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An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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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.
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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.
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Linfócitos T CD8-Positivos/citologia , Linhagem da Célula , Animais , Antígenos CD/imunologia , Biomarcadores , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Divisão Celular , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
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Divisão Celular Assimétrica , Células-Tronco Hematopoéticas , Animais , Diferenciação Celular/genética , Divisão Celular , Endossomos , Humanos , CamundongosRESUMO
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.
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Técnicas de Cultura de Células , Citocinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas , Antígenos Comuns de Leucócito/biossíntese , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , MasculinoRESUMO
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.
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Diferenciação Celular , Linhagem da Célula , Fator de Transcrição GATA1/metabolismo , Células Mieloides/citologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Eritrócitos/citologia , Retroalimentação Fisiológica , Feminino , Genes Reporter , Granulócitos/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Masculino , Megacariócitos/citologia , Camundongos , Modelos Biológicos , Monócitos/citologia , Reprodutibilidade dos Testes , Análise de Célula Única , Processos EstocásticosRESUMO
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.
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Divisão Celular Assimétrica , Diferenciação Celular , Autorrenovação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Autofagossomos , Proliferação de Células , Metabolismo Energético , Humanos , Lisossomos , Mitofagia , Transdução de SinaisRESUMO
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.
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Hematopoese , Análise de Célula Única/métodos , Animais , Contagem de Células/métodos , Rastreamento de Células/métodos , Biologia Computacional/métodos , Células-Tronco Hematopoéticas/citologia , Humanos , Células Mieloides/citologiaRESUMO
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.
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Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Mielopoese , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células-Tronco Hematopoéticas/patologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Nicho de Células-TroncoRESUMO
Differentiation alters molecular properties of stem and progenitor cells, leading to changes in their shape and movement characteristics. We present a deep neural network that prospectively predicts lineage choice in differentiating primary hematopoietic progenitors using image patches from brightfield microscopy and cellular movement. Surprisingly, lineage choice can be detected up to three generations before conventional molecular markers are observable. Our approach allows identification of cells with differentially expressed lineage-specifying genes without molecular labeling.
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Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Redes Neurais de Computação , Imagem com Lapso de Tempo/métodos , Animais , Área Sob a Curva , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Técnicas de Introdução de Genes , Aprendizado de Máquina , Masculino , Camundongos Mutantes , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/genética , Transativadores/metabolismoAssuntos
Folclore , Mitógenos , Humanos , Envelhecimento , Pontos de Checagem do Ciclo Celular , Proliferação de CélulasRESUMO
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.
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Anticorpos/farmacologia , Células Imobilizadas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptores de Hialuronatos/antagonistas & inibidores , Leucossialina/antagonistas & inibidores , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Imobilizadas/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Masculino , CamundongosRESUMO
Recent advances in single-cell technologies have permitted the investigation of heterogeneous cell populations at previously unattainable resolution. Here we apply such approaches to resolve the molecular mechanisms driving disease in mouse hematopoietic stem cells (HSCs), using JAK2V617F mutant myeloproliferative neoplasms (MPNs) as a model. Single-cell gene expression and functional assays identified a subset of JAK2V617F mutant HSCs that display defective self-renewal. This defect is rescued at the single HSC level by crossing JAK2V617F mice with mice lacking TET2, the most commonly comutated gene in patients with MPN. Single-cell gene expression profiling of JAK2V617F-mutant HSCs revealed a loss of specific regulator genes, some of which were restored to normal levels in single TET2/JAK2 mutant HSCs. Of these, Bmi1 and, to a lesser extent, Pbx1 and Meis1 overexpression in JAK2-mutant HSCs could drive a disease phenotype and retain durable stem cell self-renewal in functional assays. Together, these single-cell approaches refine the molecules involved in clonal expansion of MPNs and have broad implications for deconstructing the molecular network of normal and malignant stem cells.
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Autorrenovação Celular , Regulação Neoplásica da Expressão Gênica , Neoplasias Hematológicas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Janus Quinase 2/metabolismo , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Substituição de Aminoácidos , Animais , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Células-Tronco Hematopoéticas/patologia , Janus Quinase 2/genética , Camundongos , Camundongos Transgênicos , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologiaRESUMO
Controlled regulation of lineage decisions is imperative for hematopoiesis. Yet, the molecular mechanisms underlying hematopoietic lineage choices are poorly defined. Colony-stimulating factor 1 (CSF-1), the cytokine acting as the principal regulator of monocyte/macrophage (M) development, has been shown to be able to instruct the lineage choice of uncommitted granulocyte M (GM) progenitors toward an M fate. However, the intracellular signaling pathways involved are unknown. CSF-1 activates a multitude of signaling pathways resulting in a pleiotropic cellular response. The precise role of individual pathways within this complex and redundant signaling network is dependent on cellular context, and is not well understood. Here, we address which CSF-1-activated pathways are involved in transmitting the lineage-instructive signal in primary bone marrow-derived GM progenitors. Although its loss is compensated for by alternative signaling activation mechanisms, Src family kinase (SFK) signaling is sufficient to transmit the CSF-1 lineage instructive signal. Moreover, c-Src activity is sufficient to drive M fate, even in nonmyeloid cells.
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Linhagem da Célula , Fator Estimulador de Colônias de Macrófagos/fisiologia , Monócitos/citologia , Transdução de Sinais , Quinases da Família src/metabolismo , Animais , Células Cultivadas , Células Precursoras de Granulócitos/citologia , Hematopoese , CamundongosRESUMO
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.
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Rastreamento de Células/métodos , Células-Tronco Hematopoéticas/citologia , Dispositivos Lab-On-A-Chip , Células-Tronco Embrionárias Murinas/citologia , Análise de Célula Única/métodos , Animais , Adesão Celular , Células Cultivadas , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/metabolismo , Estudo de Prova de Conceito , Reprodutibilidade dos Testes , Imagem com Lapso de TempoRESUMO
MOTIVATION: Quantitative large-scale cell microscopy is widely used in biological and medical research. Such experiments produce huge amounts of image data and thus require automated analysis. However, automated detection of cell outlines (cell segmentation) is typically challenging due to, e.g. high cell densities, cell-to-cell variability and low signal-to-noise ratios. RESULTS: Here, we evaluate accuracy and speed of various state-of-the-art approaches for cell segmentation in light microscopy images using challenging real and synthetic image data. The results vary between datasets and show that the tested tools are either not robust enough or computationally expensive, thus limiting their application to large-scale experiments. We therefore developed fastER, a trainable tool that is orders of magnitude faster while producing state-of-the-art segmentation quality. It supports various cell types and image acquisition modalities, but is easy-to-use even for non-experts: it has no parameters and can be adapted to specific image sets by interactively labelling cells for training. As a proof of concept, we segment and count cells in over 200 000 brightfield images (1388 × 1040 pixels each) from a six day time-lapse microscopy experiment; identification of over 46 000 000 single cells requires only about two and a half hours on a desktop computer. AVAILABILITY AND IMPLEMENTATION: C ++ code, binaries and data at https://www.bsse.ethz.ch/csd/software/faster.html . CONTACT: oliver.hilsenbeck@bsse.ethz.ch or timm.schroeder@bsse.ethz.ch. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Algoritmos , Células HeLa , HumanosRESUMO
The maintenance of hematopoietic stem cells (HSCs) during ex vivo culture is an important prerequisite for their therapeutic manipulation. However, despite intense research, culture conditions for robust maintenance of HSCs are still missing. Cultured HSCs are quickly lost, preventing their improved analysis and manipulation. Identification of novel factors supporting HSC ex vivo maintenance is therefore necessary. Coculture with the AFT024 stroma cell line is capable of maintaining HSCs ex vivo long-term, but the responsible molecular players remain unknown. Here, we use continuous long-term single-cell observation to identify the HSC behavioral signature under supportive or nonsupportive stroma cocultures. We report early HSC survival as a major characteristic of HSC-maintaining conditions. Behavioral screening after manipulation of candidate molecules revealed that the extracellular matrix protein dermatopontin (Dpt) is involved in HSC maintenance. DPT knockdown in supportive stroma impaired HSC survival, whereas ectopic expression of the Dpt gene or protein in nonsupportive conditions restored HSC survival. Supplementing defined stroma- and serum-free culture conditions with recombinant DPT protein improved HSC clonogenicity. These findings illustrate a previously uncharacterized role of Dpt in maintaining HSCs ex vivo.
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Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/farmacologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/farmacologia , Células-Tronco Hematopoéticas/citologia , Masculino , Camundongos , Camundongos Transgênicos , Células Estromais/citologia , Células Estromais/metabolismo , Fatores de TempoRESUMO
Clonal hematopoiesis (CH), the relative expansion of mutant clones, is derived from hematopoietic stem cells (HSCs) with acquired somatic or cytogenetic alterations that improve cellular fitness. Individuals with CH have a higher risk for hematological and non-hematological diseases, such as cardiovascular disease, and have an overall higher mortality rate. Originally thought to be restricted to a small fraction of elderly people, recent advances in single-cell sequencing and bioinformatics have revealed that CH with multiple expanded mutant clones is universal in the elderly population. Just a few years ago, phylogenetic reconstruction across the human lifespan and novel sensitive sequencing techniques showed that CH can start earlier in life, decades before it was thought possible. These studies also suggest that environmental factors acting through aberrant inflammation might be a common theme promoting clonal expansion and disease progression. However, numerous aspects of this phenomenon remain to be elucidated and the precise mechanisms, context-specific drivers, and pathways of clonal expansion remain to be established. Here, we review our current understanding of the cellular mechanisms driving CH and specifically focus on how pro-inflammatory factors affect normal and mutant HSC fates to promote clonal selection.
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PURPOSE OF REVIEW: Studying heterogeneous populations, such as hematopoietic stem cells (HSCs), requires continuous long-term observation of living cells at the single-cell level. The purpose of this review is to discuss recent advances in technologies required for continuous single-cell analysis and the contribution of this approach to find answers in hematopoiesis research. RECENT FINDINGS: Continuous long-term imaging at the single-cell level still requires custom-made hardware, software and manual in-depth analysis of large amounts of data. Despite these technical difficulties, continuous time-lapse imaging and single-cell tracking are increasingly used in hematopoiesis research. It has already contributed to answering decades-old questions. SUMMARY: Continuous long-term single-cell analysis is indispensable for a comprehensive analysis of dynamic processes in heterogeneous cell populations. Despite many remaining technological hurdles, this approach is increasingly used in hematopoiesis research.