Cell Division Tracking Research Articles

Identification of Quiescent Cells in a Zebrafish T-Cell Acute Lymphoblastic Leukemia Model Using Cell Proliferation Staining.

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect CSCs from antiproliferative chemotherapy drugs. In T-cell acute lymphoblastic leukemia (T-ALL) patient-derived xenograft (PDX) mouse models, quiescent cells are associated with treatment resistance and stemness. Cell proliferation dyes are popular tools for the tracking of cell division. The fluorescent dye is covalently anchored into amine groups on the membrane and macromolecules inside the cell. This allows for the tracking of labeled cells for up to 10 divisions, which can be resolved by flow cytometry. Ultimately, cells with the highest proliferation rates will have low dye retention, as it will be diluted with each cell division, while dormant, slower-dividing cells will have the highest retention. The use of cell proliferation dyes to isolate dormant cells has been optimized and described in T-ALL mouse models. Complementary to the existing mouse models, the rag2:Myc-derived zebrafish T-ALL model provides an excellent venue to interrogate self-renewal in T-ALL due to the high frequency of leukemic stem cells (LSCs) and the convenience of zebrafish for large-scale transplant experiments. Here, we describe the workflow for the staining of zebrafish T-ALL cells with a cell proliferation dye, optimizing the concentration of the dye for zebrafish cells, passaging successfully stained cells in vivo, and the collection of cells with varying levels of dye retention by live cell sorting from transplanted animals. Given the absence of well-established cell surface makers for LSCs in T-ALL, this approach provides a functional means to interrogate quiescent cells in vivo. For representative results, we describe the engraftment efficiency and the LSC frequency of high and low dye-retaining cells. This method can help investigate additional properties of quiescent cells, including drug response, transcriptional profiles, and morphology.

Microalgal cell division tracking using CFSE

Microalgal cell division tracking could unlock new research means. It could help decipher cell response to an intentional stressor, aiming at increasing an added value molecule accumulation, or an accidental stressor, in the case of environmental pollution. It could also be used to monitor asynchrony in cultures exposed to photoperiod or determine the fate of dead cells. To date, because of the lack of guidelines specific to microalgae, microalgal ecotoxicology and biotechnology communities have not yet implemented such protocol in routine. Therefore, a systematic optimization methodology has been deployed to adapt the CarboxyFluorescein DiAcetate Succinimidyl Ester (CF-DA-SE, or, in short, CFSE) lymphocytes proliferation tracking technique to the microalga Chlorella vulgaris. The toxic effect of the CFSE solvent (DMSO) was delineated (stock solution at 10 mM). Then, incubation conditions (time, probe/cell ratio, illumination) were optimized (30 min, 4.50 nmol/MCell, in the dark). Finally, CFSE washing and cell recovery were robustified (low-acceleration - 100 g - centrifugation). Using a semi-synchronous culture as a test case, the method was successfully applied to count cell divisions. Up to four generations could be discriminated. The generation-to-generation signal ratio was exactly 1/4, corresponding to the natural division of Chlorella vulgaris. Furthermore, an advanced yet easy-to-implement signal processing technique was introduced to ease generation discrimination.

Modified cell trace violet proliferation assay preserves lymphocyte viability and allows spectral flow cytometry analysis.

In this study we describe three different methods for labeling T lymphocytes with cell trace violet (CTV), in order to track cell division in mouse and human cells, in both the in vitro and in vivo setting. We identified a modified method of CTV labeling that can be applied directly to either conventional or spectral flow cytometry, that maintained lymphocyte viability and function, yet minimized dye spill-over into other fluorochrome channels. Our optimized method for CTV labeling allowed us to identify up to eight cell divisions and the replication index for in vitro-stimulated mouse and human lymphocytes, and the co-expression of T-cell subset markers. Furthermore, the homeostatic trafficking, expansion and division of CTV-labeled congenic donor T cells could be detected using spectral cytometry, in an adoptive T-cell transfer mouse model. Our optimized CTV method can be applied to both in vitro and in vivo settings to examine the behavior and phenotype of activated T cells.

DetecDiv, a generalist deep-learning platform for automated cell division tracking and survival analysis.

Automating the extraction of meaningful temporal information from sequences of microscopy images represents a major challenge to characterize dynamical biological processes. So far, strong limitations in the ability to quantitatively analyze single-cell trajectories have prevented large-scale investigations to assess the dynamics of entry into replicative senescence in yeast. Here, we have developed DetecDiv, a microfluidic-based image acquisition platform combined with deep learning-based software for high-throughput single-cell division tracking. We show that DetecDiv can automatically reconstruct cellular replicative lifespans with high accuracy and performs similarly with various imaging platforms and geometries of microfluidic traps. In addition, this methodology provides comprehensive temporal cellular metrics using time-series classification and image semantic segmentation. Last, we show that this method can be further applied to automatically quantify the dynamics of cellular adaptation and real-time cell survival upon exposure to environmental stress. Hence, this methodology provides an all-in-one toolbox for high-throughput phenotyping for cell cycle, stress response, and replicative lifespan assays.

A paternal bias in germline mutation is widespread in amniotes and can arise independently of cell division numbers.

In humans and other mammals, germline mutations are more likely to arise in fathers than in mothers. Although this sex bias has long been attributed to DNA replication errors in spermatogenesis, recent evidence from humans points to the importance of mutagenic processes that do not depend on cell division, calling into question our understanding of this basic phenomenon. Here, we infer the ratio of paternal-to-maternal mutations, α, in 42 species of amniotes, from putatively neutral substitution rates of sex chromosomes and autosomes. Despite marked differences in gametogenesis, physiologies and environments across species, fathers consistently contribute more mutations than mothers in all the species examined, including mammals, birds, and reptiles. In mammals, α is as high as 4 and correlates with generation times; in birds and snakes, α appears more stable around 2. These observations are consistent with a simple model, in which mutations accrue at equal rates in both sexes during early development and at a higher rate in the male germline after sexual differentiation, with a conserved paternal-to-maternal ratio across species. Thus, α may reflect the relative contributions of two or more developmental phases to total germline mutations, and is expected to depend on generation time even if mutations do not track cell divisions.

P-243 Automating tracking of cell division for human embryo development in time lapse videos

Abstract Study question Can tracking cell division and predicting human embryo cleavage stages be automated in time-lapse videos (TLV) using AI object detection methods? Summary answer We developed software predicting blastomere count and tracking cell cleavages up until 4-5 stage. The software employs object detection technique called YOLOv5 to detect cells. What is known already Embryo morphology plays an important part in determining viability. Parameters such as number of cells present following fertilization, abnormal cell division (reverse/direct) and evaluating cleavage stages have correlation with pregnancy rates. However, continuous manual evaluation can be time-consuming, and automation will assist in embryo viability assessment. YOLOv5 has proven to accurately detect objects in videos. YOLOv5 uses mean average precision (mAP) as a metric to quantify the portions of frames in videos having the correct count of the objects. Study design, size, duration We have developed a software that uses YOLOv5 to detect cells present in frames of TLV, then marks each cell boundary with different colored circular overlays using OpenCV. We trained YOLOv5 to detect objects: cell, morula and blastocyst using 150 images of different cell-stages, morula, blastocyst. For object cell mAP was 0.65. Annotated location of objects in images and YOLOv5 predictions were reviewed by embryologists. We evaluated the software on TLV from 11 patients. Participants/materials, setting, methods After YOLOv5 detects cells in frames of TLV, our software computes cell count and assigns each cell a different color which is maintained until cell division into daughter cells. Later, daughter cells were also assigned different colors. If the frame has a preceding frame, software calculates detected cells' proximity with each cell in the preceding frame and copies color scheme provided proximity is within some threshold. The software provides TLV with colored overlays as output. Main results and the role of chance In starting frames of TLV with single cell, software accurately detected 1-cell (high precision=0.99, high recall=0.83, high F1-score=0.90). We observed some misclassification between 1-cell and morula. The reason could be that compacted morula looks like 1-cell. Best performance is observed for 2-cells (high precision=0.91, high recall=0.98, high F1-score=0.95). 4-cells were sometimes misclassified with 3 or 5-cells (high precision=0.88, low recall=0.59, high F1-score=0.71). One reason for the misclassification can be that overlapping between cells increases with number of cells. 3-cell and 5-cell are confused with other stages, still cleavage stage detection is better than random: 3-cell (average precision=0.43, high recall=0.83, average F1-score=0.49), 5-cell (average precision=0.44, average recall=0.40, average F1-score=0.40). For cell-stages>5, YOLOv5 detects less cells than actual count and software predicts cleavage later than actual by 9-10 frames on average. The proximity threshold used was 0.10 for cell-count<4 and 0.05 for count>4. In 5 TLV, overlay color for cells changes abruptly between frames, possibly because once YOLOv5 detected a stage, in consecutive frames less cell-number was recorded, and then again reported correct count. Sometimes, software selected the wrong parent for daughter cells (incorrect colored overlay). 2 TLV had direct and reverse cleavages and software could detect these two patterns. Limitations, reasons for caution Overall, our software can precisely detect cells, cell divisions and cleavage stages up to 4-cell stages. We hypothesize that training YOLOv5 on a bigger dataset and including several focal plane information will enable our software to detect overlapping cells and cleavage stages > =5. Wider implications of the findings Object detection proved to be pragmatic for ART and tracking cell division using our software will reduce time consumed in manual annotations, easier prediction of abnormal cleavages and more objective assessments. Qualitative evaluation by embryologists resulted in the overall verdict that this is useful and promising for further development. Trial registration number not applicable

CeLaVi: an interactive cell lineage visualization tool.

Recent innovations in genetics and imaging are providing the means to reconstruct cell lineages, either by tracking cell divisions using live microscopy, or by deducing the history of cells using molecular recorders. A cell lineage on its own, however, is simply a description of cell divisions as branching events. A major goal of current research is to integrate this description of cell relationships with information about the spatial distribution and identities of the cells those divisions produce. Visualizing, interpreting and exploring these complex data in an intuitive manner requires the development of new tools. Here we present CeLaVi, a web-based visualization tool that allows users to navigate and interact with a representation of cell lineages, whilst simultaneously visualizing the spatial distribution, identities and properties of cells. CeLaVi’s principal functions include the ability to explore and manipulate the cell lineage tree; to visualise the spatial distribution of cell clones at different depths of the tree; to colour cells in the 3D viewer based on lineage relationships; to visualise various cell qualities on the 3D viewer (e.g. gene expression, cell type) and to annotate selected cells/clones. All these capabilities are demonstrated with four different example data sets. CeLaVi is available at http://www.celavi.pro.

A random-sampling approach to track cell divisions in time-lapse fluorescence microscopy

BackgroundParticle-tracking in 3D is an indispensable computational tool to extract critical information on dynamical processes from raw time-lapse imaging. This is particularly true with in vivo time-lapse fluorescence imaging in cell and developmental biology, where complex dynamics are observed at high temporal resolution. Common tracking algorithms used with time-lapse data in fluorescence microscopy typically assume a continuous signal where background, recognisable keypoints and independently moving objects of interest are permanently visible. Under these conditions, simple registration and identity management algorithms can track the objects of interest over time. In contrast, here we consider the case of transient signals and objects whose movements are constrained within a tissue, where standard algorithms fail to provide robust tracking.ResultsTo optimize 3D tracking in these conditions, we propose the merging of registration and tracking tasks into a registration algorithm that uses random sampling to solve the identity management problem. We describe the design and application of such an algorithm, illustrated in the domain of plant biology, and make it available as an open-source software implementation. The algorithm is tested on mitotic events in 4D data-sets obtained with light-sheet fluorescence microscopy on growing Arabidopsis thaliana roots expressing CYCB::GFP. We validate the method by comparing the algorithm performance against both surrogate data and manual tracking.ConclusionThis method fills a gap in existing tracking techniques, following mitotic events in challenging data-sets using transient fluorescent markers in unregistered images.

Cellbow: a robust customizable cell segmentation program

BackgroundTime‐lapse live cell imaging of a growing cell population is routine in many biological investigations. A major challenge in imaging analysis is accurate segmentation, a process to define the boundaries of cells based on raw image data. Current segmentation methods relying on single boundary features have problems in robustness when dealing with inhomogeneous foci which invariably happens in cell population imaging.MethodsCombined with a multi‐layer training set strategy, we developed a neural‐network‐based algorithm — Cellbow.ResultsCellbow can achieve accurate and robust segmentation of cells in broad and general settings. It can also facilitate long‐term tracking of cell growth and division. To facilitate the application of Cellbow, we provide a website on which one can online test the software, as well as an ImageJ plugin for the user to visualize the performance before software installation.ConclusionCellbow is customizable and generalizable. It is broadly applicable to segmenting fluorescent images of diverse cell types with no further training needed. For bright‐field images, only a small set of sample images of the specific cell type from the user may be needed for training.

Long-term in vitro persistence of magnetic properties after magnetic bead-based cell separation of T cells.

Magnetic‐activated cell sorting (MACS) using magnetic nanoparticles coated with specific antibodies is commonly used in immunology research. For in vitro isolation purposes, it is important to know to what extent the magnetic properties remain present in the isolated cell populations and whether it has consequences for sequential isolations. We hypothesized that only upon cell division, cells will lose their magnetic properties via dilution of the particles in/on their daughter cells. We analysed residual magnetic properties of cells that divided vs cells that did not divide after magnetic bead‐based cell separation. As a model, we isolated T cells using beads targeting the non‐modulating surface molecule CD45RO. Cells were labelled with the cell division tracking dye PKH and cultured under different conditions to induce variable degrees of cell division. We demonstrate that T cells that underwent no, or only minimal, cell divisions after MACS retained magnetic properties for up to at least 2 weeks of in vitro culture. The presence of nanoparticles was detected on their cell surface and intracellularly using Labeling Check reagent. These results have important consequences for procedures requiring repetitive isolation rounds after in vitro culture.

Distinct host immune programs in lung macrophages elicit cell-type specific Mycobacterium tuberculosis transcriptional responses

Abstract During Mycobacterium tuberculosis (Mtb) infection, bacteria are inhaled into the lung where they first infect resident, alveolar macrophages (AM). The majority of the infection remains in AM until D14, when other cells, including recruited monocyte-derived macrophages (MDM) begin to become infected. By the peak of infection (D28) MDM represent the major infected cell type. However, even at this time point, a small population of infected AM remain. This led us to ask how these cell types control Mtb. Using microscopy, we observed that AM harbor more bacteria than MDM on a per cell basis at multiple time points, including after the initiation of the T cell response. Using RNA-seq, we identified multiple differentially expressed pathways between the two cell types. While infected MDM upregulate proinflammatory signaling pathways associated with Mtb control, infected AM are enriched for proliferation and fatty acid metabolism pathways. We performed validation studies using dyes to track cell division and mitochondrial metabolism and observed that AM were more proliferative and had sustained engagement of mitochondrial metabolism relative to MDM. In parallel, we analyzed the bacterial transcriptome and found that Mtb in MDM have a signature associated with late hypoxia. These bacterial responses also suggested that Mtb may differ in its drug tolerance in a cell type specific manner, which we are currently investigating. Together, this work is in agreement with other studies demonstrating differences in the response to Mtb by tissue resident and recruited macrophages and suggests that these differences may lead to distinct transcriptional changes in the bacteria, which could have important implications for therapeutic interventions.

Intravital imaging of mouse embryos.

Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution.

Quantitative analysis of Lactobacillus delbrueckii subsp. bulgaricus cell division and death using fluorescent dye tracking

Cell growth of lactic acid bacteria is of vital importance in starter culture manufacture in the dairy industry. However, one of the major stumbling blocks in the understanding of bacterial cell growth relates to challenges in the quantification of the cell division or death. To overcome these shortcomings, the intracellular fluorescent cell tracking assay was implemented to monitor Lactobacillus delbrueckii subsp. bulgaricus sp1.1 cell division and death. Optimization of fluorescent dye concentration suggested it could be applied in the tracking of cell division without cytotoxicity. Technical validation of the fluorescent tracking demonstrated this assay was accurate for quantitatively analyzing cell division. Furthermore, the cell death was quantified using the precursor cohort distribution of the time-series fluorescence data in batch culture. The results indicated a dynamic and unbalanced relationship between bacterial cell division and death after exponential phase in the batch culture. These findings suggested that fluorescent dye tracking is a powerful tool for monitoring L. bulgaricus sp1.1 cell division and death and can provide valuable information for bacterial growth behavior in batch culture.

Protein Amounts of the MYC Transcription Factor Determine Germinal Center B Cell Division Capacity

High-affinity B cell selection in the germinal center (GC) is governed by signals delivered by follicular helper T (Tfh) cells to B cells. Selected B cells undergo clonal expansion and affinity maturation in the GC dark zone in direct proportion to the amount of antigen they capture and present to Tfh cells in the light zone. Here, we examined the mechanisms whereby Tfh cells program the number of GC B cell divisions. Gene expression analysis revealed that Tfh cells induce Myc expression in light-zone B cells in direct proportion to antigen capture. Conditional Myc haplo-insufficiency or overexpression combined with cell division tracking showed that MYC expression produces a metabolic reservoir in selected light-zone B cells that is proportional to the number of cell divisions in the dark zone. Thus, MYC constitutes the GC B cell division timer that when deregulated leads to emergence of B cell lymphoma.

Gene signatures of quiescent glioblastoma cells reveal mesenchymal shift and interactions with niche microenvironment.

BackgroundGlioblastoma (GBM), a highly malignant brain tumor, invariably recurs after therapy. Quiescent GBM cells represent a potential source of tumor recurrence, but little is known about their molecular underpinnings.MethodsPatient-derived GBM cells were engineered by CRISPR/Cas9-assisted knock-in of an inducible histone2B-GFP (iH2B-GFP) reporter to track cell division history. We utilized an in vitro 3D GBM organoid approach to isolate live quiescent GBM (qGBM) cells and their proliferative counterparts (pGBM) to compare stem cell properties and therapy resistance. Gene expression programs of qGBM and pGBM cells were analyzed by RNA-Seq and NanoString platforms.FindingsH2B-GFP-retaining qGBM cells exhibited comparable self-renewal capacity but higher therapy resistance relative to pGBM. Quiescent GBM cells expressed distinct gene programs that affect cell cycle control, metabolic adaptation, and extracellular matrix (ECM) interactions. Transcriptome analysis also revealed a mesenchymal shift in qGBM cells of both proneural and mesenchymal GBM subtypes. Bioinformatic analyses and functional assays in GBM organoids established hypoxia and TGFβ signaling as potential niche factors that promote quiescence in GBM. Finally, network co-expression analysis of TCGA glioma patient data identified gene modules that are enriched for qGBM signatures and also associated with survival rate.InterpretationOur in vitro study in 3D GBM organoids supports the presence of a quiescent cell population that displays self-renewal capacity, high therapy resistance, and mesenchymal gene signatures. It also sheds light on how GBM cells may acquire and maintain quiescence through ECM organization and interaction with niche factors such as TGFβ and hypoxia. Our findings provide a starting point for developing strategies to tackle the quiescent population of GBM.FundNational Institutes of Health (NIH) and Deutsche Forschungsgemeinschaft (DFG).

Week-long imaging of cell divisions in the Arabidopsis root meristem

BackgroundCharacterizing the behaviors of dynamic systems requires capturing them with high temporal and spatial resolution. Owing to its transparency and genetic tractability, the Arabidopsis thaliana root lends itself well to live imaging when combined with cell and tissue-specific fluorescent reporters. We developed a novel 4D imaging method that utilizes simple confocal microscopy and readily available components to track cell divisions in the root stem cell niche and surrounding region for up to 1 week.ResultsUsing this method, we performed a direct measurement of cell division intervals within and around the root stem cell niche. The results reveal a short, steep gradient of cell division rates in proximal stem cells, with progressively more rapid cell division rates from quiescent center (QC), to cells in direct contact with the QC (initials), to their immediate daughters, after which division rates appear to become more homogeneous.ConclusionsThese results provide a baseline to study how perturbations in signaling could affect cell division patterns in the root meristem. This new setup further allows us to finely analyze meristematic cell division rates that lead to patterning.

Cell proliferation during hair cell regeneration induced by Math1 in vestibular epithelia in vitro.

Hair cell regeneration is the fundamental method of correcting hearing loss and balance disorders caused by hair cell damage or loss. How to promote hair cell regeneration is a hot focus in current research. In mammals, cochlear hair cells cannot be regenerated and few vestibular hair cells can be renewed through spontaneous regeneration. However, Math1 gene transfer allows a few inner ear cells to be transformed into hair cells in vitro or in vivo. Hair cells can be renewed through two possible means in birds: supporting cell differentiation and transdifferentiation with or without cell division. Hair cell regeneration is strongly associated with cell proliferation. Therefore, this study explored the relationship between Math1-induced vestibular hair cell regeneration and cell division in mammals. The mouse vestibule was isolated to harvest vestibular epithelial cells. Ad-Math1-enhanced green fluorescent protein (EGFP) was used to track cell division during hair cell transformation. 5-Bromo-2'-deoxyuridine (BrdU) was added to track cell proliferation at various time points. Immunocytochemistry was utilized to determine cell differentiation and proliferation. Results demonstrated that when epithelial cells were in a higher proliferative stage, more of these cells differentiated into hair cells by Math1 gene transfer. However, in the low proliferation stage, no BrdU-positive cells were seen after Math1 gene transfer. Cell division always occurred before Math1 transfection but not during or after Math1 transfection, when cells were labeled with BrdU before and after Ad-Math1-EGFP transfection. These results confirm that vestibular epithelial cells with high proliferative potential can differentiate into new hair cells by Math1 gene transfer, but this process is independent of cell proliferation.

Exercise and hippocampal neurogenesis: a dogma re-examined and lessons learned.

Exercise and hippocampal neurogenesis: a dogma re-examined and lessons learned.

Superior properties of CellTrace Yellow™ as a division tracking dye for human and murine lymphocytes.

The discovery of cell division tracking properties of 5‐(and‐6)‐carboxyfluorescein diacetate succinimidyl ester (CFSE) by Lyons and Parish in 1994 led to a broad range of new methods and numerous important biological discoveries. After labeling, CFSE is attached to free amine groups and intracellular proteins in the cytoplasm and nucleus of a cell, and halves in fluorescence intensity with each round of cell division, enabling enumeration of the number of divisions a cell has undergone. A range of popular division tracking dyes were subsequently developed, including CellTrace Violet (CTV), making available the green fluorescent channel previously occupied by CFSE. More recently, CellTrace Yellow (CTY) and CellTrace Far Red (CTFR), each with unique fluorescence properties, were introduced. In a comparison, we found that the fluorescence values of both dyes were well separated from autofluorescence, and enabled a greater number of divisions to be identified than CTV, before this limit was reached. These new dyes provided clear and well‐separated peaks for both murine and human B lymphocytes, and should find wide application. The range of excitation/emission spectra available for division tracking dyes now also facilitates multiplexing, that is, the labeling of cells with different combinations of dyes to give a unique fluorescence signature, allowing single cell in vitro and in vivo tracking. The combinatorial possibilities are significantly increased with these additional dyes.

Interpreting the Dependence of Mutation Rates on Age and Time.

Mutations can originate from the chance misincorporation of nucleotides during DNA replication or from DNA lesions that arise between replication cycles and are not repaired correctly. We introduce a model that relates the source of mutations to their accumulation with cell divisions, providing a framework for understanding how mutation rates depend on sex, age, and cell division rate. We show that the accrual of mutations should track cell divisions not only when mutations are replicative in origin but also when they are non-replicative and repaired efficiently. One implication is that observations from diverse fields that to date have been interpreted as pointing to a replicative origin of most mutations could instead reflect the accumulation of mutations arising from endogenous reactions or exogenous mutagens. We further find that only mutations that arise from inefficiently repaired lesions will accrue according to absolute time; thus, unless life history traits co-vary, the phylogenetic “molecular clock” should not be expected to run steadily across species.