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9.9
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Cells
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Single Cell Analysis
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Single Cell Analysis

A special issue of Cells (ISSN 2073-4409).

Deadline for manuscript submissions: closed (30 April 2020) | Viewed by 98832

Printed Edition Available!
A printed edition of this Special Issue is available here.

Special Issue Editors

Dr. Tuhin Subhra Santra

E-Mail Website
Guest Editor
Department of Engineering Design, Indian Institute of Technology Madras, Chennai 600036, India
Interests: MEMS; bio-NEMS; single-cell technology; biomedical micro/nano devices; micro/nanofluidics; nanomedicine
Special Issues, Collections and Topics in MDPI journals
Prof. Dr. Fan-Gang Tseng

E-Mail Website
Guest Editor
Department of Engineering and System Science, National Tsing Hua University (NTHU), Academia Sinica, Hsinchu, Taiwan
Interests: organ on a chip; microfluidic systems; biosensors; CTCs/CTM diagnosis; single cell analysis
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Cells are the most fundamental building block of all living organisms. The investigation of any type of disease mechanism and its progression still remains challenging due to cellular heterogeneity characteristics and physiological state of cells in a given population. The bulk measurement of millions of cells together can provide an average information of cells, but it cannot evolve the cellular heterogeneity and molecular dynamics in a certain cell population. Compared to this bulk or average measurement of a large number of cells together, single-cell analysis can provide detailed information of each cell, which could assist in developing an understanding of the specific biological context of cells, such as tumor progression or issues around stem cells. Single-cell omics can provide valuable information about functional mutation and copy number variations of cells. Information from single-cell investigations can help to produce a better understanding of intracellular interactions and environmental responses of cellular organelles, which can be beneficial for therapeutics development and diagnostics purposes. This Special Issue is inviting articles related to single-cell analysis and its advantages, limitations, and future prospects regarding health benefits.

Dr. Tuhin Subhra Santra
Prof. Fan-Gang Tseng
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Cells is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • single-cell manipulation, separation, lysis
  • single-cell therapeutics and diagnostics
  • single-cell omics
  • single-cell in system biology
  • single-cell imaging and applications

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Related Special Issue

Published Papers (12 papers)

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Editorial

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7 pages, 379 KiB  
Editorial
Single-Cell Analysis
by Tuhin Subhra Santra and Fan-Gang Tseng
Cells 2020, 9(9), 1993; https://doi.org/10.3390/cells9091993 - 29 Aug 2020
Cited by 11 | Viewed by 3456
Abstract
Cells are known to be the most fundamental building block of life [...] Full article
(This article belongs to the Special Issue Single Cell Analysis)
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Research

Jump to: Editorial, Review

19 pages, 3750 KiB  
Communication
Genomic Analysis of Localized High-Risk Prostate Cancer Circulating Tumor Cells at the Single-Cell Level
by Aline Rangel-Pozzo, Songyan Liu, Gabriel Wajnberg, Xuemei Wang, Rodney J. Ouellette, Geoffrey G. Hicks, Darrel Drachenberg and Sabine Mai
Cells 2020, 9(8), 1863; https://doi.org/10.3390/cells9081863 - 8 Aug 2020
Cited by 18 | Viewed by 4915
Abstract
Accurate risk classification of men with localized high-risk prostate cancer directly affects treatment management decisions and patient outcomes. A wide range of risk assessments and classifications are available. However, each one has significant limitations to distinguish between indolent and aggressive prostate cancers. Circulating [...] Read more.
Accurate risk classification of men with localized high-risk prostate cancer directly affects treatment management decisions and patient outcomes. A wide range of risk assessments and classifications are available. However, each one has significant limitations to distinguish between indolent and aggressive prostate cancers. Circulating tumor cells (CTCs) may provide an alternate additional source, beyond tissue biopsies, to enable individual patient-specific clinical assessment, simply because CTCs can reveal both tumor-derived and germline-specific genetic information more precisely than that gained from a single diagnostic biopsy. In this study, we combined a filtration-based CTC isolation technology with prostate cancer CTC immunophenotyping to identify prostate cancer CTCs. Next, we performed 3-D telomere profiling prior to laser microdissection and single-cell whole-exome sequencing (WES) of 21 CTCs and 4 lymphocytes derived from 10 localized high-risk prostate cancer patient samples. Localized high-risk prostate cancer patient CTCs present a high number of telomere signals with lower signal intensities (short telomeres). To capture the genetic diversity/heterogeneity of high-risk prostate cancer CTCs, we carried out whole-exome sequencing. We identified 202,241 single nucleotide variants (SNVs) and 137,407 insertion-deletions (indels), where less than 10% of these genetic variations were within coding regions. The genetic variation (SNVs + indels) and copy number alteration (CNAs) profiles were highly heterogeneous and intra-patient CTC variation was observed. The pathway enrichment analysis showed the presence of genetic variation in nine telomere maintenance pathways (patients 3, 5, 6, and 7), including an important gene for telomere maintenance called telomeric repeat-binding factor 2 (TRF2). Using the PharmGKB database, we identified nine genetic variations associated with response to docetaxel. A total of 48 SNVs can affect drug response for 24 known cancer drugs. Gene Set Enrichment Analysis (GSEA) (patients 1, 3, 6, and 8) identified the presence of CNAs in 11 different pathways, including the DNA damage repair (DDR) pathway. In conclusion, single-cell approaches (WES and 3-D telomere profiling) showed to be useful in unmasking CTC heterogeneity. DDR pathway mutations have been well-established as a target pathway for cancer therapy. However, the frequent CNA amplifications found in localized high-risk patients may play critical roles in the therapeutic resistance in prostate cancer. Full article
(This article belongs to the Special Issue Single Cell Analysis)
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13 pages, 4452 KiB  
Article
A Microfluidic Single-Cell Cloning (SCC) Device for the Generation of Monoclonal Cells
by Chuan-Feng Yeh, Ching-Hui Lin, Hao-Chen Chang, Chia-Yu Tang, Pei-Tzu Lai and Chia-Hsien Hsu
Cells 2020, 9(6), 1482; https://doi.org/10.3390/cells9061482 - 18 Jun 2020
Cited by 5 | Viewed by 7734
Abstract
Single-cell cloning (SCC) is a critical step in generating monoclonal cell lines, which are widely used as in vitro models and for producing proteins with high reproducibility for research and the production of therapeutic drugs. In monoclonal cell line generation, the development time [...] Read more.
Single-cell cloning (SCC) is a critical step in generating monoclonal cell lines, which are widely used as in vitro models and for producing proteins with high reproducibility for research and the production of therapeutic drugs. In monoclonal cell line generation, the development time can be shortened by validating the monoclonality of the cloned cells. However, the validation process currently requires specialized equipment that is not readily available in general biology laboratories. Here, we report a disposable SCC device, in which single cells can be isolated, validated, and expanded to form monoclonal cell colonies using conventional micropipettes and microscopes. The monoclonal cells can be selectively transferred from the SCC chip to conventional culture plates, using a tissue puncher. Using the device, we demonstrated that monoclonal colonies of actin-GFP (green fluorescent protein) plasmid-transfected A549 cells could be formed in the device within nine days and subsequently transferred to wells in plates for further expansion. This approach offers a cost-effective alternative to the use of specialized equipment for monoclonal cell generation. Full article
(This article belongs to the Special Issue Single Cell Analysis)
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14 pages, 4241 KiB  
Article
Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin
by Renjie Liao, Thai Pham, Diego Mastroeni, Paul D. Coleman, Joshua Labaer and Jia Guo
Cells 2020, 9(4), 852; https://doi.org/10.3390/cells9040852 - 1 Apr 2020
Cited by 11 | Viewed by 5333
Abstract
The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this [...] Read more.
The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin-labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin-conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced at least 10-fold, compared with the current in-situ proteomics methods. After imaging, the fluorophore and the biotin unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be profiled in individual cells at the optical resolution. Applying this approach, we have demonstrated that multiple proteins are unambiguously detected in the same set of cells, regardless of the protein analysis order. We have also shown that this method can be successfully applied to quantify proteins in formalin-fixed paraffin-embedded (FFPE) tissues. Full article
(This article belongs to the Special Issue Single Cell Analysis)
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11 pages, 3047 KiB  
Article
Total mRNA Quantification in Single Cells: Sarcoma Cell Heterogeneity
by Emma Jonasson, Lisa Andersson, Soheila Dolatabadi, Salim Ghannoum, Pierre Åman and Anders Ståhlberg
Cells 2020, 9(3), 759; https://doi.org/10.3390/cells9030759 - 19 Mar 2020
Cited by 5 | Viewed by 4894
Abstract
Single-cell analysis enables detailed molecular characterization of cells in relation to cell type, genotype, cell state, temporal variations, and microenvironment. These studies often include the analysis of individual genes and networks of genes. The total amount of RNA also varies between cells due [...] Read more.
Single-cell analysis enables detailed molecular characterization of cells in relation to cell type, genotype, cell state, temporal variations, and microenvironment. These studies often include the analysis of individual genes and networks of genes. The total amount of RNA also varies between cells due to important factors, such as cell type, cell size, and cell cycle state. However, there is a lack of simple and sensitive methods to quantify the total amount of RNA, especially mRNA. Here, we developed a method to quantify total mRNA levels in single cells based on global reverse transcription followed by quantitative PCR. Standard curve analyses of diluted RNA and sorted cells showed a wide dynamic range, high reproducibility, and excellent sensitivity. Single-cell analysis of three sarcoma cell lines and human fibroblasts revealed cell type variations, a lognormal distribution of total mRNA levels, and up to an eight-fold difference in total mRNA levels among the cells. The approach can easily be combined with targeted or global gene expression profiling, providing new means to study cell heterogeneity at an individual gene level and at a global level. This method can be used to investigate the biological importance of variations in the total amount of mRNA in healthy as well as pathological conditions. Full article
(This article belongs to the Special Issue Single Cell Analysis)
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6 pages, 1744 KiB  
Communication
Single-Nucleus Sequencing of an Entire Mammalian Heart: Cell Type Composition and Velocity
by Markus Wolfien, Anne-Marie Galow, Paula Müller, Madeleine Bartsch, Ronald M. Brunner, Tom Goldammer, Olaf Wolkenhauer, Andreas Hoeflich and Robert David
Cells 2020, 9(2), 318; https://doi.org/10.3390/cells9020318 - 28 Jan 2020
Cited by 36 | Viewed by 10739
Abstract
Analyses on the cellular level are indispensable to expand our understanding of complex tissues like the mammalian heart. Single-nucleus sequencing (snRNA-seq) allows for the exploration of cellular composition and cell features without major hurdles of single-cell sequencing. We used snRNA-seq to investigate for [...] Read more.
Analyses on the cellular level are indispensable to expand our understanding of complex tissues like the mammalian heart. Single-nucleus sequencing (snRNA-seq) allows for the exploration of cellular composition and cell features without major hurdles of single-cell sequencing. We used snRNA-seq to investigate for the first time an entire adult mammalian heart. Single-nucleus quantification and clustering led to an accurate representation of cell types, revealing 24 distinct clusters with endothelial cells (28.8%), fibroblasts (25.3%), and cardiomyocytes (22.8%) constituting the major cell populations. An additional RNA velocity analysis allowed us to study transcription kinetics and was utilized to visualize the transitions between mature and nascent cellular states of the cell types. We identified subgroups of cardiomyocytes with distinct marker profiles. For example, the expression of Hand2os1 distinguished immature cardiomyocytes from differentiated cardiomyocyte populations. Moreover, we found a cell population that comprises endothelial markers as well as markers clearly related to cardiomyocyte function. Our velocity data support the idea that this population is in a trans-differentiation process from an endothelial cell-like phenotype towards a cardiomyocyte-like phenotype. In summary, we present the first report of sequencing an entire adult mammalian heart, providing realistic cell-type distributions combined with RNA velocity kinetics hinting at interrelations. Full article
(This article belongs to the Special Issue Single Cell Analysis)
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19 pages, 3987 KiB  
Article
Single Cell Mass Cytometry of Non-Small Cell Lung Cancer Cells Reveals Complexity of In Vivo and Three-Dimensional Models over the Petri-Dish
by Róbert Alföldi, József Á. Balog, Nóra Faragó, Miklós Halmai, Edit Kotogány, Patrícia Neuperger, Lajos I. Nagy, Liliána Z. Fehér, Gábor J. Szebeni and László G. Puskás
Cells 2019, 8(9), 1093; https://doi.org/10.3390/cells8091093 - 16 Sep 2019
Cited by 21 | Viewed by 7885
Abstract
Single cell genomics and proteomics with the combination of innovative three-dimensional (3D) cell culture techniques can open new avenues toward the understanding of intra-tumor heterogeneity. Here, we characterize lung cancer markers using single cell mass cytometry to compare different in vitro cell culturing [...] Read more.
Single cell genomics and proteomics with the combination of innovative three-dimensional (3D) cell culture techniques can open new avenues toward the understanding of intra-tumor heterogeneity. Here, we characterize lung cancer markers using single cell mass cytometry to compare different in vitro cell culturing methods: two-dimensional (2D), carrier-free, or bead-based 3D culturing with in vivo xenografts. Proliferation, viability, and cell cycle phase distribution has been investigated. Gene expression analysis enabled the selection of markers that were overexpressed: TMEM45A, SLC16A3, CD66, SLC2A1, CA9, CD24, or repressed: EGFR either in vivo or in long-term 3D cultures. Additionally, TRA-1-60, pan-keratins, CD326, Galectin-3, and CD274, markers with known clinical significance have been investigated at single cell resolution. The described twelve markers convincingly highlighted a unique pattern reflecting intra-tumor heterogeneity of 3D samples and in vivo A549 lung cancer cells. In 3D systems CA9, CD24, and EGFR showed higher expression than in vivo. Multidimensional single cell proteome profiling revealed that 3D cultures represent a transition from 2D to in vivo conditions by intermediate marker expression of TRA-1-60, TMEM45A, pan-keratin, CD326, MCT4, Gal-3, CD66, GLUT1, and CD274. Therefore, 3D cultures of NSCLC cells bearing more putative cancer targets should be used in drug screening as the preferred technique rather than the Petri-dish. Full article
(This article belongs to the Special Issue Single Cell Analysis)
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17 pages, 4310 KiB  
Article
Single-Cell Transcriptomes Reveal Characteristic Features of Mouse Hepatocytes with Liver Cholestatic Injury
by Na Chang, Lei Tian, Xiaofang Ji, Xuan Zhou, Lei Hou, Xinhao Zhao, Yuanru Yang, Lin Yang and Liying Li
Cells 2019, 8(9), 1069; https://doi.org/10.3390/cells8091069 - 11 Sep 2019
Cited by 15 | Viewed by 6907
Abstract
Hepatocytes are the main parenchymal cells of the liver and play important roles in liver homeostasis and disease process. The heterogeneity of normal hepatocytes has been reported, but there is little knowledge about hepatocyte subtype and distinctive functions during liver cholestatic injury. Bile [...] Read more.
Hepatocytes are the main parenchymal cells of the liver and play important roles in liver homeostasis and disease process. The heterogeneity of normal hepatocytes has been reported, but there is little knowledge about hepatocyte subtype and distinctive functions during liver cholestatic injury. Bile duct ligation (BDL)-induced mouse liver injury model was employed, and single-cell RNA sequencing was performed. Western blot and qPCR were used to study gene expression. Immunofluoresence was employed to detect the expressions of marker genes in hepatocytes. We detected a specific hepatocyte cluster (BDL-6) expressing extracellular matrix genes, indicating these hepatocytes might undergo epithelia-mesenchymal transition. Hepatocytes of BDL-6 also performed tissue repair functions (such as angiogenesis) during cholestatic injury. We also found that four clusters of cholestatic hepatocytes (BDL-2, BDL-3, BDL-4, and BDL-5) were involved in inflammatory process in different ways. To be specific, BDL-2/3/5 were inflammation-regulated hepatocytes, while BDL-4 played a role in cell chemotaxis. Among these four clusters, BDL-5 was special. because the hepatocytes of BDL-5 were proliferating hepatocytes. Our analysis provided more knowledge of hepatocyte distinctive functions in injured liver and gave rise to future treatment aiming at hepatocytes. Full article
(This article belongs to the Special Issue Single Cell Analysis)
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Review

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45 pages, 13022 KiB  
Review
A Single-Neuron: Current Trends and Future Prospects
by Pallavi Gupta, Nandhini Balasubramaniam, Hwan-You Chang, Fan-Gang Tseng and Tuhin Subhra Santra
Cells 2020, 9(6), 1528; https://doi.org/10.3390/cells9061528 - 23 Jun 2020
Cited by 25 | Viewed by 9726
Abstract
The brain is an intricate network with complex organizational principles facilitating a concerted communication between single-neurons, distinct neuron populations, and remote brain areas. The communication, technically referred to as connectivity, between single-neurons, is the center of many investigations aimed at elucidating pathophysiology, anatomical [...] Read more.
The brain is an intricate network with complex organizational principles facilitating a concerted communication between single-neurons, distinct neuron populations, and remote brain areas. The communication, technically referred to as connectivity, between single-neurons, is the center of many investigations aimed at elucidating pathophysiology, anatomical differences, and structural and functional features. In comparison with bulk analysis, single-neuron analysis can provide precise information about neurons or even sub-neuron level electrophysiology, anatomical differences, pathophysiology, structural and functional features, in addition to their communications with other neurons, and can promote essential information to understand the brain and its activity. This review highlights various single-neuron models and their behaviors, followed by different analysis methods. Again, to elucidate cellular dynamics in terms of electrophysiology at the single-neuron level, we emphasize in detail the role of single-neuron mapping and electrophysiological recording. We also elaborate on the recent development of single-neuron isolation, manipulation, and therapeutic progress using advanced micro/nanofluidic devices, as well as microinjection, electroporation, microelectrode array, optical transfection, optogenetic techniques. Further, the development in the field of artificial intelligence in relation to single-neurons is highlighted. The review concludes with between limitations and future prospects of single-neuron analyses. Full article
(This article belongs to the Special Issue Single Cell Analysis)
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28 pages, 2510 KiB  
Review
The Role of Single-Cell Technology in the Study and Control of Infectious Diseases
by Weikang Nicholas Lin, Matthew Zirui Tay, Ri Lu, Yi Liu, Chia-Hung Chen and Lih Feng Cheow
Cells 2020, 9(6), 1440; https://doi.org/10.3390/cells9061440 - 10 Jun 2020
Cited by 18 | Viewed by 6376
Abstract
The advent of single-cell research in the recent decade has allowed biological studies at an unprecedented resolution and scale. In particular, single-cell analysis techniques such as Next-Generation Sequencing (NGS) and Fluorescence-Activated Cell Sorting (FACS) have helped show substantial links between cellular heterogeneity and [...] Read more.
The advent of single-cell research in the recent decade has allowed biological studies at an unprecedented resolution and scale. In particular, single-cell analysis techniques such as Next-Generation Sequencing (NGS) and Fluorescence-Activated Cell Sorting (FACS) have helped show substantial links between cellular heterogeneity and infectious disease progression. The extensive characterization of genomic and phenotypic biomarkers, in addition to host–pathogen interactions at the single-cell level, has resulted in the discovery of previously unknown infection mechanisms as well as potential treatment options. In this article, we review the various single-cell technologies and their applications in the ongoing fight against infectious diseases, as well as discuss the potential opportunities for future development. Full article
(This article belongs to the Special Issue Single Cell Analysis)
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20 pages, 6389 KiB  
Review
Advances of Single-Cell Protein Analysis
by Lixing Liu, Deyong Chen, Junbo Wang and Jian Chen
Cells 2020, 9(5), 1271; https://doi.org/10.3390/cells9051271 - 20 May 2020
Cited by 31 | Viewed by 9223
Abstract
Proteins play a significant role in the key activities of cells. Single-cell protein analysis provides crucial insights in studying cellular heterogeneities. However, the low abundance and enormous complexity of the proteome posit challenges in analyzing protein expressions at the single-cell level. This review [...] Read more.
Proteins play a significant role in the key activities of cells. Single-cell protein analysis provides crucial insights in studying cellular heterogeneities. However, the low abundance and enormous complexity of the proteome posit challenges in analyzing protein expressions at the single-cell level. This review summarizes recent advances of various approaches to single-cell protein analysis. We begin by discussing conventional characterization approaches, including fluorescence flow cytometry, mass cytometry, enzyme-linked immunospot assay, and capillary electrophoresis. We then detail the landmark advances of microfluidic approaches for analyzing single-cell protein expressions, including microfluidic fluorescent flow cytometry, droplet-based microfluidics, microwell-based assay (microengraving), microchamber-based assay (barcoding microchips), and single-cell Western blotting, among which the advantages and limitations are compared. Looking forward, we discuss future research opportunities and challenges for multiplexity, analyte, throughput, and sensitivity of the microfluidic approaches, which we believe will prompt the research of single-cell proteins such as the molecular mechanism of cell biology, as well as the clinical applications for tumor treatment and drug development. Full article
(This article belongs to the Special Issue Single Cell Analysis)
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34 pages, 38768 KiB  
Review
Single-Cell RNA Sequencing and Its Combination with Protein and DNA Analyses
by Jane Ru Choi, Kar Wey Yong, Jean Yu Choi and Alistair C. Cowie
Cells 2020, 9(5), 1130; https://doi.org/10.3390/cells9051130 - 4 May 2020
Cited by 50 | Viewed by 16939
Abstract
Heterogeneity in cell populations poses a significant challenge for understanding complex cell biological processes. The analysis of cells at the single-cell level, especially single-cell RNA sequencing (scRNA-seq), has made it possible to comprehensively dissect cellular heterogeneity and access unobtainable biological information from bulk [...] Read more.
Heterogeneity in cell populations poses a significant challenge for understanding complex cell biological processes. The analysis of cells at the single-cell level, especially single-cell RNA sequencing (scRNA-seq), has made it possible to comprehensively dissect cellular heterogeneity and access unobtainable biological information from bulk analysis. Recent efforts have combined scRNA-seq profiles with genomic or proteomic data, and show added value in describing complex cellular heterogeneity than transcriptome measurements alone. With the rising demand for scRNA-seq for biomedical and clinical applications, there is a strong need for a timely and comprehensive review on the scRNA-seq technologies and their potential biomedical applications. In this review, we first discuss the latest state of development by detailing each scRNA-seq technology, including both conventional and microfluidic technologies. We then summarize their advantages and limitations along with their biomedical applications. The efforts of integrating the transcriptome profile with highly multiplexed proteomic and genomic data are thoroughly reviewed with results showing the integrated data being more informative than transcriptome data alone. Lastly, the latest progress toward commercialization, the remaining challenges, and future perspectives on the development of scRNA-seq technologies are briefly discussed. Full article
(This article belongs to the Special Issue Single Cell Analysis)
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