Project description:A systems biology approach in which qualitative modeling based on combining boolean networks and in silico perturbation experiments were employed to identify 15 pluripotency regulating cytokines or cytokine related genes. IL-11 was validated as a novel factor capable of maintaing the undifferentiated state of human embryonic stem cells in the absence of exogenously added bFGF to the culture acting via a different mechanims than bFGF. Transcriptomic microarray data of eight overexpression and knock-down experiments were used for qualitative modeling based on boolean networks to predict novel factors - mainly cytokines - that maintain pluripotent human embryonic stem cells in the absence of bFGF in culture. The culture was maintained for at least 9 passages using and stained positive for alcaline phosphatase staining, OCT3/4, SOX2, NANOG, TRA1-60. Microarray based gene expression profiling showed that the IL-11 treatment occupies an intermediate state, between bFGF treatment and the negative control which is no cytokine treatment as judged by hierarchical clustering. Moreover, KEGG pathway analysis indicates common and additional distinct mechanisms of bFGF and IL-11 dependant pluripotency dependant mechanisms.
Project description:Many transcriptional and epigenetic networks must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. Here, we explore the role of Zfp217 as a key transcriptional factor in maintaining ES cell self-renewal by permorming genome-wide ChIP-Seq analyses. Examination of Zfp217 binding profiling by high throughput sequencing in mouse stem cells
Project description:Many transcriptional and epigenetic networks must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. Here, we explore the role of Zfp217 as a key transcriptional factor in maintaining ES cell self-renewal by performing meRIP analysis in control and Zfp217-depleted mouse stem cells. Examination of m6A levels from total RNA in control and Zfp217 shRNA infected mouse stem cells
Project description:SXO2 is one of the factors involved in maintaining self-renewal and pluripotency in hESCs. This study was performed to reveal the effects of reduced SOX2 levels in self-renewing pluripotent hESCs.
Project description:Pluripotent stem cells are defined by their self-renewal capacity, which is the ability of the stem cells to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into any somatic cell lineage. However, understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. To investigate the interplay between these two aspects of pluripotency, we performed four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSC self-renewal conditions, and the dissolution of the primed pluripotency identity during early differentiation. Comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further discovered a core set of factors that control both stem cell fitness and pluripotent identity, including a network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus cell fitness, and offer a valuable model for categorizing gene function in broad biological contexts.
Project description:Many transcriptional and epigenetic networks must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. Here, we explore the role of Zfp217 as a key transcriptional factor in maintaining ES cell self-renewal by permorming genome-wide ChIP-Seq analyses.
Project description:Many transcriptional and epigenetic networks must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. Here, we explore the role of Zfp217 as a key transcriptional factor in maintaining ES cell self-renewal by performing meRIP analysis in control and Zfp217-depleted mouse stem cells.
Project description:A systems biology approach in which qualitative modeling based on combining boolean networks and in silico perturbation experiments were employed to identify 15 pluripotency regulating cytokines or cytokine related genes. IL-11 was validated as a novel factor capable of maintaing the undifferentiated state of human embryonic stem cells in the absence of exogenously added bFGF to the culture acting via a different mechanims than bFGF.
Project description:Oct4 is considered a master transcription factor for pluripotent cell self-renewal and embryo development. It primarily collaborates with other transcriptional factors or coregulators to maintain pluripotency. However, it is still unclear how Oct4 interacts with its partners. Here, we show that the Oct4 linker interface mediates competing and balanced Oct4 protein interactions which are crucial for maintaining pluripotency. Linker mutant ESCs maintain the key pluripotency genes expression, but show decreased expression of self-renewal genes and increased expression of differentiation genes which result in impaired ESCs self-renewal and early embryonic lethality. Linker mutation dose not affect Oct4 genomic binding and transactivation potential, but breaks the balanced Oct4 interactome. In mutant ESCs, the interaction between Oct4 and Klf5 was decreased, but interactions between Oct4 and Cbx1, Ctr9, Cdc73 were increased which disrupt the epigenetic state of ESCs. Overexpression of Klf5 or knockdown Cbx1, Cdc73 rescue the cellular phenotype of linker mutant ESCs by rebalancing Oct4 interactome indicating that different partners interact with Oct4 competitively. Thus, by showing how Oct4 interacts with different partners, we provide novel molecular insights to explain how Oct4 contributes to the maintenance of pluripotency.
Project description:Hematopoietic stem cells (HSCs) are identified by their ability to sustain prolonged blood cell production in vivo, although recent evidence suggests that durable self-renewal (DSR) is shared by HSC subtypes with distinct self-perpetuating differentiation programs. Net expansions of DSR-HSCs occur in vivo, but molecularly defined conditions that support similar responses in vitro are lacking. We hypothesized that this might require a combination of factors that differentially promote HSC viability, proliferation and self-renewal. We now demonstrate that HSC survival and maintenance of DSR potential is variably supported by different Steel factor (SF)-containing cocktails with similar HSC-mitogenic activities. In addition, stromal cells produce other factors, including nerve growth factor and collagen 1, that can antagonize the apoptosis of initially quiescent adult HSCs and, in combination with SF and interleukin-11, produce >15-fold net expansions of DSR-HSCs ex vivo within 7 days. These findings suggest a new molecular basis for HSC control and expansion. Adult mouse bone marrow CD45+EPCR+CD48-CD150+ cells were used in a total of 4 conditions (fresh, 6 hour stimulation with SF+IL-11+UG26 CM, UG26 CM only, SF+IL-11) with 2 technical replicates per condition.