Cerebral organoids are a prolific research topic and an emerging model system for neurological diseases in human neurobiology. However, the batch-to-batch reproducibility of current cultivation protocols is challenging and thus requires a high-throughput methodology to comprehensively characterize cerebral organoid cytoarchitecture and neural development. We report a mass spectrometry-based protocol to quantify neural tissue cell markers, cell surface lipids, and housekeeping proteins in a single organoid. Profiled traits probe the development of neural stem cells, radial glial cells, neurons, and astrocytes. We assessed the cell population heterogeneity in individually profiled organoids in the early and late neurogenesis stages. Here, we present a unifying view of cell-type specificity of profiled protein and lipid traits in neural tissue. Our workflow characterizes the cytoarchitecture, differentiation stage, and batch cultivation variation on an individual cerebral organoid level.

Department of Chemistry Faculty of Pharmacy Charles University Hradec Králové 500 05 Czech Republic

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno 625 00 Czech Republic

International Clinical Research Center St Anne's University Hospital Brno 656 91 Czech Republic

RECETOX Faculty of Science Masaryk University Brno 625 00 Czech Republic

Zobrazit více v PubMed

Kelava I.; Lancaster M. A. Stem Cell Models of Human Brain Development. Cell Stem Cell 2016, 18, 736–748. 10.1016/j.stem.2016.05.022. PubMed DOI

Lancaster M. A.; Renner M.; Martin C. A.; Wenzel D.; Bicknell L. S.; Hurles M. E.; Homfray T.; Penninger J. M.; Jackson A. P.; Knoblich J. A. Cerebral Organoids Model Human Brain Development and Microcephaly. Nature 2013, 501, 373–379. 10.1038/nature12517. PubMed DOI PMC

Dang J.; Tiwari S. K.; Lichinchi G.; Qin Y.; Patil V. S.; Eroshkin A. M.; Rana T. M. Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate Immune Receptor TLR3. Cell Stem Cell 2016, 19, 258–265. 10.1016/j.stem.2016.04.014. PubMed DOI PMC

Zhang B.-Z.; Chu H.; Han S.; Shuai H.; Deng J.; Hu Y.; Gong H.; Lee A. C.-Y.; Zou Z.; Yau T.; Wu W.; Hung I. F.-N.; Chan J. F.-W.; Yuen K.-Y.; Huang J.-D. SARS-CoV-2 Infects Human Neural Progenitor Cells and Brain Organoids. Cell Res. 2020, 30, 928–931. 10.1038/s41422-020-0390-x. PubMed DOI PMC

Chiaradia I.; Lancaster M. A. Brain Organoids for the Study of Human Neurobiology at the Interface of in Vitro and in Vivo. Nat. Neurosci. 2020, 23, 1496–1508. 10.1038/s41593-020-00730-3. PubMed DOI

Dzwonek J.; Wilczyński G. M. CD44: Molecular Interactions, Signaling and Functions in the Nervous System. Front. Cell. Neurosci. 2015, 9, 17510.3389/fncel.2015.00175. PubMed DOI PMC

Prez K.; Fan L. Structural Basis for S100B Interaction with Its Target Proteins. J. Mol. Genet. Med. 2018, 12, 36610.4172/1747-0862.1000366. PubMed DOI PMC

Geisert E. E.; Johnson H. G.; Binder L. I. Expression of Microtubule-Associated Protein 2 by Reactive Astrocytes. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3967–3971. 10.1073/pnas.87.10.3967. PubMed DOI PMC

Zhang J.; Jiao J.. Molecular Biomarkers for Embryonic and Adult Neural Stem Cell and Neurogenesis. In BioMed Research International; Hindawi Limited, 2015; pp 1–14. PubMed PMC

Bohaciakova D.; Hruska-Plochan M.; Tsunemoto R.; Gifford W. D.; Driscoll S. P.; Glenn T. D.; Wu S.; Marsala S.; Navarro M.; Tadokoro T.; Juhas S.; Juhasova J.; Platoshyn O.; Piper D.; Sheckler V.; Ditsworth D.; Pfaff S. L.; Marsala M. A Scalable Solution for Isolating Human Multipotent Clinical-Grade Neural Stem Cells from ES Precursors. Stem Cell Res. Ther. 2019, 10, 8310.1186/s13287-019-1163-7. PubMed DOI PMC

Alemi M.; Gaiteiro C.; Ribeiro C. A.; Santos L. M.; Gomes J. R.; Oliveira S. M.; Couraud P. O.; Weksler B.; Romero I.; Saraiva M. J.; Cardoso I. Transthyretin Participates in Beta-Amyloid Transport from the Brain to the Liver- Involvement of the Low-Density Lipoprotein Receptor-Related Protein 1?. Sci. Rep. 2016, 6, 2016410.1038/srep20164. PubMed DOI PMC

Regina Todeschini A.; Hakomori S. Functional Role of Glycosphingolipids and Gangliosides in Control of Cell Adhesion, Motility, and Growth, through Glycosynaptic Microdomains. Biochim. Biophys. Acta, Gen. Subj. 2008, 1780, 421–433. 10.1016/j.bbagen.2007.10.008. PubMed DOI PMC

Kolter T. Ganglioside Biochemistry. ISRN Biochem. 2012, 2012, 1–36. 10.5402/2012/506160. PubMed DOI PMC

Chiricozzi E.; Lunghi G.; Di Biase E.; Fazzari M.; Sonnino S.; Mauri L. GM1 Ganglioside Is A Key Factor in Maintaining the Mammalian Neuronal Functions Avoiding Neurodegeneration. Int. J. Mol. Sci. 2020, 21, 86810.3390/ijms21030868. PubMed DOI PMC

Kim H.; Xu R.; Padmashri R.; Dunaevsky A.; Liu Y.; Dreyfus C. F.; Jiang P. Pluripotent Stem Cell-Derived Cerebral Organoids Reveal Human Oligodendrogenesis with Dorsal and Ventral Origins. Stem Cell Rep. 2019, 12, 890–905. 10.1016/j.stemcr.2019.04.011. PubMed DOI PMC

Hale J. E. Advantageous Uses of Mass Spectrometry for the Quantification of Proteins. Int. J. Proteomics 2013, 2013, 1–6. 10.1155/2013/219452. PubMed DOI PMC

Li L.; Han J.; Wang Z.; Liu J.; Wei J.; Xiong S.; Zhao Z. Mass Spectrometry Methodology in Lipid Analysis. Int. J. Mol. Sci. 2014, 15, 10492–10507. 10.3390/ijms150610492. PubMed DOI PMC

Allende M. L.; Cook E. K.; Larman B. C.; Nugent A.; Brady J. M.; Golebiowski D.; Sena-Esteves M.; Tifft C. J.; Proia R. L. Cerebral Organoids Derived from Sandhoff Disease-Induced Pluripotent Stem Cells Exhibit Impaired Neurodifferentiation. J. Lipid Res. 2018, 59, 550–563. 10.1194/JLR.M081323. PubMed DOI PMC

Latour Y. L.; Yoon R.; Thomas S. E.; Grant C.; Li C.; Sena-Esteves M.; Allende M. L.; Proia R. L.; Tifft C. J. Human GLB1 Knockout Cerebral Organoids: A Model System for Testing AAV9-Mediated GLB1 Gene Therapy for Reducing GM1 Ganglioside Storage in GM1 Gangliosidosis. Mol. Genet. Metab. Rep. 2019, 21, 10051310.1016/J.YMGMR.2019.100513. PubMed DOI PMC

Boutry M.; Branchu J.; Lustremant C.; Pujol C.; Pernelle J.; Matusiak R.; Seyer A.; Poirel M.; Chu-Van E.; Pierga A.; Dobrenis K.; Puech J. P.; Caillaud C.; Durr A.; Brice A.; Colsch B.; Mochel F.; El Hachimi K. H.; Stevanin G.; Darios F. Inhibition of Lysosome Membrane Recycling Causes Accumulation of Gangliosides That Contribute to Neurodegeneration. Cell Rep. 2018, 23, 3813–3826. 10.1016/J.CELREP.2018.05.098. PubMed DOI PMC

Shi T.; Song E.; Nie S.; Rodland K. D.; Liu T.; Qian W.-J.; Smith R. D. Advances in Targeted Proteomics and Applications to Biomedical Research. Proteomics 2016, 16, 2160–2182. 10.1002/pmic.201500449. PubMed DOI PMC

Hájek R.; Jirásko R.; Lísa M.; Cífková E.; Holčapek M. Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry Characterization of Gangliosides in Biological Samples. Anal. Chem. 2017, 89, 12425–12432. 10.1021/acs.analchem.7b03523. PubMed DOI

Vidova V.; Stuchlikova E.; Vrbova M.; Almasi M.; Klanova J.; Thon V.; Spacil Z. Multiplex Assay for Quantification of Acute Phase Proteins and Immunoglobulin A in Dried Blood Spots. J. Proteome Res. 2019, 18, 380–391. 10.1021/acs.jproteome.8b00657. PubMed DOI

Paşca A. M.; Sloan S. A.; Clarke L. E.; Tian Y.; Makinson C. D.; Huber N.; Kim C. H.; Park J. Y.; O’Rourke N. A.; Nguyen K. D.; Smith S. J.; Huguenard J. R.; Geschwind D. H.; Barres B. A.; Pasca S. P. Functional Cortical Neurons and Astrocytes from Human Pluripotent Stem Cells in 3D Culture. Nat. Methods 2015, 12, 671–678. 10.1038/nmeth.3415. PubMed DOI PMC

Nascimento J. M.; Saia-Cereda V. M.; Sartore R. C.; da Costa R. M.; Schitine C. S.; Freitas H. R.; Murgu M.; de Melo Reis R. A.; Rehen S. K.; Martins-de-Souza D. Human Cerebral Organoids and Fetal Brain Tissue Share Proteomic Similarities. Front. Cell Dev. Biol. 2019, 7, 30310.3389/fcell.2019.00303. PubMed DOI PMC

Luo C.; Lancaster M. A.; Castanon R.; Nery J. R.; Knoblich J. A.; Ecker J. R. Cerebral Organoids Recapitulate Epigenomic Signatures of the Human Fetal Brain. Cell Rep. 2016, 17, 3369–3384. 10.1016/j.celrep.2016.12.001. PubMed DOI PMC

Fair S. R.; Julian D.; Hartlaub A. M.; Pusuluri S. T.; Malik G.; Summerfied T. L.; Zhao G.; Hester A. B.; Ackerman W. E.; Hollingsworth E. W.; Ali M.; McElroy C. A.; Buhimschi I. A.; Imitola J.; Maitre N. L.; Bedrosian T. A.; Hester M. E. Electrophysiological Maturation of Cerebral Organoids Correlates with Dynamic Morphological and Cellular Development. Stem Cell Rep. 2020, 15, 855–868. 10.1016/j.stemcr.2020.08.017. PubMed DOI PMC

Sipione S.; Monyror J.; Galleguillos D.; Steinberg N.; Kadam V. Gangliosides in the Brain: Physiology, Pathophysiology and Therapeutic Applications. Front. Neurosci. 2020, 14, 100410.3389/fnins.2020.572965. PubMed DOI PMC

Nakatani Y.; Yanagisawa M.; Suzuki Y.; Yu R. K. Characterization of GD3 Ganglioside as a Novel Biomarker of Mouse Neural Stem Cells. Glycobiology 2010, 20, 78–86. 10.1093/glycob/cwp149. PubMed DOI PMC

Bond A. M.; Ming G.; Song H. Adult Mammalian Neural Stem Cells and Neurogenesis: Five Decades Later. Cell Stem Cell 2015, 17, 385–395. 10.1016/j.stem.2015.09.003. PubMed DOI PMC

Ryu J.-S.; Ko K.; Ko K.; Kim J.-S.; Kim S.-U.; Chang K.-T.; Choo Y.-K. Roles of Gangliosides in the Differentiation of Mouse Pluripotent Stem Cells to Neural Stem Cells and Neural Cells. Mol. Med. Rep. 2017, 16, 987–993. 10.3892/mmr.2017.6719. PubMed DOI

Schengrund C.-L. Gangliosides: Glycosphingolipids Essential for Normal Neural Development and Function. Trends Biochem. Sci. 2015, 40, 397–406. 10.1016/j.tibs.2015.03.007. PubMed DOI

Marconi S.; De Toni L.; Lovato L.; Tedeschi E.; Gaetti L.; Acler M.; Bonetti B. Expression of Gangliosides on Glial and Neuronal Cells in Normal and Pathological Adult Human Brain. J. Neuroimmunol. 2005, 170, 115–121. 10.1016/j.jneuroim.2005.03.025. PubMed DOI

Saito M.; Wu G.; Hui M.; Masiello K.; Dobrenis K.; Ledeen R. W.; Saito M. Ganglioside Accumulation in Activated Glia in the Developing Brain: Comparison between WT and GalNAcT KO Mice. J. Lipid Res. 2015, 56, 1434–1448. 10.1194/jlr.M056580. PubMed DOI PMC

Zhang Y.; Lukacova V.; Reindl K.; Balaz S. Quantitative Characterization of Binding of Small Molecules to Extracellular Matrix. J. Biochem. Biophys. Methods 2006, 67, 107–122. 10.1016/j.jbbm.2006.01.007. PubMed DOI PMC

Johnson J.; Sharick J. T.; Skala M. C.; Li L. Sample Preparation Strategies for High-Throughput Mass Spectrometry Imaging of Primary Tumor Organoids. J. Mass Spectrom. 2020, 55, e445210.1002/jms.4452. PubMed DOI PMC

Domino-like effect of C112R mutation on ApoE4 aggregation and its reduction by Alzheimer's Disease drug candidate

Effects of all-trans and 9-cis retinoic acid on differentiating human neural stem cells in vitro