MetaboLightsapplication/xml KhalilMetaboLightsPublicMS Imaging -<p>A high resolution MSI platform for visualizing brain organoid lipids was implemented by mounting a MALDI ion source containing a dual-ion funnel interface (Spectroglyph LLC, United States) to a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Massachusetts, United States)<strong>[1][2]</strong>. An attached Q-switched frequency tripled Nd:YLF laser of 349-nm wavelength was used at a repetition rate of 1 KHz and pulse energy of ~1.3-1.4 μJ. The laser was focused and controlled to a spot size of ~15 μm in diameter. The sample was attached to the MALDI injector stage and the high-pressure ion funnel was maintained at 7.4-7.5 Torr. The low-pressure ion funnel was maintained at 1.6-1.8 Torr.We applied 604 kHz, 80 V0-peak and 780 kHz, 191 V0-peak radio frequency voltages to the high and lowpressure ion funnels, respectively. The Orbitrap mass spectrometry was operated at ion injection time of 250 ms and Fourier Transform MS (FTMS) spectra were acquired in a profile mode using a target mass resolution of 70,000 (Full Width Half Maximum (FWHM) at m/z 400, m/z = mass-to-charge ratio). During MALDI imaging, mass spectrometer starts to acquire data after switching on to contact closure. The signal is sent from the MALDI injector and communicated via the ‘Peripheral Control’ input connection at the side of the Q Exactive MS, and the automatic gain control was switched off.</p><p><br></p><p>Data were acquired in the m/z range of 200-1000 and brain organoid image generation was done using an HR-MALDI-MSI system in both positive and negative ion modes. Samples were scanned with lateral resolution of 25 μm pixel size for 14 μm organoid sections of 2-4 mm (h) x 1-1.5 mm (w) size. The Q Exactive MS was operated in positive-ion mode with the mass range of m/z = 400-1000 whereas in negative ion mode, the mass range was m/z = 200-1000. DAN matrix peaks were used for internal calibration resulting in a mass accuracy of better than ±5 ppm. The laser beam was focused carefully to avoid oversampling. Automatic gain control was turned off and C-trap injection time was fixed to 250 ms.</p><p><br></p><p><strong>Refs:</strong></p><p><strong>[1]</strong> Belov ME, Ellis SR, Dilillo M, Paine MRL, Danielson WF, Anderson GA, de Graaf EL, Eijkel GB, Heeren RMA, McDonnell LA. Design and Performance of a Novel Interface for Combined Matrix-Assisted Laser Desorption Ionization at Elevated Pressure and Electrospray Ionization with Orbitrap Mass Spectrometry. Anal Chem. 2017 Jul 18;89(14):7493-7501. doi:10.1021/acs.analchem.7b01168. Epub 2017 Jun 28. PMID:28613836.</p><p><strong>[2]</strong> Ellis SR, Paine MRL, Eijkel GB, Pauling JK, Husen P, Jervelund MW, Hermansson M, Ejsing CS, Heeren RMA. Automated, parallel mass spectrometry imaging and structural identification of lipids. Nat Methods. 2018 Jul;15(7):515-518. doi:10.1038/s41592-018-0010-6. Epub 2018 May 21. PMID:29786091.</p>Mass spectrometry imaging as an emerging tool for studying metabolism in human brain organoids. 10.3389/fmolb.2023.1181965.Baylor college of medicineHomo sapiensmass spectrometry Osenberg.Saleh Khalil.Mirjana Maletic-Savatic. Cappuccio.Feng Li.<p>SCiLS Lab software version 2022b (SCiLS GmbH, Bremen, Germany) was used for processing of MSI data. Thermo Q Exactive MS spectral data were first imported into SCiLS Lab followed by baseline correction (convolution algorithm) and total ion count (TIC) normalization<strong>[1]</strong>. This software package was used to generate mass images from raw data files with a bin width of Δm/z = 0.01 or ±5 ppm to discriminate m/z images based on mass defect and pixel coverage. Mass spectra from 25 μm/pixel were obtained and all lipid ions were assigned within a mass accuracy of ±5 ppm. False color images (Jet) or RGB (red-greenblue) images were generated from individual lipid ion species.</p><p><br></p><p><strong>Ref:</strong></p><p><strong>[1]</strong> Claes BSR, Krestensen KK, Yagnik G, Grgic A, Kuik C, Lim MJ, Rothschild KJ, Vandenbosch M, Heeren RMA. MALDI-IHC-Guided In-Depth Spatial Proteomics: Targeted and Untargeted MSI Combined. Anal Chem. 2023 Jan 31;95(4):2329-2338. doi:10.1021/acs.analchem.2c04220. Epub 2023 Jan 13. PMID:36638208.</p>Embedding<p><strong>Human dorsal forebrain organoids</strong></p><p>To generate dorsal human forebrain organoids, we adapted the Pasca protocol for its advantages<strong>[1]</strong>: 1) It recapitulates with considerable accuracy the development of the human cortex and the organization of neuroprogenitor zones<strong>[2]-[4]</strong>; 2) It has been widely used, leading to numerous publications that have characterized it thoroughly<strong>[1][4]-[9]</strong> 3) It is highly efficient and consistent, resulting in highly reproducible organoids both within and between experiments<strong>[10]</strong>, and iv) It can be easily scaled-up to generate large numbers (thousands) of organoids, allowing us to produce sufficient analyses, which contributes to the statistical power of our material in a short period of time. In this organoid model, iPSC cultures are subsequently transferred into low-attachment AggreWell 800 to develop about 300 embryoid bodies. To achieve rapid and efficient neural induction, both the Bone morphogenetic proteins (BMP) and transforming growth factor beta (TGF-β) signaling pathways are inhibited with small molecules, dorsomorphin and SB-431542, respectively. Here, we used induced pluripotent stem cells (iPSCs) generated by reprogramming of control fibroblast cells (GM01888,;Product=CC) with Sendai virus<strong>[11]</strong>. We assessed the derived iPSC pluripotency using Karyostat, pluripotency score (Thermo Fisher), alkaline phosphatase staining (Vector Red Substrate Kit, Alkaline Phosphatase, SK-5100), and differentiation into each of the three germ layers (Human Pluripotent Stem Cell Functional Identification Kit, R&amp;D system Catalog: SC027B). iPSCs of high purity were placed in the AggreWell plate and on day eight, the floating spheroids were moved to suspension dishes and serum-free media containing fibroblast growth factor 2 (FGF2) and ascorbic acid. To promote differentiation of neuroprogenitors into neurons, FGF2 was replaced with brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3) starting on day 22-60, after which we used only neural medium without growth factors. Using this method, we can produce functional organoids with mature neurons and glial cells, preserving tissue architecture and orientation<strong>[12]</strong>.</p><p><br></p><p><strong>Refs:</strong></p><p><strong>[1]</strong> Pașca AM, Park JY, Shin HW, Qi Q, Revah O, Krasnoff R, O'Hara R, Willsey AJ, Palmer TD, Pașca SP. Human 3D cellular model of hypoxic brain injury of prematurity. Nat Med. 2019 May;25(5):784-791. doi:10.1038/s41591-019-0436-0. Epub 2019 May 6. PMID:31061540.</p><p><strong>[2]</strong> Paşca AM, Sloan SA, Clarke LE, Tian Y, Makinson CD, Huber N, Kim CH, Park JY, O'Rourke NA, Nguyen KD, Smith SJ, Huguenard JR, Geschwind DH, Barres BA, Paşca SP. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat Methods. 2015 Jul;12(7):671-8. doi:10.1038/nmeth.3415. Epub 2015 May 25. PMID:26005811.</p><p><strong>[3]</strong> Qian X, Song H, Ming GL. Brain organoids: advances, applications and challenges. Development. 2019 Apr 16;146(8):dev166074. doi:10.1242/dev.166074. PMID:30992274.</p><p><strong>[4]</strong> Birey F, Andersen J, Makinson CD, Islam S, Wei W, Huber N, Fan HC, Metzler KRC, Panagiotakos G, Thom N, O'Rourke NA, Steinmetz LM, Bernstein JA, Hallmayer J, Huguenard JR, Paşca SP. Assembly of functionally integrated human forebrain spheroids. Nature. 2017 May 4;545(7652):54-59. doi:10.1038/nature22330. Epub 2017 Apr 26. PMID:28445465.</p><p><strong>[5]</strong> Liu J, Kim YS, Richardson CE, Tom A, Ramakrishnan C, Birey F, Katsumata T, Chen S, Wang C, Wang X, Joubert LM, Jiang Y, Wang H, Fenno LE, Tok JB, Pașca SP, Shen K, Bao Z, Deisseroth K. Genetically targeted chemical assembly of functional materials in living cells, tissues, and animals. Science. 2020 Mar 20;367(6484):1372-1376. doi:10.1126/science.aay4866. PMID:32193327.</p><p><strong>[6]</strong> Cao Y, Hjort M, Chen H, Birey F, Leal-Ortiz SA, Han CM, Santiago JG, Paşca SP, Wu JC, Melosh NA. Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring. Proc Natl Acad Sci U S A. 2017 Mar 7;114(10):E1866-E1874. doi:10.1073/pnas.1615375114. Epub 2017 Feb 21. PMID:28223521.</p><p><strong>[7]</strong> Madelaine R, Sloan SA, Huber N, Notwell JH, Leung LC, Skariah G, Halluin C, Paşca SP, Bejerano G, Krasnow MA, Barres BA, Mourrain P. MicroRNA-9 Couples Brain Neurogenesis and Angiogenesis. Cell Rep. 2017 Aug 15;20(7):1533-1542. doi:10.1016/j.celrep.2017.07.051. PMID:28813666.</p><p><strong>[8]</strong> Trevino AE, Sinnott-Armstrong N, Andersen J, Yoon SJ, Huber N, Pritchard JK, Chang HY, Greenleaf WJ, Pașca SP. Chromatin accessibility dynamics in a model of human forebrain development. Science. 2020 Jan 24;367(6476):eaay1645. doi:10.1126/science.aay1645. PMID:31974223.</p><p><strong>[9]</strong> Uzquiano A, Kedaigle AJ, Pigoni M, Paulsen B, Adiconis X, Kim K, Faits T, Nagaraja S, Antón-Bolaños N, Gerhardinger C, Tucewicz A, Murray E, Jin X, Buenrostro J, Chen F, Velasco S, Regev A, Levin JZ, Arlotta P. Proper acquisition of cell class identity in organoids allows definition of fate specification programs of the human cerebral cortex. Cell. 2022 Sep 29;185(20):3770-3788.e27. doi:10.1016/j.cell.2022.09.010. PMID:36179669.</p><p><strong>[10]</strong> Yoon SJ, Elahi LS, Pașca AM, Marton RM, Gordon A, Revah O, Miura Y, Walczak EM, Holdgate GM, Fan HC, Huguenard JR, Geschwind DH, Pașca SP. Reliability of human cortical organoid generation. Nat Methods. 2019 Jan;16(1):75-78. doi:10.1038/s41592-018-0255-0. Epub 2018 Dec 20. PMID:30573846.</p><p><strong>[11]</strong> Chen IP, Fukuda K, Fusaki N, Iida A, Hasegawa M, Lichtler A, Reichenberger EJ. Induced pluripotent stem cell reprogramming by integration-free Sendai virus vectors from peripheral blood of patients with craniometaphyseal dysplasia. Cell Reprogram. 2013 Dec;15(6):503-13. doi:10.1089/cell.2013.0037. Epub 2013 Nov 12. PMID:24219578.</p><p><strong>[12]</strong> Manganas LN, Durá I, Osenberg S, Semerci F, Tosun M, Mishra R, Parkitny L, Encinas JM, Maletic-Savatic M. BASP1 labels neural stem cells in the neurogenic niches of mammalian brain. Sci Rep. 2021 Mar 10;11(1):5546. doi:10.1038/s41598-021-85129-1. Erratum in: Sci Rep. 2021 Oct 4;11(1):20008. PMID:33692421.</p>Metabolomics<p>Classical hematoxylin/eosin (HE) staining and immunostainings of adjacent sections were used to correlate the MSI images with the organoid anatomy. The tissues from the adjacent MSI-sections were visualized at x10 and x63 magnification using Leica (SP8X) fluorescence microscope and Nikon spinning disc microscope.</p><p><br></p><p><strong>Hematoxylin eosin staining (HE)</strong></p><p>Histological sections extracted from each block underwent routine HE staining following standard protocol<strong>[1]</strong>. We used commercially available Instant Hematoxylin from Thermo Scientific.</p><p><br></p><p><strong>Immunostaining</strong></p><p>Tissue sections were fixed in 150 μL 4% paraformaldehyde per section for 10 min, before incubation with the primary antibodies diluted in 0.3% Triton-X at 4 °C overnight (goat polyclonal IgG DCX sc-271390 (Santa Cruz), rabbit polyclonal IgG PAX6 901301 (Biolegend)). Secondary antibodies were applied at 1:500 dilution for 120 min at room temperature in the dark (Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647, AB_2762835, Donkey Anti-Goat IgG H&amp;L Alexa Fluor 555 preabsorbed (ab150134)). To stain the nuclei 1 mg/mL 4′,6-diamidino-2-phenylindole (DAPI, Life Tech, 62248) stock solution was diluted in PBS (1:2000) before adding to the slide. After 10 min, the DAPI was washed off twice with PBS before mounting the coverslips on glass slides with the anti-fading reagent (Molecular probes, P36930).</p><p><br></p><p><strong>Ref:</strong></p><p><strong>[1]</strong> Wittekind D. Traditional staining for routine diagnostic pathology including the role of tannic acid. 1. Value and limitations of the hematoxylin-eosin stain. Biotech Histochem. 2003 Oct;78(5):261-70. doi:10.1080/10520290310001633725. PMID:14989644.</p><p><strong>Brain organoid section preparation</strong></p><p>Brain organoids derived from iPSC were collected at 60 days using wide bore tips (ART Wide Bore Filtered Pipette Tips). They were washed 3x times with 1x Dulbecco’s phosphate-buffered saline (DPBS), without CaCl2 and MgCl2 (Life Technologies, cat. no. 14190-144) and then with distilled water. The organoids were then put firmly in the center of the Cryomolds cast blocks (25 mm x 20 mm x 5 mm) and embedding solution (10% of gelatin from fish (Sigma-Aldrich G7041-100G) or porcine skin (Sigma-Aldrich, cat. no. G1890), or culture-grade distilled water) was poured over and allowed to solidify. Next, 10% of gelatin solutions were prepared by stirring and heating the respective gelatin at 70-80 °C for 2 h, and then moving to 37 °C incubator for 30 min to remove bubbles and to equilibrate to the temperature of the incubator where organoids were grown. The molds with the organoid-gelatin/water suspension was rapidly frozen by placing the molds into a Petri dish sitting on dry ice and containing cold 100% ethanol. When completely frozen, evident by a change in color to solid white, the organoid-gelatin/water blocks were taken out and stored in aluminum foil or tin cups at -80 °C. Tissue samples were cut in 14 μm sections with a cryotome from -20 to -25 °C. Structurally uniform sections were thaw-mounted onto glass slides and indium tin oxide (ITO) conductive-coated slides (Hudson Surface Technology, New York, United States).</p><p><br></p><p><strong>Slide matrix deposition for MALDI-MSI</strong></p><p>Tissue sections were put from the -80 °C directly into a desiccator for 20 min to minimize condensation of atmospheric water on their surfaces. For MSI in positive and negative ion modes, 1,5 Diaminonaphthalene (DAN) matrix (9 mg/mL in 50% acetonitrile (ACN) and 0.4% N,N-Dimethylformamide (DMF)) was sprayed with the HTX M5+ sprayer (HTX Technologies LLC, Carrboro), respectively. Spraying parameters were as follows: temperature = 65 °C, nozzle velocity = 1200 mm/min, pump flow rate = 100 μL/min, number of passes = 4, track spacing = 2.5 mm, and 10 psi nitrogen gas pressure. The amount of matrix (Wm) deposited was 0.00055 mg/mm^2 of tissue area and the linear flow rate was 0.00008 mL/min.</p>imaging mass spectrometryneuronOrganoiduntargeted metabolitesmatrix-assisted laser desorption-ionisation mass spectrometryneuroprogenitorsimaging mass spectrometryneuronOrganoiduntargeted metabolitesmatrix-assisted laser desorption-ionisation mass spectrometryneuroprogenitorsforebrain<p>Theoretical m/z values of each lipid species were obtained from the database LIPIDMAPS ( and ALEX123 lipid calculator (, which were used for image generation with a bin width of ±5 ppm relative to the theoretical value. Various lipid species in the brain organoids were detected and identified based on the exact masses of lipid molecules.</p>PE 42:9FA 22:2FA 22:1FA 22:4FA 22:3PE 42:5PE 42:6PE 42:7PE 42:8PE 42:2PE 42:3PE 42:4PG 38:2PE 30:1PE 30:0PC O-32:1PG 38:5PE O-32:2PG 38:4PA 32:1PA 32:2PG 38:6PE 38:5PI 40:5PE 38:4PE 38:3PE 38:2PE 38:9PE 38:7PE 38:6PE 38:1PA 44:5PA 44:6PS 34:2PS 34:1PS 34:0PC 36:5;O2PC 30:1PC 38:2PC 38:1PC 38:6PC 38:7PC 38:4PC 38:5PC 38:8PS O-38:2PS O-38:4Hex2Cer 34:1;O2PS O-38:5FA 18:1FA 18:0FA 18:3FA 18:2PE 40:1PE 40:2HexCer 38:2;O3PI 38:6HexCer34:1;O4PI 38:5FA 22:6PI 38:4FA 22:5PI 38:3PI 38:2PI 38:1HexCer 40:2;O3PC 36:6;O2SM 34:1;2SM 34:1;3Cer 36:1;O2PC O-38:5PE O-38:5PE O-38:6PE O-38:7PE O-38:1PE O-38:2PE O-38:3LPE 22:4PA 34:1Hex2Cer 32:0;3PE O-30:1LPC 16:0LPC 16:1PE 32:3DGCC 36:5PE 32:2PE 32:1PG 32:1PE 32:0PG 32:0CerPE 40:2;4HexCer 36:2;O3LPI 18:0PE O-42:7PS 32:1SM 40:2;2PE O-42:5PE O-42:6LPC 20:5LPC 20:3LPC 20:4SM 40:2;O2PC 36:1PS 44:4PC 36:4PC 36:5PS 44:7SM 42:1;O2SM 36:3;O2PC 36:3PC 36:6PC 36:7SM 38:1;2SM 38:1;O2PC 40:3PC 40:4PC 40:7PC 40:5PI 36:4PI 36:3PI 36:2SM 40:1;2PI 36:1SHexCer 34:1;O3PI 36:0SHexCer 38:1;O3PA 36:0PA O-36:1PA 36:1LPE 20:4PE O-36:6PG 34:1LPG 16:0PG 34:0PG 34:3PG 34:2LPC 18:4PE O-36:2PE O-36:4LPC 18:2LPC 18:3PE O-36:5LPE 20:5PE O-36:0PE O-36:1PC O-36:2LPC 18:0LPC 18:1CerPE 36:1;O2PE 34:1PE 34:0PE 34:5PE 34:4PE 34:3PE 34:2PS 38:5PS 38:4PS 38:3PS 38:2PS 38:1PA 40:2PE O-40:5PE O-40:6PG O-40:7PE O-40:7PE O-40:8PI 34:1PI 34:0PS 42:2PC 34:2LPE 18:4HexCer 30:1;O2PC 34:3PS 42:5PS 42:6PC 34:1PC 34:6PS 42:8CerPE 34:1;O3PC 34:4PC 34:5PE 42:10SHexCer 36:1;O3PS O-34:0PS O-34:1LPE 18:0LPE 18:1LPE 18:2LPG 20:4LPE 18:3PI 34:3PI 34:2FA 20:0FA 20:4FA 20:3LPS 18:1PE 40:7PE 40:8PE 40:9PE 40:3PE 40:4PE 40:5PE 40:6PG 36:3PG 36:2PG 36:4PA 38:3PG 36:1LPG 18:1SM 40:1;O2PC O-34:2PE O-34:1PE O-34:2PE O-34:3PA O-38:0PC O-34:4PE 36:6PE 36:5Cer 34:1;O2PE 36:4CerPE 40:2;O3PE 36:3PE 36:2PE 36:1PG 40:6PS 36:4PS 36:3PS 36:2PG 40:7PS 36:1CerPE 38:2;O3PC 32:0PS 40:2PC 32:1PI 32:1PS 40:3Cer 34:0;O2PI 32:0PS 40:4PS 40:5PC 32:4PS 40:6PC 32:5PS 40:7PC 32:2PC 32:3FA 16:1PS O-36:1FA 16:0PS 40:0PC 44:7PS 37:1LPG 22:6SM 36:1;2LPE 16:1projections, IPP2A2, single-organism developmental process, ion, Prospecting, D15N2e, Feature, histology, Visible Light, Ceramide, 5730420M11Rik, AI448246, dmTAF[[II]]230, Readability, primary metabolites, Light Microscopy, diseases, Roles, hnu, Cer, Concepts, diseases and disorders, synganglion, cytopathology, PAST, Fs(3)Hor, anatomy and histology, foton, INSL3R, portion of tissue, SET, DmelCG2684, human disease, TFIID TAF250, mKIAA1668, TAF-I, cel, FATE, Simple, phosphatase 2A inhibitor I2PP2A, Tissue, SIDNA, Hand-Held Microscopy, suprasegmental levels of nervous system, NTef2, free, Drug Prospecting., DmelCG4299, set, IGAAD, GREAT, Organoid, DmelCG10574, Hand-Held, 260, papilla, sample, Pathologies, Role Concepts, Great, Homo sapiens disease, HPAST1, gamma, phapii, dTAF[[II]]230, suprasegmental structures, Radiation, anatomical protrusion, Arts, lamina, TAF200, StF-IT-1, flanges, Light, TAFII-250, TAF250/230, Optical Microscopy, Fs(3)Sz11, TAFII250, EST573322, LIGHT, Role Concept, Ionen, Playthings and Play, N-acylated sphingoid, Lipid, shelf, CT43, Diseases, Monoethanolamine, Role, tissue portion, Plaything, simple tissue, DMSIDNA, Visible Radiations, LGR8, Industrial, Visible Radiation, Lgr8, histopathology, Industrial Arts, HLA-DR-associated protein II, HVEML, DI-2, I-2Dm, shelves, Encephalon, a ceramide, Toys, expanded, CG4299, inhibitor of granzyme A-activated DNase, PAST1, Features, study protocol, CG17603, projection, TAF[[II]], ridge, human, early, Hand Held Microscopy, experimental procedures, organ system, I-2PP1, data analysis, disease, light quantum, TAF-IBETA, Taf250, enlarged, spine, SR3-5, Horka, metabolites, CG2684, Fs(3)Horka, Microscopy, TAF-Ibeta, anon-EST:Liang-2.35, i2pp2a, TAF230, the brain, EST820961, big, lipids, other disease, d230, Plays, human being, localised, CG5099, experimental, H-PAST, lamellae, ceramides, secondary metabolites, dTAFII250, EfW1, body system, terminal differentiation, process of organ, PHAPII, Ly113, protrusion, lamella, method, morphology, large, template-activating factor I, dmTAF1, Taf230, method used in an experiment, disease or disorder, system, DmF2, 2 Aminoethanol, Toy, TAF250, lod, study, Colamine, Taf200, anatomy, anatomical systems, methods, FOCUS, dTAF[[II]]250, pattern, Gpr106, Playthings, distribution, cell, iones, experimental section, Lichtquant, ipp2a2, 2pp2a, Taf1p, RXFPR2, ridges, Visible, ions, man, CG10574, Solution, non-neoplastic, Msi, dTAF250, Optical, 2PP2A, Puppets, data processing, taf-ibeta, 2-Aminoethanol, great, dSET, dSet, photon, Play, disorder, Characteristics, species, TR2, TAF, laminae, GPR106, Puppet, data, TAF[[II]]250, clone 2.35, anatomical process, igaad, disorders, l(3)84Ab, BG:DS00004.13, medical condition, CD258, encephalon, DmelCG5099, Cell, Concept, dTAF230, development, phosphoethanolamines, Characteristic, Discovery, I-2PP2A, p230, Simple Microscopy, Dm I-2, I2PP2A, TAF[[II]]250/230, TFIID, condition, focal, connected anatomical system, MIRab13, flange, organ process, Radiations, Taf[[II]]250, TAF[[II]]230, Compound Microscopy, HVEM-L, D15Mit260, Photoradiation, metabolite, TAF[II]250, Understanding, LGR8.1, Lds, sample population, LTg, plan specification, Drug, processes, process, dSET/TAF-Ibeta, 2610030F17Rik, DmelCG17603, Photoradiations, Ion, Compound, processus, biopsy, AA407739, TAF1biochemical pathways, the brain, multicellular organism metabolic process, Metabolic Process, suprasegmental structures, human being, biodegradation, Metabolic, degradation, Process, catabolism, Processes, metabolism resulting in cell growth, Encephalon, Metabolic Concepts, Metabolic Concept, metabolic process resulting in cell growth, Metabolic Processes, suprasegmental levels of nervous system, man, Organoid., encephalon, human, Concept, Metabolic Phenomena, Metabolism Concepts, single-organism metabolic process, Metabolism, Phenomena, Concepts, biotransformation, secretion, synganglion, Metabolism Concept, Phenomenon, metabolism, Metabolism Phenomena, Catabolism, Metabolic Phenomenon, AnabolismfalseMass spectrometry imaging as an emerging tool for studying metabolism in human brain organoids<p>Human brain organoids are emerging models to study human brain development and pathology as they recapitulate the development and characteristics of major neural cell types, and enable manipulation through an <em>in vitro</em> system. Over the past decade, with the advent of spatial technologies, mass spectrometry imaging (MSI) has become a prominent tool for metabolic microscopy, providing label-free, non-targeted molecular and spatial distribution information of the metabolites within tissue, including lipids. This technology has never been used for studies of brain organoids and here, we set out to develop a standardized protocol for preparation and mass spectrometry imaging of human brain organoids. We present an optimized and validated sample preparation protocol, including sample fixation, optimal embedding solution, homogenous deposition of matrices, data acquisition and processing to maximize the molecular information derived from mass spectrometry imaging. We focus on lipids in organoids, as they play critical roles during cellular and brain development. Using high spatial and mass resolution in positive- and negative-ion modes, we detected 260 lipids in the organoids. Seven of them were uniquely localized within the neurogenic niches or rosettes as confirmed by histology, suggesting their importance for neuroprogenitor proliferation. We observed a particularly striking distribution of ceramide-phosphoethanolamine CerPE 36:1; O2 restricted within rosettes and of phosphatidyl-ethanolamine PE 38:3, which was distributed throughout the organoid tissue but not in rosettes. This suggests that ceramide in this particular lipid species might be important for neuroprogenitor biology, while its removal may be important for terminal differentiation of their progeny. Overall, our study establishes the first optimized experimental pipeline and data processing strategy for mass spectrometry imaging of human brain organoids, allowing direct comparison of lipid signal intensities and distributions in these tissues. Further, our data shed new light on the complex processes that govern brain development by identifying specific lipid signatures that may play a role in metabolic cell fate trajectories. mass spectrometry imaging thus has great potential in advancing our understanding of early brain development as well as disease modeling and drug discovery.</p>2023-05-232023-03-24MTBLS7565