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Single-cell RNA-seq of hiPSCs under 5iLAF naïve culture conditions

ABSTRACT: Single-cell RNA-seq of hiPSCs under 5iLAF naïve culture conditions

PROVIDER: PRJNA812605 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18215263_1.fastq.gz	Fastqsanger.gz
	SRR18215263_2.fastq.gz	Fastqsanger.gz
	SRR18215264_1.fastq.gz	Fastqsanger.gz
	SRR18215264_2.fastq.gz	Fastqsanger.gz
	SRR18215265_1.fastq.gz	Fastqsanger.gz

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Single-cell RNA-seq of hiPSCs under 5iLAF naïve culture conditions

Project description:We applied single-cell RNA sequencing, to define heterogenous cell populations present in human naïve 5iLAF cultures. Noteworthily, although Epiblast-like cells represented the main cell population, we detected a population with a trophectoderm-like gene expression profile which could be subclustered according to three different stages of early trophectoderm maturation. Furthermore, we provided evidence for the existence of an 8-cell-like cell population, equivalent to the 8-cell human embryo. Thus, the 5iLAF naïve culture condition emerges as an excellent system to model gene regulation in the earliest stages of human embryogenesis, including zygotic genome activation..

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Project description:In mammalian preimplantation development, pluripotent cells are set aside from cells that contribute to extra-embryonic tissues. Although the pluripotent cell population of mouse and human embryos can be cultured as embryonic stem cells, little is known about the pathways involved in formation of a bovine pluripotent cell population, nor how to maintain these cells in vitro. The objective of this study was to determine the transcriptomic profile related to bovine pluripotency. Therefore, in vitro derived embryos were cultured in various culture media that recently have been reported capable of maintaining the naïve pluripotent state of human embryonic cells. Gene expression profiles of embryos cultured in these media were compared using microarray analysis and quantitative RT-PCR. Compared to standard culture conditions, embryo culture in 'naïve' media reduced mRNA expression levels of the key pluripotency markers NANOG and POU5F1. A relatively high percentage of genes with differential expression levels were located on the X-chromosome. In addition, reduced XIST expression was detected in embryos cultured in naïve media and female embryos contained fewer cells with H3K27me3 foci, indicating a delay in X-chromosome inactivation. Whole embryos cultured in one of the media, 5iLA, could be maintained until 23 days post fertilization. Together these data indicate that 'naïve' conditions do not lead to altered expression of known genes involved in pluripotency. Interestingly, X-chromosome inactivation and development of bovine embryos were dependent on the culture conditions.

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Project description:It is now well recognized that human embryonic stem cells (hESCs)1 closely resemble mouse epiblast stem cells (mEpiSCs)2-5 exhibiting primed pluripotency unlike mouse ESCs (mESCs) which acquire a naïve pluripotent state4-8. Efforts have been made to trigger naïve pluripotency in hESCs9-11 for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines12. We report here a novel culture medium facilitating rapid induction of naïve pluripotency in established hESCs. Our medium also allows derivation of naïve mESCs from blastocyst stage which has not been shown earlier. The established naïve hESCs could survive long-term single cell passaging, maintain a normal karyotype, exhibit upregulation of naïve pluripotency genes and were dependent on signaling pathways similar to naïve mESCs. Also, they undergo global DNA demethylation, cluster together with previously described naïve hESCs13 and show a distinctive long non-coding RNA profile. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs which is fast, reproducible, can be employed to derive naïve mESCs and can induce efficient differentiation. Three primed and matching naive human embryonic stem cell lines were profiled in duplo.

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