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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., Journal of Experimental Medicine, 219 (1), ISSN:0022-1007, ISSN:1540-0069, ISSN:1540-9538

Blood-borne metastasis of breast cancer involves a series of tightly regulated sequential steps, including the growth of a primary tumor lesion, intravasation of circulating tumor cells (CTC), and adaptation in various distant metastatic sites. The genes orchestrating each of these steps are poorly understood in physiologically relevant contexts, owing to the rarity of experimental models that faithfully recapitulate the biology, growth kinetics, and tropism of human breast cancer. Here, we conducted an in vivo loss-of-function CRISPR screen in newly derived CTC xenografts, unique in their ability to spontaneously mirror the human disease, and identified specific genetic dependencies for each step of the metastatic process. Validation experiments revealed sensitivities to inhibitors that are already available, such as PLK1 inhibitors, to prevent CTC intravasation. Together, these findings present a new tool to reclassify driver genes involved in the spread of human cancer, providing insights into the biology of metastasis and paving the way to test targeted treatment approaches., Cancer Research, 82 (4), ISSN:0008-5472, ISSN:1538-7445

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 naïve 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 imagingin vitroto track the division speed and genealogical connections of all descendants derived from a single naïve 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 novel 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 vs. effector fate.SignificanceRapid clonal expansion of antigen-specific T cells is a fundamental feature of adaptive immune responses. Here, we utilize continuous live-cell imagingin vitroto track the division speed and genealogical connections of all descendants derived from a single naïve CD8+T cell throughout up to ten divisions of activation-induced proliferation. Bayesian inference of tree-structured data reveals that clonal expansion is divided into a homogenously fast burst phase encompassing two to three divisions and a subsequent diversification phase during which T cells segregate into quickly dividing effector T cells and more slowly cycling memory precursors. Our work highlights cell cycle speed as a major heritable property that is regulated in parallel to key lineage decisions of activated T cells.

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., Nature Communications, 13, ISSN:2041-1723

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., Journal of Experimental Medicine, 218 (6), ISSN:0022-1007, ISSN:1540-0069, ISSN:1540-9538

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.

Summary The generation of humanized ectopic ossicles (hOss) in mice has been proposed as an advanced translational and fundamental model to study the human hematopoietic system. The approach relies on the presence of human bone marrow-derived mesenchymal stromal cells (hMSCs) supporting the engraftment of transplanted human hematopoietic stem and progenitor cells (HSPCs). However, the functional distribution of hMSCs within the humanized microenvironment remains to be investigated. Here, we combined genetic tools and quantitative confocal microscopy to engineer and subsequently analyze hMSCs′ fate and distribution in hOss. Implanted hMSCs reconstituted a humanized environment including osteocytes, osteoblasts, adipocytes, and stromal cells associated with vessels. By imaging full hOss, we identified rare physical interactions between hMSCs and human CD45+/CD34+/CD90+ cells, supporting a functional contact-triggered regulatory role of hMSCs. Our study highlights the importance of compiling quantitative information from humanized organs, to decode the interactions between the hematopoietic and the stromal compartments., Graphical Abstract, Highlights • Mesenchymal cells can generate human bone organs with tailored molecular signature • Mesenchymal cells reconstitute a human niche environment capable of regulating HSPCs, Biological Sciences; Stem Cells Research; Tissue Engineering

Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation in vivo by reducing lipid peroxides to the respective alcohols thereby stabilizing oxidation products of unsaturated fatty acids. During reticulocyte maturation, lipid peroxidation mediated by 15-lipoxygenase in humans and rabbits and by 12/15-lipoxygenase (ALOX15) in mice was considered the initiating event for the elimination of mitochondria but is now known to occur through mitophagy. Yet, genetic ablation of the Alox15 gene in mice failed to provide evidence for this hypothesis. We designed a different genetic approach to tackle this open conundrum. Since either other lipoxygenases or non-enzymatic autooxidative mechanisms may compensate for the loss of Alox15, we asked whether ablation of Gpx4 in the hematopoietic system would result in the perturbation of reticulocyte maturation. Quantitative assessment of erythropoiesis indices in the blood, bone marrow (BM) and spleen of chimeric mice with Gpx4 ablated in hematopoietic cells revealed anemia with an increase in the fraction of erythroid precursor cells and reticulocytes. Additional dietary vitamin E depletion strongly aggravated the anemic phenotype. Despite strong extramedullary erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. Gpx4-deficiency in hematopoietic cells led to systemic hepatic iron overload and simultaneous severe iron demand in the erythroid system. Despite extremely high erythropoietin and erythroferrone levels in the plasma, hepcidin expression remained unchanged. Conclusively, perturbed reticulocyte maturation in response to Gpx4 loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation., Haematologica, 105 (4), ISSN:0390-6078, ISSN:1592-8721

We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone. © 2021 American Society of Hematology ISSN:0006-4971 ISSN:1528-0020

G protein–coupled receptor 182 (GPR182) has been shown to be expressed in endothelial cells; however, its ligand and physiological role has remained elusive. We found GPR182 to be expressed in microvascular and lymphatic endothelial cells of most organs and to bind with nanomolar affinity the chemokines CXCL10, CXCL12, and CXCL13. In contrast to conventional chemokine receptors, binding of chemokines to GPR182 did not induce typical downstream signaling processes, including Gq- and Gi-mediated signaling or β-arrestin recruitment. GPR182 showed relatively high constitutive activity in regard to β-arrestin recruitment and rapidly internalized in a ligand-independent manner. In constitutive GPR182-deficient mice, as well as after induced endothelium-specific loss of GPR182, we found significant increases in the plasma levels of CXCL10, CXCL12, and CXCL13. Global and induced endothelium-specific GPR182-deficient mice showed a significant decrease in hematopoietic stem cells in the bone marrow as well as increased colony-forming units of hematopoietic progenitors in the blood and the spleen. Our data show that GPR182 is a new atypical chemokine receptor for CXCL10, CXCL12, and CXCL13, which is involved in the regulation of hematopoietic stem cell homeostasis., Proceedings of the National Academy of Sciences of the United States of America, 118 (17), ISSN:0027-8424, ISSN:1091-6490

SummaryIt is critical to understand how quiescent long-term hematopoietic stem cells (LT-HSC) sense demand from daily and stress-mediated cues and transition into bioenergetically active progeny to differentiate and meet these cellular needs. Here, we show that lysosomes, which are sophisticated nutrient sensing and signaling centers, are dichotomously regulated by the Transcription Factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSC and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, which promotes quiescence, self-renewal and governs erythroid-myeloid commitment. By contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism to drive LT-HSC activation. Collectively, our study identifies lysosomes as a central regulatory hub for proper and coordinated stem cell fate determination.

E-cadherin is a calcium-dependent cell-cell adhesion molecule extensively studied for its involvement in tissue formation, epithelial cell behavior, and suppression of cancer. However, E-cadherin expression in the hematopoietic system has not been fully elucidated. Combining single-cell RNA-sequencing analyses and immunophenotyping, we revealed that progenitors expressing high levels of E-cadherin and contained within the granulocyte-monocyte progenitors (GMPs) fraction have an enriched capacity to differentiate into basophils and mast cells. We detected E-cadherin expression on committed progenitors before the expression of other reported markers of these lineages. We named such progenitors pro-BMPs (pro-basophil and mast cell progenitors). Using RNA sequencing, we observed transcriptional priming of pro-BMPs to the basophil and mast cell lineages. We also showed that GATA-2 directly regulates E-cadherin expression in the basophil and mast cell lineages, thus providing a mechanistic connection between the expression of this cell surface marker and the basophil and mast cell fate specification. ISSN:2470-9468

Hausmann et al. describe a sentinel intercrypt macrophage population in the intestine, which detects Gram-negative microbes breaching the epithelial barrier and elicits epithelial NF-κB signaling in neighboring crypts. The tunability of this crypt-scale response allows the induction of appropriately scaled defenses according to the localization and intensity of microbial triggers., Intestinal epithelial cell (IEC) NF-κB signaling regulates the balance between mucosal homeostasis and inflammation. It is not fully understood which signals tune this balance and how bacterial exposure elicits the process. Pure LPS induces epithelial NF-κB activation in vivo. However, we found that in mice, IECs do not respond directly to LPS. Instead, tissue-resident lamina propria intercrypt macrophages sense LPS via TLR4 and rapidly secrete TNF to elicit epithelial NF-κB signaling in their immediate neighborhood. This response pattern is relevant also during oral enteropathogen infection. The macrophage–TNF–IEC axis avoids responses to luminal microbiota LPS but enables crypt- or tissue-scale epithelial NF-κB responses in proportion to the microbial threat. Thereby, intercrypt macrophages fulfill important sentinel functions as first responders to Gram-negative microbes breaching the epithelial barrier. The tunability of this crypt response allows the induction of defense mechanisms at an appropriate scale according to the localization and intensity of microbial triggers., Graphical Abstract

Cells constantly sense their environment, allowing the adaption of cell behavior to changing needs. Fine-tuned responses to complex inputs are computed by signaling pathways, which are wired in complex connected networks. Their activity is highly context-dependent, dynamic, and heterogeneous even between closely related individual cells. Despite lots of progress, our understanding of the precise implementation, relevance, and possible manipulation of cellular signaling in health and disease therefore remains limited. Here, we discuss the requirements, potential, and limitations of the different current technologies for the analysis of hematopoietic stem and progenitor cell signaling and its effect on cell fates., Journal of Experimental Medicine, 218 (7), ISSN:0022-1007, ISSN:1540-0069, ISSN:1540-9538

It is critical to understand how human quiescent long-term hematopoietic stem cells (LT-HSCs) sense demand from daily and stress-mediated cues and then transition into bioenergetically active progeny to differentiate and meet these cellular needs. However, the demand-adapted regulatory circuits of these early steps of hematopoiesis are largely unknown. Here we show that lysosomes, sophisticated nutrient-sensing and signaling centers, are regulated dichotomously by transcription factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSCs and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, promoting quiescence and self-renewal, and governing erythroid-myeloid commitment. In contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism, driving LT-HSC activation. Our study identifies TFEB-mediated control of lysosomal activity as a central regulatory hub for proper and coordinated stem cell fate determination., Cell Stem Cell, 28 (10), ISSN:1934-5909, ISSN:1875-9777

Acute myeloid leukemia (AML) is a caricature of normal hematopoiesis driven from leukemia stem cells (LSC) that share some hematopoietic stem cell (HSC) programs including responsiveness to inflammatory signaling. Although inflammation dysregulates mature myeloid cells and influences stemness programs and lineage determination in HSCs by activating stress myelopoiesis, such roles in LSCs are poorly understood. Here, we show that S1PR3, a receptor for the bioactive lipid sphingosine-1-phosphate, is a central regulator that drives myeloid differentiation and activates inflammatory programs in both HSCs and LSCs. S1PR3-mediated inflammatory signatures varied in a continuum from primitive to mature myeloid states across cohorts of patients with AML, each with distinct phenotypic and clinical properties. S1PR3 was high in LSCs and blasts of mature myeloid samples with linkages to chemosensitivity, whereas S1PR3 activation in primitive samples promoted LSC differentiation leading to eradication. Our studies open new avenues for therapeutic target identification specific for each AML subset. Significance: S1PR3 is a novel regulator of myeloid fate in normal hematopoiesis that is heterogeneously expressed in AML. S1PR3 marks a subset of less primitive AML cases with a distinct inflammatory signature and therefore has clinical implications as both a therapeutic target and a biomarker to distinguish primitive from mature AML. See related commentary by Yang et al., p. 3. This article is highlighted in the In This Issue feature, p. 1

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.

SummaryLoss of hematopoietic stem cell (HSC) quiescence and resulting clonal expansion are common initiating events in the development of hematological malignancy. Likewise, chronic inflammation related to aging, disease and/or tissue damage is associated with leukemia progression, though its role in oncogenesis is not clearly defined. Here, we show that PU.1-dependent repression of protein synthesis and cell cycle genes in HSC enforces homeostatic protein synthesis levels and HSC quiescence in response to IL-1 stimulation. These genes are constitutively de-repressed in PU.1-deficient HSC, leading to activation of protein synthesis, loss of quiescence and aberrant expansion of HSC. Taken together, our data identify a mechanism whereby HSC regulate their cell cycle activity and pool size in response to chronic inflammatory stress.

Summary 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., Highlights • A novel GATA2VENUS fusion mouse line to report GATA2 protein expression • VENUS fusion does not alter GATA2 expression or disturb development or homeostasis • GATA2 expression identifies functionally distinct HSPC subpopulations • GATA2 expression unveils an earlier monocyte-mast cell lineage bifurcation point, In this study, Schroeder and colleagues generated a GATA2VENUS protein reporter mouse line with normal GATA2 expression and localization, e.g., in the urogenital, auditory, and nervous systems. The authors also profiled GATA2 protein expression across hematopoietic cell types, identified heterogeneity within populations currently assumed to be homogeneous, and demonstrate an earlier monocyte-mast cell lineage bifurcation point.

Intestinal epithelial cell (IEC) NFκB signaling regulates the fine balance between mucosal homeostasis and inflammation. It is not fully understood which signals tune this balance, and how bacterial exposure elicits the process. Pure LPS induces epithelial NFκB activation in vivo. We found that in mice, IECs do not respond directly to LPS. Instead, CD11b+ CD103- intercrypt DCs sense LPS via TLR4 and secrete TNF to elicit epithelial NFκB signaling in their immediate neighborhood. This response is relevant also during oral enteropathogen infection. The DC-TNF-IEC axis avoids responses to luminal microbiota LPS, but enables localized or tissue-wide epithelial NFκB responses in proportion to the microbial threat. Thereby, the CD11b+ CD103- intercrypt DCs serve as important sentinels for Gram-negative microbes breaching the epithelial barrier. The tunability of this crypt response allows induction of defense mechanisms at an appropriate scale according to localization and intensity of microbial triggers.

Due to the inherent imperfections in the optical path, microscopy images, particularly fluorescence microscopy images, are often skewed by uneven illumination and hence have spurious intensity variation, also known as shading or vignetting effect. Besides spatial intensity inhomogeneity, time-lapse microscopy imaging further suffers from background variation in time, mostly due to photo-bleaching of the background medium. Moreover, the temporal background variation is often experiment-specific and hence cannot be easily corrected, in contrast to shading, where a prospective calibration method can be used. Existing retrospective illumination correction methods, ranging from simple multi-image averaging to sophisticated optimisation based methods such as CIDRE and BaSiC, all assume that the foreground of all images is uncorrelated between each other. However, this assumption is violated in e.g. long-term time-lapse microscopy imaging of adherent stem cells, in which a strong foreground correlation is observed from frame to frame. In this paper, we propose a new illumination and background correction method for time-lapse imaging, based on low-rank and sparse decomposition. We incorporate binary segmentation masks that inform the weighting scheme of our reweighted \(L_1\) norm minimisation about foreground vs background pixels in the image. This yields a better separation of the low-rank and sparse component, hence improving the estimation of illumination profiles. Experiments on both simulated and real time-lapse data demonstrate that our approach is superior to existing illumination correction methods and improves single cell quantification.

Highlights Highlights • Statistical tool for single cell lineage tree analysis • Identification of stem cell-specific lineage trees • Statistically identified murine hematopoietic stem cells display known stem cell characteristics • First step towards novel accurate in-vitro hematopoietic stem cell identification, Stem cells play a central role in the regeneration and repair of multicellular organisms. However, it remains far from trivial to reliably identify them. Despite decades of work, current techniques to isolate hematopoietic stem cells (HSCs) based on cell-surface markers only result in 50% purity, i.e. half of the sorted cells are not stem cells when functionally tested. Modern microscopy techniques allow us to follow single cells and their progeny for up to weeks in vitro, while recording the cell fates and lifetime of each individual cell. This cell tracking generates so-called lineage trees. Here, we propose statistical techniques to determine if the initial cell in a lineage tree was a HSC. We apply these techniques to murine hematopoietic lineage trees, revealing that 18% of the trees in our HSC dataset display a unique signature, and this signature is compatible with these trees having started from a true stem cell. Assuming 50% purity of HSC empirical datasets, this corresponds to a 0.35 power of the test, and the type-1-error is estimated to be 0.047. In summary, this study shows that statistical analysis of lineage trees could improve the classification of cells, which is currently done based on bio-markers only. Our statistical techniques are not limited to mammalian stem cell biology. Any type of single cell lineage trees, be it from bacteria, single cell eukaryotes, or single cells in a multicellular organism can be investigated. We expect this to contribute to a better understanding of the molecules influencing cellular dynamics at the single cell level.

Summary Circulating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, and the latter display an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the shedding of CTC clusters from a primary cancerous lesion are poorly understood. Here, when dynamically labeling breast cancer cells along cancer progression, we observe that the majority of CTC clusters are undergoing hypoxia, while single CTCs are largely normoxic. Strikingly, we find that vascular endothelial growth factor (VEGF) targeting leads to primary tumor shrinkage, but it increases intra-tumor hypoxia, resulting in a higher CTC cluster shedding rate and metastasis formation. Conversely, pro-angiogenic treatment increases primary tumor size, yet it dramatically suppresses the formation of CTC clusters and metastasis. Thus, intra-tumor hypoxia leads to the formation of clustered CTCs with high metastatic ability, and a pro-angiogenic therapy suppresses metastasis formation through prevention of CTC cluster generation., Graphical Abstract, Highlights • Hypoxia leads to cell-cell junction upregulation and intravasation of CTC clusters • Hypoxic CTC clusters are highly metastatic compared to normoxic CTCs • Increase in intra-tumor hypoxia leads to accelerated metastasis • Treatment with EpB2 reduces hypoxia and prevents CTC cluster formation, Donato et al. show that intra-tumor hypoxia leads to cell-cell junction upregulation and formation of hypoxic CTC clusters with high metastatic ability. Treatment with EphrinB2 improves tumor vascularization and decreases hypoxia, leading to a reduced CTC cluster shedding rate and suppression of metastasis.

Keeping track of individual cell identifications is imperative to the study of dynamic single-cell behavior over time. Highly motile hematopoietic stem and progenitor cells (HSPCs) migrate quickly and do not adhere, and thus must be imaged very frequently to keep cell identifications. Even worse, they are also flushed away during medium exchange. To overcome these limitations, we tested antibody coating for reducing HSPC motility in vitro. Anti-CD43- and anti-CD44-antibody coating reduced the cell motility of mouse and human HSPCs in a concentration-dependent manner. This enables 2-dimensional (2D) colony formation without cell mixing in liquid cultures, massively increases time-lapse imaging throughput, and also maintains cell positions during media exchange. Anti-CD43 but not anti-CD44 coating reduces mouse HSPC proliferation with increasing concentrations. No relevant effects on cell survival or myeloid and megakaryocyte differentiation of hematopoietic stem cells and multipotent progenitors 1-5 were detected. Human umbilical cord hematopoietic CD34+ cell survival, proliferation, and differentiation were not affected by either coating. This approach both massively simplifies and accelerates continuous analysis of suspension cells, and enables the study of their behavior in dynamic rather than static culture conditions over time.

Recent advances in engineering complex organs in vitro inspire the development of human bone marrow equivalents to foster scientific discovery and innovative therapeutics. Here, we discuss challenges in generating relevant human bone marrow proxies, potential design principles, and future directions.

Stem cells play a central role in the regeneration and repair of multicellular organisms. However, it remains far from trivial to reliably identify them. Despite decades of work, current techniques to isolate hematopoietic stem cells (HSCs) based on cell-surface markers only result in 50% purity, i.e. half of the sorted cells are not stem cells when functionally tested. Modern microscopy techniques allow us to follow single cells and their progeny for up to weeks in vitro, while recording the cell fates and lifetime of each individual cell. This cell tracking generates so-called lineage trees. Here, we propose statistical techniques to determine if the initial cell in a lineage tree was a HSC. We apply these techniques to murine hematopoietic lineage trees, revealing that 18% of the trees in our HSC dataset display a unique signature, and this signature is compatible with these trees having started from a true stem cell. Assuming 50% purity of HSC empirical datasets, this corresponds to a 0.35 power of the test, and the type-1-error is estimated to be 0.047. In summary, this study shows that statistical analysis of lineage trees could improve the classification of cells, which is currently done based on bio-markers only. Our statistical techniques are not limited to mammalian stem cell biology. Any type of single cell lineage trees, be it from bacteria, single cell eukaryotes, or single cells in a multicellular organism can be investigated. We expect this to contribute to a better understanding of the molecules influencing cellular dynamics at the single cell level., Journal of Theoretical Biology, 439, ISSN:0022-5193, ISSN:1095-8541

Multicolor 3D quantitative imaging of large tissue volumes is necessary to understand tissue development and organization as well as interactions between distinct cell types in situ. However, tissue imaging remains technically challenging, particularly imaging of bone and marrow. Here, we describe a pipeline to reproducibly generate high-dimensional quantitative data from bone and bone marrow that may be extended to any tissue. We generate thick bone sections from adult mouse femurs with preserved tissue microarchitecture and demonstrate eight-color imaging using confocal microscopy without linear unmixing. We introduce XiT, an open-access software for fast and easy data curation, exploration and quantification of large imaging data sets with single-cell resolution. We describe how XiT can be used to correct for potential artifacts in quantitative 3D imaging, and we use the pipeline to measure the spatial relationship between hematopoietic cells, bone matrix and marrow Schwann cells.

Inflammation underlies many chronic diseases of aging, involves dysregulated myeloid cells, and has emerged as a critical process regulating both normal hematopoietic stem cell (HSC) function and myeloid malignancy. However, few of the signaling pathways that underlie these proposed linkages are known. Here, we show that bioactive lipid signaling through sphingosine 1-phosphate receptor 3 (S1PR3) regulates human myeloid lineage determination via inflammatory programs and is dysregulated in acute myeloid leukemia (AML). S1PR3 is myeloid-restricted and not expressed by quiescent HSC, but upon overexpression (OE) promoted myelopoiesis and activated inflammatory signatures. S1PR3 is deficient in primitive AML, but enriched in more mature cases. Importantly, in functionally-validated primary leukemia stem cells (LSC) sorted for high S1PR3 expression or when differentiation was induced through S1PR3OE exhibited reduced xenograft repopulating ability. Treatment of recipient mice engrafted with the S1P prodrug FTY720 reduced patient AML LSC frequency. Thus, S1PR3 marks a subset of less primitive AML cells with a distinct inflammatory signature, and activation of S1PR3 may represent a novel therapeutic approach to disrupt LSC function through induction of differentiation.

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.

Summary Technological limitations have hampered understanding of how individual molecules, including putative stem cell regulators, are distributed throughout tissues and stem cell niches. Here, we report adaptation of the proximity ligation assay (PLA) for large-volume, in situ imaging of individual proteins with multiple additional fluorescent channels with integrated 3D quantification strategies and software. Using this platform, we quantified the bone marrow (BM) distribution of individual CXCL12 chemokine proteins, both before and after their depletion by granulocyte-colony stimulating factor (G-CSF) treatment. We found ubiquitous CXCL12 distributions with local enrichments but no long-range gradients, in contrast to current assumptions about how CXCL12 controls migration of hematopoietic stem and progenitor cells (HSPCs) within BM. This pipeline for discrete digital quantitative, large-volume, multicolor imaging, with up to single-molecule sensitivity, may be broadly applied to any antibody epitope and tissue, enabling further insights into molecular organization of tissues and cellular interactions.

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. The cellular degradative machinery can be asymmetrically inherited upon haematopoietic-stem-cell division, which predicts the future metabolic and translational activation of their daughter cells.

Continuous analysis of single cells, over several cell divisions and for up to weeks at a time, is crucial to deciphering rare, dynamic and heterogeneous cell responses, which would otherwise be missed by population or single-cell snapshot analysis. Although the field of long-term single-cell imaging, tracking and analysis is constantly advancing, several technical challenges continue to hinder wider implementation of this important approach. This is a particular problem for mammalian cells, where in vitro observation usually remains the only possible option for uninterrupted long-term, single-cell observation. Efforts must focus not only on identifying and maintaining culture conditions that support normal cellular behavior while allowing high-resolution imaging over time, but also on developing computational methods that enable semiautomatic analysis of the data. Solutions in microscopy hard- and software, computer vision and specialized theoretical methods for analysis of dynamic single-cell data will enable important discoveries in biology and beyond.

Software tools for single-cell tracking and quantification of cellular and molecular properties

Stem cell identity depends on the integration of extrinsic and intrinsic signals, which directly influence the maintenance of their epigenetic state. Although Myc transcription factors play a major role in stem cell self-renewal and pluripotency, their integration with signalling pathways and epigenetic regulators remains poorly defined. We addressed this point by profiling the gene expression and epigenetic pattern in ESCs whose growth depends on conditional Myc activity. Here we show that Myc potentiates the Wnt/β-catenin signalling pathway, which cooperates with the transcriptional regulatory network in sustaining ESC self-renewal. Myc activation results in the transcriptional repression of Wnt antagonists through the direct recruitment of PRC2 on these targets. The consequent potentiation of the autocrine Wnt/β-catenin signalling induces the transcriptional activation of the endogenous Myc family members, which in turn activates a Myc-driven self-reinforcing circuit. Thus, our data unravel a Myc-dependent self-propagating epigenetic memory in the maintenance of ESC self-renewal capacity., The Myc transcription factor is a major regulator of stem cell (SC) self-renewal and pluripotency but how this integrates signals from other pathways is unclear. Here, the authors show that Myc activation triggers epigenetic memory in self renewing embryonic SCs via PRC2-mediated potentiation of the Wnt pathway.

Despite the widespread interest in direct neuronal reprogramming, the mechanisms underpinning fate conversion remain largely unknown. Our study revealed a critical time point after which cells either successfully convert into neurons or succumb to cell death. Co-transduction with Bcl-2 greatly improved negotiation of this critical point by faster neuronal differentiation. Surprisingly, mutants with reduced or no affinity for Bax demonstrated that Bcl-2 exerts this effect by an apoptosis-independent mechanism. Consistent with a caspase-independentrole, ferroptosis inhibitors potently increased neuronal reprogramming by inhibiting lipid peroxidation occurring during fate conversion. Genome-wide expression analysis confirmed that treatments promoting neuronal reprogramming elicit an anti-oxidative stress response. Importantly, co-expression of Bcl-2 and anti-oxidative treatments leads to an unprecedented improvement in glial-to-neuron conversion after traumatic brain injury invivo, underscoring the relevance of these pathways in cellular reprograming irrespective of cell type invitro and invivo.

Maintaining cellular redox balance is vital for cell survival and tissue homoeostasis because imbalanced production of reactive oxygen species (ROS) may lead to oxidative stress and cell death. The antioxidant enzyme glutathione peroxidase 4 (Gpx4) is a key regulator of oxidative stress-induced cell death. We show that mice with deletion of Gpx4 in hematopoietic cells develop anemia and that Gpx4 is essential for preventing receptor-interacting protein 3 (RIP3)-dependent necroptosis in erythroid precursor cells. Absence of Gpx4 leads to functional inactivation of caspase 8 by glutathionylation, resulting in necroptosis, which occurs independently of tumor necrosis factor α activation. Although genetic ablation of Rip3 normalizes reticulocyte maturation and prevents anemia, ROS accumulation and lipid peroxidation in Gpx4-deficient cells remain high. Our results demonstrate that ROS and lipid hydroperoxides function as not-yet-recognized unconventional upstream signaling activators of RIP3-dependent necroptosis.

Hematopoietic stem cells (HSCs) maintain blood cell production life-long by their unique abilities of self-renewal and differentiation into all blood cell lineages. Growth arrest and DNA-damage-inducible 45 alpha (GADD45A) is induced by genotoxic stress in HSCs. GADD45A has been implicated in cell cycle control, cell death and senescence, as well as in DNA-damage repair. In general, GADD45A provides cellular stability by either arresting the cell cycle progression until DNA damage is repaired or, in cases of fatal damage, by inducing apoptosis. However, the function of GADD45A in hematopoiesis remains controversial. We revealed the changes in murine HSC fate control orchestrated by the expression of GADD45A at single cell resolution. In contrast to other cellular systems, GADD45A expression did not cause a cell cycle arrest or an alteration in the decision between cell survival and apoptosis in HSCs. Strikingly, GADD45A strongly induced and accelerated the differentiation program in HSCs. Continuous tracking of individual HSCs and their progeny via time-lapse microscopy elucidated that once GADD45A was expressed, HSCs differentiate into committed progenitors within 29 hours. GADD45A-expressing HSCs failed to long-term reconstitute the blood of recipients by inducing multilineage differentiation in vivo. Importantly, γ-irradiation of HSCs induced their differentiation by upregulating endogenous GADD45A. The differentiation induction by GADD45A was transmitted by activating p38 Mitogen-activated protein kinase (MAPK) signaling and allowed the generation of megakaryocytic-erythroid, myeloid, and lymphoid lineages. These data indicate that genotoxic stress-induced GADD45A expression in HSCs prevents their fatal transformation by directing them into differentiation and thereby clearing them from the system.

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. [Figure: see text]

Human long-term hematopoietic stem cells (LT-HSC) residing at the top of the hematopoietic hierarchy must meet enormous daily demand (~10e11 cells daily) while also sustaining life-long maintenance of the stem cell pool through self-renewal. This hierarchical organization is widely thought to protect LT-HSC from exhaustion by maintaining them in a quiescent and undifferentiated state, activating only in response to microenvironment signals to generate highly proliferative but more short-lived populations including short-term HSC (ST-HSC) and committed progenitors. When called upon to exit this dormant state, HSC must respond and adapt their metabolism and nutrient uptake to meet increased bioenergetic demands for cell growth and differentiation. At the same time, the events underlying cellular and metabolic activation must also be suppressed to allow LT-HSC to re-enter quiescence and ultimately maintain the LT-HSC pool through self-renewal. Thus, proper sensing of cellular output demands must be coordinated with the cell cycle and metabolic machinery of LT-HSC to balance stem cell fates and maintain hematopoietic homeostasis. However, the regulatory circuits of this demand-adapted regulation of early hematopoiesis are largely unknown. The ability of cells to receive signals or take up nutrients depends on proteins that are embedded within the plasma membrane. These proteins move to the cell's interior through endocytosis and can be degraded in the lysosomes or rerouted back to the cell surface and reused. Moreover, lysosomes are the terminal catabolic stations of the autophagy pathway that is essential for preserving stem cell function through clearance of toxic cellular components. However, little is known about the regulation and role of lysosomes in the stem cell context. Here, we describe the unexpected finding that lysosomes, whose activity is intricately balanced by TFEB and MYC, are instrumental for regulating the stemness and differentiation properties of human LT-HSC. Furthermore, we found that TFEB, which is normally implicated in stress response, induces a constitutive lysosomal flux in unperturbed LT-HSC that actively maintains quiescence, preserves self-renewal and governs lineage commitment. These effects are accompanied by endolysosomal degradation of membrane receptors, such as the transferrin receptor 1 (TfR1), pointing to a role for TFEB in coordinating how LT-HSC sense environmental changes and initiate the earliest steps of their fate transitions and lineage commitment decisions. These transitions are regulated by a TFEB/MYC dichotomy where MYC is a driver of LT-HSC anabolism and activation and counteracts TFEB function by serving as a negative transcriptional regulator of lysosomes. Moreover, our findings further suggest that active suppression of TFEB and its downstream lysosomal degradation of TfR1 within LT-HSC is required for commitment along the erythroid lineage: activation of TFEB can abolish erythroid differentiation even after lineage commitment has occurred. In summary, we uncovered a MYC-TFEB-mediated dichotomous regulation of lysosomal activity that is required to balance anabolic and catabolic processes that ultimately impact human LT-HSC fate determination. Figure Disclosures Takayanagi: Kirin Holdings Company, Ltd: Current Employment. Dick:Bristol-Myers Squibb/Celgene: Research Funding.

Hematopoietic stem cell (HSC) quiescence supports lifelong blood regeneration and guards against pre-leukemic clonal expansion. Acute exposure to the pro-inflammatory cytokine interleukin (IL)-1 drives myeloid cell production and HSC cell cycle entry. However, HSC return to a quiescent state suggesting the presence of a ‘braking’ mechanism that limits HSC proliferative capacity during chronic inflammation. To identify mechanism(s) regulating HSC cell cycle activity, we injected mice with IL-1 for 20 days modeling chronic inflammation in vivo. RNA-seq analysis of HSC following IL-1 exposure revealed repression of cell cycle and protein synthesis genes, suggesting the activation of a ‘growth arrest’ gene program. This gene program coincided with increased PU.1 expression, and ChIP-seq analysis identified PU.1 binding on nearly all repressed genes, suggesting PU.1 enforces HSC quiescence during chronic inflammation. Strikingly, HSC from IL-1 treated PU.1-deficient mice exhibited loss of quiescence associated with aberrant myeloid expansion in the bone marrow and spleen. Together, our results suggest PU.1 induction maintains HSC quiescence under chronic inflammatory stress. They also suggest IL-1 may confer a competitive advantage to HSC with impaired PU.1 function, triggering aberrant proliferation and myeloid expansion. Several oncogenic mutations found in acute myelogenous leukemia (AML) impair the expression and/or function of PU.1. As AML pre-dominantly affects elderly individuals and leukemogenesis is often associated with chronic inflammation, our data supports a model where chronic inflammation triggers the selective expansion of HSC harboring oncogenic mutations, leading to a pre-leukemic state. Thus, blockade of inflammation may be a tractable approach to delay and/or prevent leukemia.

Effective manipulation of clinically relevant umbilical cord blood-derived human hematopoietic stem cell (hHSC) fate decisions remains an important goal for improved therapies. Based on observations in other model organisms, it was postulated that asymmetric cell division (ACD) regulates hHSC fate decisions of self-renewal and differentiation. In ACD, daughter cell fates are determined during cell division, e.g., by the asymmetric inheritance of cell fate determinants. Previous studies suggested the asymmetric inheritance of factors during progenitor divisions. However, due to the lack of tools for sufficient prospective hHSC enrichment, quantification of asymmetric inheritance and future daughter cell behaviors in the same experiment, hHSC ACD remained hypothetical. Here, we use continuous quantitative long-term single-cell imaging of highly purified hHSCs to identify factors and organelles that are asymmetrically inherited during hHSC divisions. Importantly, this asymmetric inheritance correlates with future asymmetric adhesion, motility, cell cycle progression, marker production, and metabolic activity in hHSC daughters. We further show, using novel real-time computational differentiation landscapes and in-silico hHSC purification, that this asymmetric inheritance also correlates with future expression of stem cell markers, and that culture conditions known to expand hHSCs increase symmetric cell divisions. We thus provide the first direct quantitative evidence that ACD exists in human hematopoietic stem and progenitor cells and modulates future daughter cells fates by the inheritance of factors during division. We provide insights into the underlying molecular mechanisms, and the exciting possibility to manipulate ACD and hHSC fate decision for improved therapy.

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.