Wnt inhibition confers hESCs a unique transcriptional state compatible with differentiation, but not with derivation
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ABSTRACT:
PROVIDER: PRJNA415005 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6190370.fastq.gz | Fastqsanger.gz | |||
| SRR6190380.fastq.gz | Fastqsanger.gz | |||
| SRR6190390.fastq.gz | Fastqsanger.gz | |||
| SRR6190400.fastq.gz | Fastqsanger.gz | |||
| SRR6190410.fastq.gz | Fastqsanger.gz |
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Project description:Human embryonic stem cells (hESCs) hold great value for future clinical applications. However, standard culture conditions maintain hESCs in a primed state, which bears heterogeneity and a tendency for spontaneous differentiation. To counter these drawbacks, hESCs have been converted to a naive state, but this has in turn restricted the efficiency of existing directed differentiation protocols. We show that adaptation of hESCs to Wnt-inhibiting condition (Wnt-i) conferred a unique transcriptional signature with high levels of pluripotency markers and reduced levels of differentiation markers. Moreover, neuronal and cardiac differentiation of Wnt-i hESCs progressed similarly to primed hESCs, unlike their naïve counterparts. Remarkably, in contrast to mouse, Wnt-i conditions failed to support direct derivation of hESCs, suggesting that active Wnt signaling is required for the transition from inner cell mass to hESCs. Wnt-i hESCs retained advantages of both primed and naive counterparts and may therefore be a better starting point for differentiation towards clinically desired cell types.
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Project description:Deciphering the regulatory network for human naïve and primed pluripotency is of fundamental theoretical and applicable significance. Here, by combining quantitative proteomics, phosphoproteomics and acetylproteomics analyses, we revealed RNA processing and translation as the most differentially-regulated processes between naïve and primed human embryonic stem cells (hESCs). While glycolytic primed hESCs rely predominantly on eIF4E-mediated cap-dependent pathway for protein translation, naïve hESCs with reduced mTORC1 activity are more tolerant to blockage of eIF4E-dependent translation, and their bivalent metabolism allows for translating selective mRNAs via both eIF4E-dependent and eIF4E-independent/eIF4A2-dependent pathways to form a more compact naïve proteome. Globally up-regulated proteostasis system and down-regulated post-translational modification system help to further refine and maintain the naïve proteome that is compatible with the more rapid cycling of naïve hESCs, where CDK1 plays an indispensable coordinative role.
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Project description:X chromosome inactivation (XCI) is a dosage compensation mechanism in female cells to regulate X-linked gene expression. We report here that subcultures from established lines of female hESCs displayed variations (0-100%) in the expression of XCI markers such as XIST RNA coating and enrichment of histone H3 lysine 27 trimethylation (H3K27me3) on inactive X chromosome. Surprisingly, regardless of the presence or absence of XCI markers in different cultures, all female hESCs we examined (H7, H9, and HSF6 cells) exhibit a mono-allelic expression pattern for a majority of X-linked genes. Our results suggest that these established female hESCs have completed XCI during the process of derivation and/or propagation, and the XCI pattern of lines we investigated is already non-random. However, XIST gene expression in subsets of female hESCs is unstable and subject to epigenetic silencing through DNA methylation. Concomitant with the loss of XCI markers including XIST expression and H3K27me3, approximately 12% of X-linked CpG islands become hypomethylated and a subset of previously silenced X-linked alleles are reactivated, resulting a significant elevation of gene expression dosage. Because changes in dosage compensation of X-linked genes could impair somatic cell function, we propose that XCI status should be routinely checked in subcultures of female hESCs, with cultures displaying XCI markers better suited for use in regenerative medicine. Keywords: Genotyping, gene expression and DNA methylation We used Affymetrix Genotyping array for looking for X-linked SNPs with HSF6, H7 and H9 genomic DNA, Agilent Gene expression array for comparing gene expression patten changes between HSF6 hESCs with X-inactiation and HSF6 hESCs without X-inactivation (3 repeats). Finally, mDIP-Chip method was used to detect X-linked CpG island methylation changes differences between hESCs with X-inactivation and hESCs without X-inactivation using Agilent CpG island array (3 repeats, and one of them has dye swap)
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Project description:X chromosome inactivation (XCI) is a dosage compensation mechanism in female cells to regulate X-linked gene expression. We report here that subcultures from established lines of female hESCs displayed variations (0-100%) in the expression of XCI markers such as XIST RNA coating and enrichment of histone H3 lysine 27 trimethylation (H3K27me3) on inactive X chromosome. Surprisingly, regardless of the presence or absence of XCI markers in different cultures, all female hESCs we examined (H7, H9, and HSF6 cells) exhibit a mono-allelic expression pattern for a majority of X-linked genes. Our results suggest that these established female hESCs have completed XCI during the process of derivation and/or propagation, and the XCI pattern of lines we investigated is already non-random. However, XIST gene expression in subsets of female hESCs is unstable and subject to epigenetic silencing through DNA methylation. Concomitant with the loss of XCI markers including XIST expression and H3K27me3, approximately 12% of X-linked CpG islands become hypomethylated and a subset of previously silenced X-linked alleles are reactivated, resulting a significant elevation of gene expression dosage. Because changes in dosage compensation of X-linked genes could impair somatic cell function, we propose that XCI status should be routinely checked in subcultures of female hESCs, with cultures displaying XCI markers better suited for use in regenerative medicine. Keywords: Genotyping, gene expression and DNA methylation
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Project description:Histones modulate gene expression by chromatin compaction, regulating numerous processes such as differentiation. However, the mechanisms underlying histone degradation remain elusive. When compared with their differentiated counterparts, immortal human embryonic stem cells (hESCs) have a unique chromatin architecture and low levels of trimethylated histone H3 at lysine 9 (H3K9me3), a heterochromatin-associated modification. Here we assess a link between the intrinsic epigenetic landscape and ubiquitin-proteasome system of hESCs. We find that hESCs exhibit high expression of UBE2K, a ubiquitin-conjugating enzyme. Loss of UBE2K increases the levels of H3K9 trimethyltransferase SETDB1, resulting in H3K9 trimethylation and repression of neurogenic genes during differentiation. Concomitantly, loss of UBE2K impairs the ability of hESCs to differentiate into neural progenitors with neurogenic properties. Besides H3K9 trimethylation, we find that UBE2K binds histone H3 to induce its polyubiquitination and degradation by the proteasome. Notably, ubc-20, the worm orthologue of UBE2K, also regulates both histone H3 levels and H3K9 trimethylation in C. elegans germline. Thus, our results indicate that UBE2K crosses evolutionary boundaries to promote histone H3 degradation and reduce H3K9me3 repressive marks in immortal cells.
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Project description:Currently, there is only minimal information elucidating the transcriptional changes occurring during the in vitro transition of the inner cell mass (ICM) towards the human embryonic stem cell (hESC) stage and the role played by the post inner cell mass intermediate (PICMI). In this study, we perform in-depth analysis of the transcriptional heterogeneity between these three stages of hESC derivation in order to correlate their downstream effects on pluripotency states and differentiation. We reveal that although PICMI is transcriptionally in close proximity to the hESC profile and distinct from ICM, it exhibits upregulation of primordial germ cell markers, dependence on LIF signaling, upregulation of naïve pluripotency-specific signaling networks and appears to be a switching point from naïve to primed pluripotency. This indicates similarities with mouse epiblast-like cells displaying a transient state of pluripotency, between naïve and primed pluripotent states. Overall, we highlight the need to gain insight into the molecular and transcriptional landscape during hESC derivation, which will further help in the derivation of human PGCs and naïve hESCs in vitro which remains inefficient.
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