Unknown,Transcriptomics,Genomics,Proteomics

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A developmental coordinate of the spectrum of pluripotency among mice, monkeys, and humans

ABSTRACT: The epiblast (EPI) is the origin of all somatic and germ cells in mammals, and of the spectrum of pluripotent stem cells (PSCs) in vitro. To explore the ontogeny of human/primate pluripotency, we performed comprehensive single-cell RNA sequencing for pre- and post-implantation EPI development in cynomolgus monkeys. Here we show that after specification in the blastocysts [embryonic day (E)7], cyEPI undergoes major transcriptome changes upon implantation. Thereafter, cyEPI, while generating gastrulating cells (~E13), maintains its transcriptome relatively stably over a week, retaining a unique set of pluripotency genes while acquiring properties for âneuron differentiation.â h/cyPSCs show the highest similarity to post-implantation late cyEPI (~E17), which, despite co-existing with gastrulating cells, bears characteristics of pre-gastrulating mouse EPI (E5.5) and epiblast-like cells (EpiLCs) in vitro. These findings not only reveal divergence/coherence of EPI development, but also identify a developmental coordinate of the spectrum of pluripotency among key species, providing a basis for better regulation of human pluripotency in vitro. Single cell transcriptome analysis of cynomolgus monkey embryo E6-17 and mouse embryo E4.5 - E6.5 as well as of cynomogus monkey embryonic stem cells (ESCs) and mouse ESC, epiblast like cells (EpiLCs), using SC3-seq technology.

ORGANISM(S): Mus musculus

SUBMITTER: Yukihiro Yabuta

PROVIDER: E-GEOD-74767 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A developmental coordinate of pluripotency among mice, monkeys and humans.

Nakamura Tomonori T Okamoto Ikuhiro I Sasaki Kotaro K Yabuta Yukihiro Y Iwatani Chizuru C Tsuchiya Hideaki H Seita Yasunari Y Nakamura Shinichiro S Yamamoto Takuya T Saitou Mitinori M

Nature 20160824 7618

The epiblast (EPI) is the origin of all somatic and germ cells in mammals, and of pluripotent stem cells in vitro. To explore the ontogeny of human and primate pluripotency, here we perform comprehensive single-cell RNA sequencing for pre- and post-implantation EPI development in cynomolgus monkeys (Macaca fascicularis). We show that after specification in the blastocysts, EPI from cynomolgus monkeys (cyEPI) undergoes major transcriptome changes on implantation. Thereafter, while generating gast ...[more]

PMID: 27556940

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Project description:The potential for epigenetic changes in host cells following microbial infection has been widely suggested, but few examples have been reported. We assessed genome-wide patterns of DNA methylation in human macrophage-like U937 cells following infection with Burkholderia pseudomallei, an intracellular bacterial pathogen and the causative agent of human melioidosis. Our analyses revealed significant changes in host cell DNA methylation, at multiple CpG sites in the host cell genome, following infection. Infection induced differentially methylated probes (iDMPs) showing the greatest changes in DNA methylation were found to be in the vicinity of genes involved in inflammatory responses, intracellular signalling, apoptosis and pathogen-induced signalling. A comparison of our data with reported methylome changes in cells infected with M. tuberculosis revealed commonality of differentially methylated genes, including genes involved in T cell responses (BCL11B, FOXO1, KIF13B, PAWR, SOX4, SYK), actin cytoskeleton organisation (ACTR3, CDC42BPA, DTNBP1, FERMT2, PRKCZ, RAC1), and cytokine production (FOXP1, IRF8, MR1). Overall our findings show that pathogenic-specific and pathogen-common changes in the methylome occur following infection. The human leukemic monocyte lymphoma cell line (U937, ATCC CRL-1593.2) was maintained in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) at 37Â°C. U937 cells were differentiated to macrophage-like cells following exposure to 20 ng/ml (final concentration) of phorbol 12-myristate 13-acetate (PMA) for 48 hours at 37Â°C and differentiation evidenced by increased adherence to tissue culture flasks. Overnight cultures of B. pseudomallei K96243 were diluted in L-15 medium and added to differentiated U937 cells at a multiplicity of infection (MOI) of 10. Uninfected controls were overlaid with L15 medium only. The cells were then incubated at 37Â°C to allow infection. The cells were washed 3 times with PBS and incubated with fresh L15 medium containing 1mg/ml kanamycin to kill extracellular bacteria. At appropriate time points the cells were washed 3 times in warm PBS and lysed with 0.1% (vol/vol) Triton X-100. DNA was isolated using an AllPrep kit (Qiagen) and stored at -80ÂºC until required. DNA yield was measured using a Nanodrop instrument with measurements between 22.8 â 50.6 ng/ul. Two experiments were carried out using the above procedure. In Experiment 1, DNA was collected from uninfected and infected cells at 2 hours (T2), and 4 hours (T4) post infection (2 biological replicates and 2 technical replicates from each group). In Experiment 2, DNA was collected from uninfected and infected cells at 1 hour (T1), 2 hours (T2), 3 hours (T3) and 4 hours (T4) post infection (1 sample from each group). The DNA methylation profile was determined using the Infinium HumanMethylation450 BeadChip (450K) (Illumina Inc.) following the manufacturerâs instructions. The data was extracted and the initial analysis was performed using GenomeStudio (2010.3) methylation module (1.8.5). Quality control checks and quantile normalisation were implemented using WateRmelon. Samples with more than 1% of sites with a detection p-value greater than 0.05 were removed as were probes with 1% of samples with a detection p-value greater than 0.05. Probes were removed if they had a bead count less than 3 in 1% of samples. Cross-hybridizing probes were removed, leaving 425496 probes for analysis. Here we report DNA methylation profile of 18 samples (10 infected and 8 control).

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A developmental coordinate of the spectrum of pluripotency among mice, monkeys, and humans

Publications

A developmental coordinate of pluripotency among mice, monkeys and humans.

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