Single Cell Fate Research Articles

TAMI-02. ALTERATIONS IN C-X3-C MOTIF CHEMOKINE RECEPTOR 1 (CX3CR1) EXPRESSION INFLUENCE MICROGLIAL AND MACROPHAGE RESPONSE IN DIFFERENT STEPS OF CEREBRAL METASTASIS FORMATION

Abstract Metastasis to the brain is a frequent complication in lung cancer and is still associated with a dismal prognosis. Current treatment strategies not only target tumor cells but also focus on cells of the tumor microenvironment like tumor associated microglia/macrophages (TAMs). The interactions between tumor cells and TAMs during different steps of cerebral metastasis formation of lung cancer brain metastasis are poorly characterized. Moreover, the role of CX3CR1 in this process remains unclear. We established a syngeneic cerebral metastasis mouse model by combining a chronic cranial window and two-photon laser scanning microscopy (TPLSM), which allows the tracking of single fluorescent metastasizing tumor cells and the tumor microenvironment on a cellular resolution in vivoover time for a period of weeks. Transgenic CX3CR1 proficient and deficient mice (CX3CRGFP/wt and CX3CR1GFP/GFP) were injected with red fluorescent Lewis lung carcinoma cells. During different steps of metastasis formation (extravasation, formation of micro- and macrometastasis) the density and cell body volume of TAMs, their interaction with tumor cells and possible influence on the fate of single metastatic tumor cells were investigated using serial TPLSM. We found that during metastasis formation TAM density was significantly lower in CX3CR1 deficient mice. However, activation as assessed by TAM morphology did not differ in the absence of CX3CR1. Strikingly, CX3CR1 deficiency was associated with a significant increase of tumor cells successfully extravasating the cerebral vasculature. However, subsequent steps (mirco- and macrometastasis formation) were observed less frequent in CX3CR1 deficient mice. In summary, our results highlight a complex role of CX3CR1 for TAMs during cerebral metastasis formation, indicating anti-tumorous properties of CX3CR1 at early steps and possible pro-tumorous effects at later stages (micro- and macrometastasis formation).

3020 – THE CLONAL AND MOLECULAR AETIOLOGY OF EMERGENCY DENDRITIC CELL DEVELOPMENT

Extrinsic regulation of single haematopoietic stem and progenitor cell (HSPC) fate is crucial for immune cell development. Here, we examine the aetiology of Flt3 ligand (Flt3L)-mediated emergency development of type 1 conventional dendritic cells (cDC1s), which results in enhanced immunity against infections and cancer. Using cellular barcoding, we demonstrate a predominant role of enhanced clonal expansion and moderate contribution via recruitment of additional cDC1-generating HSPCs. The selective cDC1 expansion occurs primarily via multi-/oligo-potent clones, without compromising output to other lineages. To understand the molecular hallmarks early during a Flt3L response, we develop Divi-Seq to simultaneously profile cell division history, surface phenotype and transcriptional state of single HSPCs. We discover that Flt3L-responsive HSPCs maintain a proliferative ‘early progenitor'-like state, which leads to selective emergence of CD11c+cKit+ transitional precursors with high cellular output to cDC1s. These findings inform the mechanistic action of Flt3L in natural immunity and immunotherapy at a clonal level.

Single-cell lineage tracing approaches in hematology research: technical considerations

The coordinated differentiation of hematopoietic stem and progenitor cells (HSPCs) into the various mature blood cell types is responsible for sustaining blood and immune system homeostasis. The cell fate decisions underlying this important biological process are made at the level of single cells. Methods to trace the fate of single cells are therefore essential for understanding hematopoietic system activity in health and disease and have had a major impact on how we understand and represent hematopoiesis. Here, we discuss the basic methodologies and technical considerations for three important clonal assays: single-cell transplantation, lentiviral barcoding, and Sleeping Beauty barcoding. This perspective is a synthesis of presentations and discussions from the 2019 International Society for Experimental Hematology (ISEH) Annual Meeting New Investigator Technology Session and the 2019 ISEH Winter Webinar.

Cardiac fibroblast diversity in health and disease

The cardiac stroma plays essential roles in health and following cardiac damage. The major player of the stroma with respect to extracellular matrix deposition, maintenance and remodeling is the poorly defined fibroblast. It has long been recognized that there is considerable variability to the fibroblast phenotype. With the advent of new, high throughput analytical methods our understanding and appreciation of this heterogeneity has grown dramatically. This review aims to explore the diversity of cardiac fibroblasts and highlights new insights into the diverse nature of these cells and their progenitors as revealed by single cell sequencing and fate mapping studies. We propose that at least in part the observed heterogeneity is related to the existence of a differentiation cascade within stromal cells. Beyond in-organ heterogeneity, we also discuss how the stromal response to damage differs between non-regenerating organs such as the heart and regenerating organs such as skeletal muscle. In exploring possible causes for these differences, we outline that although fibrogenic cells from different organs overlap in many properties, they still possess organ-specific transcriptional signatures and differentiation biases that make them functionally distinct.

Single-cell fate decisions of bipotential hematopoietic progenitors.

In hematopoiesis, rapid cell fate decisions are necessary for timely responses to environmental stimuli resulting in the production of diverse types of blood cells. Early studies have led to a hierarchical, tree-like view of hematopoiesis with hematopoietic stem cells residing at the apex and serially branching out to give rise to bipotential progenitors with increasingly restricted lineage potential. Recent single-cell studies have challenged some aspects of the classical model of hematopoiesis. Here, we review the latest articles on cell fate decision in hematopoietic progenitors, highlighting single-cell studies that have questioned previously established concepts and those that have reaffirmed them. The hierarchical organization of hematopoiesis and the importance of transcription factors have been largely validated at the single-cell level. In contrast, single-cell studies have shown that lineage commitment is progressive rather than switch-like as originally proposed. Furthermore, the reconstruction of cell fate paths suggested the existence of a gradient of hematopoietic progenitors that are in a continuum of changing fate probabilities rather than in a static bipotential state, leading us to reconsider the notion of bipotential progenitors. Single-cell transcriptomic and proteomic studies have transformed our view of lineage commitment and offer a drastically different perspective on hematopoiesis.

Hydrogel Network Dynamics Regulate Vascular Morphogenesis

Increasingly, matrix dynamics is being recognized as an indispensable characteristic impacting single cell behavior and fate. However, it is unknown how matrix dynamics influence individual cells to ultimately make up a complex multicellular tissue, such as vascular networks. Here we develop hydrogels with identical polymer components but different cross-links, enabling to investigate how matrix dynamics modulate vascular morphogenesis. We show that the dynamics of hydrogel networks alone, independent of stiffness, promote clustering of integrin β1 and the recruitment of vinculin, leading to the formation of focal adhesions and the phosphorylation of the focal adhesion kinase (FAK). We further demonstrate that dynamic-mediated FAK activation leads to an upregulation in metalloproteinases (MMP) expression, enabling rapid sprouting and robust assembly of vasculature. These results emphasize the importance of matrix dynamics in vascular tissue formation and the corresponding molecular pathways, which have implications for mechanistic studies and applications for tissue engineering and vascularization.

Reconstructed Single-Cell Fate Trajectories Define Lineage Plasticity Windows during Differentiation of Human PSC-Derived Distal Lung Progenitors.

Alveolar epithelial type 2 cells (AEC2s) are the facultative progenitors responsible for maintaining lung alveoli throughout life but are difficult to isolate from patients. Here, we engineer AEC2s from human pluripotent stem cells (PSCs) invitro and use time-series single-cell RNA sequencing with lentiviral barcoding to profile the kinetics of their differentiation in comparison to primary fetal and adult AEC2 benchmarks. We observe bifurcating cell-fate trajectories as primordial lung progenitors differentiate invitro, with some progeny reaching their AEC2 fate target, while others diverge to alternative non-lung endodermal fates. We develop a Continuous State Hidden Markov model to identify the timing and type of signals, such as overexuberant Wnt responses, that induce some early multipotent NKX2-1+ progenitors to lose lung fate. Finally, we find that this initial developmental plasticity is regulatable and subsides over time, ultimately resulting in PSC-derived AEC2s that exhibit a stable phenotype and nearly limitless self-renewal capacity.

Computational Tools for Quantifying Concordance in Single-Cell Fate.

Cells are dynamic biological systems that interact with each other and their surrounding environment. Understanding how cell extrinsic and intrinsic factors control cell fate is fundamental to many biological experiments. However, due to transcriptional heterogeneity or microenvironmental fluctuations, cell fates appear to be random. Individual cells within well-defined subpopulations vary with respect to their proliferative potential, survival, and lineage potency. Therefore, methods to quantify fate outcomes for heterogeneous populations that consider both the stochastic and deterministic features of single-cell dynamics are required to develop accurate models of cell growth and differentiation. To study random versus deterministic cell behavior, one requires a probabilistic modelling approach to estimate cumulative incidence functions relating the probability of a cell's fate to its lifetime and to model the deterministic effect of cell environment and inheritance, i.e., nature versus nurture. We have applied competing risks statistics, a branch of survival statistics, to quantify cell fate concordance from cell lifetime data. Competing risks modelling of cell fate concordance provides an unbiased, robust statistical modelling approach to model cell growth and differentiation by estimating the effect of cell extrinsic and heritable factors on the cause-specific cumulative incidence function.

Novel fluorescence techniques to quantitate renal cell biology.

Fluorescence microscopy techniques are powerful tools to study tissue dynamics, cellular function and biology both in vivo and in vitro. These tools allow for functional assessment and quantification along with qualitative analysis, thus providing a comprehensive understanding of various cellular processes under normal physiological and disease conditions. The main focus of this chapter is the recently developed method of serial intravital multiphoton microscopy that has helped shed light on the dynamic alterations of the spatial distribution and fate of single renal cells or cell populations and their migration patterns in the same tissue region over several days in response to various stimuli within the living kidney. This technique is very useful for studying in vivo the molecular and cellular mechanisms of tissue remodeling and repair after injury. In addition, complementary in vitro imaging tools are also described and discussed, like tissue clearing techniques and protein synthesis measurement in tissues in situ that provide an in depth assessment of changes at the cellular level. Thus, these novel fluorescence techniques can be effectively leveraged for different tissue types, experimental conditions as well as disease models to improve our understanding of renal cell biology.

Label free, quantitative single-cell fate tracking of time-lapse movies

Historically, the ability to perform multi-day time-lapse imaging of adherent cells required expensive and specialized microscopy equipment. As byproduct of this cost, many labs would synchronize cells using inhibitors such as hydroxyurea and thymidine, and or use fluorescent biosensors to minimize time required on the microscope. These methods introduce significant artefacts including phototoxicity, increased DNA replication stress and mitotic defects, thereby limiting the ability to characterize various cell cycle phenotypes. However, increased access to low cost live cell microscopes has removed many of the economic barriers thereby allowing multi-day imaging on asynchronous cells on a regular basis. Here we describe our protocol for manually tracking individual cell fates across multiple generations of random daughter cells using only low toxicity brightfield based imaging. Importantly, our pipeline relies on the free open-source software ImageJ/Fiji and an easy to use Microsoft Excel spreadsheet. Furthermore, annotated files can be saved to allow later recall of any individual cell. In summary, our method provides quantitative data on interphase and mitotic transit time, points of cell cycle arrest and critically, the ability to link these events with cell fate.

Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation.

Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (Ø: 80 µm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g., in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.

Genotoxic Stress-Induced Senescence.

A cell's genomic integrity is at risk when DNA-damaging stress, evoked by mitogenic oncogenes or genotoxic treatment modalities such as radiation or chemotherapy, apply. If the DNA repair machinery fails to fix the damaged site during a temporary cell-cycle arrest, or if massive genotoxic stress overwhelmed the repair capacity, cellular failsafe programs such as apoptosis or senescence will be triggered to limit aberrant propagation of these damaged and potentially harmful cells. After decades of scientific focusing on apoptosis, cellular senescence is increasingly recognized as an equally important but biologically andfundamentally different type of ultimate cell-cycle exit program, because of its lastingly persistent nature and cell-intrinsic and extrinsic roles within the tissue and tumor microenvironment. We established primary apoptosis-compromised, Bcl2-expressing Eμ-myc transgenic mouse lymphomas as a versatile and clinically relevant model system to study therapy-induced senescence (TIS). Given the lack of a single specific senescence-defining marker, we previously exploited co-staining of senescence-associated β-galactosidase (SA-β-gal) activity with immunohistochemical detection of trimethylated histone H3 lysine 9 (H3K9me3), an established S-phase gene expression-controlling, repressive chromatin mark, and the proliferation marker Ki67. This biomarker panel is instrumental to characterize cells as senescent via their high SA-β-gal activity, strong nuclear H3K9me3 expression and Ki67-negative profile. In this chapter, we demonstrate the detection of viable senescent cells by novel methods based on a fluorescent version of the SA-β-gal (fSA-β-gal) assay, combined with immuno-fluoroscencestaining of H3K9me3 or Ki67, or analysis of the DNA replication status by incorporating 5-ethynyl-2'-deoxyuridine (EdU) detection into the protocol. Notably, while most senescence markers, irrespective of their specificity and sensitivity, may only be assessed in endpoint assays, we wouldlike to emphasize here the strength of viablefSA-β-gal to track single-cell fate in senescent populations over time.

High-throughput single-cell fate potential assay of murine hematopoietic progenitors in vitro

The advent of single-cell transcriptomics has led to the proposal of a number of novel high-resolution models for the hematopoietic system. Testing the predictions generated by such models requires cell fate potential assays of matching, single-cell resolution. Here we detail the development of an in vitro high-throughput single-cell culture assay using flow cytometrically sorted single murine bone marrow progenitors, which measures their differentiation into any of five myeloid lineages. We identify critical parameters for single-cell culture outcome, including the choice of sorter nozzle size and pressure, culture media, and the coating of culture dishes with extracellular matrix proteins. Further, we find that accurate assay readout requires the titration of antibodies specifically for their use under low-cell-number conditions. Our approach may be used as a template for the development of single-cell fate potential assays for a variety of blood cell progenitors.

Intravital Imaging of Tumor Cell Motility in the Tumor Microenvironment Context.

Cancer cell motility and invasion are key features of metastatic tumors. Both are highly linked to tumor microenvironmental parameters, such as collagen architecture or macrophage density. However, due to the genetic, epigenetic and microenvironmental heterogeneities, only a small portion of tumor cells in the primary tumor are motile and furthermore, only a small portion of those will metastasize. This creates a challenge in predicting metastatic fate of single cells based on the phenotype they exhibit in the primary tumor. To overcome this challenge, tumor cell subpopulations need to be monitored at several timescales, mapping their phenotype in primary tumor as well as their potential homing to the secondary tumor site. Additionally, to address the spatial heterogeneity of the tumor microenvironment and how it relates to tumor cell phenotypes, large numbers of images need to be obtained from the same tumor. Finally, as the microenvironment complexity results in nonlinear relationships between tumor cell phenotype and its surroundings, advanced statistical models are required to interpret the imaging data. Toward improving our understanding of the relationship between cancer cell motility, the tumor microenvironment context and successful metastasis, we have developed several intravital approaches for continuous and longitudinal imaging, as well as data classification via support vector machine (SVM) algorithm. We also describe methods that extend the capabilities of intravital imaging by postsacrificial microscopy of the lung as well as correlative immunofluorescence in the primary tumor.

MiR-221-regulated KIT level by wild type or leukemia mutant RUNX1: a determinant of single myeloblast fate decisions that - collectively - drives or hinders granulopoiesis.

RUNX1, a master transcription factor of hematopoiesis, was shown to orchestrate both cell proliferation and differentiation during granulopoiesis by regulating microRNAs (miRs). In this study, taking advantage of the miR-ON reporter system, we monitored first, how the granulocyte colony stimulation factor (GCSF) temporally modulates the concomitant level variation of miR-221 and one of its prototypic targets, the stem cell factor receptor KIT, in single 32DmiR-ON-221 myeloblasts expressing wild type RUNX1. Second, with the same reporter system we assessed how these temporal dynamics are affected by the t(8;21)(q22;q22) acute myelogenous leukemia mutant RUNX1-MTG8 (RM8) in single 32D-RM8miR-ON-221 myeloblasts. Depending on either wild type, or mutant, RUNX1 transcriptional regulation, the cell-context specific miR-221-regulated KIT level translates into differential single cell fate decisions. Collectively, single cell fate choices translate into either initial expansion of undifferentiated myeloblasts followed by terminal granulocyte differentiation, as it happens in normal granulopoiesis, or aggressive growth of undifferentiated myeloblasts, as it happens in RUNX1-MTG8-positive acute myelogenous leukemia. Increasing knowledge of biological changes, due to altered miRNA dynamics, is expected to have relevant translational implications for leukemia detection and treatment.

NucliTrack: an integrated nuclei tracking application.

SummaryLive imaging studies give unparalleled insight into dynamic single cell behaviours and fate decisions. However, the challenge of reliably tracking single cells over long periods of time limits both the throughput and ease with which such studies can be performed. Here, we present NucliTrack, a cross platform solution for automatically segmenting, tracking and extracting features from fluorescently labelled nuclei. NucliTrack performs similarly to other state-of-the-art cell tracking algorithms, but NucliTrack’s interactive, graphical interface makes it significantly more user friendly.Availability and implementationNucliTrack is available as a free, cross platform application and open source Python package. Installation details and documentation are at: http://nuclitrack.readthedocs.io/en/latest/ A video guide can be viewed online: https://www.youtube.com/watch?v=J6e0D9F-qSU Source code is available through Github: https://github.com/samocooper/nuclitrack. A Matlab toolbox is also available at: https://uk.mathworks.com/matlabcentral/fileexchange/61479-samocooper-nuclitrack-matlab.Supplementary information Supplementary data are available at Bioinformatics online.

Tumor microtubes convey resistance to surgical lesions and chemotherapy in gliomas.

Primary and adaptive resistance against chemo- and radiotherapy and local recurrence after surgery limit the benefits from these standard treatments in glioma patients. Recently we found that glioma cells can extend ultra-long membrane protrusions, "tumor microtubes" (TMs), for brain invasion, proliferation, and interconnection of single cells to a syncytium that is resistant to radiotherapy. We wondered whether TMs also convey resistance to the other 2 standard treatment modalities. Patient-derived glioblastoma stemlike cell (GBMSC) lines were implanted under a cranial window in mice. Longitudinal in vivo two-photon laser scanning microscopy was used to follow tumor growth, including the fate of single glioma cells over months. After a cylindrical surgical lesion, GBMSCs increasingly extended TMs toward the lesion area, which contributed to the repopulation of this area over many weeks. In fact, an excessive "healing response" was observed in which tumor cell densities significantly exceeded those of unlesioned brain regions over time. Inhibition of TM formation and function by genetic targeting of growth associated protein-43 robustly suppressed this surgery-induced tumor growth reaction, in contrast to standard postsurgical anti-inflammatory treatment with dexamethasone. After one cycle of temozolomide chemotherapy, intra- and intertumoral heterogeneity of TM formation and interconnection was strongly associated with therapy response: when tumor cells were integrated in TM networks, they were more likely to resist chemotherapy. TMs can contribute to the resistance against standard treatment modalities in gliomas. Specific inhibition of TMs is a promising approach to reduce local recurrence after surgery and lower resistance to chemotherapy.

Morphogen and community effects determine cell fates in response to BMP4 signaling in human embryonic stem cells.

Paracrine signals maintain developmental states and create cell fate patterns in vivo and influence differentiation outcomes in human embryonic stem cells (hESCs) in vitro Systematic investigation of morphogen signaling is hampered by the difficulty of disentangling endogenous signaling from experimentally applied ligands. Here, we grow hESCs in micropatterned colonies of 1-8 cells ('µColonies') to quantitatively investigate paracrine signaling and the response to external stimuli. We examine BMP4-mediated differentiation in µColonies and standard culture conditions and find that in µColonies, above a threshold concentration, BMP4 gives rise to only a single cell fate, contrary to its role as a morphogen in other developmental systems. Under standard culture conditions BMP4 acts as a morphogen but this requires secondary signals and particular cell densities. We find that a 'community effect' enforces a common fate within µColonies, both in the state of pluripotency and when cells are differentiated, and that this effect allows a more precise response to external signals. Using live cell imaging to correlate signaling histories with cell fates, we demonstrate that interactions between neighbors result in sustained, homogenous signaling necessary for differentiation.

Single cell-laden protease-sensitive microniches for long-term culture in 3D.

Single cell-laden three-dimensional (3D) microgels that can serve to mimic stem cell niches in vitro, and are therefore termed microniches, can be efficiently fabricated by droplet-based microfluidics. In this technique an aqueous polymer solution along with a highly diluted cell solution is injected into a microfluidic device to create monodisperse pre-microgel droplets that are then solidified by a polymer crosslinking reaction to obtain monodisperse single cell-laden microniches. However, problems limiting this approach studying the fate of single cells include Poisson encapsulation statistics that result in mostly empty microniches, and cells egressing from the microniches during subsequent cell culture. Here, we present a strategy to bypass Poisson encapsulation statistics in synthetic microniches by selective crosslinking of only cell-laden pre-microgel droplets. Furthermore, we show that we can position cells in the center of the microniches, and that even in protease-sensitive microniches this greatly reduces cell egress. Collectively, we present the development of a versatile protocol that allows for unprecedented efficiency in creation of synthetic protease-sensitive microniches for probing single stem cell fate in 3D.

Long-term Live Single Cell Quantification of Transcription Factor Dynamics

Hematopoiesis is highly complex and dynamic, and consist of large numbers of different cells expressing many molecules. Despite intensive research, many long-standing questions in hematopoiesis research remain disputed. One major reason is the fact that we usually only analyze populations of cells - rather than individual cells - at very few time points of an experiment. Tracking of individual cells would be an extremely powerful approach to improve our understanding of molecular cell fate control. We are therefore developing imaging systems to follow the fate of single cells over many generations. We program new software to help recording and displaying the divisional history, position, properties, interaction, etc. of all individual cells over many generations. In addition, novel microfluidics devices are designed and produced to allow improved observation and manipulation of cells. Our technologies allow continuous long-term quantification of protein expression or activity in living cells. Among other approaches, we generate knock in models expressing transcription factor to fluorescent protein fusions from endogenous gene loci. This enables non-invasive long-term live quantification of transcription factor protein dynamics in single stem and progenitor cells throughout their differentiation. The resulting novel kind of continuous quantitative single cell data is used for the generation and falsification of models describing the molecular control of hematopoietic cell fates. DisclosuresNo relevant conflicts of interest to declare.