Single Mouse Embryonic Stem Cells Research Articles

Simultaneous profiling of chromatin architecture and transcription in single cells.

The three-dimensional structure of chromatin plays a crucial role in development and disease, both of which are associated with transcriptional changes. However, given the heterogeneity in single-cell chromatin architecture and transcription, the regulatory relationship between the three-dimensional chromatin structure and gene expression is difficult to explain based on bulk cell populations. Here we develop a single-cell, multimodal, omics method allowing the simultaneous detection of chromatin architecture and messenger RNA expression by sequencing (single-cell transcriptome sequencing (scCARE-seq)). Applying scCARE-seq to examine chromatin architecture and transcription from 2i to serum single mouse embryonic stem cells, we observe improved separation of cell clusters compared with single-cell chromatin conformation capture. In addition, after defining the cell-cycle phase of each cell through chromatin architecture extracted by scCARE-seq, we find that periodic changes in chromatin architecture occur in parallel with transcription during the cell cycle. These findings highlight the potential of scCARE-seq to facilitate comprehensive analyses that may boost our understanding of chromatin architecture and transcription in the same single cell.

Deciphering Hierarchical Chromatin Domains and Preference of Genomic Position Forming Boundaries in Single Mouse Embryonic Stem Cells.

The exploration of single-cell 3D genome maps reveals that chromatin domains are indeed physical structures presenting in single cells, and domain boundaries vary from cell to cell. However, systematic analysis of the association between regulatory factor binding and elements and the formation of chromatin domains in single cells has not yet emerged. To this end, a hierarchical chromatin domain structure identification algorithm (named as HiCS) is first developed from individual single-cell Hi-C maps, with superior performance in both accuracy and efficiency. The results suggest that in addition to the known CTCF-cohesin complex, Polycomb, TrxG, pluripotent protein families, and other multiple factors also contribute to shaping chromatin domain boundaries in single embryonic stem cells. Different cooperation patterns of these regulatory factors drive genomic position categories with differential preferences forming boundaries, and the most extensive six types of retrotransposons are differentially distributed in these genomic position categories with preferential localization. The above results suggest that these different retrotransposons within genomic regions interplay with regulatory factors navigating the preference of genomic positions forming boundaries, driving the formation of higher-order chromatin structures, and thus regulating cell functions in single mouse embryonic stem cells.

Self-organizing in vitro mouse neural tube organoids mimic embryonic development.

The embryonic neural tube is the origin of the entire adult nervous system, and disturbances in its development cause life-threatening birth defects. However, the study of mammalian neural tube development is limited by the lack of physiologically realistic three-dimensional (3D) in vitro models. Here, we report a self-organizing 3D neural tube organoid model derived from single mouse embryonic stem cells that exhibits an in vivo-like tissue architecture, cell type composition and anterior-posterior (AP) patterning. Moreover, maturation of the neural tube organoids showed the emergence of multipotent neural crest cells and mature neurons. Single-cell transcriptome analyses revealed the sequence of transcriptional events in the emergence of neural crest cells and neural differentiation. Thanks to the accessibility of this model, phagocytosis of migrating neural crest cells could be observed in real time for the first time in a mammalian model. We thus introduce a tractable in vitro model to study some of the key morphogenetic and cell type derivation events during early neural development.

U-net structures for segmentation of single mouse embryonic stem cells using three-dimensional confocal microscopy images.

Cell segmentation at a single cell resolution is required to provide insights for basic biology and application study. However, there are issues of low signal-to-noise ratio, weak fluorescence response, and insufficient resolution along the image stacking direction in 3D confocal images (volume). It has been difficult to segment out single cells from close or contacted cells in a cell volume using image processing methods or together with geometric processing methods. Recently, 3D deep learning methods have been used to avoid tedious parameter settings in the image and geometric processing, but still not easy to segment out close or contacted single cells. This paper proposes a 2D U-net to segment cell regions in high accuracy and computing performance. Better 3D cell images and single cell segmentation for close or contacted cells are achieved by combining a 3D U-net to detect the centers of single cells in the volume.

Bookend: precise transcript reconstruction with end-guided assembly

We developed Bookend, a package for transcript assembly that incorporates data from different RNA-seq techniques, with a focus on identifying and utilizing RNA 5′ and 3′ ends. We demonstrate that correct identification of transcript start and end sites is essential for precise full-length transcript assembly. Utilization of end-labeled reads present in full-length single-cell RNA-seq datasets dramatically improves the precision of transcript assembly in single cells. Finally, we show that hybrid assembly across short-read, long-read, and end-capture RNA-seq datasets from Arabidopsis thaliana, as well as meta-assembly of RNA-seq from single mouse embryonic stem cells, can produce reference-quality end-to-end transcript annotations.

Geometrically defined environments direct cell division rate and subcellular YAP localization in single mouse embryonic stem cells

Mechanotransduction via yes-associated protein (YAP) is a central mechanism for decision-making in mouse embryonic stem cells (mESCs). Nuclear localization of YAP is tightly connected to pluripotency and increases the cell division rate (CDR). How the geometry of the extracellular environment influences mechanotransduction, thereby YAP localization, and decision-making of single isolated mESCs is largely unknown. To investigate this relation, we produced well-defined 2D and 2.5D microenvironments and monitored CDR and subcellular YAP localization in single mESCs hence excluding cell–cell interactions. By systematically varying size and shape of the 2D and 2.5D substrates we observed that the geometry of the growth environment affects the CDR. Whereas CDR increases with increasing adhesive area in 2D, CDR is highest in small 2.5D micro-wells. Here, mESCs attach to all four walls and exhibit a cross-shaped cell and nuclear morphology. This observation indicates that changes in cell shape are linked to a high CDR. Inhibition of actomyosin activity abrogate these effects. Correspondingly, nuclear YAP localization decreases in inhibitor treated cells, suggesting a relation between cell shape, intracellular forces, and cell division rate. The simplicity of our system guarantees high standardization and reproducibility for monitoring stem cell reactions and allows addressing a variety of fundamental biological questions on a single cell level.

Integrated spatial genomics reveals global architecture of single nuclei.

Identifying the relationships between chromosome structures, nuclear bodies, chromatin states, and gene expression is an overarching goal of nuclear organization studies1–4. Because individual cells appear to be highly variable at all these levels5, it is essential to map different modalities in the same cells. Here, we report the imaging of 3,660 chromosomal loci in single mouse embryonic stem cells (mESCs) by DNA seqFISH+, along with 17 chromatin marks and subnuclear structures by sequential immunofluorescence (IF) and the expression profile of 70 RNAs. We found many loci were invariantly associated with IF marks in single mESCs. These loci form “fixed points” in the nuclear organizations in single cells and often appear on the surfaces of nuclear bodies and zones defined by combinatorial chromatin marks. Furthermore, highly expressed genes appear to be pre-positioned to active nuclear zones, independent of bursting dynamics in single cells. Our analysis also uncovered several distinct mESCs subpopulations with characteristic combinatorial chromatin states. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3K27me3 and macroH2A1 (mH2A1), are heritable over at least 3–4 generations, whereas other marks fluctuate on a faster time scale. This seqFISH+ based spatial multimodal approach can be used to explore nuclear organization and cell states in diverse biological systems.

Single-cell replication profiling to measure stochastic variation in mammalian replication timing

Mammalian DNA replication is regulated via multi-replicon segments that replicate in a defined temporal order during S-phase. Further, early/late replication of RDs corresponds to active/inactive chromatin interaction compartments. Although replication origins are selected stochastically, variation in replication timing is poorly understood. Here we devise a strategy to measure variation in replication timing using DNA copy number in single mouse embryonic stem cells. We find that borders between replicated and unreplicated DNA are highly conserved between cells, demarcating active and inactive compartments of the nucleus. Fifty percent of replication events deviated from their average replication time by ± 15% of S phase. This degree of variation is similar between cells, between homologs within cells and between all domains genomewide, regardless of their replication timing. These results demonstrate that stochastic variation in replication timing is independent of elements that dictate timing or extrinsic environmental variation.

Reconstitution of a Patterned Neural Tube from Single Mouse Embryonic Stem Cells.

The recapitulation of tissue development and patterning in three-dimensional (3D) culture is an important dimension of stem cell research. Here, we describe a 3D culture protocol in which single mouse ES cells embedded in Matrigel under neural induction conditions clonally form a lumen containing, oval-shaped epithelial structure within 3 days. By Day 7 an apicobasally polarized neuroepithelium with uniformly dorsal cell identity forms. Treatment with retinoic acid at Day 2 results in posteriorization and self-organization of dorsal-ventral neural tube patterning. Neural tube organoid growth is also supported by pure laminin gels as well as poly(ethylene glycol) (PEG)-based artificial extracellular matrix hydrogels, which can be fine-tuned for key microenvironment characteristics. The rapid generation of a simple, patterned tissue in well-defined culture conditions makes the neural tube organoid a tractable model for studying neural stem cell self-organization.

Bidirectional Promoter Engineering for Single Cell MicroRNA Sensors in Embryonic Stem Cells.

MicroRNAs have emerged as important markers and regulators of cell identity. Precise measurements of cellular miRNA levels rely traditionally on RNA extraction and thus do not allow to follow miRNA expression dynamics at the level of single cells. Non-invasive miRNA sensors present an ideal solution but they critically depend on the performance of suitable ubiquitous promoters that reliably drive expression both in pluripotent and differentiated cell types. Here we describe the engineering of bidirectional promoters that drive the expression of precise ratiometric fluorescent miRNA sensors in single mouse embryonic stem cells (mESCs) and their differentiated derivatives. These promoters are based on combinations of the widely used CAG, EF1α and PGK promoters as well as the CMV and PGK enhancers. miR-142-3p, which is known to be bimodally expressed in mESCs, served as a model miRNA to gauge the precision of the sensors. The performance of the resulting miRNA sensors was assessed by flow cytometry in single stable transgenic mESCs undergoing self-renewal or differentiation. EF1α promoters arranged back-to-back failed to drive the robustly correlated expression of two transgenes. Back-to-back PGK promoters were shut down during mESC differentiation. However, we found that a back-to-back arrangement of CAG promoters with four CMV enhancers provided both robust expression in mESCs undergoing differentiation and the best signal-to-noise for measurement of miRNA activity in single cells among all the sensors we tested. Such a bidirectional promoter is therefore particularly well suited to study the dynamics of miRNA expression during cell fate transitions at the single cell level.

Single-Cell Analyses of ESCs Reveal Alternative Pluripotent Cell States and Molecular Mechanisms that Control Self-Renewal

SummaryAnalyses of gene expression in single mouse embryonic stem cells (mESCs) cultured in serum and LIF revealed the presence of two distinct cell subpopulations with individual gene expression signatures. Comparisons with published data revealed that cells in the first subpopulation are phenotypically similar to cells isolated from the inner cell mass (ICM). In contrast, cells in the second subpopulation appear to be more mature. Pluripotency Gene Regulatory Network (PGRN) reconstruction based on single-cell data and published data suggested antagonistic roles for Oct4 and Nanog in the maintenance of pluripotency states. Integrated analyses of published genomic binding (ChIP) data strongly supported this observation. Certain target genes alternatively regulated by OCT4 and NANOG, such as Sall4 and Zscan10, feed back into the top hierarchical regulator Oct4. Analyses of such incoherent feedforward loops with feedback (iFFL-FB) suggest a dynamic model for the maintenance of mESC pluripotency and self-renewal.

Precisely delivered nanomechanical forces induce blebbing in undifferentiated mouse embryonic stem cells

The aim of this study was to probe the morphological response of single mouse embryonic stem cells (mESC) to precisely delivered nanomechanical forces. Plating mESC as single cells gave rise to either round compact or flattened fibroblastic morphologies. The expression of OCT4 and Nanog was reduced in flattened cells, indicating that this population had begun to differentiate. Upon application of.> nN of force, using atomic force microscopy and simultaneous laser scanning confocal microscopy, round cells, but not flattened cells, were capable of forming mechanically induced blebs (miBlebs). Flattened cells appeared to have a more highly developed cytoskeleton than undifferentiated stem cells as characterized by the distribution of phospho-ezrin-radixin-moesin (pERM). Higher levels of pERM and an inability to form miBlebs in flattened cells imply that the earliest stages of embryonic stem cell differentiation are associated with the development of stronger mechanical links between the plasma membrane and the cytoskeleton © 2011 Hemsley et al, publisher and licensee Dove Medical Press Ltd.

High-Density Microwell Chip for Culture and Analysis of Stem Cells

With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.

Low Oxygen Enhances Primitive and Definitive Neural Stem Cell Colony Formation by Inhibiting Distinct Cell Death Pathways

Neural stem cells (NSCs) can be derived from single mouse embryonic stem cells (ESCs) in the absence of instructive factors. Clonal primitive NSC (pNSC) colonies are formed first, and then give rise to clonal, fibroblast growth factor-dependent definitive neural stem cells (dNSCs). We tested low-oxygen culture as a potential method of alleviating the extensive cell death seen in pNSCs and dNSCs. Culture in low (4%) oxygen promoted survival of pNSCs by inhibiting apoptosis-inducing factor (AIF)-dependent cell death, although pNSCs undergo both AIF- and caspase-mediated cell death in 20% oxygen. In contrast, survival of dNSCs in low oxygen was increased by inhibition of caspase-dependent cell death. In normoxia, AIF is implicated in promoting dNSC survival. Neither survival effect was dependent on the main transcriptional effector of hypoxia, hypoxia-inducible factor 1. Low-oxygen concentrations may be involved in expansion of early NSC populations by inhibiting cell death through different pathways in these sequential pNSC and dNSC populations. Stem Cells 2009;27:1879–1886