bioorthogonal-chemical-imaging-cell-metabolism-regulated-aromatic

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, ...

University of California San Diego

Pluripotent stem cell-derived organoids have transformed our ability to recreate complex three-dimensional models of human tissue. However, the directed differentiation methods used to create them do not afford the ability to introduce cross-germ-layer cell types. Here, we present a bottom-up engineering approach to building vascularized human tissue by combining genetic reprogramming with chemically directed organoid differentiation. As a proof of concept, we created neuro-vascular and myo-vascular organoids via transcription factor overexpression in vascular organoids. We comprehensively characterized neuro-vascular organoids in terms of marker gene expression and composition, and demonstrated that the organoids maintain neural and vascular function for at least 45 days in culture. Finally, we demonstrated chronic electrical stimulation of myo-vascular organoid aggregates as a potential path toward engineering mature and large-scale vascularized skeletal muscle tissue from organoids. Our approach offers a roadmap to build diverse vascularized tissues of any type derived entirely from pluripotent stem cells.

Programmatic introduction of parenchymal cell types into blood vessel organoids.

Oxidative imbalance plays an essential role in the progression of many diseases that include cancer and neurodegenerative diseases. Aromatic amino acids (AAA) such as phenylalanine and tryptophan have the capability of escalating oxidative stress because of their involvement in the production of Reactive Oxygen Species (ROS) Here, we use DO (heavy water) probed stimulated Raman scattering microscopy (DO-SRS) and two Photon Excitation Fluorescence (2PEF) microscopy as a multimodal imaging approach to visualize metabolic changes in HeLa cells under excess AAA such as phenylalanine or trytophan in culture media. The cellular spatial distribution of  lipogenesis, new protein synthesis, NADH, Flavin, unsaturated lipids, and saturated lipids were all imaged and quantified in this experiment. Our studies reveal ∼10% increase in  lipogenesis and the ratio of NADH to flavin, and ∼50% increase of the ratio of unsaturated lipids to saturated lipid in cells treated with excess phenylalanine or trytophan. In contrast, these cells exhibited a decrease in the protein synthesis rate by ∼10% under these AAA treatments. The cellular metabolic activities of these biomolecules are indicators of elevated oxidative stress and mitochondrial dysfunction. Furthermore, 3D reconstruction images of lipid droplets were acquired and quantified to observe their spatial distribution around cells' nuceli under different AAA culture media. We observed a higher number of lipid droplets in excess AAA conditions. Our study showcases that DO-SRS imaging can be used to quantitatively study how excess AAA regulates metabolic activities of cells with subcellular resolution .

Visualizing Cancer Cell Metabolic Dynamics Regulated With Aromatic Amino Acids Using DO-SRS and 2PEF Microscopy.

Emerging studies have shown that lipids and proteins play versatile roles in various aspects of aging. High-resolution  optical imaging provides a powerful approach to study the metabolic dynamics of lipids and proteins during aging. Here, we integrated DO probing and stimulated Raman scattering (DO-SRS) microscopy to directly visualize metabolic changes in aging  ovary. The subcellular spatial distribution of  protein synthesis and lipogenesis in ovary was quantitatively imaged and examined. Our Raman spectra showed that early stages follicles were protein-enriched whereas mature eggs were lipid-enriched. DO-SRS imaging showed a higher protein synthesis in the earlier developing stages and an increased lipid turned over at the late stage. Aged (35 days) flies exhibited a dramatic decrease in metabolic turnover activities of both proteins and lipids, particularly, in the germ stem cell niche of germarium. We found an accumulation of unsaturated lipids in the nurse cells and oocytes in old flies, suggesting that unsaturated lipids may play an important role in the processes of oocyte maturation. We further detected changes in mitochondrial morphology and accumulation of Cytochrome c during aging. To our knowledge, this is the first study that directly visualizes spatiotemporal changes in lipid and protein metabolism in  ovary during development and aging processes. Our study not only demonstrates the application of a new imaging platform in visualizing metabolic dynamics of lipids and proteins  but also unravels how the metabolic activity and lipid distribution change in  ovary during aging.

Direct Imaging of Lipid Metabolic Changes in  Ovary During Aging Using DO-SRS Microscopy.

Stimulated Raman scattering (SRS) offers the ability to image metabolic dynamics with high signal-to-noise ratio. However, its spatial resolution is limited by the numerical aperture of the imaging objective and the scattering cross-section of molecules. To achieve super-resolved SRS imaging, we developed a deconvolution algorithm, adaptive moment estimation (Adam) optimization-based pointillism deconvolution (A-PoD) and demonstrated a spatial resolution of lower than 59 nm on the membrane of a single lipid droplet (LD). We applied A-PoD to spatially correlated multiphoton fluorescence imaging and deuterium oxide (DO)-probed SRS (DO-SRS) imaging from diverse samples to compare nanoscopic distributions of proteins and lipids in cells and subcellular organelles. We successfully differentiated newly synthesized lipids in LDs using A-PoD-coupled DO-SRS. The A-PoD-enhanced DO-SRS imaging method was also applied to reveal metabolic changes in brain samples from Drosophila on different diets. This new approach allows us to quantitatively measure the nanoscopic colocalization of biomolecules and metabolic dynamics in organelles.

Article	Year
Programmatic introduction of parenchymal cell types into blood vessel organoids. Stem cell reports\| PubMed ID: 34559998	2021
Visualizing Cancer Cell Metabolic Dynamics Regulated With Aromatic Amino Acids Using DO-SRS and 2PEF Microscopy. Frontiers in molecular biosciences\| PubMed ID: 34977157	2021
Direct Imaging of Lipid Metabolic Changes in Ovary During Aging Using DO-SRS Microscopy. Frontiers in aging\| PubMed ID: 35822015	2021
Super-resolution SRS microscopy with A-PoD. Nature methods\| PubMed ID: 36797410	2023

Pegah Bagheri

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