Transient Cell State Research Articles

Generalizing RNA velocity to transient cell states through dynamical modeling.

RNA velocity has opened up new ways of studying cellular differentiation in single-cell RNA-sequencing data. It describes the rate of gene expression change for an individual gene at a given time point based on the ratio of its spliced and unspliced messenger RNA (mRNA). However, errors in velocity estimates arise if the central assumptions of a common splicing rate and the observation of the full splicing dynamics with steady-state mRNA levels are violated. Here we present scVelo, a method that overcomes these limitations by solving the full transcriptional dynamics of splicing kinetics using a likelihood-based dynamical model. This generalizes RNA velocity to systems with transient cell states, which are common in development and in response to perturbations. We apply scVelo to disentangling subpopulation kinetics in neurogenesis and pancreatic endocrinogenesis. We infer gene-specific rates of transcription, splicing and degradation, recover each cell's position in the underlying differentiation processes and detect putative driver genes. scVelo will facilitate the study of lineage decisions and gene regulation.

Parallel analysis of tri-molecular biosynthesis with cell identity and function in single cells

Cellular products derived from the activity of DNA, RNA, and protein synthesis collectively control cell identity and function. Yet there is little information on how these three biosynthesis activities are coordinated during transient and sparse cellular processes, such as activation and differentiation. Here, we describe Simultaneous Overview of tri-Molecule Biosynthesis (SOM3B), a molecular labeling and simultaneous detection strategy to quantify DNA, RNA, and protein synthesis in individual cells. Comprehensive interrogation of biosynthesis activities during transient cell states, such as progression through cell cycle or cellular differentiation, is achieved by partnering SOM3B with parallel quantification of select biomolecules with conjugated antibody reagents. Here, we investigate differential de novo DNA, RNA, and protein synthesis dynamics in transformed human cell lines, primary activated human immune cells, and across the healthy human hematopoietic continuum, all at a single-cell resolution.

New insights into human primordial germ cells and early embryonic development from single-cell analysis.

Human preimplantation developmental studies are difficult to accomplish due to associated ethical and moral issues. Preimplantation cells are rare and exist only in transient cell states. From a single cell, it is very challenging to analyse the origination of the heterogeneity and complexity inherent to the human body. However, recent advances in single-cell technology and data analysis have provided new insights into the process of early human development and germ cell specification. In this Review, we examine the latest single-cell datasets of human preimplantation embryos and germ cell development, compare them to bulk cell analyses, and interpret their biological implications.

Co-expression of Tbx6 and Sox2 identifies a novel transient neuromesoderm progenitor cell state

Elongation of the body axis is a key aspect of body plan development. Bipotential neuromesoderm progenitors (NMPs) ensure axial growth of embryos by contributing both to the spinal cord and mesoderm. The current model for the mechanism controlling NMP deployment invokes Tbx6, a T-box factor, to drive mesoderm differentiation of NMPs. Here, we identify a new population of Tbx6+ cells in a subdomain of the NMP niche in mouse embryos. Based on co-expression of a progenitor marker, Sox2, we identify this population as representing a transient cell state in the mesoderm-fated NMP lineage. Genetic lineage tracing confirms the presence of the Tbx6+ NMP cell state. Furthermore, we report a novel aspect of the documented Tbx6 mutant phenotype, namely an increase from two to four ectopic neural tubes, corresponding to the switch in NMP niche, thus highlighting the importance of Tbx6 function in NMP fate decision. This study emphasizes the function of Tbx6 as a bistable switch that turns mesoderm fate 'on' and progenitor state 'off', and thus has implications for the molecular mechanism driving NMP fate choice.

MERVL/Zscan4 Network Activation Results in Transient Genome-wide DNA Demethylation of mESCs

SummaryMouse embryonic stem cells are dynamic and heterogeneous. For example, rare cells cycle through a state characterized by decondensed chromatin and expression of transcripts, including the Zscan4 cluster and MERVL endogenous retrovirus, which are usually restricted to preimplantation embryos. Here, we further characterize the dynamics and consequences of this transient cell state. Single-cell transcriptomics identified the earliest upregulated transcripts as cells enter the MERVL/Zscan4 state. The MERVL/Zscan4 transcriptional network was also upregulated during induced pluripotent stem cell reprogramming. Genome-wide DNA methylation and chromatin analyses revealed global DNA hypomethylation accompanying increased chromatin accessibility. This transient DNA demethylation was driven by a loss of DNA methyltransferase proteins in the cells and occurred genome-wide. While methylation levels were restored once cells exit this state, genomic imprints remained hypomethylated, demonstrating a potential global and enduring influence of endogenous retroviral activation on the epigenome.

Abstract B32: Single-cell non-genetic heterogeneity confers drug resistance in melanoma

Abstract Melanoma is the most aggressive and deadliest form of skin cancer. Historically, treatment has consisted of harsh chemotherapeutics that kill all rapidly proliferating cells in the body. In 2011, however, the first targeted therapy, named vemurafenib, was approved for metastatic melanoma. While this drug had an unprecedented success at shrinking tumor mass, drug resistance arose in the majority of patients. Importantly, the fact that patients initially respond positively to this drug suggests that the majority of the tumor is drug sensitive (and therefore killed by the drug); however, the fact that the tumor returns suggests that a few cells survive the drug treatment and give rise to resistance. Generally, researchers believe that drug resistance has a genetic basis, in which cells survive drug treatment because they have a secondary mutation to overcome the drug effects. In this framework, resistance is a Darwinian trait, passed on during each cell division. An alternative hypothesis is that drug resistance is Lamarckian and comes from a non-heritable or reversible phenotype, such as a transient cell state. Using patient derived melanoma cell lines, we tested these hypotheses to determine if resistance to vemurafenib is Darwinian or Lamarckian. Drawing experimental inspiration from Luria and Delbruck with E. coli and resistance to T1 phage, we found that resistance to vemurafenib is not heritable prior to treatment with drug, consistent with a Lamarckian hypothesis. This result suggests that resistance is not mediated by a genetic change, but rather that vemurafenib resistance is transient and not heritable. With these phenomenological results in hand, we then sought to characterize the molecular underpinnings of resistance. First, we performed high throughput RNA sequencing on 38 resistant melanoma colonies and 20 non-resistant clones to identify alterations in gene expression that are associated with drug resistance. Next, we used high throughput imaging and iterative cycles of RNA FISH in situ hybridizations to measure the resistance marker transcriptome for >10,000 individual melanoma cells at multiple time points after drug treatment. In drug naive cells, we find that there is a sub-population of individual cells that already express these resistance markers and that there is tremendous single-cell heterogeneity in expression for these resistance markers. For example, if the mean expression level for a population is 0-2 RNA per cell, we also find a few cells (1:100 to 1:2000) that have hundreds of the same RNA. Surprisingly, we find that some of these cells have many of the resistance markers suggesting that the resistance state is coordinated, while other cells in the same population can have high levels of only one marker. These experiments lead us to EGFR as a marker of pre-existing resistance and a known vemurafenib resistance mechanism in literature. Taken together, our results suggest that random, but coordinated fluctuations in gene expression allow melanoma cells to overcome vemurafenib treatment, potentially giving rise to disease relapse in vivo. Citation Format: Sydney M. Shaffer, Andrew G. Biaesch, Stefan Torborg, Clemens Krepler, Meenhard Herlyn, Arjun Raj. Single-cell non-genetic heterogeneity confers drug resistance in melanoma. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B32.

Abstract 4752: Genomic cytometry: Single cell mutation analysis combining PCR with flow cytometry

Abstract Genomic cytometry is a new technology field combining the high-throughput capabilities of flow cytometry and cell sorting with single-cell multiplex genomic analysis in microfluidic drops. The genomic cytometry method we will describe is PCR-activated cell sorting (PACS). PACS is analogous to Fluorescence-Activated Cell Sorting (FACS) in throughput and the ability to analyze single cells; however, unlike conventional flow cytometric immunophenotyping, PACS uses extremely sensitive microdroplet TaqMan PCR reactions to identify and, if needed, sort cells of interest for additional downstream molecular characterization. This unique approach enables PACS to identify cells based on the presence of transcripts, genomic DNA sequences, mRNA splice variants, SNPs, non-coding RNAs, and other biomarkers not amenable to antibody-based probe detection. PACS can be used to sort and enrich cells targeting single-copy genomic DNA for the analysis of circulating tumor cells (CTCs), intra-tumor heterogeneity, rare stem cells, viral or pathogen infections and genomic characterization of cancer immune system response and disorder. By analyzing individual cells within a population, it is possible to identify rare CTC populations or transient cell states, critical to human health and cancer progression, that are otherwise unobservable by ensemble measurements. With PACS, we identified and sorted prostate cancer cells from a heterogeneous population by performing >132,000 simultaneous single-cell TaqMan RT-PCR reactions targeting vimentin mRNA. Following vimentin-positive droplet sorting and downstream analysis of recovered nucleic acids, we found that cancer-specific genomes and transcripts were significantly enriched. Citation Format: William C. Hyun, Adam Sciambi, Maurizio Pellegrino, Dennis Eastburn. Genomic cytometry: Single cell mutation analysis combining PCR with flow cytometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4752. doi:10.1158/1538-7445.AM2015-4752