Abstract
Stem cell technologies have facilitated the development of human cellular disease models that can be used to study pathogenesis and test therapeutic candidates. These models hold promise for complex neurological diseases such as Alzheimer's disease (AD), because existing animal models have been unable to fully recapitulate all aspects of pathology. We recently reported the characterization of a novel 3D culture system that exhibits key events in AD pathogenesis, including extracellular aggregation of amyloid-β (Aβ) and accumulation of hyperphosphorylated tau. Here we provide instructions for the generation and analysis of 3D human neural cell cultures, including the production of genetically modified human neural progenitor cells (hNPCs) with familial AD mutations, the differentiation of the hNPCs in a 3D matrix and the analysis of AD pathogenesis. The 3D culture generation takes 1–2 d. The aggregation of Aβ is observed after 6 weeks of differentiation, followed by robust tau pathology after 10–14 weeks.
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Change history
01 July 2015
In the version of this article initially published online, the y-axis labels in Figure 5b–d were incorrectly given as pmol/ml. The correct label is pmol/liter. The error has been corrected for the print, PDF and HTML versions of this article.
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Acknowledgements
This work is supported by grants from the Cure Alzheimer's Fund to D.Y.K. and R.E.T., from the US National Institutes of Health (NIH) 1RF1AG048080-01 (D.Y.K. and R.E.T.), 5P01AG15379 (D.Y.K. and R.E.T.), 2R01AG014713 (D.Y.K.) and 5R37MH060009 (R.E.T.), from the German Federal Ministry for Education and Research BioPharma-NeuroAllianz 0315608A (O.B.), from EU SCR&Tox HEALTH-F5-2010-266753 (O.B.), and from Bio & Medical Technology Development Program of the National Research Foundation (funded by the Korean government) (Y.H.K.). We appreciate B.T. Hyman, O. Berezovska, D.M. Kovacs (MGH), J. Hardy (NIH) and P. Davies (Albert Einstein College of Medicine) for providing cDNAs and antibodies. We also acknowledge B.A. Tannos at MGH Viral Vector Core (supported by NIH/National Institute of Neurological Disorders and Stroke P30NS04776), M. Waring at Ragon Institute's Imaging Core facility (part of the Harvard Center for AIDS Research Immunology Core), and M.L. McKee and D. Capen at MGH Microscopy Core of the Center for Systems Biology for providing the detailed protocols and for revising the manuscript.
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D.Y.K. and R.E.T. were equally responsible for experimental design and supervising the whole project. Y.H.K., S.H.C., C.D. and D.Y.K. mainly contributed to writing and revising the manuscript. C.D., E.B., K.J.W., M.H. and D.Y.K. performed the experiments. M.H., O.B., J.B.K. and C.S. contribute to writing the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Pictures showing each steps of our 3D culture protocol.
The numbers in each picturematch with those of 3D culture steps described within the protocol.
Supplementary Figure 2 Extracellular Aβ deposits increased in 3D-differentiated ReN VM cells withFAD mutations.
a. Detection of amyloid plaques with Amylo-Glo, a fluorescent amyloidspecificdye, in control ReN-G and FAD HReN-mGAP cells 3D-differentiated for 7 and 17weeks. HReN-mGAP cells, ReN-G cells were transfected with APPSL/PSΔE9/mCherryvectors, enriched with FACS to achieve high Aβ secretion; Blue, Amylo-Glo; Red,Propidium Iodide (P.I.); Scale bar, 50 μm. b. The number and size of Amylo-Glo-stainedamyloid structures were increased in 17-week differentiated FAD HReN-mGAP cells ascompared to 7-week 3D cultures (Blue, Amylo-Glo).
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Supplementary Figures 1–2 (PDF 466 kb)
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Kim, Y., Choi, S., D'Avanzo, C. et al. A 3D human neural cell culture system for modeling Alzheimer's disease. Nat Protoc 10, 985–1006 (2015). https://doi.org/10.1038/nprot.2015.065
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DOI: https://doi.org/10.1038/nprot.2015.065
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