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 shown
. 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 division
. 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.
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.
•Gpx4 is essential for preventing anemia in mice via inhibiting RIP3-dependent necroptosis in erythroid precursor cells.•ROS accumulation and lipid peroxidation in erythroid precursor cells trigger receptor-independent activation of necroptosis.
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.
Analysis of the mechanisms underlying cell fates requires the molecular quantification of cellular features. Classical techniques use population average readouts at single time points. However, these ...approaches mask cellular heterogeneity and dynamics and are limited for studying rare and heterogeneous cell populations like stem cells. Techniques for single-cell analyses, ideally allowing non-invasive quantification of molecular dynamics and cellular behaviour over time, are required for studying stem cells. Here, we review the development and application of these techniques to stem cell research.
Hematopoietic Cytokines Can Instruct Lineage Choice Rieger, Michael A; Hoppe, Philipp S; Smejkal, Benjamin M ...
Science (American Association for the Advancement of Science),
07/2009, Letnik:
325, Številka:
5937
Journal Article
Recenzirano
The constant regeneration of the blood system during hematopoiesis requires tightly controlled lineage decisions of hematopoietic progenitor cells (HPCs). Because of technical limitations, ...differentiation of individual HPCs could not previously be analyzed continuously. It was therefore disputed whether cell-extrinsic cytokines can instruct HPC lineage choice or only allow survival of cells that are already lineage-restricted. Here, we used bioimaging approaches that allow the continuous long-term observation of individual differentiating mouse HPCs. We demonstrate that the physiological cytokines, macrophage colony-stimulating factor and granulocyte colony-stimulating factor, can instruct hematopoietic lineage choice.
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.
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 ∼315,000 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.
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.
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.
•TNF integrates inflammatory signals to directly regulate the lineage instructing transcription factor PU.1 in HSCs.
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Transcription factor (TF) networks are thought to regulate embryonic stem cell (ESC) pluripotency. However, TF expression dynamics and regulatory mechanisms are poorly understood. We use reporter ...mouse ESC lines allowing non-invasive quantification of Nanog or Oct4 protein levels and continuous long-term single-cell tracking and quantification over many generations to reveal diverse TF protein expression dynamics. For cells with low Nanog expression, we identified two distinct colony types: one re-expressed Nanog in a mosaic pattern, and the other did not re-express Nanog over many generations. Although both expressed pluripotency markers, they exhibited differences in their TF protein correlation networks and differentiation propensities. Sister cell analysis revealed that differences in Nanog levels are not necessarily accompanied by differences in the expression of other pluripotency factors. Thus, regulatory interactions of pluripotency TFs are less stringently implemented in individual self-renewing ESCs than assumed at present.
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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•Hepatocytes throughout the liver contribute to liver homeostasis and repair•AXIN2+ pericentral hepatocytes do not comprise bona fide liver stem cells•Hepatocytes upregulate AXIN2 and LGR5 during regeneration•Ablation of AXIN2+ hepatocytes only transiently disrupts the pericentral zone
The existence of specialized liver stem cells, like AXIN2+ pericentral hepatocytes, is controversial. Sun and colleagues now show that hepatocytes throughout the liver contribute to liver homeostasis and upregulate the tissue stem cell markers AXIN2 and LGR5 during regeneration from injury, with no superior proliferative capacity of AXIN2+ pericentral hepatocytes.
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