Project description:Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C signalling sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signalling, are phenotypically stable and karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed in reset cells with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground state transcription factors, TFCP2L1 or KLF4 has marginal impact on conventional human pluripotent stem cells, but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground state pluripotency in human cells. DNA methylation analysis in Conventional and Reset human embryonic stem cells by whole genome bisulfite sequencing, in triplicate, using the Illumina platform
Project description:Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question whether an earlier ‘naive’ state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support autonomous self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate a homogeneous population of human pluripotent stem cells in which transcription factors associated with the ground state of pluripotency are highly upregulated. Comparison with previously reported naive human ESCs indicates that our kinase inhibitor cocktail captures a novel pluripotent state in humans that closely resembles mouse ESCs. ChIP-seq data from human embryonic stem cells in naive and primed conditions were generated by deep sequencing using Illumina Hi-Seq 2000.
Project description:Time series embryonic gonadal transcriptome of the painted turtle Chrysemys picta prior-to, at the onset of, during, and end of the thermosensitive period. Raw sequence reads
Project description:We apply deep small-RNA sequencing technology for high-throughput profiling of microRNAs in ground state embryonic stem cells (ESCs). We provide global expression signatures of microRNAs in ESCs cultured under serum, 2i, and R2i conditions. We report that microRNAs are significantly differentially expressed when ESCs are cultured under different conditions, and that ground state pluripotency features a uniqure microRNA signature which is mainly encoded by microRNA-coding sequences within the developmentally important DLK1-Dio3 locus. Finally, we indicate that microRNA upregulated in ground state pluripotent cells (i.e. 2i/R2i) contribute to the maintenace of ground state pluripotency through stimulating self-renewal and inhibiting multi-lineague differentiation.
Project description:Deciphering the mechanisms that control the pluripotent ground state is key for understanding embryonic development. Nonetheless, the epigenetic regulation of ground-state mouse embryonic stem cells (mESCs) is not fully understood. Here, we identify the epigenetic protein MPP8 as being essential for ground-state pluripotency. Its depletion leads to cell cycle arrest and spontaneous differentiation. MPP8 has been suggested to repress LINE1 elements by recruiting the human silencing hub (HUSH) complex to H3K9me3-rich regions. Unexpectedly, we find that LINE1 elements are efficiently repressed by MPP8 lacking the chromodomain, while the unannotated C-terminus is essential for its function. Moreover, we show that SETDB1 recruits MPP8 to its genomic target loci, whereas transcriptional repression of LINE1 elements is maintained without retaining H3K9me3 levels. Taken together our findings demonstrate that MPP8 protects the DNA-hypomethylated pluripotent ground state through its association with the HUSH core complex, however, independently of detectable chromatin binding and maintenance of H3K9me3.