Single-cell Phenotyping Research Articles

ERK and RSK regulate distinct steps of a cellular program that induces transition from multicellular epithelium to single cell phenotype

The ERK (extracellular signal-regulated kinases) cascade has an evolutionarily conserved three tier architecture consisting of protein kinases Raf, MEK (MAPK/ERK kinase) and ERK. Following activation, ERK phosphorylates various cellular elements leading to diverse cellular responses. Downstream of ERK the family of p90 ribosomal S6 kinases (RSKs) has been proven to be an important conveyor of ERK signaling, however, little is known if ERK and RSK coordinate their functions to generate a specific biological response. Here we show that in epithelial cells conditional activation of the ERK pathway causes phenotypic conversion of epithelial cells to autonomously migrating cells. This process involves two sequential steps characterized by loss of apical–basal polarity followed by cell scattering. The activation of ERK, but not RSK, is sufficient for the execution of the first step and it requires calpain mediated remodeling of actin cytoskeleton. Conversely, RSK regulates the successive stage characterized by cell–cell contact weakening and increased cellular migration. Thus, ERK and RSK regulate different cellular subprograms and coordinated execution of these subprograms in time generates a relevant biological response. Our data also suggest that the mechanism by which the ERK pathway controls a cellular response may be distributed between ERK and RSK, rather than being elicited by a single effector kinase.

Single cell expression quantitative trait loci and complex traits

The recently developed ability to quantify mRNA abundance and noise in single cells has allowed the effect of heritable variations on gene function to be re-evaluated. A recent study has shown that major sources of variation are masked when gene expression is averaged over many cells. Heritable variations that determine single-cell expression phenotypes may exert a regulatory function in specific cellular processes underlying disease. Masked effects on gene expression should therefore be modeled, not ignored.

The Autographa californica Multiple Nucleopolyhedrovirus ORF78 Is Essential for Budded Virus Production and General Occlusion Body Formation

ORF78 (ac78) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a baculovirus core gene of unknown function. To determine the role of ac78 in the baculovirus life cycle, an AcMNPV mutant with ac78 deleted, Ac78KO, was constructed. Quantitative PCR analysis revealed that ac78 is a late gene in the viral life cycle. After transfection into Spodoptera frugiperda cells, Ac78KO produced a single-cell infection phenotype, indicating that no infectious budded viruses (BVs) were produced. The defect in BV production was also confirmed by both viral titration and Western blotting. However, viral DNA replication was unaffected, and occlusion bodies were formed. An analysis of BVs and occlusion-derived viruses (ODVs) revealed that AC78 is associated with both forms of the virions and is an envelope structural protein. Electron microscopy revealed that AC78 also plays an important role in the embedding of ODV into the occlusion body. The results of this study demonstrate that AC78 is a late virion-associated protein and is essential for the viral life cycle.

Promoting Single-Cell Invasion

EphA2 can promote collective or single-cell invasive phenotypes depending on its cleavage status.

Mapping genetic interactions in human cancer cells with RNAi and multiparametric phenotyping

Genetic interactions influence many phenotypes and can be used as a powerful experimental tool to discover functional relationships between genes. Here we describe a robust and scalable method to systematically map genetic interactions in human cancer cells using combinatorial RNAi and high-throughput imaging. Through automated, single-cell phenotyping, we measured genetic interactions across a broad spectrum of phenotypes, including cell count, cell eccentricity and nuclear area. We mapped genetic interactions of epigenetic regulators in colon cancer cells, recovering known protein complexes. Our study also revealed the prospects and challenges of studying genetic interactions in human cells using multiparametric phenotyping.

Memory phenotype and polyfunctional T cells in kidney transplant patients

Allospecific memory T cells are a barrier against long-term graft survival. Production of multiple cytokines by a single T cell is considered a sign of an active ongoing immune response, the presence of these polyfunctional cells has not been addressed in transplanted patients accordingly to graft outcome. Memory phenotype, based on the expression of CD45RO and CD27, and polyfunctional T cells were evaluated in long-term graft survival patients (LTS), short-term survival patients (STS), chronic rejection patients (ChrRx), dialysis patients (DIAL) and healthy controls (Ctrls). Memory T cells were quantified ex vivo, after allogeneic and anti-CD3 plus anti-CD28 stimulation, in cells proliferating or not to these stimuli. The percentages of cells producing IFNγ, IL-2 and/or TNFα after allogeneic stimulation and the memory phenotype of single cytokine producing cells were evaluated. Ex vivo CD8+CD45RO−CD27− effector cells were decreased in transplanted patients compared to non-transplanted individuals. After allogeneic stimulation, CD4+CD45RO+CD27+, central memory cells in LTS and CD4+CD45RO−CD27− effector cells in Dial were augmented compared to Ctrls and ChrRx, and CD8+CD45RO−CD27− effector cells were increased in ChrRx. There were no differences in the percentage of single cytokine producing cells among the groups. IFNγ+TNFα+CD4 and CD8 cells were detected in Ctrls, STS and ChrRx and no cells positive for the three cytokines were found. The phenotype of cytokine producing cells was mainly effector memory. Interestingly, in LTS there was an increase in effector cells producing IFNγ and IL-2. Changes in subpopulation distribution in patients with different outcomes may be a reflection of the graft acceptance or rejection status.

From cell phenotype to epigenetic mechanisms: new insights into regenerating myocardium

The self-regenerating property of the adult myocardium is not a new discovery. Even though we could not confirm that the adult myocardium is a post-mitotic tissue, we should consider that its plasticity is extremely low. Studies are still in progress to decipher the mechanisms underlying the abovementioned potential fetal features of the adult heart. The modest results of several clinical trials based on the transplantation of millions of autologous stem cells into the dysfunctional heart have confirmed that the cross-talk of different signals, such as the microenvironment, promotes the regeneration of adult myocardium. Recent scientific evidence has revealed that cellular cross-talk does not depend on the action of a single cell phenotype. It is conceivable that the limited turnover of cardiomyocytes is ensured by the interplay of adult cardiac cells in response to environmental changes. The epigenetic state of a cell serves as a dynamic interface between the environment and phenotype. The epigenetic modulation of the adult cardiac cells by natural active compounds encourages further studies to improve myocardial plasticity. In this review, we will highlight the most relevant studies demonstrating the epigenetic modulation of myocardial regeneration without the use of stem cell transplantation.

Metabolic Imaging of Colon Cancer Tumors In Vivo by Phasor Fluorescence Lifetime Microscopy of NADH

Here we use a non-invasive method to measure the metabolic phenotype of single colon cancer cells in vivo. By using NADH as optical biomarker and the phasor approach to Fluorescence Lifetime microscopy (FLIM) we identify cancer metabolism related to different rates of glycolysis, cell growth and proliferation of cells. We perform label-free Phasor FLIM on living and actively-perfused xenograft tumors. Colon cancer cells are injected subcutaneously into immune deficient (NSG) mice and tumors are allowed to grow for three weeks. The tumor vasculature is labeled by injecting TRITC Dextran into the tail vein and xenografts are exposed via a skin flap, still perfused by their feeder vessels.FLIM distinguishes collagen fibers, the tumor stroma, adipocytes, blood vessels and single cancer cells within the living tumor. By measuring NADH lifetime, we quantify the relative concentration of free and bound NADH in single cancer cells, which reflects the cellular redox NADH/NAD+ ratio and balance of oxidative phosphorylation and glycolysis. We investigate the tumor microenvironment by characterizing the distribution of the metabolic fingerprint of single colon cancer cells and by mapping the three-dimensional metabolic heterogeneity of the tumor at different distances from blood vessels.Our method permits a non invasive measurement of single cancer cell metabolism in a living intact tumor microenvironment. It allows monitoring of spatial and temporal dynamic changes of tumor metabolism upon different physiological conditions such as blood flow, tissue oxygenation levels, nutrient availability and drug delivery.Work supported by NIH-P41 P41-RRO3155, P50-GM076516, NIH RO1, HD49488, NIH PO1 HD47675.

The National DNA Data Bank of Canada: a Quebecer perspective

The Canadian National DNA Database was created in 1998 and first used in the mid-2000. Under management by the RCMP, the National DNA Data Bank of Canada offers each year satisfactory reported statistics for its use and efficiency. Built on two indexes (convicted offenders and crime scene indexes), the database not only provides increasing matches to offenders or linked traces to the various police forces of the nation, but offers a memory repository for cold cases. Despite these achievements, the data bank is now facing new challenges that will inevitably defy the way the database is currently used. These arise from the increasing power of detection of DNA traces, the diversity of demands from police investigators and the growth of the bank itself. Examples of new requirements from the database now include familial searches, low-copy-number analyses and the correct interpretation of mixed samples. This paper aims to develop on the original way set in Québec to address some of these challenges. Nevertheless, analytic and technological advances will inevitably lead to the introduction of new technologies in forensic laboratories, such as single cell sequencing, phenotyping, and proteomics. Furthermore, it will not only request a new holistic/global approach of the forensic molecular biology sciences (through academia and a more investigative role in the laboratory), but also new legal developments. Far from being exhaustive, this paper highlights some of the current use of the database, its potential for the future, and opportunity to expand as a result of recent technological developments in molecular biology, including, but not limited to DNA identification.

Memory Phenotype and Polyfunctional T Cells in Kidney Transplant Patients

Introduction: Immunological memory is a main feature of adaptive immunity; however, allospecific memory T cells represent a barrier against long-term graft survival due to their prolonged half-life, access to lymphoid and not-lymphoid tissues and less activation requirements, compared to naïve T cells. Also, polyfunctional T cells, which are single cell producing multiple cytokines, present during active immune responses may be deleterious to graft survival. Determining these subpopulations may help to monitor the immunological status of transplanted patients. Aims: To evaluated T cell memory subpopulations and cytokine producing polyfunctional T cells after allogeneic stimulation of PBMCs from patients with long-term survival renal grafts (LTS), short-term graft survival (STS), undergoing chronic rejection (ChrRx), dialysis patients (Dial) and healthy controls (Ctrls) Methods: Circulating CD4+ and CD8+ cells, ex vivo and after stimulation with allogeneic cells or anti-CD3+anti-CD28 were evaluated for their memory phenotype according to the expression of CD45RO and CD27 in proliferating and non-proliferating cells. These cells were also evaluated for the production of IFNg, IL-2 and TNFa by intracellular staining. Also the memory phenotype of single cytokine producing cells was evaluated Results:ex vivo CD8+CD45RO-CD27+ cells were decreased in transplanted patients compared to non-transplanted individuals. After allogeneic stimulation, CD4+CD45RO+CD27+ cells were augmented in LTS and CD4+CD45RO-CD27- were higher in Dial compared to Ctrls and ChrRx. CD8+CD45RO-CD27- cells were increased in transplanted patients and CD8+CD45RO-CD27+ were augmented in Dial compared to the other groups. The response to polyclonal stimulation was very homogeneous among the groups and the main population responding to this stimulus in CD4 and CD8 cells was the CD45RO+CD27+ population. There were not differences among the groups in the percentage of cells producing IFNγ, IL-2 or TNFα only. IFNγ+TNFα+ cells, either CD4+ or CD8+, were detected in Ctrls and STS patients, but cells positive for the 3 cytokines were not detected in any group. CD45RO+CD27- cells, either CD4+ or CD8+, were the more frequent producers of IFNγ, IL2 or TNFα, followed by CD45RO-CD27- cells, particularly for CD8+IFNγ+ and CD8+IL-2+ cells. Of note, transplanted patients had decreased percentage of CD4+IFNγ+ cells compared with non-transplanted individuals, also CD45RO-CD27- cells producing either IFNγ or IL-2 were elevated in LTS patients. The presence of alloreactive polyfunctional T cells may be indicative of the potential immune responsiveness, as suggested by the finding that double positive cells were detected mainly in healthy controls and dialysis patients, but not in ChrRx or LTS patients. Contrariwise, the predominance of single cytokine producing cells in the transplanted patients may be a consequence of the chronic stimulation exerted by the graft on the T cells from these patients. How these T cell subpopulation affects the transplant outcome remains to be defined. Supported by Colciencias grant 11154592-1497

High-Content Imaging-Based Screening of Microenvironment-Induced Changes to Stem Cells

Effective screening methodologies for cells are challenged by the divergent and heterogeneous nature of phenotypes inherent to stem cell cultures, particularly on engineered biomaterial surfaces. In this study, we showcase a high-content, confocal imaging-based methodology to parse single-cell phenotypes by quantifying organizational signatures of specific subcellular reporter proteins and applied this profiling approach to three human stem cell types (embryonic–human embryonic stem cell [hESC], induced pluripotent–induced pluripotent stem cell [iPSC], and mesenchymal–human mesenchymal stem cell [hMSC]). We demonstrate that this method could distinguish self-renewing subpopulations of hESCs and iPSCs from heterogeneous populations. This technique can also provide insights into how incremental changes in biomaterial properties, both physiochemical and mechanical, influence stem cell fates by parsing the organization of stem cell proteins. For example, hMSCs cultured on polymeric films with varying degrees of poly(ethylene glycol) to modulate osteogenic differentiation were parsed using high-content organization of the cytoskeletal protein F-actin. In addition, hMSCs cultured on a self-assembled monolayer platform featuring compositional gradients were screened and descriptors obtained to correlate substrate variations with adipogenic lineage commitment. Taken together, high-content imaging of structurally sensitive proteins can be used as a tool to identify stem cell phenotypes at the single-cell level across a diverse range of culture conditions and microenvironments.

AcMNPV Core Gene ac109 Is Required for Budded Virion Transport to the Nucleus and for Occlusion of Viral Progeny

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac109 core gene has been previously characterized as an essential late gene. Our results showed that budded virions could be detected in supernatants of infected Sf-9 cells, even when ac109 knockout viruses displayed a single-cell infection phenotype. Moreover, confocal microscopy analysis revealed that budded virions can enter the cytoplasm but are unable to enter the cell nucleus. This defect could be repaired by complementing ac109 in trans. In addition, polyhedra of normal size could be detected in Sf-9 nuclei infected with ac109 knockout viruses. However, electron microscopy demonstrated that these occlusion bodies were empty. Altogether, these results indicate that ac109 is required for infectivity of both phenotypes of virus.

Metabolic trajectory of cellular differentiation in small intestine by Phasor Fluorescence Lifetime Microscopy of NADH

There is a lack of fast and high resolution methods to measure metabolic activity of single cells in their native environment. Here we develop a straightforward, non-invasive and sensitive method to measure metabolic phenotype of single cells in a live tissue. By using NADH as optical biomarker and the phasor approach to Fluorescence Lifetime microscopy (FLIM) we identify cellular metabolic fingerprints related to different rates of oxidative phosphorylation and glycolysis. For the first time we measure a three dimensional metabolic gradient in the small intestine (SI) epithelia that appears tightly associated with epithelial cell proliferation, differentiation and the Wnt gradient. The highest free/bound NADH ratios are measured at the base of the crypt within the highly proliferative stem cells, indicating high levels of glycolysis. For the first time mouse small intestinal stem cells in intact live crypts are identified within the tissue by their metabolic fingerprint.

Deletion of AcMNPV ac146 eliminates the production of budded virus

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac146 is a highly conserved gene in the Alpha- and Betabaculovirus genera that has an unknown function. Northern blot analysis and transcript mapping showed that ac146 is transcribed at late times post infection as a 1.2kb mRNA. To determine the role of ac146 in the baculovirus life cycle ac146 knock out viruses were constructed. Transfection and plaque assays showed that all the ac146 deletions produced a single cell phenotype indicating that no infectious budded virus (BV) was produced, however occlusion bodies were formed. The lack of BV production was confirmed by viral titration utilizing both qPCR and TCID50. Analysis of BV and occlusion derived virus (ODV) revealed that AC146 is associated with both forms of the virus and is modified specifically in ODV. This study therefore demonstrates that AC146 is a late virion associated protein and is essential for the viral life cycle.

Molecular phenotyping of aging in single yeast cells using a novel microfluidic device

Budding yeast has served as an important model organism for aging research, and previous genetic studies have led to the discovery of conserved genes/pathways that regulate lifespan across species. However, the molecular causes of aging and death remain elusive, because it is very difficult to directly observe the cellular and molecular events accompanying aging in single yeast cells by the traditional approach based on micromanipulation. We have developed a microfluidic system to track individual mother cells throughout their lifespan, allowing automated lifespan measurement and direct observation of cell cycle dynamics, cell/organelle morphologies, and various molecular markers. We found that aging of the wild-type cells is characterized by an increased general stress and a progressive lengthening of the cell cycle for the last few cell divisions; these features are much less apparent in the long-lived FOB1 deletion mutant. Following the fate of individual cells revealed that there are different forms of cell death that are characterized by different terminal cell morphologies, and associated with different levels of stress and lifespan. We have identified a molecular marker - the level of the expression of Hsp104, as a good predictor for the lifespan of individual cells. Our approach allows detailed molecular phenotyping of single cells in the process of aging and thus provides new insight into its mechanism.

Live Cell Imaging of <em>Bacillus subtilis</em> and <em>Streptococcus pneumoniae</em> using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy). Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of <em>Bacillus subtilis</em> and <em>Streptococcus pneumoniae</em> using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Deterministic and Stochastic Allele Specific Gene Expression in Single Mouse Blastomeres

Stochastic and deterministic allele specific gene expression (ASE) might influence single cell phenotype, but the extent and nature of the phenomenon at the onset of early mouse development is unknown. Here we performed single cell RNA-Seq analysis of single blastomeres of mouse embryos, which revealed significant changes in the transcriptome. Importantly, over half of the transcripts with detectable genetic polymorphisms exhibit ASE, most notably, individual blastomeres from the same two-cell embryo show similar pattern of ASE. However, about 6% of them exhibit stochastic expression, indicated by altered expression ratio between the two alleles. Thus, we demonstrate that ASE is both deterministic and stochastic in early blastomeres. Furthermore, we also found that 1,718 genes express two isoforms with different lengths of 3′UTRs, with the shorter one on average 5–6 times more abundant in early blastomeres compared to the transcripts in epiblast cells, suggesting that microRNA mediated regulation of gene expression acquires increasing importance as development progresses.

Potent High-Affinity Antibodies for Treatment and Prophylaxis of Respiratory Syncytial Virus Derived from B Cells of Infected Patients

Native human Abs represent attractive drug candidates; however, the low frequency of B cells expressing high-quality Abs has posed a barrier to discovery. Using a novel single-cell phenotyping technology, we have overcome this barrier to discover human Abs targeting the conserved but poorly immunogenic central motif of respiratory syncytial virus (RSV) G protein. For the entire cohort of 24 subjects with recent RSV infection, B cells producing Abs meeting these stringent specificity criteria were rare, <10 per million. Several of the newly cloned Abs bind to the RSV G protein central conserved motif with very high affinity (K(d) 1-24 pM). Two of the Abs were characterized in detail and compared with palivizumab, a humanized mAb against the RSV F protein. Relative to palivizumab, the anti-G Abs showed improved viral neutralization potency in vitro and enhanced reduction of infectious virus in a prophylaxis mouse model. Furthermore, in a mouse model for postinfection treatment, both anti-G Abs were significantly more effective than palivizumab at reducing viral load. The combination of activity in mouse models for both prophylaxis and treatment makes these high-affinity human-derived Abs promising candidates for human clinical testing.

CellClassifier: supervised learning of cellular phenotypes

CellClassifier is a tool for classifying single-cell phenotypes in microscope images. It includes several unique and user-friendly features for classification using multiclass support vector machines Source code, user manual and SaveObjectSegmentation CellProfiler module available for download at www.cellclassifier.ethz.ch under the GPL license (implemented in Matlab).