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Special Issue "Single Cell Analysis"

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

Deadline for manuscript submissions: closed (30 April 2020) | Viewed by 62476

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Dr. Tuhin Subhra Santra

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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

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Guest Editor

Department of Engineering and System Science, Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu 30013, Taiwan
Interests: MEMS/NEMS; microfluidics; 3D tissue culture; electrochemical sensors; nanotechnology in biomedical applications
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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

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

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 2400 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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Single Cell Analysis 2.0 in Cells (13 articles)

Published Papers (12 papers)

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Editorial

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Open AccessEditorial

Single-Cell Analysis

Tuhin Subhra Santra

and

Fan-Gang Tseng

Cells 2020, 9(9), 1993; https://doi.org/10.3390/cells9091993 - 29 Aug 2020

Cited by 9 | Viewed by 2164

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

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Open AccessCommunication

Genomic Analysis of Localized High-Risk Prostate Cancer Circulating Tumor Cells at the Single-Cell Level

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Cells 2020, 9(8), 1863; https://doi.org/10.3390/cells9081863 - 08 Aug 2020

Cited by 11 | Viewed by 3102

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 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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Open AccessArticle

A Microfluidic Single-Cell Cloning (SCC) Device for the Generation of Monoclonal Cells

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Cells 2020, 9(6), 1482; https://doi.org/10.3390/cells9061482 - 18 Jun 2020

Cited by 3 | Viewed by 4616

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 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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Open AccessArticle

Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin

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Cells 2020, 9(4), 852; https://doi.org/10.3390/cells9040852 - 01 Apr 2020

Cited by 8 | Viewed by 3289

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 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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Open AccessArticle

Total mRNA Quantification in Single Cells: Sarcoma Cell Heterogeneity

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Cells 2020, 9(3), 759; https://doi.org/10.3390/cells9030759 - 19 Mar 2020

Cited by 3 | Viewed by 3173

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 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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Single-Nucleus Sequencing of an Entire Mammalian Heart: Cell Type Composition and Velocity

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Cells 2020, 9(2), 318; https://doi.org/10.3390/cells9020318 - 28 Jan 2020

Cited by 24 | Viewed by 7887

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 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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Open AccessArticle

Single Cell Mass Cytometry of Non-Small Cell Lung Cancer Cells Reveals Complexity of In Vivo and Three-Dimensional Models over the Petri-Dish

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Cells 2019, 8(9), 1093; https://doi.org/10.3390/cells8091093 - 16 Sep 2019

Cited by 14 | Viewed by 5573

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 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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Open AccessArticle

Single-Cell Transcriptomes Reveal Characteristic Features of Mouse Hepatocytes with Liver Cholestatic Injury

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Cells 2019, 8(9), 1069; https://doi.org/10.3390/cells8091069 - 11 Sep 2019

Cited by 9 | Viewed by 4833

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 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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Open AccessFeature PaperReview

A Single-Neuron: Current Trends and Future Prospects

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 16 | Viewed by 5427

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 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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Open AccessReview

The Role of Single-Cell Technology in the Study and Control of Infectious Diseases

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Cells 2020, 9(6), 1440; https://doi.org/10.3390/cells9061440 - 10 Jun 2020

Cited by 13 | Viewed by 3681

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 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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Open AccessReview

Advances of Single-Cell Protein Analysis

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Cells 2020, 9(5), 1271; https://doi.org/10.3390/cells9051271 - 20 May 2020

Cited by 23 | Viewed by 6233

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 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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Open AccessReview

Single-Cell RNA Sequencing and Its Combination with Protein and DNA Analyses

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Cells 2020, 9(5), 1130; https://doi.org/10.3390/cells9051130 - 04 May 2020

Cited by 30 | Viewed by 10618

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 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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