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

2019 ◽  
Vol 9 (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.

scholarly journals Defining totipotency using criteria of increasing stringency

2020 ◽  
Author(s):  
Eszter Posfai ◽  
John Paul Schell ◽  
Adrian Janiszewski ◽  
Isidora Rovic ◽  
Alexander Murray ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Differentiated Cells ◽  
Totipotent Cell

AbstractTotipotency is the ability of a single cell to give rise to all the differentiated cells that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies upon a variety of assays of variable stringency. Here we describe criteria to define totipotency. We illustrate how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in the mouse, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbor increased totipotent potential relative to conventional embryonic stem cells under in vivo conditions.

scholarly journals Profiling Epigenetic Age in Single Cells

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. 677-677
Author(s):  
Alexandre Trapp ◽  
Csaba Kerepesi ◽  
Vadim Gladyshev
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Single Cells ◽  
Probabilistic Approach ◽  
Embryonic Stem ◽  
Chronological Age ◽  
Cell Type ◽  
Cpg Dinucleotides ◽  
Epigenetic Age ◽  
Quantitative Changes

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.

scholarly journals Profiling epigenetic age in single cells

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.

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

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.

scholarly journals Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads

2017 ◽  
Author(s):  
Yohei Sasagawa ◽  
Hiroki Danno ◽  
Hitomi Takada ◽  
Masashi Ebisawa ◽  
Kaori Tanaka ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Embryonic Stem ◽  
Stromal Vascular Fraction ◽  
Rna Seq ◽  
Sequencing Method ◽  
Unique Molecular Identifier

AbstractHigh-throughput single-cell RNA-seq methods assign limited unique molecular identifier (UMI) counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts. We thus developed a high-throughput single-cell RNA-seq method, Quartz-Seq2, to overcome these issues. Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts (at a rate of 30%–50%) and detect more genes. To demonstrate the power of Quartz-Seq2, we analyzed approximately 10,000 transcriptomes in total from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads.

scholarly journals Time-course single-cell RNA sequencing reveals transcriptional dynamics and heterogeneity of limbal stem cells derived from human pluripotent stem cells

2020 ◽  
Author(s):  
Changbin Sun ◽  
Hailun Wang ◽  
Qiwang Ma ◽  
Chao Chen ◽  
Jianhui Yue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Cell Fate ◽  
Time Course ◽  
Embryonic Stem ◽  
Limbal Stem Cells ◽  
Developmental Origin ◽  
Differentiated Cells ◽  
Corneal Transplants

AbstractHuman pluripotent stem cell-derived limbal stem cells (hPSC-derived LSCs) provide a promising cell source for corneal transplants and ocular surface reconstruction. Although recent efforts in the identification of LSC markers have increased our understanding of the biology of LSCs, the lack of knowledge of the developmental origin, cell fate determination, and identity of human LSCs hindered the establishment of differentiation protocols for hPSC-derived LSCs and hold back their clinical application. Here, we performed a time-course single-cell RNA-seq to investigate transcriptional heterogeneity and expression changes of LSCs derived from human embryonic stem cells. Based on current protocol, expression heterogeneity of reported LSC markers were identified in subpopulations of differentiated cells. EMT has been shown to occur during differentiation process, which could possibly result in generation of untargeted cells. Pseudotime trajectory analysis revealed transcriptional changes and signatures of commitment of hPSCs-derived LSCs and their progeny - the transit amplifying cells. Furthermore, several new makers of LSCs were identified, which could facilitate elucidating the identity and developmental origin of human LSCs in vivo.

scholarly journals mRNA-Driven Generation of Transgene-Free Neural Stem Cells from Human Urine-Derived Cells

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1043 ◽  
Author(s):  
Phil Jun Kang ◽  
Daryeon Son ◽  
Tae Hee Ko ◽  
Wonjun Hong ◽  
Wonjin Yun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Human Urine ◽  
Neurological Disorders ◽  
Therapeutic Potential ◽  
Embryonic Stem ◽  
Global Gene Expression ◽  
Patient Specific ◽  
Human Neural Stem Cells

Human neural stem cells (NSCs) hold enormous promise for neurological disorders, typically requiring their expandable and differentiable properties for regeneration of damaged neural tissues. Despite the therapeutic potential of induced NSCs (iNSCs), a major challenge for clinical feasibility is the presence of integrated transgenes in the host genome, contributing to the risk for undesired genotoxicity and tumorigenesis. Here, we describe the advanced transgene-free generation of iNSCs from human urine-derived cells (HUCs) by combining a cocktail of defined small molecules with self-replicable mRNA delivery. The established iNSCs were completely transgene-free in their cytosol and genome and further resembled human embryonic stem cell-derived NSCs in the morphology, biological characteristics, global gene expression, and potential to differentiate into functional neurons, astrocytes, and oligodendrocytes. Moreover, iNSC colonies were observed within eight days under optimized conditions, and no teratomas formed in vivo, implying the absence of pluripotent cells. This study proposes an approach to generate transplantable iNSCs that can be broadly applied for neurological disorders in a safe, efficient, and patient-specific manner.

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

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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