Stem cells reside in specialized niches that are critical for their function. Upon activation, hair follicle stem cells (HFSCs) exit their niche to generate the outer root sheath (ORS), but a subset of ORS progeny returns to the niche to resume an SC state. Mechanisms of this fate reversibility are unclear. We show that the ability of ORS cells to return to the SC state requires suppression of a metabolic switch from glycolysis to oxidative phosphorylation and glutamine metabolism that occurs during early HFSC lineage progression. HFSC fate reversibility and glutamine metabolism are regulated by the mammalian target of rapamycin complex 2 (mTORC2)-Akt signaling axis within the niche. Deletion of mTORC2 results in a failure to re-establish the HFSC niche, defective hair follicle regeneration, and compromised long-term maintenance of HFSCs. These findings highlight the importance of spatiotemporal control of SC metabolic states in organ homeostasis.
Glutamine/metabolism , Hair Follicle/metabolism , Stem Cells/metabolism , Animals , Cells, Cultured , Hair Follicle/cytology , Male , Mice , Mice, Inbred C57BL , Optical Imaging , Stem Cells/cytology
Calcific aortic valve disease (CAVD) is the most common form of valve disease where the only available treatment strategy is surgical valve replacement. Technologies for the early detection of CAVD would benefit the development of prevention, mitigation and alternate therapeutic strategies. Two-photon excited fluorescence (TPEF) microscopy is a label-free, non-destructive imaging technique that has been shown to correlate with multiple markers for cellular differentiation and phenotypic changes in cancer and wound healing. Here we show how specific TPEF markers, namely, the optical redox ratio and mitochondrial fractal dimension, correlate with structural, functional and phenotypic changes occurring in the aortic valve interstitial cells (VICs) during osteogenic differentiation. The optical redox ratio, and fractal dimension of mitochondria were assessed and correlated with gene expression and nuclear morphology of VICs. The optical redox ratio decreased for VICs during early osteogenic differentiation and correlated with biological markers for CAVD progression. Fractal dimension correlated with structural and osteogenic markers as well as measures of nuclear morphology. Our study suggests that TPEF imaging markers, specifically the optical redox ratio and mitochondrial fractal dimension, can be potentially used as a tool for assessing early CAVD progression in vitro.
Aortic Valve Stenosis/diagnosis , Aortic Valve/pathology , Calcinosis/diagnosis , Intravital Microscopy/methods , Osteoblasts/pathology , Animals , Aortic Valve/cytology , Aortic Valve Stenosis/pathology , Calcinosis/pathology , Cell Differentiation , Cell Nucleus/pathology , Cells, Cultured , Disease Progression , Feasibility Studies , Humans , Male , Microscopy, Fluorescence, Multiphoton/methods , Mitochondria/pathology , Osteoblasts/cytology , Oxidation-Reduction , Primary Cell Culture , Swine
SIGNIFICANCE: Optical imaging using the endogenous fluorescence of metabolic cofactors has enabled nondestructive examination of dynamic changes in cell and tissue function both in vitro and in vivo. Quantifying NAD(P)H and FAD fluorescence through an optical redox ratio and fluorescence lifetime imaging (FLIM) provides sensitivity to the relative balance between oxidative phosphorylation and glucose catabolism. Since its introduction decades ago, the use of NAD(P)H imaging has expanded to include applications involving almost every major tissue type and a variety of pathologies. Recent Advances: This review focuses on the use of two-photon excited fluorescence and NAD(P)H fluorescence lifetime techniques in cancer, neuroscience, tissue engineering, and other biomedical applications over the last 5 years. In a variety of cancer models, NAD(P)H fluorescence intensity and lifetime measurements demonstrate a sensitivity to the Warburg effect, suggesting potential for early detection or high-throughput drug screening. The sensitivity to the biosynthetic demands of stem cell differentiation and tissue repair processes indicates the range of applications for this imaging technology may be broad. CRITICAL ISSUES: As the number of applications for these fluorescence imaging techniques expand, identifying and characterizing additional intrinsic fluorophores and chromophores present in vivo will be vital to accurately measure and interpret metabolic outcomes. Understanding the full capabilities and limitations of FLIM will also be key to future advances. FUTURE DIRECTIONS: Future work is needed to evaluate whether a combination of different biochemical and structural outcomes using these imaging techniques can provide complementary information regarding the utilization of specific metabolic pathways.
Cells/metabolism , Flavin-Adenine Dinucleotide/analysis , Molecular Imaging , NAD/analysis , Optical Imaging , Animals , Flavin-Adenine Dinucleotide/metabolism , Humans , NAD/metabolism , Neoplasms/metabolism , Signal Transduction
An improved technique for fractal characterization called the modified blanket method is introduced that can quantify surrounding fractal structures on a pixel by pixel basis without artifacts associated with scale-dependent image features such as object size. The method interprets images as topographical maps, obtaining information regarding the local surface area as a function of image resolution. Local fractal dimension (FD) can be quantified from the power law exponent derived from the surface area and image resolution relationship. We apply this technique on simulated cell images of known FD and compared the obtained values to power spectral density (PSD) analysis. Our method is sensitive to a wider FD range (2.0-4.5), having a mean error of 1.4% compared to 6% for PSD analysis. This increased sensitivity and an ability to compute regional FD properties enabled the discrimination of the differences in radiation resistant cancer cell responses that could not be detected using PSD analysis.
