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A 3D human neural cell culture system for modeling Alzheimer's disease

Nature Protocols volume 10, pages 985–1006 (2015)Cite this article

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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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Figure 1: Polycistronic lentiviral vectors used in this study.
Figure 2: Overview of the ReN VM cell 3D culture protocol.
Figure 3: FACS enrichment of ReN-G and ReN-GA cells for higher expressions of APP with FAD mutations.
Figure 4: Various 3D culture formats.
Figure 5: Analysis of soluble Aβ levels in the enriched FAD ReN cells by western blotting and Aβ ELISA.
Figure 6: Images of ReN-G2-differentiated neural cell populations in 3D cultures after 2- and 4-week differentiation.
Figure 7: Reconstitution of Aβ aggregates in 3D-cultured FAD ReN cells.
Figure 8: p-Tau accumulation in cell body and neurites of the enriched ReN-mAP cells.
Figure 9: Detection of aggregated p-tau in the enriched ReN-G and HReN-mGAP cells.

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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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Author notes

  1. Young Hye Kim, Se Hoon Choi and Carla D'Avanzo: These authors contributed equally to this work.

Authors and Affiliations

  1. Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA

    Young Hye Kim, Se Hoon Choi, Carla D'Avanzo, Matthias Hebisch, Christopher Sliwinski, Enjana Bylykbashi, Kevin J Washicosky, Justin B Klee, Rudolph E Tanzi & Doo Yeon Kim

  2. Biomedical Omics Group, Korea Basic Science Institute, Cheongju-si, Chungbuk, Republic of Korea.,

    Young Hye Kim

  3. Institute of Reconstructive Neurobiology, Life and Brain Center, University of Bonn and Hertie Foundation, Bonn, Germany

    Matthias Hebisch & Oliver Brüstle

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Contributions

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.

Corresponding authors

Correspondence to Rudolph E Tanzi or Doo Yeon Kim.

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The authors declare no competing financial interests.

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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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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