Embryonic stem cell ERK, AKT, plus STAT3 response dynamics combinatorics are heterogeneous but NANOG state independent.

Signaling is central in cell fate regulation, and relevant information is encoded in its activity over time (i.e., dynamics). However, simultaneous dynamics quantification of several pathways in single mammalian stem cells has not yet been accomplished. Here we generate mouse embryonic stem cell (ESC) lines simultaneously expressing fluorescent reporters for ERK, AKT, and STAT3 signaling activity, which all control pluripotency. We quantify their single-cell dynamics combinations in response to different self-renewal stimuli and find striking heterogeneity for all pathways, some dependent on cell cycle but not pluripotency states, even in ESC populations currently assumed to be highly homogeneous. Pathways are mostly independently regulated, but some context-dependent correlations exist. These quantifications reveal surprising single-cell heterogeneity in the important cell fate control layer of signaling dynamics combinations and raise fundamental questions about the role of signaling in (stem) cell fate control.

Optogenetic manipulation identifies the roles of ERK and AKT dynamics in controlling mouse embryonic stem cell exit from pluripotency.

ERK and AKT signaling control pluripotent cell self-renewal versus differentiation. ERK pathway activity over time (i.e., dynamics) is heterogeneous between individual pluripotent cells, even in response to the same stimuli. To analyze potential functions of ERK and AKT dynamics in controlling mouse embryonic stem cell (ESC) fates, we developed ESC lines and experimental pipelines for the simultaneous long-term manipulation and quantification of ERK or AKT dynamics and cell fates. We show that ERK activity duration or amplitude or the type of ERK dynamics (e.g., transient, sustained, or oscillatory) alone does not influence exit from pluripotency, but the sum of activity over time does. Interestingly, cells retain memory of previous ERK pulses, with duration of memory retention dependent on duration of previous pulse length. FGF receptor/AKT dynamics counteract ERK-induced pluripotency exit. These findings improve our understanding of how cells integrate dynamics from multiple signaling pathways and translate them into cell fate cues.

Open-source personal pipetting robots with live-cell incubation and microscopy compatibility.

Liquid handling robots have the potential to automate many procedures in life sciences. However, they are not in widespread use in academic settings, where funding, space and maintenance specialists are usually limiting. In addition, current robots require lengthy programming by specialists and are incompatible with most academic laboratories with constantly changing small-scale projects. Here, we present the Pipetting Helper Imaging Lid (PHIL), an inexpensive, small, open-source personal liquid handling robot. It is designed for inexperienced users, with self-production from cheap commercial and 3D-printable components and custom control software. PHIL successfully automates pipetting (incl. aspiration) for e.g. tissue immunostainings and stimulations of live stem and progenitor cells during time-lapse microscopy using 3D printed peristaltic pumps. PHIL is cheap enough to put a personal pipetting robot within the reach of most labs and enables users without programming skills to easily automate a large range of experiments.

Heritable changes in division speed accompany the diversification of single T cell fate.

Rapid clonal expansion of antigen-specific T cells is a fundamental feature of adaptive immune responses. It enables the outgrowth of an individual T cell into thousands of clonal descendants that diversify into short-lived effectors and long-lived memory cells. Clonal expansion is thought to be programmed upon priming of a single naive T cell and then executed by homogenously fast divisions of all of its descendants. However, the actual speed of cell divisions in such an emerging "T cell family" has never been measured with single-cell resolution. Here, we utilize continuous live-cell imaging in vitro to track the division speed and genealogical connections of all descendants derived from a single naive CD8+ T cell throughout up to ten divisions of activation-induced proliferation. This comprehensive mapping of T cell family trees identifies a short burst phase, in which division speed is homogenously fast and maintained independent of external cytokine availability or continued T cell receptor stimulation. Thereafter, however, division speed diversifies, and model-based computational analysis using a Bayesian inference framework for tree-structured data reveals a segregation into heritably fast- and slow-dividing branches. This diversification of division speed is preceded already during the burst phase by variable expression of the interleukin-2 receptor alpha chain. Later it is accompanied by selective expression of memory marker CD62L in slower dividing branches. Taken together, these data demonstrate that T cell clonal expansion is structured into subsequent burst and diversification phases, the latter of which coincides with specification of memory versus effector fate.

Asymmetric organelle inheritance predicts human blood stem cell fate.

Understanding human hematopoietic stem cell fate control is important for its improved therapeutic manipulation. Asymmetric cell division, the asymmetric inheritance of factors during division instructing future daughter cell fates, was recently described in mouse blood stem cells. In human blood stem cells, the possible existence of asymmetric cell division remained unclear because of technical challenges in its direct observation. Here, we use long-term quantitative single-cell imaging to show that lysosomes and active mitochondria are asymmetrically inherited in human blood stem cells and that their inheritance is a coordinated, nonrandom process. Furthermore, multiple additional organelles, including autophagosomes, mitophagosomes, autolysosomes, and recycling endosomes, show preferential asymmetric cosegregation with lysosomes. Importantly, asymmetric lysosomal inheritance predicts future asymmetric daughter cell-cycle length, differentiation, and stem cell marker expression, whereas asymmetric inheritance of active mitochondria correlates with daughter metabolic activity. Hence, human hematopoietic stem cell fates are regulated by asymmetric cell division, with both mechanistic evolutionary conservation and differences to the mouse system.

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.

Symmetric and asymmetric activation of hematopoietic stem cells.

PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) are in an inactive quiescent state for most of their life. To replenish the blood system in homeostasis and after injury, they activate and divide. HSC daughter cells must then decide whether to return to quiescence and metabolic inactivity or to activate further to proliferate and differentiate and replenish lost blood cells. Although the regulation of HSC activation is not well understood, recent discoveries shed new light on involved mechanisms including asymmetric cell division (ACD). RECENT FINDINGS: HSC metabolism has emerged as a regulator of cell fates. Recent evidence suggests that cellular organelles mediating anabolic and catabolic processes can be asymmetrically inherited during HSC divisions. These include autophagosomes, mitophagosomes, and lysosomes, which regulate HSC quiescence. Their asymmetric inheritance has been linked to future metabolic and translational activity in HSC daughters, showing that ACD can regulate the balance between HSC (in)activity. SUMMARY: We discuss recent insights and remaining questions in how HSCs balance activation and quiescence, with a focus on ACD.

Cytokine combinations for human blood stem cell expansion induce cell-type- and cytokine-specific signaling dynamics.

How hematopoietic stem cells (HSCs) integrate signals from their environment to make fate decisions remains incompletely understood. Current knowledge is based on either averages of heterogeneous populations or snapshot analyses, both missing important information about the dynamics of intracellular signaling activity. By combining fluorescent biosensors with time-lapse imaging and microfluidics, we measured the activity of the extracellular-signal-regulated kinase (ERK) pathway over time (ie, dynamics) in live single human umbilical cord blood HSCs and multipotent progenitor cells (MPPs). In single cells, ERK signaling dynamics were highly heterogeneous and depended on the cytokines, their combinations, and cell types. ERK signaling was activated by stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand in HSCs but SCF, interleukin 3, and granulocyte colony-stimulating factor in MPPs. Different cytokines and their combinations led to distinct ERK signaling dynamics frequencies, and ERK dynamics in HSCs were more transient than those in MPPs. A combination of 5 cytokines recently shown to maintain HSCs in long-term culture, had a more-than-additive effect in eliciting sustained ERK dynamics in HSCs. ERK signaling dynamics also predicted future cell fates. For example, CD45RA expression increased more in HSC daughters with intermediate than with transient or sustained ERK signaling. We demonstrate heterogeneous cytokine- and cell-type-specific ERK signaling dynamics, illustrating their relevance in regulating hematopoietic stem and progenitor (HSPC) cell fates.

PU.1 enforces quiescence and limits hematopoietic stem cell expansion during inflammatory stress.

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.

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.

Pitfalls and requirements in quantifying asymmetric mitotic segregation.

The asymmetric inheritance of NUMB during mitosis determines future daughter cell fates in multiple model organisms. NUMB asymmetric inheritance has also been postulated for hematopoietic stem cell (HSC) divisions but remained controversial until recently. To reconcile conflicting reports, we revisited the evidence for asymmetric inheritance of NUMB during HSC divisions. We demonstrate that previously used strategies to identify dividing cells in fixed samples suffer from multiple systematic errors. Nonmitotic cells in close proximity are frequently mistaken as dividing cells, while mitotic cells are not detected. Furthermore, microtubule depolymerization by either nocodazole or low temperatures prevents the reliable detection of mitosis and introduces mitotic artifacts. Without artificial microtubule depolymerization and by the use of reliable mitotic markers, we find NUMB differences in daughter cells to be reduced and restricted to cells with low NUMB expression and thus low signal over background. This bias fits the expected random distribution of simulated noise data, suggesting that the putative asymmetric inheritance of NUMB in HSCs could be merely technical noise. We conclude that functionally relevant asymmetric inheritance of NUMB and other factors in mitotic HSCs and other cells cannot be conclusively demonstrated using snapshot data and requires alternative approaches, such as continuous quantitative single-cell analysis.

Determination of non-extractable residues in soils: Towards a standardised approach.

After exposure of soils to anthropogenic organic chemicals non-extractable residues (NER) can be formed. The quantitative proportion of a compound which remains non-extractable is operationally defined by the extraction procedure employed and can be quantified only when using isotope labelled compounds (e.g. 14C or 13C). In Germany and the EU, there is no standardised procedure, how to determine NER, especially when different legal regulations apply. Consequently, the comparability of NER data is low. Hence, a major task of this study was the development of a general approach for the quantification of non-extractable residues (NER) in soils using radiotracer analysis. For that, extraction efficiencies were determined for 42 non-labelled organic chemicals spiked onto 3 soils applying a number of extraction techniques and conditions, developing an extraction procedure which provides high extraction efficiencies and a low variability for a broad spectrum of analytes. Additionally, NER generated within soil transformation studies according to OECD 307 using 14C-triclosan, 14C-fenoxycarb and 14C-acetaminophen were analysed using sequential batch extraction and pressurised liquid extraction (PLE). Depending on the extraction procedure used, the NER fraction related to 14C-triclosan in a soil varied greatly between 96% and 28%. In this study a widely universal extraction procedure was developed to improve the comparability of the NER data and limit overestimation of NER, which can be of enormous consequence for the assessment of persistence and environmental risk of organic chemicals. Furthermore, silylation, EDTA-extraction and HCl-treatment were compared regarding a further analysis of NER using radiotracer analysis.

Publisher Correction: Asymmetric lysosome inheritance predicts activation of haematopoietic stem cells.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Asymmetric lysosome inheritance predicts activation of haematopoietic stem cells.

Haematopoietic stem cells self-renew and differentiate into all blood lineages throughout life, and can repair damaged blood systems upon transplantation. Asymmetric cell division has previously been suspected to be a regulator of haematopoietic-stem-cell fate, but its existence has not directly been shown1. In asymmetric cell division, asymmetric fates of future daughter cells are prospectively determined by a mechanism that is linked to mitosis. This can be mediated by asymmetric inheritance of cell-extrinsic niche signals by, for example, orienting the divisional plane, or by the asymmetric inheritance of cell-intrinsic fate determinants. Observations of asymmetric inheritance or of asymmetric daughter-cell fates alone are not sufficient to demonstrate asymmetric cell division2. In both cases, sister-cell fates could be controlled by mechanisms that are independent of division. Here we demonstrate that the cellular degradative machinery-including lysosomes, autophagosomes, mitophagosomes and the protein NUMB-can be asymmetrically inherited into haematopoietic-stem-cell daughter cells. This asymmetric inheritance predicts the asymmetric future metabolic and translational activation and fates of haematopoietic-stem-cell daughter cells and their offspring. Therefore, our studies provide evidence for the existence of asymmetric cell division in haematopoietic stem cells.

Understanding cell fate control by continuous single-cell quantification.

Cells and the molecular processes underlying their behavior are highly dynamic. Understanding these dynamic biological processes requires noninvasive continuous quantitative single-cell observations, instead of population-based average or single-cell snapshot analysis. Ideally, single-cell dynamics are measured long-term in vivo; however, despite progress in recent years, technical limitations still prevent such studies. On the other hand, in vitro studies have proven to be useful for answering long-standing questions. Although technically still demanding, long-term single-cell imaging and tracking in vitro have become valuable tools to elucidate dynamic molecular processes and mechanisms, especially in rare and heterogeneous populations. Here, we review how continuous quantitative single-cell imaging of hematopoietic cells has been used to solve decades-long controversies. Because aberrant cell fate decisions are at the heart of tissue degeneration and disease, we argue that studying their molecular dynamics using quantitative single-cell imaging will also improve our understanding of these processes and lead to new strategies for therapies.

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.