NfκB signaling dynamics and their target genes differ between mouse blood cell types and induce distinct cell behavior.

Cells can use signaling pathway activity over time (ie, dynamics) to control cell fates. However, little is known about the potential existence and function of signaling dynamics in primary hematopoietic stem and progenitor cells (HSPCs). Here, we use time-lapse imaging and tracking of single murine HSPCs from green fluorescent protein-p65/H2BmCherry reporter mice to quantify their nuclear factor κB (NfκB) activity dynamics in response to tumor necrosis factor α and interleukin 1ß. We find response dynamics to be heterogeneous between individual cells, with cell type-specific dynamics distributions. Transcriptome sequencing of single cells physically isolated after live dynamics quantification shows activation of different target gene programs in cells with different dynamics. Finally, artificial induction of oscillatory NfκB activity causes changes in granulocyte/monocyte progenitor behavior. Thus, HSPC behavior can be influenced by signaling dynamics, which are tightly regulated during hematopoietic differentiation and enable cell type-specific responses to the same signaling inputs.

Blood stem cell PU.1 upregulation is a consequence of differentiation without fast autoregulation.

Transcription factors (TFs) regulate cell fates, and their expression must be tightly regulated. Autoregulation is assumed to regulate many TFs' own expression to control cell fates. Here, we manipulate and quantify the (auto)regulation of PU.1, a TF controlling hematopoietic stem and progenitor cells (HSPCs), and correlate it to their future fates. We generate transgenic mice allowing both inducible activation of PU.1 and noninvasive quantification of endogenous PU.1 protein expression. The quantified HSPC PU.1 dynamics show that PU.1 up-regulation occurs as a consequence of hematopoietic differentiation independently of direct fast autoregulation. In contrast, inflammatory signaling induces fast PU.1 up-regulation, which does not require PU.1 expression or its binding to its own autoregulatory enhancer. However, the increased PU.1 levels induced by inflammatory signaling cannot be sustained via autoregulation after removal of the signaling stimulus. We conclude that PU.1 overexpression induces HSC differentiation before PU.1 up-regulation, only later generating cell types with intrinsically higher PU.1.

An automated microfluidic system for efficient capture of rare cells and rapid flow-free stimulation.

Cell fates are controlled by environmental stimuli that rapidly change the activity of intracellular signaling. Studying these processes requires rapid manipulations of micro-environmental conditions while continuously observing single cells over long periods of time. Current microfluidic devices are unable to simultaneously i) efficiently capture and concentrate rare cells, ii) conduct automated rapid media exchanges via diffusion without displacing non-adherent cells, and iii) allow sensitive high-throughput long-term time-lapse microscopy. Hematopoietic stem and progenitor cells pose a particular challenge for these types of experiments as they are impossible to obtain in very large numbers and are displaced by the fluid flow usually used to change culture media, thus preventing cell tracking. Here, we developed a programmable automated system composed of a novel microfluidic device for efficient capture of rare cells in independently addressable culture chambers, a custom incubation system, and user-friendly control software. The chip's culture chambers are optimized for efficient and sensitive fluorescence microscopy and their media can be individually and quickly changed by diffusion without non-adherent cell displacement. The chip allows efficient capture, stimulation, and sensitive high-frequency time-lapse observation of rare and sensitive murine and human primary hematopoietic stem cells. Our 3D-printed humidification and incubation system minimizes gas consumption, facilitates chip setup, and maintains stable humidity and gas composition during long-term cell culture. This approach now enables the required continuous long-term single-cell quantification of rare non-adherent cells with rapid environmental manipulations, e.g. of rapid signaling dynamics and the later stem cell fate choices they control.

A Novel GATA2 Protein Reporter Mouse Reveals Hematopoietic Progenitor Cell Types.

The transcription factor (TF) GATA2 plays a key role in organ development and cell fate control in the central nervous, urogenital, respiratory, and reproductive systems, and in primitive and definitive hematopoiesis. Here, we generate a knockin protein reporter mouse line expressing a GATA2VENUS fusion from the endogenous Gata2 genomic locus, with correct expression and localization of GATA2VENUS in different organs. GATA2VENUS expression is heterogeneous in different hematopoietic stem and progenitor cell populations (HSPCs), identifies functionally distinct subsets, and suggests a novel monocyte and mast cell lineage bifurcation point. GATA2 levels further correlate with proliferation and lineage outcome of hematopoietic progenitors. The GATA2VENUS mouse line improves the identification of specific live cell types during embryonic and adult development and will be crucial for analyzing GATA2 protein dynamics in TF networks.

Inflammatory signals directly instruct PU.1 in HSCs via TNF.

The molecular mechanisms governing the transition from hematopoietic stem cells (HSCs) to lineage-committed progenitors remain poorly understood. Transcription factors (TFs) are powerful cell intrinsic regulators of differentiation and lineage commitment, while cytokine signaling has been shown to instruct the fate of progenitor cells. However, the direct regulation of differentiation-inducing hematopoietic TFs by cell extrinsic signals remains surprisingly difficult to establish. PU.1 is a master regulator of hematopoiesis and promotes myeloid differentiation. Here we report that tumor necrosis factor (TNF) can directly and rapidly upregulate PU.1 protein in HSCs in vitro and in vivo. We demonstrate that in vivo, niche-derived TNF is the principal PU.1 inducing signal in HSCs and is both sufficient and required to relay signals from inflammatory challenges to HSCs.

Automated Microfluidic System for Dynamic Stimulation and Tracking of Single Cells.

Dynamic environments determine cell fate decisions and function. Understanding the relationship between extrinsic signals on cellular responses and cell fate requires the ability to dynamically change environmental inputs in vitro, while continuously observing individual cells over extended periods of time. This is challenging for nonadherent cells, such as hematopoietic stem and progenitor cells, because media flow displaces and disturbs such cells, preventing culture and tracking of single cells. Here, we present a programmable microfluidic system designed for the long-term culture and time-lapse imaging of nonadherent cells in dynamically changing cell culture conditions without losing track of individual cells. The dynamic, valve-controlled design permits targeted seeding of cells in up to 48 independently controlled culture chambers, each providing sufficient space for long-term cell colony expansion. Diffusion-based media exchange occurs rapidly and minimizes displacement of cells and eliminates shear stress. The chip was successfully tested with long-term culture and tracking of primary hematopoietic stem and progenitor cells, and murine embryonic stem cells. This system will have important applications to analyze dynamic signaling inputs controlling fate choices.

Illuminating stem cell transcription factor dynamics: long-term single-cell imaging of fluorescent protein fusions.

Most single-cell approaches to date are based on destructive snapshot measurements which do not permit to correlate a current molecular state with future fate. However, to understand how cell fate choices are established by transcription factor networks (TFNs) regulating cell fates, TFN dynamics must be continuously monitored in single cells. Here we review how quantitative time-lapse imaging can contribute to understanding TFN dependent cell fate regulation at the single-cell level. We outline potentials of the technology and highlight challenges for interpreting the dynamics of fluorescent protein reporters that may interfere with endogenous TF function. We provide an outlook on how continuous observation of TF dynamics and single-cell fates may be complemented by perturbation studies and be linked to multidimensional molecular profiling in the future.

Cellular Decision Making by Non-Integrative Processing of TLR Inputs.

Cells receive a multitude of signals from the environment, but how they process simultaneous signaling inputs is not well understood. Response to infection, for example, involves parallel activation of multiple Toll-like receptors (TLRs) that converge on the nuclear factor κB (NF-κB) pathway. Although we increasingly understand inflammatory responses for isolated signals, it is not clear how cells process multiple signals that co-occur in physiological settings. We therefore examined a bacterial infection scenario involving co-stimulation of TLR4 and TLR2. Independent stimulation of these receptors induced distinct NF-κB dynamic profiles, although surprisingly, under co-stimulation, single cells continued to show ligand-specific dynamic responses characteristic of TLR2 or TLR4 signaling rather than a mixed response, comprising a cellular decision that we term "non-integrative" processing. Iterating modeling and microfluidic experiments revealed that non-integrative processing occurred through interaction of switch-like NF-κB activation, receptor-specific processing timescales, cell-to-cell variability, and TLR cross-tolerance mediated by multilayer negative feedback.

Preservation of cell-survival mechanisms by the presenilin-1 K239N mutation may cause its milder clinical phenotype.

Presenilin 1 (PSEN1) mutations are the main cause of monogenic Alzheimer's disease. We studied the functional effects of the mutation K239N, which shows incomplete penetrance at the age of 65 years and compared it with the more aggressive mutation E120G. We engineered stable cell lines expressing human PSEN1 wild type or with K239N or E120G mutations. Both mutations induced dysfunction of γ-secretase in the processing of amyloid-ß protein precursor, leading to an increase in the amyloid ß42/amyloid ß40 ratio. Analysis of homeostatic mechanisms showed that K239N induced lower basal and hydrogen peroxide induced intracellular levels of reactive oxygen species than E120G. Similarly, K239N induced lower vulnerability to apoptosis by hydrogen peroxide injury than E120G. Accordingly, the proapoptotic signaling pathways c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase maintained PSEN1-mediated negative regulation in K239N but not in E120G-bearing cells. Furthermore, the activation of the prosurvival signaling pathways mitogen-activated protein kinase/extracellular signal-regulated kinase and phosphoinositide 3-kinase/Akt was lower in E120G-bearing cells. Therefore, preservation of mechanisms regulating cell responses independent of amyloid-ß protein precursor processing may account for the milder phenotype induced by the PSEN1 K239N mutation.

Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios.

The mechanisms underlying haematopoietic lineage decisions remain disputed. Lineage-affiliated transcription factors with the capacity for lineage reprogramming, positive auto-regulation and mutual inhibition have been described as being expressed in uncommitted cell populations. This led to the assumption that lineage choice is cell-intrinsically initiated and determined by stochastic switches of randomly fluctuating cross-antagonistic transcription factors. However, this hypothesis was developed on the basis of RNA expression data from snapshot and/or population-averaged analyses. Alternative models of lineage choice therefore cannot be excluded. Here we use novel reporter mouse lines and live imaging for continuous single-cell long-term quantification of the transcription factors GATA1 and PU.1 (also known as SPI1). We analyse individual haematopoietic stem cells throughout differentiation into megakaryocytic-erythroid and granulocytic-monocytic lineages. The observed expression dynamics are incompatible with the assumption that stochastic switching between PU.1 and GATA1 precedes and initiates megakaryocytic-erythroid versus granulocytic-monocytic lineage decision-making. Rather, our findings suggest that these transcription factors are only executing and reinforcing lineage choice once made. These results challenge the current prevailing model of early myeloid lineage choice.

Seamless Combination of Fluorescence-Activated Cell Sorting and Hanging-Drop Networks for Individual Handling and Culturing of Stem Cells and Microtissue Spheroids.

Open microfluidic cell culturing devices offer new possibilities to simplify loading, culturing, and harvesting of individual cells or microtissues due to the fact that liquids and cells/microtissues are directly accessible. We present a complete workflow for microfluidic handling and culturing of individual cells and microtissue spheroids, which is based on the hanging-drop network concept: The open microfluidic devices are seamlessly combined with fluorescence-activated cell sorting (FACS), so that individual cells, including stem cells, can be directly sorted into specified culturing compartments in a fully automated way and at high accuracy. Moreover, already assembled microtissue spheroids can be loaded into the microfluidic structures by using a conventional pipet. Cell and microtissue culturing is then performed in hanging drops under controlled perfusion. On-chip drop size control measures were applied to stabilize the system. Cells and microtissue spheroids can be retrieved from the chip by using a parallelized transfer method. The presented methodology holds great promise for combinatorial screening of stem-cell and multicellular-spheroid cultures.

Quantitative single-cell approaches to stem cell research.

Understanding the molecular control of cell fates is central to stem cell research. Such insight requires quantification of molecular and cellular behavior at the single-cell level. Recent advances now permit high-throughput molecular readouts from single cells as well as continuous, noninvasive observation of cell behavior over time. Here, we review current state-of-the-art approaches used to query stem cell fate at the single-cell level, including advances in lineage tracing, time-lapse imaging, and molecular profiling. We also offer our perspective on the advantages and drawbacks of available approaches, key technical limitations, considerations for data interpretation, and future innovation.

Instruction of hematopoietic lineage choice by cytokine signaling.

Hematopoiesis is the cumulative consequence of finely tuned signaling pathways activated through extrinsic factors, such as local niche signals and systemic hematopoietic cytokines. Whether extrinsic factors actively instruct the lineage choice of hematopoietic stem and progenitor cells or are only selectively allowing survival and proliferation of already intrinsically lineage-committed cells has been debated over decades. Recent results demonstrated that cytokines can instruct lineage choice. However, the precise function of individual cytokine-triggered signaling molecules in inducing cellular events like proliferation, lineage choice, and differentiation remains largely elusive. Signal transduction pathways activated by different cytokine receptors are highly overlapping, but support the production of distinct hematopoietic lineages. Cellular context, signaling dynamics, and the crosstalk of different signaling pathways determine the cellular response of a given extrinsic signal. New tools to manipulate and continuously quantify signaling events at the single cell level are therefore required to thoroughly interrogate how dynamic signaling networks yield a specific cellular response.

MicroRNA-mediated control of macrophages and its implications for cancer.

Deregulation of microRNAs (miRNAs) can drive oncogenesis, tumor progression, and metastasis by acting cell-autonomously in cancer cells. However, solid tumors are also infiltrated by large amounts of non-neoplastic stromal cells, including macrophages, which express several active miRNAs. Tumor-associated macrophages (TAMs) enhance angiogenic, immunosuppressive, invasive, and metastatic programming of neoplastic tissue and reduce host survival. Here, we review the role of miRNAs (including miR-155, miR-146, and miR-511) in the control of macrophage production and activation, and examine whether reprogramming miRNA activity in TAMs and/or their precursors might be effective for controlling tumor progression.

Angiotensin II drives the production of tumor-promoting macrophages.

Macrophages frequently infiltrate tumors and can enhance cancer growth, yet the origins of the macrophage response are not well understood. Here we address molecular mechanisms of macrophage production in a conditional mouse model of lung adenocarcinoma. We report that overproduction of the peptide hormone Angiotensin II (AngII) in tumor-bearing mice amplifies self-renewing hematopoietic stem cells (HSCs) and macrophage progenitors. The process occurred in the spleen but not the bone marrow, and was independent of hemodynamic changes. The effects of AngII required direct hormone ligation on HSCs, depended on S1P(1) signaling, and allowed the extramedullary tissue to supply new tumor-associated macrophages throughout cancer progression. Conversely, blocking AngII production prevented cancer-induced HSC and macrophage progenitor amplification and thus restrained the macrophage response at its source. These findings indicate that AngII acts upstream of a potent macrophage amplification program and that tumors can remotely exploit the hormone's pathway to stimulate cancer-promoting immunity.

Myocardial infarction accelerates atherosclerosis.

During progression of atherosclerosis, myeloid cells destabilize lipid-rich plaques in the arterial wall and cause their rupture, thus triggering myocardial infarction and stroke. Survivors of acute coronary syndromes have a high risk of recurrent events for unknown reasons. Here we show that the systemic response to ischaemic injury aggravates chronic atherosclerosis. After myocardial infarction or stroke, Apoe-/- mice developed larger atherosclerotic lesions with a more advanced morphology. This disease acceleration persisted over many weeks and was associated with markedly increased monocyte recruitment. Seeking the source of surplus monocytes in plaques, we found that myocardial infarction liberated haematopoietic stem and progenitor cells from bone marrow niches via sympathetic nervous system signalling. The progenitors then seeded the spleen, yielding a sustained boost in monocyte production. These observations provide new mechanistic insight into atherogenesis and provide a novel therapeutic opportunity to mitigate disease progression.

Regulation of monocyte functional heterogeneity by miR-146a and Relb.

Monocytes serve as a central defense system against infection and injury but can also promote pathological inflammatory responses. Considering the evidence that monocytes exist in at least two subsets committed to divergent functions, we investigated whether distinct factors regulate the balance between monocyte subset responses in vivo. We identified a microRNA (miRNA), miR-146a, which is differentially regulated both in mouse (Ly-6C(hi)/Ly-6C(lo)) and human (CD14(hi)/CD14(lo)CD16(+)) monocyte subsets. The single miRNA controlled the amplitude of the Ly-6C(hi) monocyte response during inflammatory challenge whereas it did not affect Ly-6C(lo) cells. miR-146a-mediated regulation was cell-intrinsic and depended on Relb, a member of the noncanonical NF-κB/Rel family, which we identified as a direct miR-146a target. These observations not only provide mechanistic insights into the molecular events that regulate responses mediated by committed monocyte precursor populations but also identify targets for manipulating Ly-6C(hi) monocyte responses while sparing Ly-6Clo monocyte activity.

Demyelinating diseases: myeloperoxidase as an imaging biomarker and therapeutic target.

PURPOSE: To evaluate myeloperoxidase (MPO) as a newer therapeutic target and bis-5-hydroxytryptamide-diethylenetriaminepentaacetate-gadolinium (Gd) (MPO-Gd) as an imaging biomarker for demyelinating diseases such as multiple sclerosis (MS) by using experimental autoimmune encephalomyelitis (EAE), a murine model of MS. MATERIALS AND METHODS: Animal experiments were approved by the institutional animal care committee. EAE was induced in SJL mice by using proteolipid protein (PLP), and mice were treated with either 4-aminobenzoic acid hydrazide (ABAH), 40 mg/kg injected intraperitoneally, an irreversible inhibitor of MPO, or saline as control, and followed up to day 40 after induction. In another group of SJL mice, induction was performed without PLP as shams. The mice were imaged by using MPO-Gd to track changes in MPO activity noninvasively. Imaging results were corroborated by enzymatic assays, flow cytometry, and histopathologic analyses. Significance was computed by using the t test or Mann-Whitney U test. RESULTS: There was a 2.5-fold increase in myeloid cell infiltration in the brain (P = .026), with a concomitant increase in brain MPO level (P = .0087). Inhibiting MPO activity with ABAH resulted in decrease in MPO-Gd-positive lesion volume (P = .012), number (P = .009), and enhancement intensity (P = .03) at MR imaging, reflecting lower local MPO activity (P = .03), compared with controls. MPO inhibition was accompanied by decreased demyelination (P = .01) and lower inflammatory cell recruitment in the brain (P < .0001), suggesting a central MPO role in inflammatory demyelination. Clinically, MPO inhibition significantly reduced the severity of clinical symptoms (P = .0001) and improved survival (P = .0051) in mice with EAE. CONCLUSION: MPO may be a key mediator of myeloid inflammation and tissue damage in EAE. Therefore, MPO could represent a promising therapeutic target, as well as an imaging biomarker, for demyelinating diseases and potentially for other diseases in which MPO is implicated.

Regulation of macrophage and dendritic cell responses by their lineage precursors.

Tissue macrophages (Mø) and dendritic cells (DC) are thought to derive from hematopoietic stem cells, which exist in the bone marrow and generate intermediate precursor populations with increasingly restricted lineage potentials. There exists several precursors committed to the Mø and DC lineages; these cells exhibit distinct tropism and function and respond differentially in pathophysiologic conditions. In this review, we consider experimental contexts in which Mø and DC responses in tissue are not only dictated by the local environment, but also by the quantity and quality of newly recruited lineage precursor cells. Consequently, we discuss whether therapeutic control of Mø and DC responses in tissue may be achieved through manipulation of their lineage precursors.