Ground state of pluripotency cannot be maintained in mouse embryonic stem cells upon prolonged inhibition of GSK3
ABSTRACT: We compare gene expression changes in mESCs culture in serum/LIF or 2i/LIF which favour the ground state pluripotency. Published RNASeq data where compared with newly generated data set in order to indentify transciptional signatures associated with pluripotency of mouse ES cells Examination of dynamic gene expression in mouse ES cells culture in convetional media (serum/LIF) and in 2i/LIF which favour the gorund state pluripotency
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.
Project description:Nanog null neural stem (NS) cells were reprogrammed to naive pluripotency in 2i/LIF conditions with mouse (m) Nanog and human (h) Nanog. Global gene expression in resulting iPS cells was compared to embryonic stem (ES) cells and nanog null NS cells. Murine iPS cells derived with mouse nanog iPS and human nanog iPS and then compared to embryonic stem cells and nanog null neural stem cells (3 replicates each).
Project description:This study describes the DNA methylation profiling using whole-genome bisulfite sequencing of mouse ES cells, either derived and maintained in 2i serum-free NDiff medium, or in the presence of serum and LIF, or maintained and derived in the presence of serum and LIF and subsequently adapted to 2i serum-free NDiff medium, or maintained and derived in the presence of 2i and LIF and subsequently adapted to 2i serum. DNA methylation profiling using whole-genome bisulfite sequencing of 14 samples, 3 different lines (E14, XT67E1, Rex/GFP-2i) of pluripotent mouse ES cells as well during conversion from 2i to serum and vice versa.
Project description:Following implantation, mouse epiblast cells transit from a naïve to a primed state in which they are competent for both somatic and primordial germ cell (PGC) specification. Using mouse embryonic stem cells (mESC) as an in vitro model to study the transcriptional regulatory principles orchestrating peri-implantation development, here we show that the transcription factor Foxd3 is necessary for the exit from naïve pluripotency and the progression to a primed pluripotent state. During this transition, Foxd3 acts as a repressor that dismantles a significant fraction of the naïve pluripotency expression program through the decommissioning of active enhancers associated with key naïve pluripotency and early germline genes. Subsequently, Foxd3 needs to be silenced in primed pluripotent cells to allow the reactivation of relevant genes required for proper PGC specification. Our findings uncover a wave of activation-deactivation of Foxd3 as a crucial step for the exit from naïve pluripotency and subsequent PGC specification. mRNA profiles were generated by RNA-seq in duplicates for each of the following mESC lines: Foxd3fl/fl;Cre-ER mESC maintained in "Serum+LIF" (SL) treated with TM for three days (SL Foxd3-/-); untreated Foxd3fl/fl;Cre-ER SL mESC (SL Foxd3fl/fl); tetON Foxd3 SL mESC treated with Dox for three days; WT SL mESC treated with Dox for three days; Foxd3fl/fl;Cre-ER mESC maintained in "2i+LIF" (2i) treated with TM for three days (2i Foxd3-/-); untreated Foxd3fl/fl;Cre-ER 2i mESC (2i Foxd3fl/fl).
Project description:In this study we have analyzed the global gene expression of naïve mouse embryonic stem cells in different culture conditions including R2i (PD0325901+SB431542), 2i (PD0325901+CHIR99021), and also PD0325901+LIF and SB431542+LIF to show the similarities and differences between the conditions in maintaining pluripotency. Since the first generation of mouse embryonic stem (ES) cells, extrinsic regulation of pluripotency has been at the focus of attention. Here we show that suppression of transforming growth factor β (TGFβ) signaling could have impressive effects on self-renewal and pluripotency of mouse ES cells. We introduce a chemically defined medium with inhibitors of TGFβ and mitogen-activated protein kinase (MAPK) kinase, designated here as R2i (Royan 2 inhibitors), for highly efficient establishment of ES cell lines from various mouse strains. R2i also supports homogenous expression of Nanog and Dppa3 proteins in ES cells and shows minimal differentiation leakage. Our results uncover an appropriate condition for ES cell generation from single blastomeres of various cleavage-stage embryos. Further, the high accuracy of genome integrity after long-term cultivation in R2i illustrates an optimal condition for ES cell culture. Global transcriptomic analysis of R2i cells demonstrates that bone morphogenetic protein 4 (BMP4) signaling becomes overrepresented pursuant to TGFβ repression, which may confer further robustness to pluripotency through shielding cells from differentiation stimuli. These findings point to a new facet of ground state pluripotency by blocking differentiation pathways, without influencing the pluripotency regulatory circuitry of mouse ES cells. Whole gene expression study of 4 different mouse embryonic stem cell lines maintained under 4 different chemically defined conditions: R2i, 2i, PD and SB
Project description:We performed an integrated analysis of RNA and proteins at the transition between naïve ES cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators were specifically released from translational inhibition mediated by RNA-Induced Silencing Complex (RISC). This suggests that, prior to differentiation, the propensity of ES cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators was reinstated following acute inactivation of RISC, and it correlated with loss of stemness markers and activation of early cell differentiation markers in treated ES cells. We evaluated global miRNA profiles of embryonic stem cells cultured in either 10%FCS+LIF or 2i+LIF and of Epiblast-Like Aggregates derived from ES cells in 10%FCS+LIF or 2iL+LIF.
Project description:Nanog null neural stem (NS) cells were reprogrammed to naive pluripotency in 2i/LIF conditions with chick (c) and zebrafish (z) Nanog orthologs. Global gene expression was compared to iPS cells derived with mouse (m) Nanog. Murine iPS cells derived with zebrafish nanog, chick nanog, and mouse nanog orthologs (2 replicates each).
Project description:Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. While it is known that the addition of inhibitors of GSK3β and MEK (so-called 2i conditions) push ESC cultures towards a more homogeneous naïve pluripotent state, the molecular underpinnings of this naïve transition are not completely understood. Here we demonstrate that Dazl, a RNA-binding protein previously thought to be expressed specifically in developing primordial germ cells (PGCs), marks a subpopulation of ESCs in vitro that is actively transitioning toward naïve pluripotency. In the absence of Dazl expression, ESCs fail to induce proper expression of Tet enzymes required for 5-hydroxymethylation in 2i-culture conditions. As a result, 5-hydroxymethylation of methylated cystosine residues is impaired. Indeed, we demonstrate that Tet1 and Tet2 are mRNA targets of Dazl, indicating that Dazl might play a role in protection or stabilizing these mRNA molecules. Our results provide insight in the regulation of the acquisition of naïve pluripotency and demonstrate that Dazl is required for TET-mediated cytosine hydroxymethylation in cells that are actively reprogramming to a pluripotent ground state. Two independent mouse ES cell lines, Dazl-GFP and Stella-GFP, were cultured on γ-irradiated feeder MEFs in DMEM containing 15% FBS or serum-free B27N2 medium both supplemented with leukemia inhibitory factor (LIF) (Ying 2008). For the 2i experiments, 1µM MEK inhibitor PD0325901 (Axon Medchem), and 5 µM GSK3β inhibitor Kenpaullone (Tocris), were used. Cells were harvest at 0, 3 and 10 days in each condition for expression microarray analysis.
Project description:The self-renewing pluripotent state was first captured in mouse embryonic stem cells (mESCs) over two decades ago. The standard condition requires the presence of serum and LIF, which provide growth promoting signals for cell expansion. However, there are pro-differentiation signals which destabilize the undifferentiated state of mESCs. The dual inhibition (2i) of the pro-differentiation Mek/Erk and Gsk3/Tcf3 pathways in mESCs is sufficient to establish an enhanced pluripotent “ground state” which bears features resembling the pre-implantation mouse epiblast. Gsk3 inhibition alleviates the repression of Esrrb, a transcription factor that can substitute for Nanog function in mESCs. The molecular mechanism that is mediated by Mek inhibition is however not clear. In this study, we investigate the pathway through which Mek inhibition operates to maintain ground state pluripotency. We have found that in mESCs, Kruppel-like factor 2 (Klf2) is a protein target of the Mek/Erk pathway; and that Klf2 protein is phosphorylated by Erk2 and subsequently degraded through the proteosome. It is therefore by Mek-inhibition through PD0325901 or 2i that enables the stabilization and accumulation of Klf2 to sustain ground state pluripotency. Importantly, we found that Klf2-null mESCs, while viable under LIF/Serum conditions, cannot be maintained and eventually gradually die within a few passages. Our result thus demonstrates that Klf2 is an essential factor of ground state pluripotency. Collectively, our study defines the Mek/Klf2 axis that cooperates with the Gsk3/Esrrb pathway in mediating ground state pluripotency.
Project description:Analysis of genes induced by 2I condition 2i contains glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase kinase (MEK) inhibitors: 3uM Chir99021 and 1uM PD0325901 Total RNA obtained from B6 mESCs treated with LIF or LIF/2I for 12 hours.