Overview
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Key numbers
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- and 200 nM) that covers the range of Hg concentrations typically found in human maternal blood during DE cell induction.
- the developmental effects of MeHg exposure on non-brain 4% oxygen (O2) and 10% carbon dioxide (CO2) in a tri-gas
- reached around 85%. Only hESCs under passage 40 (within MeHg by 1000 × dilution of the stock solutions in induction
- 10% carbon dioxide (CO2) with daily medium change. When Cell viability was determined by a Cell Counting Kit 8
- the cell confluency reached around 20%–30%, the growth (WST-8/CCK8) (Catalog no. ab228554, Abcam Inc.)
- fixing with 4% paraformaldehyde (Santa Cruz Biotechnol- exposed to MeHg as described above. At the end of induc-
- 5% bovine serum albumin (BSA) diluted in PBS containing gently lifted by StemPro Accutase cell dissociation reagent
- 0.3% Triton X-100 (Sigma-Aldrich). The cells were, respec- after rinsing by PBS. Then the cells were centrifuged and
- diluted at 1:500 in 5% BSA at 4 °C, overnight. Prolong Gold point (before induction, during induction and after induc-
- MeHg chloride (purity: 99.9%) was obtained from Sigma- MeHg doses used for RNA sequencing were chosen
- 0.3, 0.4, 0.5, 1, 5 and 10 mM). hESCs were seeded in six- maternal blood (Table 1). The final doses of 0, 10, 100,
- fluency of around 20%–30%. The cells were then exposed sequencing as these doses did not induce significant cell
- to 0 (DMSO as vehicle control), 0.001, 0.01, 0.1, 0.2, 0.3, death and covered the range of Hg concentrations typi-
- Table 1 Maternal blood concentrations of Hg in different populations
- Plus containing 10% β-mercaptoethanol (β-ME). The cell bioinformatics.babraham.ac.uk/projects/fastqc/). All analy-
- 10,000 rpm, 70% ethanol was added to the flow-through, the “edgeR” package (Robinson et al. 2010). Boxplots
- mental toxicity of MeHg on hESCs while differentiating Cell lysate samples were electrophoresed on 4–20%
- et al. 2021), to calculate the benchmark doses (BMD). The ranged from 20 to 24 μg between different batches. Gels
- Reverse transcription quantitative polymerase with 5% non-fat dry milk (NFDM, Catalog no. 1706404;
- chain reaction (RT‑qPCR) Bio-Rad) or 3% bovine serum albumin (BSA, Catalog no.
- centration was determined using NanoDrop™ 2000 spec- with antibodies of interest (Table S2) diluted in their corre-
- ment of the relative intensities of the 28 and 18 s ribosomal containing 0.1% Tween 20 (TBST) on a shaker for 5 min and
- and reverse primers (Table S1), and 0.5 µl cDNA template ware (Bio-Rad) was used to quantify the band intensities,
- for all primer pairs was between 90 and 110%. Final data stem cells (Sharma et al. 2022), the expression of autophagic
- were normalized to the geometric mean of the three refer- protein markers (LC3B and SQSTM1, antibody information
- ence genes (gapdh, hddc2 and znf324b) and the 0 nM group is shown in Table S2) was also measured by western blots
- a total of 1163 DEGs were identified (Excel Table S1), with ing negative regulation of cell differentiation (GO:0045596),
- being considered as significant. Data are presented as means ± SEMs et al. 2021; Roos et al. 2012), such as calcium signaling
- age rank of DEGs based on their BMDs (Excel Table S3), LEFTY2 were also major genes involved in the endoderm
- gered by increasing doses of MeHg (Excel Table S4). verification. In addition to the above genes, two other genes
- embryonic stem cells (hESCs) during definitive endoderm showed close to 50% reduction in cell viability when
- mal models (Bittencourt et al. 2019). This is supported by consistent, as 85% of DEGs in the 10 nM MeHg-dosed
- findings in this study with respect to the effects of MeHg at group and 93% of DEGs in the 100 nM MeHg-dosed group
- cells to DE cell differentiation. Data are presented as means ± SEMs differences between treatment and control groups with p < 0.05 being
- with N = 4–6 for each protein. Each batch was first normalized to considered as significant marked by asterisk
- promoted, abnormal organ development, and subsequently (Table 1). Our results suggest that the risk of MeHg during
Methods (brief)
- RNA sample preparation and next‑generation
- induced, and treated as described above. RNA samples Sequencing data analysis and marker selection
- and mixed well by pipetting. The mixed sample solutions showed filtered data to reflect quantile normalization, and
- for 15 s at 10,000 rpm. The flow-through was discarded, strate the clustering of samples in different groups. The R
- possible residual Buffer RPE. To elute the RNA samples, (GO) functional enrichment and Kyoto Encyclopedia of
- for 1 min, and the eluted RNA samples were stored imme- used as the cutoff for KEGG pathway analysis. Data were
- The RNA samples were sent to Genome Quebec for GO functional enrichment analysis identified 32 GO
- reads for each sample and three biological replicates for related GO terms was conducted using Cytoscape (https://
- downstream qPCR and western blot verification. 100 °C for 4 min after mixing with 4 × Laemmli sample
- mental toxicity of MeHg on hESCs while differentiating Cell lysate samples were electrophoresed on 4–20%
- RNA samples were prepared as described above. RNA con- the protein targets for 1 h at room temperature and incubated
- samples were run for every primer pair. Reaction efficiency As autophagy plays a critical role in the differentiation of
- malization of the samples (Fig. 3a, b). Based on the cutoff tiation (GO:0006413), and protein targeting to membrane
- digestion and absorption (hsa04974) and calcium signaling 2015; Teo et al. 2011; Xuan and Sussel 2016), these genes
- genes (DEGs). a Boxplot for transcriptomic samples after quan- represents relatively high gene expression levels. d–f Volcano plots
- tile normalization. b PCA for transcriptomic samples after quantile of DEGs identified from 10 nM, 100 nM, and 200 nM MeHg expo-
- responding to a gene and each column corresponding to a sample. (color figure online)
Implications
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Wiki pages this source may touch
- Fish — marine, non-predatory (sardines, anchovies, salmon, cod)
- Mercury
- Mercury
- Cadmium
- Arsenic
- Chromium
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Update history
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