Project description:Accurate Protein Characterization and Quantification of Xenograft Tumors Requires Separating Human and Mouse Cells prior to Proteomics Profiling
Project description:We developed a scalable assay permitting the simultaneous quantification of hundreds of proteins and the full transcriptome in thousands of individual cells from samples where cell number is limiting. The RNA Expression and Protein Sequencing assay (REAP-seq) uses DNA-labeled antibodies to permit DNA sequencing of both mRNA and antibodies in a single workflow using droplet microfluidics. We describe the development and validation of REAP-seq in human PBMCs, and use the assay to assess the costimulatory effects of an anti-CD27 agonist antibody in naïve CD8+ lymphocytes. Protein quantification using REAP-seq was more sensitive than parallel mRNA measurements and differentiated cell states with fewer analytes. Unbiased profiling of single cells without prior selection enabled identification and characterization of an unexpected cell type that would have been missed with population-averaged profiling methodologies.
Project description:Implantation is a milestone event during mammalian embryogenesis. Due to the extreme difficulty of obtaining in vivo human early post-implantation embryos, the gene regulatory network and epigenetics controlling human embryo implantation remains elusive. Here, combining an in vitro culture system for human post-implantation development and single-cell omics sequencing technologies, over 10,000 single cells at five representative stages of pre/post-implantation development were systematically analyzed. Unsupervised dimensionality reduction and clustering algorithm of the transcriptome data show stepwise implantation routes for the epiblast, primitive endoderm, and trophectoderm lineages, suggesting preparation for the establishment of a mother-to-offspring connection after implantation. Female embryos showed asynchronous progress of dosage loss of X chromosomes during implantation. Furthermore, using the single cell trio-Seq (scTrio-Seq) strategy, re-methylation of the genomes of all the three lineages was unambiguously revealed. Surprisingly, the genome re-methylation of PE lineage were much slower than both EPI and TE lineages during the implantation process, indicating distinct methylome features between EPI and PE although both of which were derived from ICM. Collectively, our work paves the way for understanding the complex molecular mechanisms that regulate human embryo implantation, informing new insights and future efforts in early embryonic development and reproductive medicine.
Project description:Members of the miR-371 cluster are of the earliest de novo synthesized by embryos and have important actions in mammalian reproduction. MiR-371a is secreted by embryos at the peri-implantation period and its release has been previously associated with poor embryo quality and lower implantation rates in women undergoing IVF. Here we investigated the potential biological role of miR-371a release in regards to embryonic implantation, and specifically the molecular effects that this miRNA exerts on the cells of the endometrial lining. Gene expression changes in endometrial cells upon miR-371a addition were therefore identified using large-scale genomic plattform that covers the whole human genome (Affymetrix).
Project description:Comprehensive quantitative proteomic study of human pre-implantation embryo stages reveal dynamic proteome landscape from M2, 8-cell and blastocyst stage, and during trophoblast stem cell (TS) differentiation. Identified key factors in early human embryos and lineage-specific trophoblast proteome profiles, correlated with transcriptomic analyses. This direct proteomic analysis provides a comprehensive analysis of the dynamic protein expression in human embryos during pre-implantation development and a powerful resource to enable further mechanistic studies on human trophoblast development and function.
Project description:Implantation is a milestone event during mammalian embryogenesis. Implantation failure is a nonnegligible cause of human early pregnancy loss. Due to the extreme difficulty of obtaining in vivo human early post-implantation embryos, it remains elusive how the gene regulatory network and epigenetic mechanisms control human embryo implantation. Here, combining an in vitro culture system for human post-implantation development and single-cell omics sequencing technologies, over 8,000 individual cells from 65 human peri-implantation embryos were systematically analyzed. Unsupervised dimensionality reduction and clustering algorithm of the transcriptome data show stepwise implantation routes for the epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE) lineages, suggesting robust preparation for the proper establishment of a mother-to-offspring connection during implantation. Female embryos showed initiation of random X chromosome inactivation based on analysis of parental allele-specific expression of X chromosome-linked genes during implantation. Surprisingly, by the single-cell Trio-Seq (scTrio-Seq) analysis, the genome re-methylation of PE lineage was shown to be much slower than those of both EPI and TE lineages during implantation process, indicating distinct DNA methylome re-establishment features between EPI and PE although both of which were derived from inner cell mass (ICM). Collectively, our work paves the way for understanding the complex molecular mechanisms that regulate human embryo implantation, offering new insights and future efforts in early embryonic development and reproductive medicine.
Project description:To determine the effect of Zika virus infection on pre-implantation embryonic development, we performed single blastocyst RNA-Seq on MOCK and ZIKV infected embryos. ZIKV infection results in an increased risk of spontaneous abortion and poor intrauterine growth although the mechanisms underlying fetal loss remain undetermined. Little is known about the impact of ZIKV infection during the earliest stages of pregnancy, or pre- and peri-implantation, because most current studies of ZIKV infection in pregnancy models focus on post-implantation stages. Here, we demonstrate that trophectoderm cells of pre-implantation human and mouse embryos can be efficiently infected with ZIKV, and that trophectoderm can propagate virus causing cell death of neural progenitors. These findings were corroborated by our demonstration that hESC-derived trophectoderm cells are infected by ZIKV in a dose dependent manner. RNAseq of single blastocysts revealed key transcriptional changes in cellular and physiologic functions upon ZIKV infection, including nervous system development and function, prior to commitment to the neural cell lineage. Finally, the pregnancy rate of mice infected pre-implantation was > 50% lower than females infected at E4.5. These results demonstrate that pre-implantation ZIKV infection of trophectoderm leads to miscarriage or spontaneous abortion. Moreover, pre- and peri-implantation ZIKV infects trophectoderm cells that propagate virus over time causing cell death in neural progenitors. Cumulatively, these data demonstrate that vertical pre- and peri-implantation ZIKV infection of trophectoderm impairs fetal development and causes neural progenitor cell death, elucidating a previously unappreciated association of pre- and peri-implantation ZIKV infection and microcephaly.
Project description:We collected over 8000 mouse embryos of each developmental stage (zygote, 2-cell, 4-cell, 8-cell, morula, blastocyst) and performed tandem mass tag (TMT)-based quantitative mass spectrometry and identified nearly 5000 proteins for each stage. In-depth analysis indicates that protein expression profiles of zygote, morula and blastocyst show apparent difference from 2- to 8-cell embryos due to the maternal-totipotent-differentiation transition. We further identified novel factors and proved that they play important roles in determining pre-implantation embryo development
Project description:Human embryogenesis is characterised by a series of coordinated cell fate specification events. Upon implantation on day 7, the three cell lineages forming the human blastocyst undergo major morphogenetic and transcriptomic changes. Using single-cell RNA sequencing, we reconstruct the molecular events driving post-implantation development of the human embryo at day 9 and 11. Our study provides the first molecular map of gene expression patterns in early post-implantation human embryos, unveiling how signalling interactions between embryonic and extra-embryonic tissues drive human embryogenesis.