Cells

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5 pages, 665 KiB

Open AccessEditorial

Single-Cell Analysis 2.0

by Tuhin Subhra Santra and Fan-Gang Tseng

Cells 2023, 12(1), 154; https://doi.org/10.3390/cells12010154 - 30 Dec 2022

Cited by 1 | Viewed by 1463

Abstract

In 1665, Robert Hooke published his revolutionary book Micrographia [...] Full article

(This article belongs to the Special Issue Single Cell Analysis 2.0)

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Research

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15 pages, 4854 KiB

Open AccessArticle

Single Cell Effects of Photobiomodulation on Mitochondrial Membrane Potential and Reactive Oxygen Species Production in Human Adipose Mesenchymal Stem Cells

by Li-Chern Pan, Nguyen-Le-Thanh Hang, Mamadi M.S Colley, Jungshan Chang, Yu-Cheng Hsiao, Long-Sheng Lu, Bing-Sian Li, Cheng-Jen Chang and Tzu-Sen Yang

Cells 2022, 11(6), 972; https://doi.org/10.3390/cells11060972 - 11 Mar 2022

Cited by 24 | Viewed by 5107

Abstract

Photobiomodulation (PBM) has recently emerged in cellular therapy as a potent alternative in promoting cell proliferation, migration, and differentiation during tissue regeneration. Herein, a single-cell near-infrared (NIR) laser irradiation system (830 nm) and the image-based approaches were proposed for the investigation of the [...] Read more.

Photobiomodulation (PBM) has recently emerged in cellular therapy as a potent alternative in promoting cell proliferation, migration, and differentiation during tissue regeneration. Herein, a single-cell near-infrared (NIR) laser irradiation system (830 nm) and the image-based approaches were proposed for the investigation of the modulatory effects in mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), and vesicle transport in single living human adipose mesenchymal stem cells (hADSCs). The irradiated-hADSCs were then stained with 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) and Rhodamine 123 (Rh123) to represent the ΔΨm and ROS production, respectively, with irradiation in the range of 2.5–10 (J/cm²), where time series of bright-field images were obtained to determine the vesicle transport phenomena. Present results showed that a fluence of 5 J/cm² of PBM significantly enhanced the ΔΨm, ROS, and vesicle transport phenomena compared to the control group (0 J/cm²) after 30 min PBM treatment. These findings demonstrate the efficacy and use of PBM in regulating ΔΨm, ROS, and vesicle transport, which have potential in cell proliferation, migration, and differentiation in cell-based therapy. Full article

(This article belongs to the Special Issue Single Cell Analysis 2.0)

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16 pages, 2706 KiB

Open AccessArticle

Nucleus Near-Infrared (nNIR) Irradiation of Single A549 Cells Induces DNA Damage and Activates EGFR Leading to Mitochondrial Fission

by Momoh Gbetuwa, Long-Sheng Lu, Tsung-Jen Wang, Yin-Ju Chen, Jeng-Fong Chiou, Tai-Yuan Su and Tzu-Sen Yang

Cells 2022, 11(4), 624; https://doi.org/10.3390/cells11040624 - 11 Feb 2022

Cited by 2 | Viewed by 2570

Abstract

There has been great interest in identifying the biological substrate for light-cell interaction and their relations to cancer treatment. In this study, a near-infrared (NIR) laser is focused into the nucleus (nNIR) or cytoplasm (cNIR) of a single living cell by a high [...] Read more.

There has been great interest in identifying the biological substrate for light-cell interaction and their relations to cancer treatment. In this study, a near-infrared (NIR) laser is focused into the nucleus (nNIR) or cytoplasm (cNIR) of a single living cell by a high numerical aperture condenser to dissect the novel role of cell nucleus in mediating NIR effects on mitochondrial dynamics of A549 non-small cell lung cancer cells. Our analysis showed that nNIR, but not cNIR, triggered mitochondrial fission in 10 min. In contrast, the fission/fusion balance of mitochondria directly exposed to cNIR does not change. While the same phenomenon is also triggered by single molecular interactions between epidermal growth factor (EGF) and its receptor EGFR, pharmacological studies with cetuximab, PD153035, and caffeine suggest EGF signaling crosstalk to DNA damaging response to mediate rapid mitochondrial fission as a result of nNIR irradiation. These results suggest that nuclear DNA integrity is a novel biological target for cellular response to NIR. Full article

(This article belongs to the Special Issue Single Cell Analysis 2.0)

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16 pages, 4470 KiB

Open AccessEditor’s ChoiceArticle

Discordant Genome Assemblies Drastically Alter the Interpretation of Single-Cell RNA Sequencing Data Which Can Be Mitigated by a Novel Integration Method

by Helen G. Potts, Madeleine E. Lemieux, Edward S. Rice, Wesley Warren, Robin P. Choudhury and Mathilda T. M. Mommersteeg

Cells 2022, 11(4), 608; https://doi.org/10.3390/cells11040608 - 10 Feb 2022

Cited by 2 | Viewed by 2870

Abstract

Advances in sequencing and assembly technology have led to the creation of genome assemblies for a wide variety of non-model organisms. The rapid production and proliferation of updated, novel assembly versions can create vexing problems for researchers when multiple-genome assembly versions are available [...] Read more.

Advances in sequencing and assembly technology have led to the creation of genome assemblies for a wide variety of non-model organisms. The rapid production and proliferation of updated, novel assembly versions can create vexing problems for researchers when multiple-genome assembly versions are available at once, requiring researchers to work with more than one reference genome. Multiple-genome assemblies are especially problematic for researchers studying the genetic makeup of individual cells, as single-cell RNA sequencing (scRNAseq) requires sequenced reads to be mapped and aligned to a single reference genome. Using the Astyanax mexicanus, this study highlights how the interpretation of a single-cell dataset from the same sample changes when aligned to its two different available genome assemblies. We found that the number of cells and expressed genes detected were drastically different when aligning to the different assemblies. When the genome assemblies were used in isolation with their respective annotations, cell-type identification was confounded, as some classic cell-type markers were assembly-specific, whilst other genes showed differential patterns of expression between the two assemblies. To overcome the problems posed by multiple-genome assemblies, we propose that researchers align to each available assembly and then integrate the resultant datasets to produce a final dataset in which all genome alignments can be used simultaneously. We found that this approach increased the accuracy of cell-type identification and maximised the amount of data that could be extracted from our single-cell sample by capturing all possible cells and transcripts. As scRNAseq becomes more widely available, it is imperative that the single-cell community is aware of how genome assembly alignment can alter single-cell data and their interpretation, especially when reviewing studies on non-model organisms. Full article

(This article belongs to the Special Issue Single Cell Analysis 2.0)

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12 pages, 1916 KiB

Open AccessArticle

Interpretable Autoencoders Trained on Single Cell Sequencing Data Can Transfer Directly to Data from Unseen Tissues

by Julie Sparholt Walbech, Savvas Kinalis, Ole Winther, Finn Cilius Nielsen and Frederik Otzen Bagger

Cells 2022, 11(1), 85; https://doi.org/10.3390/cells11010085 - 28 Dec 2021

Cited by 4 | Viewed by 2699

Abstract

Autoencoders have been used to model single-cell mRNA-sequencing data with the purpose of denoising, visualization, data simulation, and dimensionality reduction. We, and others, have shown that autoencoders can be explainable models and interpreted in terms of biology. Here, we show that such autoencoders [...] Read more.

Autoencoders have been used to model single-cell mRNA-sequencing data with the purpose of denoising, visualization, data simulation, and dimensionality reduction. We, and others, have shown that autoencoders can be explainable models and interpreted in terms of biology. Here, we show that such autoencoders can generalize to the extent that they can transfer directly without additional training. In practice, we can extract biological modules, denoise, and classify data correctly from an autoencoder that was trained on a different dataset and with different cells (a foreign model). We deconvoluted the biological signal encoded in the bottleneck layer of scRNA-models using saliency maps and mapped salient features to biological pathways. Biological concepts could be associated with specific nodes and interpreted in relation to biological pathways. Even in this unsupervised framework, with no prior information about cell types or labels, the specific biological pathways deduced from the model were in line with findings in previous research. It was hypothesized that autoencoders could learn and represent meaningful biology; here, we show with a systematic experiment that this is true and even transcends the training data. This means that carefully trained autoencoders can be used to assist the interpretation of new unseen data. Full article

(This article belongs to the Special Issue Single Cell Analysis 2.0)

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15 pages, 4598 KiB

Open AccessArticle

Gliogenic Potential of Single Pallial Radial Glial Cells in Lower Cortical Layers

by Ana Cristina Ojalvo-Sanz and Laura López-Mascaraque

Cells 2021, 10(11), 3237; https://doi.org/10.3390/cells10113237 - 19 Nov 2021

Cited by 7 | Viewed by 2386

Abstract

During embryonic development, progenitor cells are progressively restricted in their potential to generate different neural cells. A specific progenitor cell type, the radial glial cells, divides symmetrically and then asymmetrically to produce neurons, astrocytes, oligodendrocytes, and NG2-glia in the cerebral cortex. However, the [...] Read more.

During embryonic development, progenitor cells are progressively restricted in their potential to generate different neural cells. A specific progenitor cell type, the radial glial cells, divides symmetrically and then asymmetrically to produce neurons, astrocytes, oligodendrocytes, and NG2-glia in the cerebral cortex. However, the potential of individual progenitors to form glial lineages remains poorly understood. To further investigate the cell progeny of single pallial GFAP-expressing progenitors, we used the in vivo genetic lineage-tracing method, the UbC-(GFAP-PB)-StarTrack. After targeting those progenitors in embryonic mice brains, we tracked their adult glial progeny in lower cortical layers. Clonal analyses revealed the presence of clones containing sibling cells of either a glial cell type (uniform clones) or two different glial cell types (mixed clones). Further, the clonal size and rostro-caudal cell dispersion of sibling cells differed depending on the cell type. We concluded that pallial E14 neural progenitors are a heterogeneous cell population with respect to which glial cell type they produce, as well as the clonal size of their cell progeny. Full article

(This article belongs to the Special Issue Single Cell Analysis 2.0)

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20 pages, 7124 KiB

Open AccessArticle

Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells

by Po-Yu Lin, Denny Yang, Chi-Hsuan Chuang, Hsuan Lin, Wei-Ju Chen, Chia-Ying Chen, Trees-Juen Chuang, Chien-Ying Lai, Long-Yuan Li, Scott C. Schuyler, Frank Leigh Lu, Yu-Chuan Liu and Jean Lu

Cells 2021, 10(11), 3111; https://doi.org/10.3390/cells10113111 - 10 Nov 2021

Cited by 6 | Viewed by 4090

Abstract

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of [...] Read more.

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are ‘true’ totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely ‘cluster 3’, as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells. Full article

(This article belongs to the Special Issue Single Cell Analysis 2.0)

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16 pages, 3333 KiB

Open AccessArticle

Intratumoral Heterogeneity Promotes Collective Cancer Invasion through NOTCH1 Variation

by Peter Torab, Yue Yan, Mona Ahmed, Hironobu Yamashita, Joshua I. Warrick, Jay D. Raman, David J. DeGraff and Pak Kin Wong

Cells 2021, 10(11), 3084; https://doi.org/10.3390/cells10113084 - 9 Nov 2021

Cited by 8 | Viewed by 2704

Abstract

Cellular and molecular heterogeneity within tumors has long been associated with the progression of cancer to an aggressive phenotype and a poor prognosis. However, how such intratumoral heterogeneity contributes to the invasiveness of cancer is largely unknown. Here, using a tumor bioengineering approach, [...] Read more.

Cellular and molecular heterogeneity within tumors has long been associated with the progression of cancer to an aggressive phenotype and a poor prognosis. However, how such intratumoral heterogeneity contributes to the invasiveness of cancer is largely unknown. Here, using a tumor bioengineering approach, we investigate the interaction between molecular subtypes within bladder microtumors and the corresponding effects on their invasiveness. Our results reveal heterogeneous microtumors formed by multiple molecular subtypes possess enhanced invasiveness compared to individual cells, even when both cells are not invasive individually. To examine the molecular mechanism of intratumoral heterogeneity mediated invasiveness, live single cell biosensing, RNA interference, and CRISPR-Cas9 gene editing approaches were applied to investigate and control the composition of the microtumors. An agent-based computational model was also developed to evaluate the influence of NOTCH1 variation on DLL4 expression within a microtumor. The data indicate that intratumoral variation in NOTCH1 expression can lead to upregulation of DLL4 expression within the microtumor and enhancement of microtumor invasiveness. Overall, our results reveal a novel mechanism of heterogeneity mediated invasiveness through intratumoral variation of gene expression. Full article

(This article belongs to the Special Issue Single Cell Analysis 2.0)

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12 pages, 3474 KiB

Open AccessArticle

Single Cell Analysis of Stored Red Blood Cells Using Ultra-High Throughput Holographic Cytometry

by Han-Sang Park, Hillel Price, Silvia Ceballos, Jen-Tsan Chi and Adam Wax

Cells 2021, 10(9), 2455; https://doi.org/10.3390/cells10092455 - 17 Sep 2021

Cited by 24 | Viewed by 3345

Abstract

Holographic cytometry is introduced as an ultra-high throughput implementation of quantitative phase imaging of single cells flowing through parallel microfluidic channels. Here, the approach was applied for characterizing the morphology of individual red blood cells during storage under regular blood bank conditions. Samples [...] Read more.

Holographic cytometry is introduced as an ultra-high throughput implementation of quantitative phase imaging of single cells flowing through parallel microfluidic channels. Here, the approach was applied for characterizing the morphology of individual red blood cells during storage under regular blood bank conditions. Samples from five blood donors were examined, over 100,000 cells examined for each, at three time points. The approach allows high-throughput phase imaging of a large number of cells, greatly extending our ability to study cellular phenotypes using individual cell images. Holographic cytology images can provide measurements of multiple physical traits of the cells, including optical volume and area, which are observed to consistently change over the storage time. In addition, the large volume of cell imaging data can serve as training data for machine-learning algorithms. For the study here, logistic regression was used to classify the cells according to the storage time points. The analysis showed that at least 5000 cells are needed to ensure accuracy of the classifiers. Overall, results showed the potential of holographic cytometry as a diagnostic tool. Full article

(This article belongs to the Special Issue Single Cell Analysis 2.0)

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14 pages, 3706 KiB

Open AccessArticle

Mapping Surface Charge Distribution of Single-Cell via Charged Nanoparticle

by Leixin Ouyang, Rubia Shaik, Ruiting Xu, Ge Zhang and Jiang Zhe

Cells 2021, 10(6), 1519; https://doi.org/10.3390/cells10061519 - 16 Jun 2021

Cited by 14 | Viewed by 4365

Abstract

Many bio-functions of cells can be regulated by their surface charge characteristics. Mapping surface charge density in a single cell’s surface is vital to advance the understanding of cell behaviors. This article demonstrates a method of cell surface charge mapping via electrostatic cell–nanoparticle [...] Read more.

Many bio-functions of cells can be regulated by their surface charge characteristics. Mapping surface charge density in a single cell’s surface is vital to advance the understanding of cell behaviors. This article demonstrates a method of cell surface charge mapping via electrostatic cell–nanoparticle (NP) interactions. Fluorescent nanoparticles (NPs) were used as the marker to investigate single cells’ surface charge distribution. The nanoparticles with opposite charges were electrostatically bonded to the cell surface; a stack of fluorescence distribution on a cell’s surface at a series of vertical distances was imaged and analyzed. By establishing a relationship between fluorescent light intensity and number of nanoparticles, cells’ surface charge distribution was quantified from the fluorescence distribution. Two types of cells, human umbilical vein endothelial cells (HUVECs) and HeLa cells, were tested. From the measured surface charge density of a group of single cells, the average zeta potentials of the two types of cells were obtained, which are in good agreement with the standard electrophoretic light scattering measurement. This method can be used for rapid surface charge mapping of single particles or cells, and can advance cell-surface-charge characterization applications in many biomedical fields. Full article

(This article belongs to the Special Issue Single Cell Analysis 2.0)

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18 pages, 4087 KiB

Open AccessArticle

Comparative Transcriptomic Analysis of the Hematopoietic System between Human and Mouse by Single Cell RNA Sequencing

by Shouguo Gao, Zhijie Wu, Jeerthi Kannan, Liza Mathews, Xingmin Feng, Sachiko Kajigaya and Neal S. Young

Cells 2021, 10(5), 973; https://doi.org/10.3390/cells10050973 - 21 Apr 2021

Cited by 9 | Viewed by 5122

Abstract

(1) Background: mouse models are fundamental to the study of hematopoiesis, but comparisons between mouse and human in single cells have been limited in depth. (2) Methods: we constructed a single-cell resolution transcriptomic atlas of hematopoietic stem and progenitor cells (HSPCs) of human [...] Read more.

(1) Background: mouse models are fundamental to the study of hematopoiesis, but comparisons between mouse and human in single cells have been limited in depth. (2) Methods: we constructed a single-cell resolution transcriptomic atlas of hematopoietic stem and progenitor cells (HSPCs) of human and mouse, from a total of 32,805 single cells. We used Monocle to examine the trajectories of hematopoietic differentiation, and SCENIC to analyze gene networks underlying hematopoiesis. (3) Results: After alignment with Seurat 2, the cells of mouse and human could be separated by same cell type categories. Cells were grouped into 17 subpopulations; cluster-specific genes were species-conserved and shared functional themes. The clustering dendrogram indicated that cell types were highly conserved between human and mouse. A visualization of the Monocle results provided an intuitive representation of HSPC differentiation to three dominant branches (Erythroid/megakaryocytic, Myeloid, and Lymphoid), derived directly from the hematopoietic stem cell and the long-term hematopoietic stem cells in both human and mouse. Gene regulation was similarly conserved, reflected by comparable transcriptional factors and regulatory sequence motifs in subpopulations of cells. (4) Conclusions: our analysis has confirmed evolutionary conservation in the hematopoietic systems of mouse and human, extending to cell types, gene expression and regulatory elements. Full article

(This article belongs to the Special Issue Single Cell Analysis 2.0)

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Review

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16 pages, 4146 KiB

Open AccessReview

Tutorial: Guidelines for Single-Cell RT-qPCR

by Daniel Zucha, Mikael Kubista and Lukas Valihrach

Cells 2021, 10(10), 2607; https://doi.org/10.3390/cells10102607 - 30 Sep 2021

Cited by 14 | Viewed by 12028

Abstract

Reverse transcription quantitative PCR (RT-qPCR) has delivered significant insights in understanding the gene expression landscape. Thanks to its precision, sensitivity, flexibility, and cost effectiveness, RT-qPCR has also found utility in advanced single-cell analysis. Single-cell RT-qPCR now represents a well-established method, suitable for an [...] Read more.

Reverse transcription quantitative PCR (RT-qPCR) has delivered significant insights in understanding the gene expression landscape. Thanks to its precision, sensitivity, flexibility, and cost effectiveness, RT-qPCR has also found utility in advanced single-cell analysis. Single-cell RT-qPCR now represents a well-established method, suitable for an efficient screening prior to single-cell RNA sequencing (scRNA-Seq) experiments, or, oppositely, for validation of hypotheses formulated from high-throughput approaches. Here, we aim to provide a comprehensive summary of the scRT-qPCR method by discussing the limitations of single-cell collection methods, describing the importance of reverse transcription, providing recommendations for the preamplification and primer design, and summarizing essential data processing steps. With the detailed protocol attached in the appendix, this tutorial provides a set of guidelines that allow any researcher to perform scRT-qPCR measurements of the highest standard. Full article

(This article belongs to the Special Issue Single Cell Analysis 2.0)

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46 pages, 8754 KiB

Open AccessReview

A Review of Single-Cell Adhesion Force Kinetics and Applications

by Ashwini Shinde, Kavitha Illath, Pallavi Gupta, Pallavi Shinde, Ki-Taek Lim, Moeto Nagai and Tuhin Subhra Santra

Cells 2021, 10(3), 577; https://doi.org/10.3390/cells10030577 - 5 Mar 2021

Cited by 38 | Viewed by 8304

Abstract

Cells exert, sense, and respond to the different physical forces through diverse mechanisms and translating them into biochemical signals. The adhesion of cells is crucial in various developmental functions, such as to maintain tissue morphogenesis and homeostasis and activate critical signaling pathways regulating [...] Read more.

Cells exert, sense, and respond to the different physical forces through diverse mechanisms and translating them into biochemical signals. The adhesion of cells is crucial in various developmental functions, such as to maintain tissue morphogenesis and homeostasis and activate critical signaling pathways regulating survival, migration, gene expression, and differentiation. More importantly, any mutations of adhesion receptors can lead to developmental disorders and diseases. Thus, it is essential to understand the regulation of cell adhesion during development and its contribution to various conditions with the help of quantitative methods. The techniques involved in offering different functionalities such as surface imaging to detect forces present at the cell-matrix and deliver quantitative parameters will help characterize the changes for various diseases. Here, we have briefly reviewed single-cell mechanical properties for mechanotransduction studies using standard and recently developed techniques. This is used to functionalize from the measurement of cellular deformability to the quantification of the interaction forces generated by a cell and exerted on its surroundings at single-cell with attachment and detachment events. The adhesive force measurement for single-cell microorganisms and single-molecules is emphasized as well. This focused review should be useful in laying out experiments which would bring the method to a broader range of research in the future. Full article

(This article belongs to the Special Issue Single Cell Analysis 2.0)

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