Profiling Epigenetic Age in Single Cells

Abstract DNA methylation of a defined set of CpG dinucleotides emerged as a critical and precise biomarker of the aging process. Multi-variate machine learning models, known as epigenetic clocks, can exploit quantitative changes in the methylome to predict the age of bulk tissue with remarkable accuracy. However, intrinsic sparsity and digitized methylation in individual cells have so far precluded the assessment of aging in single cell data. We developed scAge, a probabilistic approach to determine the epigenetic age of single cells, and validated our results in mice. scAge tissue-specific and multi-cell type single cell clocks correctly recapitulated the chronological age of the original tissue, while uncovering the inherent heterogeneity that exists at the single-cell level. The data suggested that while cells in a tissue age in a coordinated fashion, some cells age more or less rapidly than others. We showed that individual embryonic stem cells exhibit an age close to zero, that certain stem cells in a tissue showed a reduced age compared to their chronological age, and that early embryogenesis is associated with the reduction of epigenetic age in individual cells, the latter supporting a natural rejuvenation event during gastrulation. scAge is both robust against the low coverage that is characteristic of single cell sequencing techniques and is flexible for studying any cell type and mammalian organism of interest. We demonstrate the potential for accurate epigenetic age profiling at single-cell resolution.

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Profiling epigenetic age in single cells

10.1101/2021.03.13.435247 ◽

2021 ◽

Author(s):

Alexandre Trapp ◽

Csaba Kerepesi ◽

Vadim N Gladyshev

Keyword(s):

Stem Cells ◽

Single Cell ◽

Single Cells ◽

Probabilistic Approach ◽

Embryonic Stem ◽

Chronological Age ◽

Cell Type ◽

Cpg Dinucleotides ◽

Epigenetic Age ◽

Quantitative Changes

DNA methylation of a defined set of CpG dinucleotides emerged as a critical and precise biomarker of the aging process. Multi-variate machine learning models, known as epigenetic clocks, can exploit quantitative changes in the methylome to predict the age of bulk tissue with remarkable accuracy. However, intrinsic sparsity and digitized methylation in individual cells have so far precluded the assessment of aging in single cell data. Here, we present scAge, a probabilistic approach to determine the epigenetic age of single cells, and validate our results in mice. scAge tissue-specific and multi-cell type single cell clocks correctly recapitulate chronological age of the original tissue, while uncovering the inherent heterogeneity that exists at the single-cell level. The data suggest that while tissues age in a coordinated fashion, some cells age more or less rapidly than others. We show that individual embryonic stem cells exhibit an age close to zero, that certain stem cells in a tissue show a reduced age compared to their chronological age, and that early embryogenesis is associated with the reduction of epigenetic age of individual cells, the latter supporting a natural rejuvenation event during gastrulation. scAge is both robust against the low coverage that is characteristic of single cell sequencing techniques and is flexible for studying any cell type and vertebrate organism of interest. This study demonstrates for the first time the potential for accurate epigenetic age profiling at single-cell resolution.

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Uncovering low-level mosaicism in human embryonic stem cells using high throughput single cell shallow sequencing

Scientific Reports ◽

10.1038/s41598-019-51314-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Alexander Keller ◽

Laurentijn Tilleman ◽

Dominika Dziedzicka ◽

Filippo Zambelli ◽

Karen Sermon ◽

...

Keyword(s):

Stem Cells ◽

Single Cell ◽

Single Cells ◽

Embryonic Stem ◽

Copy Number Variations ◽

Fragile Sites ◽

Low Grade ◽

Bulk Analysis ◽

Genetic Mosaicism

Abstract Human pluripotent stem cells (hPSCs) have significant levels of low-grade genetic mosaicism, which commonly used techniques fail to detect in bulk DNA. These copy number variations remain a hurdle for the clinical translation of hPSC, as their effect in vivo ranges from unknown to dangerous, and the ability to detect them will be necessary as the field advances. As such there is need for techniques which can efficiently analyse genetic content in single cells with higher throughput and lower costs. We report here on the use of the Fluidigm C1 single cell WGA platform in combination with shallow whole genome sequencing to analyse the genetic content of single hPSCs. From a hPSC line carrying an isochromosome 20, 56 single cells were analysed and found to carry a total of 50 aberrations, across 23% of cells, which could not be detected by bulk analysis. Aberrations were predominantly segmental gains, with a fewer number of segmental losses and aneuploidies. Interestingly, 40% of the breakpoints seen here correspond to known DNA fragile sites. Our results therefore demonstrate the feasibility of single cell shallow sequencing of hPSC and further expand upon the biological importance and frequency of single cell mosaicism in hPSC.

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Visualization and quantification of dynamic intercellular coupling in human embryonic stem cells using single cell sonoporation

Scientific Reports ◽

10.1038/s41598-020-75347-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Zhenzhen Fan ◽

Xufeng Xue ◽

Jianping Fu ◽

Cheri X. Deng

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Single Cell ◽

Human Embryonic Stem Cells ◽

Single Cells ◽

Critical Role ◽

Embryonic Stem ◽

Cell Permeability ◽

Dynamic Visualization ◽

Dye Transfer

Abstract Gap junctions (GJs), which are proteinaceous channels, couple adjacent cells by permitting direct exchange of intracellular molecules with low molecular weights. GJ intercellular communication (GJIC) plays a critical role in regulating behaviors of human embryonic stem cells (hESCs), affecting their proliferation and differentiation. Here we report a novel use of sonoporation that enables single cell intracellular dye loading and dynamic visualization/quantification of GJIC in hESC colonies. By applying a short ultrasound pulse to excite single microbubbles tethered to cell membranes, a transient pore on the cell membrane (sonoporation) is generated which allows intracellular loading of dye molecules and influx of Ca2+ into single hESCs. We employ live imaging for continuous visualization of intercellular dye transfer and Ca2+ diffusion in hESC colonies. We quantify cell–cell permeability based on dye diffusion using mass transport models. Our results reveal heterogeneous intercellular connectivity and a variety of spatiotemporal characteristics of intercellular Ca2+ waves in hESC colonies induced by sonoporation of single cells.

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A cell atlas of the adult Drosophila midgut

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1916820117 ◽

2020 ◽

Vol 117 (3) ◽

pp. 1514-1523 ◽

Cited By ~ 25

Author(s):

Ruei-Jiun Hung ◽

Yanhui Hu ◽

Rory Kirchner ◽

Yifang Liu ◽

Chiwei Xu ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Single Cell ◽

Rna Sequencing ◽

Cell Biology ◽

Single Cells ◽

Intestinal Stem Cells ◽

Cell Type ◽

Molecular Features ◽

Single Cell Rna Sequencing

Studies of the adult Drosophila midgut have led to many insights in our understanding of cell-type diversity, stem cell regeneration, tissue homeostasis, and cell fate decision. Advances in single-cell RNA sequencing provide opportunities to identify new cell types and molecular features. We used single-cell RNA sequencing to characterize the transcriptome of midgut epithelial cells and identified 22 distinct clusters representing intestinal stem cells, enteroblasts, enteroendocrine cells (EEs), and enterocytes. This unbiased approach recovered most of the known intestinal stem cells/enteroblast and EE markers, highlighting the high quality of the dataset, and led to insights on intestinal stem cell biology, cell type-specific organelle features, the roles of new transcription factors in progenitors and regional variation along the gut, 5 additional EE gut hormones, EE hormonal expression diversity, and paracrine function of EEs. To facilitate mining of this rich dataset, we provide a web-based resource for visualization of gene expression in single cells. Altogether, our study provides a comprehensive resource for addressing functions of genes in the midgut epithelium.

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Unveiling transposable element expression heterogeneity in cell fate regulation at the single-cell level

10.1101/2020.07.23.218800 ◽

2020 ◽

Cited By ~ 2

Author(s):

Jiangping He ◽

Isaac A. Babarinde ◽

Li Sun ◽

Shuyang Xu ◽

Ruhai Chen ◽

...

Keyword(s):

Single Cell ◽

Cell Fate ◽

Single Cells ◽

Embryonic Stem ◽

Cell Type ◽

Cell Level ◽

Single Cell Sequencing ◽

Processing Pipeline ◽

Sequencing Technologies ◽

Disease Specific

AbstractTransposable elements (TEs) make up a majority of a typical eukaryote’s genome, and contribute to cell heterogeneity and fate in unclear ways. Single cell-sequencing technologies are powerful tools to explore cells, however analysis is typically gene-centric and TE activity has not been addressed. Here, we developed a single-cell TE processing pipeline, scTE, and report the activity of TEs in single cells in a range of biological contexts. Specific TE types were expressed in subpopulations of embryonic stem cells and were dynamically regulated during pluripotency reprogramming, differentiation, and embryogenesis. Unexpectedly, TEs were expressed in somatic cells, including human disease-specific TEs that are undetectable in bulk analyses. Finally, we applied scTE to single cell ATAC-seq data, and demonstrate that scTE can discriminate cell type using chromatin accessibly of TEs alone. Overall, our results reveal the dynamic patterns of TEs in single cells and their contributions to cell fate and heterogeneity.

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Identifying transposable element expression dynamics and heterogeneity during development at the single-cell level with a processing pipeline scTE

Nature Communications ◽

10.1038/s41467-021-21808-x ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Jiangping He ◽

Isaac A. Babarinde ◽

Li Sun ◽

Shuyang Xu ◽

Ruhai Chen ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Embryonic Stem ◽

Cell Heterogeneity ◽

Cell Type ◽

Cell Level ◽

Single Cell Sequencing ◽

Processing Pipeline ◽

Sequencing Technologies ◽

Disease Specific

AbstractTransposable elements (TEs) make up a majority of a typical eukaryote’s genome, and contribute to cell heterogeneity in unclear ways. Single-cell sequencing technologies are powerful tools to explore cells, however analysis is typically gene-centric and TE expression has not been addressed. Here, we develop a single-cell TE processing pipeline, scTE, and report the expression of TEs in single cells in a range of biological contexts. Specific TE types are expressed in subpopulations of embryonic stem cells and are dynamically regulated during pluripotency reprogramming, differentiation, and embryogenesis. Unexpectedly, TEs are expressed in somatic cells, including human disease-specific TEs that are undetectable in bulk analyses. Finally, we apply scTE to single-cell ATAC-seq data, and demonstrate that scTE can discriminate cell type using chromatin accessibly of TEs alone. Overall, our results classify the dynamic patterns of TEs in single cells and their contributions to cell heterogeneity.

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Faculty Opinions recommendation of Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725508825.793514059 ◽

2016 ◽

Author(s):

Cole Trapnell ◽

Serena Liu

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Single Cell ◽

Embryonic Stem

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Single-Cell Transcriptome Analysis as a Promising Tool to Study Pluripotent Stem Cell Reprogramming

International Journal of Molecular Sciences ◽

10.3390/ijms22115988 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5988

Author(s):

Hyun Kyu Kim ◽

Tae Won Ha ◽

Man Ryul Lee

Keyword(s):

Stem Cells ◽

Single Cell ◽

Cell Fate ◽

Human Cell ◽

Transcriptome Analysis ◽

Single Cell Analysis ◽

Embryonic Stem ◽

Single Cell Level ◽

Cell Analysis ◽

Cell Level

Cells are the basic units of all organisms and are involved in all vital activities, such as proliferation, differentiation, senescence, and apoptosis. A human body consists of more than 30 trillion cells generated through repeated division and differentiation from a single-cell fertilized egg in a highly organized programmatic fashion. Since the recent formation of the Human Cell Atlas consortium, establishing the Human Cell Atlas at the single-cell level has been an ongoing activity with the goal of understanding the mechanisms underlying diseases and vital cellular activities at the level of the single cell. In particular, transcriptome analysis of embryonic stem cells at the single-cell level is of great importance, as these cells are responsible for determining cell fate. Here, we review single-cell analysis techniques that have been actively used in recent years, introduce the single-cell analysis studies currently in progress in pluripotent stem cells and reprogramming, and forecast future studies.

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Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02085-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ping Zhou ◽

Jia-Min Shi ◽

Jing-E Song ◽

Yu Han ◽

Hong-Jiao Li ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Telomerase Activity ◽

Human Pluripotent Stem Cells ◽

Cell Counting ◽

Cell Type ◽

Alizarin Red

Abstract Background Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed. Methods Monolayer cultured human embryonic stem cells and human-induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week. Results The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrate the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded. Conclusions Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a stepwise induction system in the future.

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