Project description:We explored Max ablation-mediated up-regulation of germ-related genes, especially meiosis-related genes in mouse embryonic stem cells which were cultured either under conventional mouse ES medium or 2i condition using inhibitors against MEK and GSK3b. Effect of culture conditions (conventional mouse ES medium or 2i condition) on the expression profile of germ-related genes in Max expression ablated ES cells were examined using inducible Max-null ES cells in which Max gene had been homozygously disrupted, but carries Max cDNA in ROSA26 locus with tetracycline-off system.
Project description:c-Myc is one of key players that are crucially involved in maintaining the undifferentiated state and the self-renewal of ESCs. To understand the mechanism by which c-Myc helps preserve these prominent characteristics of ESCs, we generated null-ES cells for the Max gene, which encodes the best characterized partner protein for all Myc family proteins. Although Myc/Max complexes have been widely regarded as crucial regulators of the ESC status, our data reveal that ESCs do not absolutely require these complexes in so-called ground state or related conditons and that this requirement is restricted to conventional ES culture conditions without using a MAPK inhibitor. Simply Dox-treated or Nanog (WT or D67G mutant)-rescued Max-null ESCs which were cultured under conventional culture condition and 2i/Nam-treated completely Max-null ES cells from which Dox-regulatable cDNA was removed were used for RNA source. Samples from Dox-untreated Max-null ESCs cultured under conventional culture condition were used as reference samples for Dox-treated cells and Nanog-rescued cells, while a sample from wild-type ESCs cultured under 2i/Nam condition was used as a reference sample for completely Max-null ES cells cultured with 2i/Nam.
Project description:We explored Max ablation-mediated up-regulation of germ-related genes, especially meiosis-related genes in mouse embryonic stem cells which were cultured either under conventional mouse ES medium or 2i condition using inhibitors against MEK and GSK3b.
Project description:Ablation of expression of the Max gene encoding a Myc protein partner in ES cells provoked two major phenomena, i.e. loss of pluripotency and apoptotic cell death. We found that nicotinamide (Nam) significantly alleviates these Max expression ablation-coupled phenotypes in ES cells. To see the alleviation effect of Nam on the overall expression profile of Max-null ES cells whose Max expression is controlled by the tet-off system, we eliminated Max expression by adding doxycycline (Dox) in the presence of Nam.
Project description:Ablation of expression of the Max gene encoding a Myc protein partner in ES cells provoked two major phenomena, i.e. loss of pluripotency and apoptotic cell death. We found that nicotinamide (Nam) significantly alleviates these Max expression ablation-coupled phenotypes in ES cells. To see the alleviation effect of Nam on the overall expression profile of Max-null ES cells whose Max expression is controlled by the tet-off system, we eliminated Max expression by adding doxycycline (Dox) in the presence of Nam. DNA microarray analyses were performed using total RNAs from Nam (4 mM)-treated Max-null ES cells that were cultured in the presence or absence of doxycycline for 6 days.
Project description:We explored the relationship between Myc activity and PI3K signaling in ESCs. Our data demonstrate that Myc and PI3K signaling function cooperatively for supporting pluripotent property of ESCs. Moreover, our data demonstrate that exposure of ESCs to 2i condition render both Myc and PI3K dispensable for preserving ESC status. Effect of PI3K inhibitor, LY4294002 on EBRTcH3 ESCs or their derivatives overexpressing c-Myc (wild-type or T58A mutant) was examined. Effect of LY4294002 on EBRTcH3 ESCs under 2i conditions was also examined. Furthermore, effect of Max expression ablation was compared between ESCs and those overexpressing p110 alpha.
Project description:Comparing global transcriptional profile between standard or 2i derived ES cell lines. RNA extracted from established ES cell lines either from standard derivation condition or 2i derivation condition, all cultured without 2i after derivation.
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.
Project description:Identification of transcripts in murine embryonic stem (ES) cell lines growing under 3 different self-renewing conditions; GMEM + 10% FCS, N2B27 + 2i (1 μM PD032 and 3 μM CHIR99021) and knockout serum replacement (KOSR) medium (80% Knockout DMEM, 20% Knockout Serum Replacement). Under all conditions, ES cells were grown on gelatin-coated dishes and passaged by trypsinisation. ES cells were cultured in each condition for at least 3 passages prior to sample collection. The aim of this array experiment is to identify significant differences in transcript levels of ES cells grown under different conditions. Differences of transcript levels from the different conditions should be consistent among the biological and technical replicates for each.
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. RNA-seq of sorted Hex low and high expressing ES cell populations cultured in serum/LIF or 2i/LIF as well as unsorted ES cells from 2i or 2i/LIF.