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Comparative parallel multi-omics analysis during the induction of pluripotent and trophectoderm states.

ABSTRACT: Following fertilization, it is only at the 32-64-cell stage when a clear segregation between cells of the inner cell mass and trophectoderm is observed, suggesting a 'T'-shaped model of specification. Here, we examine whether the acquisition of these two states in vitro, by nuclear reprogramming, share similar dynamics/trajectories. Using a comparative parallel multi-omics analysis (i.e., bulk RNA-seq, scRNA-seq, ATAC-seq, ChIP-seq, RRBS and CNVs) on cells undergoing reprogramming to pluripotency and TSC state we show that each reprogramming system exhibits specific trajectories from the onset of the process, suggesting 'V'-shaped model. We describe in detail the various trajectories toward the two states and illuminate reprogramming stage-specific markers, blockers, facilitators and TSC subpopulations. Finally, we show that while the acquisition of the TSC state involves the silencing of embryonic programs by DNA methylation, during the acquisition of pluripotency these regions are initially defined but retain inactive by the elimination of H3K27ac.

SUBMITTER: Jaber M

PROVIDER: S-EPMC9205865 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Comparative parallel multi-omics analysis during the induction of pluripotent and trophectoderm states.

Jaber Mohammad M Radwan Ahmed A Loyfer Netanel N Abdeen Mufeed M Sebban Shulamit S Khatib Areej A Yassen Hazar H Kolb Thorsten T Zapatka Marc M Makedonski Kirill K Ernst Aurelie A Kaplan Tommy T Buganim Yosef Y

Nature communications 20220617 1

Following fertilization, it is only at the 32-64-cell stage when a clear segregation between cells of the inner cell mass and trophectoderm is observed, suggesting a 'T'-shaped model of specification. Here, we examine whether the acquisition of these two states in vitro, by nuclear reprogramming, share similar dynamics/trajectories. Using a comparative parallel multi-omics analysis (i.e., bulk RNA-seq, scRNA-seq, ATAC-seq, ChIP-seq, RRBS and CNVs) on cells undergoing reprogramming to pluripotenc ...[more]

PMID: 35715410

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Project description:During early embryogenesis, totipotent cells undergo asymmetric cell divisions, resulting in two compartments in the early embryo: the inner cell mass (ICM) that gives rise to pluripotency cells and an outer layer of trophectoderm (TE). It is only at the 32-64- cell stage when a clear segregation between the two cell types can be observed. This proposes a ‘T’-like model where cells of each stage (2-16-cell stage) of the early embryo undergo a relatively similar changes in their transcriptome and epigenome before specification. Here, we sought to understand whether cells acquiring pluripotency or trophectoderm state, by reprogramming factors (OSKM vs GETM), share similar processes between themselves such as those of the early embryo. To that end, we conducted a comparative parallel meta-analysis on cells undergoing reprogramming to induced pluripotent stem cells (iPSCs) and induced trophoblast stem cells (iTSCs) and examined their transcriptome (i.e. Bulk RNA-seq and SC-RNA-seq), methylome (i.e. RRBS), chromatin accessibility and activity (i.e. ATAC-seq and Chip-seq for H3K4me2 and H3K27ac) and genomic stability (CNVs) along the process. Our analysis revealed that, in contrast to cells of the pre-segregation embryo that mostly resemble each other in each stage, cells undergoing reprogramming to pluripotency and TSC states, exhibit unique and specific trajectories from the beginning of the process till the end, suggesting a ‘V’-like behavior. Although similar processes such as somatic identity loss, proliferation, MET and metabolic shift occur in the two models, each of the processes mostly uses different sets of genes and regulatory elements to induce its own fate. We characterized each trajectory in details and illuminated key aspects that are either shared or distinguished between the two states. Using these analyses, we identified previously unknown markers for faithful reprogramming process and key factors that drastically inhibit both reprogramming processes. We show that this comparative parallel meta-analysis is a powerful tool to understand nuclear reprogramming and cellular plasticity at large.

Project description:BackgroundIn mammals, specification of primordial germ cells (PGCs) is established in the early post-implantation embryo. The bone morphogenetic protein (BMP)-SMAD and WNT3-β-catenin signaling initiate the gene regulatory network for PGC specification. The activation of SOX17-BLIMP1 axis is critical for human PGC program. Moreover, EpCAM and INTEGRINα6 were identified as surface markers of human PGC-like cells (PGCLCs) recently. However, the signaling mechanism for PGC specification in non-rodent mammals remains to be clarified.MethodsWe differentiated human induced pluripotent stem cells (hiPSCs) into PGCLCs in vitro in response to Activin A and BMP4. The percentage of EpCAM/INTEGRINα6 double-positive cells (PGCLCs) was analyzed by flow cytometry. The expression of PGC genes was evaluated by qRT-PCR and immunofluorescence. The expression dynamic of multi-lineage genes during the differentiation process was evaluated by qRT-PCR.ResultsUnder the stimulation for PGCLC induction, the embryoids derived from hiPSCs initiated significant upregulation of the early PGC genes (BLIMP1, TFAP2C, and NANOS3), but maintained low or no levels of DPPA3 and late PGC genes (DAZL and DDX4). The percentage of EpCAM/INTEGRINα6 double-positive PGCLCs reached the highest at day 6 of induction. After pre-induction, the incipient mesoderm-like cells (iMeLCs) upregulated most of the mesoderm genes (EOMES, T, MSXI, RUNX2, and MIXL1). The differentiating embryoids showed high levels of key pluripotency genes, OCT4 and NANOG, but became negative for SOX2. In contrast to iMeLCs, the differentiating embryoids downregulated mesoderm genes RUNX2 and EOMES, and ectoderm gene PAX6, but increased the expression of endoderm gene SOX17.ConclusionsDuring PGCLC induction process in vitro, the differentiating embryoids not only activated the PGC-related genes, but also displayed complex regulation of pluripotency genes and multi-lineage genes. These results would be meaningful for future research investigating the regulation of human early germ line development.

Dataset Information

Comparative parallel multi-omics analysis during the induction of pluripotent and trophectoderm states.

Publications

Comparative parallel multi-omics analysis during the induction of pluripotent and trophectoderm states.

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