Transcriptional Heterogeneity in Naive and Primed Human Pluripotent Stem Cells at Single-Cell Resolution

Messmer, Tobias; Meyenn, Ferdinand von; Savino, Aurora; Santos, Fátima; Mohammed, Hisham; Lun, Aaron T. L.; Marioni, John C.; Reik, Wolf

doi:10.1016/j.celrep.2018.12.099

Cited by 134 publications

(189 citation statements)

References 63 publications

(92 reference statements)

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“…Prior to differential expression (DE) analysis, we pooled transcript counts within each cell type and each donor, creating "pseudo-bulk" transcriptomes. This approach has been employed by several single cell transcriptomics studies (31)(32)(33)(34) since it provides robustness to varying numbers and library sizes of cells/nuclei among replicates (donors acting as replicates in our case), provides strong type I error control, and allows the use of bulk RNA-seq DE tools that have been optimized and validated over the course of many years. After pseudobulking, a typically DESeq2 was used to test for differentially expressed genes (DEGs), with batch as a covariate.…”

Section: Differential Expression and Pathway Analysismentioning

confidence: 99%

Single cell transcriptome profiling of the human alcohol-dependent brain

Brenner

Tiwari

Liu

et al. 2019

Preprint

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BackgroundAlcoholism remains a prevalent health concern throughout the world. Previous studies have identified transcriptomic patterns in the brain associated with alcohol dependence in both humans and animal models.But none of these studies have systematically investigated expression within the unique cell types present in the brain. ResultsWe utilized single nucleus RNA sequencing (snRNA-seq) to examine the transcriptomes of over 16,000 nuclei isolated from prefrontal cortex of alcoholic and control individuals. Each nucleus was assigned to one of seven major cell types by unsupervised clustering. Cell type enrichment patterns varied greatly among neuroinflammatory-related genes, which are known to play roles in alcohol dependence and neurodegeneration. Differential expression analysis identified cell type-specific genes with altered expression in alcoholics. The largest number of differentially expressed genes (DEGs), including both protein-coding and non-coding, were detected in astrocytes, oligodendrocytes, and microglia. ConclusionsTo our knowledge, this is the first single cell transcriptome analysis of alcohol-associated gene expression in any species, and the first such analysis in humans for any addictive substance. These findings greatly advance understanding of transcriptomic changes in the brain of alcohol-dependent individuals.1 Background Alcohol abuse is involved in over 200 pathologies and health conditions (e.g. alcohol dependence, liver cirrhosis, cancers, and injuries) and creates substantial social and economic burdens (1,2) . To develop more effective therapeutic strategies, we must first understand how alcohol affects the body at the cellular and molecular level. Previous studies of transcriptomic responses in human alcoholics (3-6) relied on RNA extracted from brain regions using tissue homogenates comprised of multiple cell types. This approach likely masks differences in gene expression patterns among specific cells, as well as heterogeneity from cell-to-cell variation within a given cell type.Single cell RNA sequencing (scRNA-seq) has recently gained attention in cell and molecular biology research for its ability to profile novel cell types and measure cell-to-cell variation in gene expression. To our knowledge, transcriptomic responses to chronic alcohol exposure or any other abused drug have not been studied at the cellular level in the human brain. We hypothesized that cell type-specific gene expression patterns associated with alcoholism will identify novel alcohol targets that were previously missed by bulk analysis of tissue homogenates, as has been shown for other neuropathologies (7,8) . Using single nucleus RNA-seq (snRNA-seq), a popular scRNA-seq alternative for analyzing frozen brain tissue (9-18) , we profiled the transcriptomes of 16,305 nuclei extracted from frozen prefrontal cortex (PFC) samples of 4 control and 3 alcohol dependent individuals. The PFC is involved in executive function and is an important substrate in the reward circuitry associated with development of a...

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Section: Differential Expression and Pathway Analysismentioning

confidence: 99%

Single cell transcriptome profiling of the human alcohol-dependent brain

Brenner

Tiwari

Liu

et al. 2019

Preprint

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“…No further quality criteria were applied. Data from [22] was downloaded from EMBL-EBI ArrayExpress under the accession number E-MTAB-6819. Human gene names were converted to mouse names using BioMart.…”

Section: Comparisons Between Different Datasetsmentioning

confidence: 99%

G2M splits mouse embryonic stem cells into naïve and formative pluripotency states

Jääger

Simpson

Kalantzaki

et al. 2019

Preprint

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Embryonic stem cells (ESCs) express heterogeneous levels of pluripotency and developmental transcription factors (TFs) and their cell cycle is unsynchronised when grown in the presence of serum. Here, we asked whether the cell cycle and developmental heterogeneities of ESCs are coordinated by determining the state identities of G1-and G2M-enriched mouse ESCs (mESCs) at single cell resolution. We found that G2M cells were not all the same and demonstrate their split into the naïve and formative (intermediate) pluripotency states marked by high or low Esrrb expression, respectively. The naïve G2M sub-state resembles 'ground' state pluripotency of the LIF/2i cultured mESCs. The naïve and formative G2M sub-states exist in the pre-and post-implantation stages of the mouse embryo, respectively, verifying developmental distinction. Moreover, the G2M sub-states partially match between the mouse and human ESCs, suggesting higher similarity of transcriptional control between these species in G2M. Our findings propose a model whereby G2M separates mESCs into naïve and formative pluripotency states. This concept of G2M-diverted pluripotency states provides new framework for understanding the mechanisms of pluripotency maintenance and lineage specification in vitro and in vivo, and the development of more efficient and clinically relevant reprogramming strategies.

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“…Epigenetic modifications are mostly associated with changes in gene expression, which were shown to differ between hPSCs grown in different microenvironments (Newman & Cooper, ), as well as between hESCs and hiPSCs (Chin et al , , ) and between primed and naïve hPSCs (Messmer et al , ). However, quantifying the transcriptional levels of genes in the pluripotent state does not directly reveal the underlying mechanism of variability, nor its level of stability and inheritance.…”

Section: Introductionmentioning

confidence: 99%

Epigenetic aberrations in human pluripotent stem cells

Bar

Benvenisty

2019

The EMBO Journal

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Human pluripotent stem cells (hPSCs) are being increasingly utilized worldwide in investigating human development, and modeling and discovering therapies for a wide range of diseases as well as a source for cellular therapy. Yet, since the first isolation of human embryonic stem cells (hESCs) 20 years ago, followed by the successful reprogramming of human‐induced pluripotent stem cells (hiPSCs) 10 years later, various studies shed light on abnormalities that sometimes accumulate in these cells in vitro. Whereas genetic aberrations are well documented, epigenetic alterations are not as thoroughly discussed. In this review, we highlight frequent epigenetic aberrations found in hPSCs, including alterations in DNA methylation patterns, parental imprinting, and X chromosome inactivation. We discuss the potential origins of these abnormalities in hESCs and hiPSCs, survey the different methods for detecting them, and elaborate on their potential consequences for the different utilities of hPSCs.

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Transcriptional Heterogeneity in Naive and Primed Human Pluripotent Stem Cells at Single-Cell Resolution

Cited by 134 publications

References 63 publications

Single cell transcriptome profiling of the human alcohol-dependent brain

Single cell transcriptome profiling of the human alcohol-dependent brain

G2M splits mouse embryonic stem cells into naïve and formative pluripotency states

Epigenetic aberrations in human pluripotent stem cells

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