During the transition from the inner cell mass (ICM) cells of blastocysts to pluripotent embryonic stem cells (ESCs) in vitro, a normal developmental program is replaced in cells that acquire a capacity for infinite self-renewal and pluripotency. We explored the underlying mechanism of this switch by using RNA-Seq transcriptome analysis at the resolution of single cells. We detected significant molecular transitions and major changes in transcript variants, which include genes for general metabolism. Furthermore, the expression of repressive epigenetic regulators increased with a concomitant decrease in gene activators that might be necessary to sustain the inherent plasticity of ESCs. Furthermore, we detected changes in microRNAs (miRNAs), with one set that targets early differentiation genes while another set targets pluripotency genes to maintain the unique ESC epigenotype. Such genetic and epigenetic events may contribute to a switch from a normal developmental program in adult cells during the formation of diseased tissues, including cancers.
Stochastic and deterministic allele specific gene expression (ASE) might influence single cell phenotype, but the extent and nature of the phenomenon at the onset of early mouse development is unknown. Here we performed single cell RNA-Seq analysis of single blastomeres of mouse embryos, which revealed significant changes in the transcriptome. Importantly, over half of the transcripts with detectable genetic polymorphisms exhibit ASE, most notably, individual blastomeres from the same two-cell embryo show similar pattern of ASE. However, about 6% of them exhibit stochastic expression, indicated by altered expression ratio between the two alleles. Thus, we demonstrate that ASE is both deterministic and stochastic in early blastomeres. Furthermore, we also found that 1,718 genes express two isoforms with different lengths of 3′UTRs, with the shorter one on average 5–6 times more abundant in early blastomeres compared to the transcripts in epiblast cells, suggesting that microRNA mediated regulation of gene expression acquires increasing importance as development progresses.
Entrance of activated T cells into the tumor after adoptive transfer is a prerequisite for the efficacy of this form of immunotherapy. Because lymphocyte binding to vascular endothelium is the critical step in which lymphocyte extravasation into the tissue is controlled, we compared adhesion of tumor-infiltrating lymphocytes (TIL) to endothelial cells in tumors, peripheral lymph nodes, mucosa-associated lymphatic tissues, and inflamed synovium. Simultaneously, expression of the known homing-associated Ags both on TIL and tumor vasculature was analyzed. All TIL strongly expressed alpha 4-integrins, LFA-1 and CD44, whereas only a low level of L-selectin expression was detected. Tumor vasculature showed signs of activation in each patient on the basis of elevated levels of intercellular adhesion molecule-1, E-selectin, vascular cell adhesion molecule-1, and/or peripheral lymph node addressin (PNAd). TIL showed significantly enhanced binding to tumor vasculature in comparison with other endothelial specificities. Increased binding was not markedly due to up-regulation of the inflammation-induced endothelial cell adhesion molecules in tumors, because binding to inflamed synovium that expressed the same adhesion molecules was not enhanced. In summary, TIL show preferential binding to tumor vasculature and the binding is partially mediated by currently unknown mechanisms. In vitro analysis of endothelial cell binding properties may help to identify those TIL populations that will have the best potential to home back to tumor tissue after adoptive transfer.
We have developed a sequencing-based gene expression profiling assay at single-cell resolution by combining a modified single-cell whole transcriptome amplification method with the next generation sequencing technique, the SOLiD system. Using this assay, we have shown that blastomeres in a four-cell stage embryo have similar gene expression, which is compatible with the fact that they have similar developmental potential. We proved that compared with cDNA microarray technique, our single-cell cDNA SOLiD sequencing assay can detect expression of thousands of more genes. Moreover, for the genes detected by microarray and SOLiD sequencing, our assay detected new transcript variants for a large proportion of them, which confirms unambiguously at single-cell resolution that the transcriptome complexity is higher than expected traditionally. Finally, by using our assay to Dicer knockout (KO) and Ago2 KO oocytes, we showed that a significant amount of transposons were up-regulated abnormally in Dicer/Ago2 KO mature oocytes compared with wild-type controls.
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