Exploring the universe of single cells using multi-omic approaches

In both unicellular and multicellular organisms, no individual cell is completely the same. The tissues of humans and other animals are composed of various cell types, each with a different function. Even within the population of a specific type of cell, all cells differ from one another on multiple molecular levels. This variation is constantly introduced in cells, making them more unique step by step. Variation can be introduced during cell division through for example DNA mutations or changes in the structure or the packaging of DNA. Diseases such as cancer can start in an individual cell when it quickly diverges from its predecessors through cell divisions and gives rise to a tumor. Chromosome loss in colorectal cancer The second chapter provides more insight into various modalities of individual cells. We use organoid models of colorectal cancer, which mimic the disease in tiny 3D structures grown in a dish. With NLAIII-seq for whole genome sequencing and viral barcoding for lineage tracing we perform and read out multiple measurements in individual cells to reconstruct the order in which chromosomal abnormalities occur in this type of cancer. Because of the integrated measurements, we are able to measure changes that occur in parallel in different cells within one population. Using this method, we found a recurring loss of chromosome 4 that only occurs after the loss of chromosome 18. These findings coincide with clinical observations in patients with colorectal cancer. Hematopoiesis In the third chapter we introduce a novel technique called sort-ChIC. This technique measures histone modifications in individual cells. Modifications in these molecules influence which parts of the DNA in that cell can be read and thereby influence the proteins that the cell produces. We apply sort-ChIC in two active (H3K4me1 and H3K4me3) and two repressive (H3K27me3 and H3K9me3) histone modifications on blood stem cells (HSPCs) and adult blood cells in the bone marrow of the mouse to gain insight into the histone modifications that occur during blood formation. Joint profiling of H3K4me1 and H3K9me3 demonstrates that cell types within the myeloid lineage have distinct active chromatin but share similar myeloid-specific heterochromatin repressed states. This suggests hierarchical chromatin regulation during hematopoiesis. State-of-the-art technology The fourth chapter introduces a new technique, scChIC-TAPS, that can measure various modalities – including histone marks and DNA structure – simultaneously in an individual cell. Our approach combines bisulfite-free conversion of methylated cytosines and targeted MNase digestion. With these integrated measurements we can resolve the local correlations of different histone modifications and DNA methylation states at base-pair resolution. We describe the validation of scChIC-TAPS and its application in the Fucci cell line, a system in which the position of every cell in the cell cycle can be measured precisely. We use this cell cycle information to integrate the data of multiple histone marks and compare their behavior throughout the cell cycle. Our data provides the first direct evidence that kinetics of replication-coupled methylation are influenced by the local chromatin environment

Exploring the universe of single cells using multi-omic approaches

In both unicellular and multicellular organisms, no individual cell is completely the same. The tissues of humans and other animals are composed of various cell types, each with a different function. Even within the population of a specific type of cell, all cells differ from one another on multiple molecular levels. This variation is constantly introduced in cells, making them more unique step by step. Variation can be introduced during cell division through for example DNA mutations or changes in the structure or the packaging of DNA. Diseases such as cancer can start in an individual cell when it quickly diverges from its predecessors through cell divisions and gives rise to a tumor. Chromosome loss in colorectal cancer The second chapter provides more insight into various modalities of individual cells. We use organoid models of colorectal cancer, which mimic the disease in tiny 3D structures grown in a dish. With NLAIII-seq for whole genome sequencing and viral barcoding for lineage tracing we perform and read out multiple measurements in individual cells to reconstruct the order in which chromosomal abnormalities occur in this type of cancer. Because of the integrated measurements, we are able to measure changes that occur in parallel in different cells within one population. Using this method, we found a recurring loss of chromosome 4 that only occurs after the loss of chromosome 18. These findings coincide with clinical observations in patients with colorectal cancer. Hematopoiesis In the third chapter we introduce a novel technique called sort-ChIC. This technique measures histone modifications in individual cells. Modifications in these molecules influence which parts of the DNA in that cell can be read and thereby influence the proteins that the cell produces. We apply sort-ChIC in two active (H3K4me1 and H3K4me3) and two repressive (H3K27me3 and H3K9me3) histone modifications on blood stem cells (HSPCs) and adult blood cells in the bone marrow of the mouse to gain insight into the histone modifications that occur during blood formation. Joint profiling of H3K4me1 and H3K9me3 demonstrates that cell types within the myeloid lineage have distinct active chromatin but share similar myeloid-specific heterochromatin repressed states. This suggests hierarchical chromatin regulation during hematopoiesis. State-of-the-art technology The fourth chapter introduces a new technique, scChIC-TAPS, that can measure various modalities – including histone marks and DNA structure – simultaneously in an individual cell. Our approach combines bisulfite-free conversion of methylated cytosines and targeted MNase digestion. With these integrated measurements we can resolve the local correlations of different histone modifications and DNA methylation states at base-pair resolution. We describe the validation of scChIC-TAPS and its application in the Fucci cell line, a system in which the position of every cell in the cell cycle can be measured precisely. We use this cell cycle information to integrate the data of multiple histone marks and compare their behavior throughout the cell cycle. Our data provides the first direct evidence that kinetics of replication-coupled methylation are influenced by the local chromatin environment

Three-dimensional analysis of single molecule FISH in human colon organoids

The culturing of mini-organs (organoids) in three-dimensions (3D) presents a simple and powerful tool to investigate the principles underlying human organ development and tissue self-organization in both healthy and diseased states. Applications of single molecule analysis are highly informative for a comprehensive understanding of the complexity underlying tissue and organ physiology. To fully exploit the potential of single molecule technologies, the adjustment of protocols and tools to 3D tissue culture is required. Single molecule RNA fluorescence in situ hybridization (smFISH) is a robust technique for visualizing and quantifying individual transcripts. In addition, smFISH can be employed to study splice variants, fusion transcripts as well as transcripts of multiple genes at the same time. Here, we develop a 3-day protocol and validation method to perform smFISH in 3D in whole human organoids.We provide a number of applications to exemplify the diverse possibilities for the simultaneous detection of distinctmRNAtranscripts, evaluation of their spatial distribution and the identification of divergent cell lineages in 3D in organoids

scChIX-seq infers dynamic relationships between histone modifications in single cells

Regulation of chromatin states involves the dynamic interplay between different histone modifications to control gene expression. Recent advances have enabled mapping of histone marks in single cells, but most methods are constrained to profile only one histone mark per cell. Here, we present an integrated experimental and computational framework, scChIX-seq (single-cell chromatin immunocleavage and unmixing sequencing), to map several histone marks in single cells. scChIX-seq multiplexes two histone marks together in single cells, then computationally deconvolves the signal using training data from respective histone mark profiles. This framework learns the cell-type-specific correlation structure between histone marks, and therefore does not require a priori assumptions of their genomic distributions. Using scChIX-seq, we demonstrate multimodal analysis of histone marks in single cells across a range of mark combinations. Modeling dynamics of in vitro macrophage differentiation enables integrated analysis of chromatin velocity. Overall, scChIX-seq unlocks systematic interrogation of the interplay between histone modifications in single cells

Sequencing metabolically labeled transcripts in single cells reveals mRNA turnover strategies

The regulation of messenger RNA levels in mammalian cells can be achieved by the modulation of synthesis and degradation rates. Metabolic RNA-labeling experiments in bulk have quantified these rates using relatively homogeneous cell populations. However, to determine these rates during complex dynamical processes, for instance during cellular differentiation, single-cell resolution is required. Therefore, we developed a method that simultaneously quantifies metabolically labeled and preexisting unlabeled transcripts in thousands of individual cells. We determined synthesis and degradation rates during the cell cycle and during differentiation of intestinal stem cells, revealing major regulatory strategies. These strategies have distinct consequences for controlling the dynamic range and precision of gene expression. These findings advance our understanding of how individual cells in heterogeneous populations shape their gene expression dynamics

Single-cell sortChIC identifies hierarchical chromatin dynamics during hematopoiesis

Post-translational histone modifications modulate chromatin activity to affect gene expression. How chromatin states underlie lineage choice in single cells is relatively unexplored. We develop sort-assisted single-cell chromatin immunocleavage (sortChIC) and map active (H3K4me1 and H3K4me3) and repressive (H3K27me3 and H3K9me3) histone modifications in the mouse bone marrow. During differentiation, hematopoietic stem and progenitor cells (HSPCs) acquire active chromatin states mediated by cell-type-specifying transcription factors, which are unique for each lineage. By contrast, most alterations in repressive marks during differentiation occur independent of the final cell type. Chromatin trajectory analysis shows that lineage choice at the chromatin level occurs at the progenitor stage. Joint profiling of H3K4me1 and H3K9me3 demonstrates that cell types within the myeloid lineage have distinct active chromatin but share similar myeloid-specific heterochromatin states. This implies a hierarchical regulation of chromatin during hematopoiesis: heterochromatin dynamics distinguish differentiation trajectories and lineages, while euchromatin dynamics reflect cell types within lineages

Molecular characterization of Barrett's esophagus at single-cell resolution

Barrett’s esophagus (BE) is categorized, based on morphological appearance, into different stages, which correlate with the risk of developing esophageal adenocarcinoma. More advanced stages are more likely to acquire chromosomal instabilities, but stage-specific markers remain elusive. Here, we performed single-cell DNA-sequencing experiments (scDNAseq) with fresh BE biopsies. Dysplastic BE cells frequently contained chromosomal instability (CIN) regions, and these CIN cells carried mutations corresponding to the COSMIC mutational signature SBS17, which were not present in biopsy-matched chromosomally stable (CS) cells or patient-matched nondiseased control cells. CS cells were predominantly found in nondysplastic BE biopsies. The single-base substitution (SBS) signatures of all CS BE cells analyzed were indistinguishable from those of nondiseased esophageal or gastric cells. Single-cell RNA-sequencing (scRNAseq) experiments with BE biopsies identified two sets of marker genes which facilitate the distinction between columnar BE epithelium and nondysplastic/dysplastic stages. Moreover, histological validation confirmed a correlation between increased CLDN2 expression and the presence of dysplastic BE stages. Our scDNAseq and scRNAseq datasets, which are a useful resource for the community, provide insight into the mutational landscape and gene expression pattern at different stages of BE development

Integration of multiple lineage measurements from the same cell reconstructs parallel tumor evolution

Summary: Organoid evolution models complemented with integrated single-cell sequencing technology provide a powerful platform to characterize intra-tumor heterogeneity (ITH) and tumor evolution. Here, we conduct a parallel evolution experiment to mimic the tumor evolution process by evolving a colon cancer organoid model over 100 generations, spanning 6 months in time. We use single-cell whole-genome sequencing (WGS) in combination with viral lineage tracing at 12 time points to simultaneously monitor clone size, CNV states, SNV states, and viral lineage barcodes for 1,641 single cells. We integrate these measurements to construct clonal evolution trees with high resolution. We characterize the order of events in which chromosomal aberrations occur and identify aberrations that recur multiple times within the same tumor sub-population. We observe recurrent sequential loss of chromosome 4 after loss of chromosome 18 in four unique tumor clones. SNVs and CNVs identified in our organoid experiments are also frequently reported in colorectal carcinoma samples, and out of 334 patients with chromosome 18 loss in a Memorial Sloan Kettering colorectal cancer cohort, 99 (29.6%) also harbor chromosome 4 loss. Our study reconstructs tumor evolution in a colon cancer organoid model at high resolution, demonstrating an approach to identify potentially clinically relevant genomic aberrations in tumor evolution

Snake Venom Gland Organoids

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland

Snake Venom Gland Organoids

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland