Dataset Information

The Differences in the Developmental Stages of the Cardiomyocytes and Endothelial Cells in Human and Mouse Embryos at the Single-Cell Level.

ABSTRACT: Our research focuses on expression patterns in human and mouse embryonic cardiomyocytes and endothelial cells at the single-cell level. We analyzed single-cell datasets containing different species, cardiac chambers, and cell types. We identified developmentally dynamic genes associated with different cellular lineages in the heart and explored their expression and possible roles during cardiac development. We used dynamic time warping, a method that aligns temporal sequences, to compare these developmental stages across two species. Our results indicated that atrial cardiomyocytes from E9.5 to E13.5 in mice corresponded to a human embryo age of approximately 5-6 weeks, whereas in ventricular cardiomyocytes, they corresponded to a human embryo age of 13-15 weeks. The endothelial cells in mouse hearts corresponded to 6-7-week-old human embryos. Next, we focused on expression changes in cardiac transcription factors over time in different species and chambers, and found that Prdm16 might be related to interspecies cardiomyocyte differences. Moreover, we compared the developmental trajectories of cardiomyocytes differentiated from human pluripotent stem cells and embryonic cells. This analysis explored the relationship between their respective developments and provided compelling evidence supporting the relevance of our dynamic time-warping results. These significant findings contribute to a deeper understanding of cardiac development across different species.

SUBMITTER: Liu C

PROVIDER: S-EPMC10970218 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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The Differences in the Developmental Stages of the Cardiomyocytes and Endothelial Cells in Human and Mouse Embryos at the Single-Cell Level.

Liu Chuyu C Shao Ning-Yi NY

International journal of molecular sciences 20240313 6

Our research focuses on expression patterns in human and mouse embryonic cardiomyocytes and endothelial cells at the single-cell level. We analyzed single-cell datasets containing different species, cardiac chambers, and cell types. We identified developmentally dynamic genes associated with different cellular lineages in the heart and explored their expression and possible roles during cardiac development. We used dynamic time warping, a method that aligns temporal sequences, to compare these d ...[more]

PMID: 38542214

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Project description:AbstractThe transition of maternal to zygotic gene expression regulation is critical for human preimplantation embryo development. In recent years, single-cell RNA sequencing (scRNA-seq) had been applied to detect the factors that regulate human oocyte maturation and early embryo development. Here, the evaluation of transcriptomes in single blastomere from the embryo collected from patients by scRNA-seq was performed. There were 20 blastomeres biopsied from 8-cell embryos of seven patients who received more than two ART cycles due to low embryo competence. Meanwhile, ten cells were collected from 8-cell embryos of four patients who received ART treatment due to male or tubal factors. The blastomeres were then evaluated using the previously established scRNA-seq method to determine the associations between their gene expression and developmental competence. The total number of genes detected in 8-cell embryos that failed to form blastocyst including maternal and zygotic mRNAs was reduced. There were 324 differently expressed genes detected among the 8-cell embryos including 65 genes that were significantly suppressed in the 8-cell embryos that failed to form blastocyst. Further analysis found these 8-cell embryos arrested at the cleavage stage due to the dysfunction of the cell cycle, DNA transcription activity, histone methylation, and cell division-related genes such as SMCO-1, ZNF271P,ZNF679, ASF1b, BEX3, DPPA2, and ORC4. The alterations of gene expression detected in human 8-cell embryos are tightly associated with its developmental competence and could be used as targets to enhance embryo development or parameters to predict the embryo's development outcomes.Lay summaryMany females are suffering infertility due to the failure of embryonic development at early stages due to unknown causes. At the very beginning of human embryo development, the embryos start to express its own genes, which should be achieved at 8-cell stage. In current research, we isolated one cell from 8-cell embryos and detected the gene expression at single-cell level. Then the remaining cells of these embryos were cultured to form blastocyst. Meanwhile, the data was analyzed according to the outcomes of embryo development. We detected 324 differently expressed genes between the 8-cell embryos that succeeded and failed to form blastocyst. Our research showed the association between the gene expression and the developmental competence of 8-cell embryos. The findings could be used to predict the embryo quality and potential therapy target to improve the efficiency of assisted reproductive techniques.

Project description:Sample Preparation | Four biological replicate of 25 dechorionated, but non-deyolked, zebrafish embryos were collected at 4 time points of embryonic development: 1-cell stage (0-hpf, hour post fertilization), 2-hph, 4-hpf, and 6-hpf. All 16 pooled embryo samples were lysed independently by adding 100 ul of Mammalian lysis buffer and 1x Protease Inhibitor cocktail. Digestion | Samples were reduced and alkylated with TCEP and 2-chloroacetamide. Six volumes of pre-chilled acetone were added, and the precipitation was let to proceed overnight. The precipitated proteins were dissolved in 100 ul of 50 mM TEAB, mass spectrometry grade trypsin (Promega Gold) was added at 1:40 w/w and the digestion was let to proceed at 37oC overnight. Pierce fluorometric peptide assay was performed on 10 ul of each digested sample. Tandem Mass Tag (TMT) Labeling | 20 ul of anhydrous acetonitrile were added to each of the 0.5mg TMTpro 16plex reagents. For each sample, 20 ug of peptides were measured out and adjusted to 100 ul with 100 mM TEAB, then mixed with the TMT reagents. The replicate samples for each of the time points were labeled for 1 hr with TMTpro-126, 127N, 127C, -128N (0-hpf, replicates 1-4); 128C, 129N, 129C, 130C (2-hpf, 1-4); 130C, 131N, 131C, 132N (4-hpf, 1-4), 132C, 133N, 133C, 134N (6-hpf, 1-4), respectively. After checking labeling efficiency (>99%), 30 ul each of the 16 differentially labeled samples were combined in a new tube and the resulting volume was reduced using a SpeedVac concentrator to less than 10 ul. High pH Reverse Phase Fractionation | 300 ul of the TMT-labeled peptide mixture (in 0.1% TFA) were loaded onto a Pierce high pH fractionation cartridge placed on a 2.0 ml sample tube. After centrifuging at 3000 x g for 2 minutes, the eluate was collected as the flow-through fraction. The loaded cartridge was placed on a new sample tube, washed with 300 ul of ddH20, and the eluate collected as wash fraction. An additional round of washing was performed using 300 ul of 5% acetonitrile in 0.1% TFA to remove unreacted TMT reagent. A total of 9 HpH RP fractions (E1-E9) were collected by sequential elution in new sample tubes using 300 ul of 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 50%, and 80% acetonitrile in 0.1% TFA. Dried samples were resolubilized in 44 ul of buffer A (5% acetonitrile in 0.1% formic acid, FA) before LC-MS analysis. Multiplexed Mass Spectrometry Analysis | TMT-labeled peptides were analyzed on an Orbitrap Eclipse Tribrid Mass Spectrometer with a FAIMS Pro interface, equipped with a Nanospray Flex Ion Source, and coupled to a Dionex UltiMate 3000 RSCLnano System. Peptides were loaded on an Acclaim PepMap 100 C18 trap cartridge (0.3 mm i.d., 5 mm length) with the U3000 loading pump via the autosampler. A 75 um i.d. analytical microcapillary column was packed in-house with 250 mm of 1.9 um ReproSil-Pur C18-AQ resin (Dr. Masch). The Orbitrap Eclipse was set up to run the TMT-SPS-MS3 method with three FAIMS compensation voltages (CV) at -40V, -55V, and -70V and a cycle time of 1 sec. Briefly, peptides were scanned from 400-1600 m/z in the Orbitrap at 120,000 resolving power before MS2 fragmentation by CID at 35% NCE and detection in the ion trap set to turbo detection. Dynamic exclusion was enabled for 45s. Real time search (RTS) was performed during the ddMS2 IT CID step against a non-redundant Danio rerio sequence database downloaded from UniProtKB 2021-03 and complemented with common contaminants. Carbamidomethyl and TMTpro16plex (304.2071 Da on Kn) were searched statically, while methionine oxidation was searched as a variable modification. Synchronous precursor scanning (SPS) selected the top 10 MS2 peptides for TMT reporter ion detection in the Orbitrap using HCD fragmentation at 65% NCE at 50,000 resolving power. In addition, RTS was also set up to reject further fragmentation of peptides mapping to contaminants and yolk proteins using an exclusion list with the keywords Contaminant, Phosvitin, and Vitellogenin. MS/MS Data Processing | The LC/MSn dataset was searched using Proteome Discoverer 2.4 against the same protein database used for RTS. SEQUEST-HT implemented through Proteome Discoverer was set up as: precursor ion mass tolerance 10 ppm, fragment mass tolerance 0.6 Dalton, up to two missed cleavage sites, static modification of cysteine, and lysine and peptide N-termini with TMT tag (+229.163 Da) and dynamic oxidation of methionine. Results were filtered to a 1% FDR at peptides levels using Percolator through Proteome Discoverer. MS3 spectra were processed to extract intensity for each reporter ion. Proteins were quantified by summing reporter ion intensities across all matching PSMs.

Dataset Information

The Differences in the Developmental Stages of the Cardiomyocytes and Endothelial Cells in Human and Mouse Embryos at the Single-Cell Level.

Publications

The Differences in the Developmental Stages of the Cardiomyocytes and Endothelial Cells in Human and Mouse Embryos at the Single-Cell Level.

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