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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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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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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
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/metabolismoRESUMO
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.
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Eritropoese , Ferro , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Reticulócitos , Vitamina E , Animais , Homeostase , Camundongos , CoelhosRESUMO
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
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.
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Apoptose , Células Eritroides/patologia , Glutationa Peroxidase/fisiologia , Necrose , Estresse Oxidativo , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Animais , Western Blotting , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Eritroides/metabolismo , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Knockout , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Espécies Reativas de Oxigênio/metabolismoRESUMO
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
Transcription factors are key regulators of hematopoietic stem cells (HSCs) and act through their ability to bind DNA and impact on gene transcription. Their functions are interpreted in the complex landscape of chromatin, but current knowledge on how this is achieved is very limited. C/EBPα is an important transcriptional regulator of hematopoiesis, but its potential functions in HSCs have remained elusive. Here we report that C/EBPα serves to protect adult HSCs from apoptosis and to maintain their quiescent state. Consequently, deletion of Cebpa is associated with loss of self-renewal and HSC exhaustion. By combining gene expression analysis with genome-wide assessment of C/EBPα binding and epigenetic configurations, we show that C/EBPα acts to modulate the epigenetic states of genes belonging to molecular pathways important for HSC function. Moreover, our data suggest that C/EBPα acts as a priming factor at the HSC level where it actively promotes myeloid differentiation and counteracts lymphoid lineage choice. Taken together, our results show that C/EBPα is a key regulator of HSC biology, which influences the epigenetic landscape of HSCs in order to balance different cell fate options.
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Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Animais , Apoptose , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Linhagem da Célula , Proliferação de Células , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/metabolismo , CamundongosRESUMO
BACKGROUND: In recent years, high-throughput microscopy has emerged as a powerful tool to analyze cellular dynamics in an unprecedentedly high resolved manner. The amount of data that is generated, for example in long-term time-lapse microscopy experiments, requires automated methods for processing and analysis. Available software frameworks are well suited for high-throughput processing of fluorescence images, but they often do not perform well on bright field image data that varies considerably between laboratories, setups, and even single experiments. RESULTS: In this contribution, we present a fully automated image processing pipeline that is able to robustly segment and analyze cells with ellipsoid morphology from bright field microscopy in a high-throughput, yet time efficient manner. The pipeline comprises two steps: (i) Image acquisition is adjusted to obtain optimal bright field image quality for automatic processing. (ii) A concatenation of fast performing image processing algorithms robustly identifies single cells in each image. We applied the method to a time-lapse movie consisting of â¼315,000 images of differentiating hematopoietic stem cells over 6 days. We evaluated the accuracy of our method by comparing the number of identified cells with manual counts. Our method is able to segment images with varying cell density and different cell types without parameter adjustment and clearly outperforms a standard approach. By computing population doubling times, we were able to identify three growth phases in the stem cell population throughout the whole movie, and validated our result with cell cycle times from single cell tracking. CONCLUSIONS: Our method allows fully automated processing and analysis of high-throughput bright field microscopy data. The robustness of cell detection and fast computation time will support the analysis of high-content screening experiments, on-line analysis of time-lapse experiments as well as development of methods to automatically track single-cell genealogies.
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Biologia Computacional/métodos , Técnicas Citológicas/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Algoritmos , Animais , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Ensaios de Triagem em Larga Escala , Camundongos , SoftwareRESUMO
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.
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Diferenciação Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Homeostase/genética , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Regulação para Cima/genética , Animais , Células Cultivadas , Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Imagem com Lapso de Tempo/métodos , Transativadores/metabolismoRESUMO
Human organoids allow the study of proliferation, lineage specification, and 3D tissue development. Here we present a genome-wide CRISPR screen in induced pluripotent stem cell (iPSC)-derived kidney organoids. The combination of inducible genome editing, longitudinal sampling, and endpoint sorting of tubular and stromal cells generated a complex, high-quality dataset uncovering a broad spectrum of insightful biology from early development to "adult" epithelial morphogenesis. Our functional dataset allows improving mesoderm induction by ROCK inhibition, contains monogenetic and complex trait kidney disease genes, confirms two additional congenital anomalies of the kidney and urinary tract (CAKUT) genes (CCDC170 and MYH7B), and provides a large candidate list of ciliopathy-related genes. Finally, identification of a cis-inhibitory effect of Jagged1 controlling epithelial proliferation shows how mosaic knockouts in pooled CRISPR screening can reveal ways of communication between heterogeneous cell populations in complex tissues. These data serve as a rich resource for the kidney research community and as a benchmark for future iPSC-derived organoid CRISPR screens.
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Células-Tronco Pluripotentes Induzidas , Organoides , Edição de Genes , Humanos , Rim , OrganogêneseRESUMO
Neutrophils play a vital role in the immune defense, which is evident by the severity of neutropenia causing life-threatening infections. Granulocyte macrophage-colony stimulating factor (GM-CSF) controls homeostatic and emergency development of granulocytes. However, little is known about the contribution of the downstream mediating transcription factors signal transducer and activator of transcription 5A and 5B (STAT5A/B). To elucidate the function of this pathway, we generated mice with complete deletion of both Stat5a/b genes in hematopoietic cells. In homeostasis, peripheral neutrophils were markedly decreased in these animals. Moreover, during emergency situations, such as myelosuppression, Stat5a/b-mutant mice failed to produce enhanced levels of neutrophils and were unable to respond to GM-CSF. Both the GM-CSF-permitted survival of mature neutrophils and the generation of granulocytes from granulocyte-macrophage progenitors (GMPs) were markedly reduced in Stat5a/b mutants. GMPs showed impaired colony-formation ability with reduced number and size of colonies on GM-CSF stimulation. Moreover, continuous cell fate analyses by time-lapse microscopy and single cell tracking revealed that Stat5a/b-null GMPs showed both delayed cell-cycle progression and increased cell death. Finally, transcriptome analysis indicated that STAT5A/B directs GM-CSF signaling through the regulation of proliferation and survival genes.
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Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Leucopoese/fisiologia , Neutrófilos/citologia , Fator de Transcrição STAT5/imunologia , Transdução de Sinais/imunologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Células Progenitoras de Granulócitos e Macrófagos/citologia , Células Progenitoras de Granulócitos e Macrófagos/imunologia , Granulócitos/citologia , Granulócitos/imunologia , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Wilms tumor is the most widespread kidney cancer in children and frequently associated with homozygous loss of the tumor suppressor WT1. Pediatric tumorigenesis is largely inaccessible in humans. Here, we develop a human kidney organoid model for Wilms tumor formation and show that deletion of WT1 during organoid development induces overgrowth of kidney progenitor cells at the expense of differentiating glomeruli and tubules. Functional and gene expression analyses demonstrate that absence of WT1 halts progenitor cell progression at a pre-epithelialized cell state and recapitulates the transcriptional changes detected in a subgroup of Wilms tumor patients with ectopic myogenesis. By "transplanting" WT1 mutant cells into wild-type kidney organoids, we find that their propagation requires an untransformed microenvironment. This work defines the role of WT1 in kidney progenitor cell progression and tumor suppression, and establishes human kidney organoids as a phenotypic model for pediatric tumorigenesis.
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Transformação Celular Neoplásica/genética , Genes Supressores de Tumor , Neoplasias Renais/etiologia , Células-Tronco Neoplásicas/metabolismo , Proteínas WT1/genética , Tumor de Wilms/etiologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Biologia Computacional/métodos , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Hiperplasia , Imunofenotipagem , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Anotação de Sequência Molecular , Células-Tronco Neoplásicas/patologia , Organoides/metabolismo , Organoides/patologia , Proteínas WT1/metabolismo , Tumor de Wilms/metabolismo , Tumor de Wilms/patologiaRESUMO
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.
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Fator de Transcrição GATA2/metabolismo , Genes Reporter , Células-Tronco Hematopoéticas/metabolismo , Envelhecimento/genética , Animais , Linhagem da Célula , Proliferação de Células , Embrião de Mamíferos/metabolismo , Fator de Transcrição GATA2/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Hematopoese , Mastócitos/citologia , Camundongos , Modelos Biológicos , Monócitos/citologia , Neutrófilos/citologia , Especificidade de Órgãos , Fatores de Transcrição/metabolismoRESUMO
The existence of specialized liver stem cell populations, including AXIN2+ pericentral hepatocytes, that safeguard homeostasis and repair has been controversial. Here, using AXIN2 lineage tracing in BAC-transgenic mice, we confirm the regenerative potential of intestinal stem cells (ISCs) but find limited roles for pericentral hepatocytes in liver parenchyma homeostasis. Liver regrowth following partial hepatectomy is enabled by proliferation of hepatocytes throughout the liver, rather than by a pericentral population. Periportal hepatocyte injury triggers local repair as well as auxiliary proliferation in all liver zones. DTA-mediated ablation of AXIN2+ pericentral hepatocytes transiently disrupts this zone, which is reestablished by conversion of pericentral vein-juxtaposed glutamine synthetase (GS)- hepatocytes into GS+ hepatocytes and by compensatory proliferation of hepatocytes across liver zones. These findings show hepatocytes throughout the liver can upregulate AXIN2 and LGR5 after injury and contribute to liver regeneration on demand, without zonal dominance by a putative pericentral stem cell population.
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Hepatócitos , Fígado , Animais , Proteína Axina , Homeostase , Regeneração Hepática , Camundongos , Células-TroncoRESUMO
Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/ß-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.
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Lesão Pulmonar Aguda/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ductos Biliares/patologia , Proteínas de Ciclo Celular/metabolismo , Células Epiteliais/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Modelos Animais de Doenças , Humanos , Regeneração Hepática , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piridinas/toxicidade , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismo , Via de Sinalização Wnt , Proteínas de Sinalização YAPRESUMO
Molecular regulation of cell fate decisions underlies health and disease. To identify molecules that are active or regulated during a decision, and not before or after, the decision time point is crucial. However, cell fate markers are usually delayed and the time of decision therefore unknown. Fortunately, dividing cells induce temporal correlations in their progeny, which allow for retrospective inference of the decision time point. We present a computational method to infer decision time points from correlated marker signals in genealogies and apply it to differentiating hematopoietic stem cells. We find that myeloid lineage decisions happen generations before lineage marker onsets. Inferred decision time points are in agreement with data from colony assay experiments. The levels of the myeloid transcription factor PU.1 do not change during, but long after the predicted lineage decision event, indicating that the PU.1/GATA1 toggle switch paradigm cannot explain the initiation of early myeloid lineage choice.
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Diferenciação Celular , Linhagem da Célula , Fator de Transcrição GATA1/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Algoritmos , Animais , Biologia Computacional/métodos , Células-Tronco Hematopoéticas/citologia , Modelos Biológicos , Células Mieloides/citologia , Células Mieloides/metabolismo , Fatores de TempoRESUMO
Embryonic stem cells (ESCs) display heterogeneous expression of pluripotency factors such as Nanog when cultured with serum and leukemia inhibitory factor (LIF). In contrast, dual inhibition of the signaling kinases GSK3 and MEK (2i) converts ESC cultures into a state with more uniform and high Nanog expression. However, it is so far unclear whether 2i acts through an inductive or selective mechanism. Here, we use continuous time-lapse imaging to quantify the dynamics of death, proliferation, and Nanog expression in mouse ESCs after 2i addition. We show that 2i has a dual effect: it both leads to increased cell death of Nanog low ESCs (selective effect) and induces and maintains high Nanog levels (inductive effect) in single ESCs. Genetic manipulation further showed that presence of NANOG protein is important for cell viability in 2i medium. This demonstrates complex Nanog-dependent effects of 2i treatment on ESC cultures.