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Gene expression in E14 TG2a cells grown with or without LIF and sorted for 5T4 expression

ABSTRACT: E14 TG2a cells grown with LIF were disaggregated and FACS sorted for cell surface 5T4 negativity versus E14 TG2a cells grown without LIF for 3 days and sorted for 5T4 positivity.

ORGANISM(S): Mus musculus

PROVIDER: GSE20372 | GEO | 2011/02/15

SECONDARY ACCESSION(S): PRJNA125475

REPOSITORIES: GEO

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Gene expression in E14 TG2a cells grown with or without LIF and sorted for 5T4 expression

Project description:E14 TG2a cells grown with LIF were disaggregated and FACS sorted for cell surface 5T4 negativity versus E14 TG2a cells grown without LIF for 3 days and sorted for 5T4 positivity. Two samples in triplicate. 5T4- vs. 5T4+

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RNA-seq analysis of ES cell populations with different functional potentials in serum/LIF and 2i/LIF, as well as analysis of the transcriptional effect of LIF in ground state (2i) culture conditions.

Project description:We report that ES cells cultured in ground state (2i and 2i/LIF) culture conditions are heterogeneous and show heterogeneus expression of extraembryonic markers. Using a highly sensitive reporter for the endoderm marker Hex we can sort Hex high and low populations from either serum/LIF or 2i/LIF and demonstrate that they have different functional properties. Here we explored the transcriptional basis of these functional differences and noted that Hex low (HV-) and Hex high (HV+) populations showed more distinct expression profiles in 2i/LIF than in serum/LIF. Additionally in 2i/LIF the HV+ population showed an upregulation of extraembryonic markers (such as trophoblast stem cell specific genes) and also imprinted genes compared to the HV- population, which is not observed when these populations are sorted from serum/LIF. We also analysed the transcriptional effect of LIF in 2i by analysing unsorted ES cells cultured in either 2i alone or 2i with LIF. We observed that the addition of LIF led to an upregulation of extraembryonic markers but did not effect the expression of pluripotency genes, other than Klf4. Additionally, the most significantly upregulated genes from 2i/LIF cultured ES cells compared to 2i cultured ES cells showed the greatest correlation to placental tissue when compared to the GNF tissue specific expression database. This analysis, alongside functional experiments, suggested that HV+ ES cells in 2i/LIF corresponded to an extraembryonically primed population of cells and that the addition of LIF supported this population.

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LIF-dependent gene expression in human neural stem cell cultures

Project description:Human cortical neural stem cells, provided by Dr. Clive Svendsen (Waisman Center, University of Wisconsin at Madison), were generated from human fetal cortex (9 weeks post conception) and grown as free-floating neurospheres as previously detailed (Wright, et al 2003. Journal of Neurochemistry 86, 179-195). At 15 weeks of growth, 10 ng/ml LIF was added to the cultures. In cases where LIF was withdrawn, neurospheres were collected, medium removed, and neurospheres washed three times with 1% N2 supplemented with 20 ng/ml EGF, and cultured into the same. Total RNA was prepared from a single human neural progenitor cell grown long-term LIF collected at 3 time points: early passage (GSM51360-GSM51362), mid passage (GSM51363-GSM51365), and late passage (GSM51366-GSM51368). Keywords: parallel sample

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RNA-seq analysis of ES cell populations with different functional potentials in serum/LIF and 2i/LIF, as well as analysis of the transcriptional effect of LIF in ground state (2i) culture conditions.

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Expression data from TOP-GFP sorted colon cancer cells

Project description:Colon cancers typically contain tumor cell populations with differential WNT signaling activity. Colon cancer cells with high WNT-activity have been attributed increase tumorigenic potential and stem cell characteristics. We extracted tumor cells with differential WNT activity using fluorescent reporters from xenografted human colon cancer cell line tumors and primary human colon cancer xenografts and analysed their gene expression profiles. Colon cancer cell lines and primary colon cancer xenografts were transduced with lentiviral TOP-GFP reporters, and grown as xenografts in NOD-SCID mice. We disaggregated these tumors, flow sorted for GFPhigh and GFPlow tumor cells and subjected these populations to gene expression profiling.

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LIF signaling-dependent gene expression

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Project description:Gene expression in E14 TG2a cells grown with or without LIF and sorted for 5T4 expression

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CC chemokine ligand 2 and LIF cooperatively promote pluripotency in mouse induced pluripotent cells

Project description:The pluripotency of mouse embryonic stem cells (ESC) and induced-pluripotent stem cells (iPSC) can be maintained by feeder cells, which secrete Leukemia Inhibitory Factor (LIF). We found that feeder cells provide a relatively low concentration (25 unit/ml) of LIF, which is insufficient to maintain the ESC/iPSC pluripotency in feeder free conditions. In order to identify additional factors involved in the maintenance of pluripotency, we carried out a global transcript expression profiling of mouse iPSC cultured on feeder cells and in feeder-free (LIF-treated) conditions. This identified 17 significantly differentially expressed genes (adjusted p-value<0.05) including 7 chemokines over-expressed in iPSC grown on feeder cells. Ectopic expression of these chemokines in iPSC revealed that CC chemokine ligand 2 (Ccl2) induced the key transcription factor genes for pluripotency, Klf4, Nanog, Sox2 and Tbx3. Further, addition of recombinant Ccl2 protein drastically increased the number of Nanog-GFP-positive iPSC grown in low LIF feeder free conditions. Interestingly, this effect was not observed in the absence of LIF. We further revealed that pluripotency promotion by Ccl2 is mediated by activating the Stat3-pathway followed by Klf4 up-regulation. We demonstrated that Ccl2 mediated increased pluripotency is independent of PI3K and MAPK pathways and that Tbx3 may be directly up-regulated by Klf4. Overall, Ccl2 cooperatively activates the Stat3-pathway with LIF in feeder free condition to maintain pluripotency for ESC/iPSC.

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