We describe a label-free imaging method to monitor stem-cell metabolism that discriminates different states of stem cells as they differentiate in living tissues. In this method we use intrinsic fluorescence biomarkers and the phasor approach to fluorescence lifetime imaging microscopy in conjunction with image segmentation, which we use to introduce the concept of the cell phasor. In live tissues we are able to identify intrinsic fluorophores, such as collagen, retinol, retinoic acid, porphyrin, flavins, and free and bound NADH. We have exploited the cell phasor approach to detect a trend in metabolite concentrations along the main axis of the Caenorhabditis elegans germ line. This trend is consistent with known changes in metabolic states during differentiation. The cell phasor approach to lifetime imaging provides a label-free, fit-free, and sensitive method to identify different metabolic states of cells during differentiation, to sense small changes in the redox state of cells, and may identify symmetric and asymmetric divisions and predict cell fate. Our method is a promising noninvasive optical tool for monitoring metabolic pathways during differentiation or disease progression, and for cell sorting in unlabeled tissues.phasor analysis | single cell metabolism T he hallmark of stem cells is their ability to produce a new stem cell by self-renewing, as they generate daughter cells that are committed to differentiation and form specialized tissues (1). The modulation of the balance between self-renewing divisions and differentiation is a central mechanism for stem cells during embryo development, adult tissue regeneration, and homeostasis. Stem-cell differentiation is a complex process mediated both by intrinsic molecular mechanisms and extrinsic signaling. Intrinsic cell polarity, subcellular localization mechanism, asymmetric centrosome and spindles, as well as cell-cycle regulators can establish self-renewing asymmetry during stem-cell division (2, 3). The influence of external chemical and physical stimuli, such as molecular gradients, extracellular matrix remodeling, and niche signaling is crucial for stem-cell plasticity and tissue development (4-6). Extrinsic signals can be propagated through intracellular signal-transduction pathways that converge to genetic networks that control pluripotency. Many different signaling and transcriptional pathways, which are important for development and cell-cycle regulation, converge on regulation of the redox state of stem cells (5,7,8). In turn, histone posttranscriptional modifications and epigenetic mechanisms, such as phosphorylation, acetylation, and methylation, sense cellular metabolism and the variation of metabolites levels (9). Hence, redox balance plays an important role in the maintenance and modulation of stem cell self-renewal and differentiation (10-12).In stem-cell research there is a high demand for noninvasive techniques to investigate self-renewal and differentiation. Methods, such as immunohistochemistry, metabolic assays, and PCR are currently...
Self-renewing organs often experience a decline in function in the course of aging. It is unclear whether chronological age or external factors control this decline, or whether it is driven by stem cell self-renewal—for example, because cycling cells exhaust their replicative capacity and become senescent. Here we assay the relationship between stem cell cycling and senescence in the Caenorhabditis elegans reproductive system, defining this senescence as the progressive decline in “reproductive capacity,” i.e. in the number of progeny that can be produced until cessation of reproduction. We show that stem cell cycling diminishes remaining reproductive capacity, at least in part through the DNA damage response. Paradoxically, gonads kept under conditions that preclude reproduction keep cycling and producing cells that undergo apoptosis or are laid as unfertilized gametes, thus squandering reproductive capacity. We show that continued activity is in fact beneficial inasmuch as gonads that are active when reproduction is initiated have more sustained early progeny production. Intriguingly, continued cycling is intermittent—gonads switch between active and dormant states—and in all likelihood stochastic. Other organs face tradeoffs whereby stem cell cycling has the beneficial effect of providing freshly-differentiated cells and the detrimental effect of increasing the likelihood of cancer or senescence; stochastic stem cell cycling may allow for a subset of cells to preserve proliferative potential in old age, which may implement a strategy to deal with uncertainty as to the total amount of proliferation to be undergone over an organism’s lifespan.
Stem cells niches are increasingly recognized as dynamic environments that play a key role in transducing signals that allow an organism to exert control on its stem cells. Live imaging of stem cell niches in their in vivo setting is thus of high interest to dissect stem cell controls. Here we report a new microfluidic design that is highly amenable to dissemination in biology laboratories that have no microfluidics expertise. This design has allowed us to perform the first time lapse imaging of the C. elegans germline stem cell niche. Our results show that this niche is strikingly dynamic, and that morphological changes that take place during development are the result of a highly active process. These results lay the foundation for future studies to dissect molecular mechanisms by which stem cell niche morphology is modulated, and by which niche morphology controls stem cell behavior.
BackgroundStem cells are thought to play a critical role in minimizing the accumulation of mutations, but it is not clear which strategies they follow to fulfill that performance objective. Slow cycling of stem cells provides a simple strategy that can minimize cell pedigree depth and thereby minimize the accumulation of replication-dependent mutations. Although the power of this strategy was recognized early on, a quantitative assessment of whether and how it is employed by biological systems is missing.ResultsHere we address this problem using a simple self-renewing organ – the C. elegans gonad – whose overall organization is shared with many self-renewing organs. Computational simulations of mutation accumulation characterize a tradeoff between fast development and low mutation accumulation, and show that slow-cycling stem cells allow for an advantageous compromise to be reached. This compromise is such that worm germ-line stem cells should cycle more slowly than their differentiating counterparts, but only by a modest amount. Experimental measurements of cell cycle lengths derived using a new, quantitative technique are consistent with these predictions.ConclusionsOur findings shed light both on design principles that underlie the role of stem cells in delaying aging and on evolutionary forces that shape stem-cell gene regulatory networks.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0148-y) contains supplementary material, which is available to authorized users.
Summary Positional information derived from local morphogen concentration plays an important role in patterning. A key question is how morphogen diffusion and gene expression regulation shape positional information into an appropriate profile with suitably low noise. We address this question using a model system — the C. elegans germ line — whose regulatory network has been well characterized genetically but whose spatiotemporal dynamics are poorly understood. We show that diffusion within the germline syncytium is a critical control of stem cell differentiation, and that semi-permeable diffusion barriers present at key locations make it possible — in combination with a feedback loop in the germline regulatory network — for mitotic zone size to be robust against spatial noise in Notch signaling. Spatial averaging within compartments defined by diffusion barriers is an advantageous patterning strategy, which attenuates noise while still allowing for sharp transitions between compartments. This strategy could apply to other organs.
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