity. Using defined small molecules in presence of gastric stromal cells as feeders, we reprogramed human gastric epithelia into induced multipotent endodermal progenitors (hiMEPs) with efficiency of up-to-6%. The hiMEPs expressed genes relative to endodermal lineages but not associating with pluripotency, and could be expanded clonogenically remaining as undifferentiated colonies. Upon induction, hiMEPs were able to give rise to multiple functional endodermal cell types, apart from ectodermal or mesodermal lineages. TGFÎ² inhibition and particular Wnt signaling activation were crucial in reprogramming process. Collective advantages of availability from donors without age restriction, capabilities in expansion and differentiation, and no concern of tumorigenesis, let hiMEPs have the considerable application potentials on cell therapies of diseases such as liver failure and diabetes, as well as personalized drug-screenings. Human gastric epithelial cells (hGECs) and gastric subepithelial myofibroblasts (GSEMFs) are isolated from human stomach, and human duodenum epithelial cells (hDECs) are isolated from human duodenum. Human Induced multipotent endodermal stem cells (hiMESCs) were reprogrammed from hGECs/hDECs by small molecules with the support of GSEMFs. Definitive endoderm cells (DEs) are derived from human embryonic stem cells by differentiation. Totally, 13 samples including two samples of hDECs, one sample of hGECs, two clones of D-hiMESCs, one clone of G-hiMESCs, four samples of DEs and three samples of GSCs were analyzed using microarray.

ORGANISM(S): Homo sapiens

SUBMITTER: Jinhua Qin

PROVIDER: E-GEOD-71301 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Previously, we described a mouse model where the well-known reproductive carcinogen, diethylstilbestrol (DES), caused uterine adenocarcinoma following neonatal treatment. Tumor incidence was dose-dependent reaching >90% by 18 mo. following 1000 µg/kg/day of DES. These tumors followed the initiation/promotion model of hormonal carcinogenesis with developmental exposure as the initiator, and exposure to ovarian hormones at puberty as the promoter. To identify molecular pathways involved in DES-initiation events, uterine gene expression profiles were examined in prepubertal mice exposed to DES (1, 10 or 1000 µg/kg/day) on days 1-5 and compared to age-matched controls. Of more than 20,000 transcripts, approximately 3% were differentially expressed in at least one DES treatment group compared to controls; several transcripts demonstrated dose-responsiveness. Assessment of gene ontology annotation revealed alterations in genes associated with cell growth, differentiation, and adhesion. When expression profiles were compared to published studies of uteri from 5 day old DES-treated mice, or adult mice treated with 17β estradiol, similarities were seen suggesting persistent differential expression of estrogen responsive genes following developmental DES exposure. Moreover, several significantly altered genes have been identified in human uterine adenocarcinomas. Four altered genes [Lactotransferrin (Ltf), Transforming growth factor beta inducible (Tgfβ1), Cyclin D1 (Ccnd1), and Secreted frizzled-related protein 4 (Sfrp4)], selected for real time RT-PCR analysis, correlated well with the directionality of the microarray data. These data suggest altered gene expression profiles observed two weeks after treatment ceased, were imprinted at the time of developmental exposure and maybe related to the initiation events resulting in carcinogenesis. Experiment Overall Design: There were 3 DES doses (1, 10 and 1000 ug/kg/day) administered to neonates on days 1-5. RNA was pooled for 10 mice, with each dose having its own matching control (corn oil). Dye-flipped hybridizations were performed for each paired comparison.

Project description:Diethylstilbestrol (DES) is a synthetic estrogen that has been banned for use in humans, but still is employed in livestock and aquaculture operations in some parts of the world. Detectable concentrations of DES in effluent and surface waters have been reported to range from slightly below 1 to greater than 10 ng/L. Little is known, however, concerning the toxicological potency of DES in fish. In this study, sexually-mature fathead minnows (Pimephales promelas) of both sexes were exposed to 1, 10 or 100 ng DES/L water in a flow-through system. Tissue concentrations of DES and changes in a number of estrogen-responsive endpoints, including alterations to the hepatic transcriptome in females, were measured in the fish at the end of a 4-d exposure, and after a 4-d depuration/recovery period in clean water. The objectives of the study were to measure accumulation of DES in fish tissues, characterize effects of the chemical on physiological endpoints related to reproductive performance, and to evaluate DES's potency relative to concentrations reported in aquatic environments. The current series includes n=11 microarrays associated with recovery-phase samples (collected from fish exposed continuously for 4 d, and then held in control water for an additional 4 days before tissues were collected). Fish were exposed to 0, 1, 10, or 100 ng DES/L delivered in a continuous flow (45 ml/min) of sand filtered, UV treated, Lake Superior Water, without the use of carrier solvents. Flow of DES into the test system was initiated without fish present and continued for 1 d. Exposures then were started by placing three male and three female sexually-mature fathead minnows into each tank. There were eight replicate tanks for the control and 100 ng/L treatments, and six replicate tanks for the 1 and 10 ng/L groups. The fish were held at 25+1M-BM-:C under a 16:8 L:D photoperiod and were fed frozen adult brine shrimp twice daily (San Francisco Bay Brand, Newark, CA, USA). Fish used for the experiment were from an on-site culture, and all procedures involving animals conformed to guidelines approved by the local Animal Care and Use Committee. After the 4-d exposure, fish from three tanks from each treatment (n=9 per sex) were sampled for determination of various biological endpoints. At this time fish from two additional tanks from the control and 100 ng/L treatments also were sampled for determination of DES tissue residues (n=6 per sex). Delivery of DES to the test system was subsequently stopped, and fish from the remaining three tanks per treatment group were sampled 4 d later (n= 9 per sex). The fathead minnows were euthanized with buffered MS-222 (Argent, Redmond, VA, USA) and weighed. Fish were scored for occurrence and relative expression of nuptial tubercles, which in males can be reduced by ER agonists. Blood was collected from the caudal vein/artery with a microhematocrit tube, and plasma was separated by centrifugation and stored at -80M-BM-:C. Gonads were removed from males and a subsample (8.5M-BM-13.0 mg; meanM-BM-1SD) was immediately immersed in tissue culture media for determination of ex vivo steroid production. Livers were removed from both sexes and flash-frozen for gene expression analyses using microarray (females) or real-time QPCR (males). Hepatic transcripts from three females per treatment group, in each phase of the experiment (except n=2 from recovery phase 10 ng/L) were analyzed using a custom 15,000 feature microarray (GEO Platform Accession GPL9248). Data sets for the exposure phase (n=12 microarrays) and recovery phase (n=11) were normalized independently using Fastlo (Ballman et al., 2004) implemented in R (http://www.r-project.org/), but analyzed using parallel approaches.

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Conversion of Human Somatic Cells to Multipotent Endoderm Stem Cells by Small Molecules [array]

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