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A 3D human triculture system modeling neurodegeneration and neuroinflammation in Alzheimer’s disease

Nature Neuroscience volume 21, pages 941–951 (2018)Cite this article

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Abstract

Alzheimer’s disease (AD) is characterized by beta-amyloid accumulation, phosphorylated tau formation, hyperactivation of glial cells, and neuronal loss. The mechanisms of AD pathogenesis, however, remain poorly understood, partially due to the lack of relevant models that can comprehensively recapitulate multistage intercellular interactions in human AD brains. Here we present a new three-dimensional (3D) human AD triculture model using neurons, astrocytes, and microglia in a 3D microfluidic platform. Our model provided key representative AD features: beta-amyloid aggregation, phosphorylated tau accumulation, and neuroinflammatory activity. In particular, the model mirrored microglial recruitment, neurotoxic activities such as axonal cleavage, and NO release damaging AD neurons and astrocytes. Our model will serve to facilitate the development of more precise human brain models for basic mechanistic studies in neural–glial interactions and drug discovery.

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Fig. 1: Construction of a 3D organotypic human AD culture model (3D Neu + AC + MG AD): a triculturing system of AD neurons, astrocytes differentiated from hNPCs, and human adult microglia in a 3D microfluidic platform.
Fig. 2: Recapitulation of pathological AD signatures: Aβ, p-tau, and IFN-γ in the 3D Neu + AC AD model.
Fig. 3: Activation of microglial inflammation: morphogenesis, marker expression, recruitment, and inflammatory mediator release.
Fig. 4: Neuronal damage is exacerbated through interactions with reactive microglial cells.
Fig. 5: Assessment of neurotoxic neuron–glia interactions mediated by TLR4 and IFN-γ receptor.
Fig. 6: Neuron–glia interactions recapitulated with human iPSC-derived AD neurons/astrocytes.

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Acknowledgements

We thank L. Quinti (MGH) for sharing preliminary results regarding 3D neuron/microglia co-culture systems, M. Busche (MGH) for helpful guidance regarding the calcium imaging, S.H. Choi (MGH) for helpful discussion regarding the data interpretation, and Y.J. Kang (UNCC) for critically reviewing our manuscript. This work was supported by the NIH/NIA (P01 AG015379 and RF1 AG048080 to D.Y.K. and R.E.T.; R01 AG014713 to D.Y.K.), the Pioneering Funding Award funded by Cure Alzheimer’s Fund (CAF; to H.C., D.Y.K., R.E.T.), BrightFocus Foundation (D.Y.K.), the Duke Energy Special Initiatives funded by Charlotte Research Institute (CRI; to H.C.), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1A6A3A03019848, to J.P.).

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Authors and Affiliations

  1. Department of Mechanical Engineering and Engineering Science, University of North Carolina at Charlotte, Charlotte, NC, USA

    Joseph Park, Isaac Wetzel & Hansang Cho

  2. Center for Biomedical Engineering and Science, University of North Carolina at Charlotte, Charlotte, NC, USA

    Joseph Park, Isaac Wetzel, Ian Marriott, Didier Dréau & Hansang Cho

  3. Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC, USA

    Joseph Park, Isaac Wetzel, Ian Marriott, Didier Dréau & Hansang Cho

  4. The Nanoscale Science Program, University of North Carolina at Charlotte, Charlotte, NC, USA

    Joseph Park, Isaac Wetzel & Hansang Cho

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

    Joseph Park, Carla D’Avanzo, Doo Yeon Kim & Rudolph E. Tanzi

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Contributions

J.P. and H.C. designed, fabricated, and tested devices; designed experiments; performed immunostaining and statistical quantification; generated figures; and wrote and edited the manuscript. I.W. performed immunostaining and statistical quantification. C.D. generated human AD NPCs derived from iPSCs and measured APP and Aβ levels. D.D. and I.M. helped with confocal imaging and immunostaining, and wrote and edited the manuscript. D.Y.K., R.E.T., and H.C. conceived the ideas and directed the work, including all experiments and data analysis, and wrote and edited the manuscript. All authors read and edited the manuscript extensively.

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Correspondence to Hansang Cho.

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Integrated supplementary information

Supplementary Figure 1 Generation of FACS-sorted ReN cells with FAD mutations.

FACS sorting of ReN cell VM human neural stem (ReN) cells that were stably transfected with polycistronic GFP lentiviral vector. The cells were then enriched based on GFP and signals by FACS (above black-dotted lines, the selected ranges of cells for the experiments GFP, area intensity of GFP signal). ReN cells stably expressing GFP alone (ReN-G), APPSL-GFP (ReN-GA). (Numberdevice = 5; All experiments were repeated ≥ 3 times, Green: GFP; scale bars: 25 μm).

Supplementary Figure 2 3D reconstructed human AD neuron/astrocyte expressing GFP and APPSL mutation in microfluidic device.

The depth is 400 um. (Numberdevice = 5; All experiments were repeated ≥ 3 times, green: GFP; scale bars: 100 μm).

Supplementary Figure 3 3-week differentiated ReN cells with dendritic marker of MAP2 and dendritic spine formation.

(a) 3-week differentiated ReN cells immunstained with dendritic marker of MAP2 (yellow) with nucleus stain (blue). (b) dendritic spine formation observed in week 3 neuron cells that were stably transfected with polycistronic GFP (Numberdevice = 5 in a and b; All experiments were repeated ≥ 3 times, scale bars: 50 μm (a), 5 μm (b)).

Supplementary Figure 4 Astrocyte marker staining of S100, S100A6 and S100β at Week 6.

(Numberdevice = 5; All experiments were repeated ≥ 3 times, Scale bars: 100 μm).

Supplementary Figure 5 Focal plane of neurons and astrocytes used to measure the Ca2 + transients in the cell indicated by the red dotted line.

Somata of stained neurons had a characteristic ring-shape appearance with the surrounding cytoplasm (Numberdevice = 5; All experiments were repeated ≥ 3 times, Scale bar: 100 μm).

Supplementary Figure 6 Pie charts fractions of hyperactive neurons (red) in week 3 and week 9.

(Numberneurons = 50; All experiments were repeated ≥ 3 times at each condition, t = 2.31, d.f. = 7.71, P = 0.032).

Supplementary Figure 7 Released amount of Abeta40 and Abeta42 ratio in week 9.

Conditioned media from each individual device from 3D Neu + AC (blue), 2D Neu + AC AD (purple) and 3D Neu + AC AD (red) were measured and quantified by dividing Abeta40 or 42 at week0 by showing the released amount of soluble Abeta40 and Abeta42 in week 9. Graph showed increased amount of soluble Abeta40 in conditioned media while Abeta42 was slightly decreased by comparison with Week 0. Two-way ANOVA revealed significant differences in between 3D Neu + AC vs 3D Neu + AC AD and 2D Neu + AC AD vs 3D Neu + AC AD (all **p < 0.0001, F (2, 8) = 100.8), All experiments were repeated ≥ 3 times. Each data point is the average ± SEM. of five different devices.

Supplementary Figure 8 Analysis of SDS soluble Abeta levels in the enriched model by western blotting.

6E10-Abeta detected ~4 kDa a monomer band in the medium collected from the 3D Neu + AC, 2D Neu + AC AD and 3D Neu + AC AD cells. The 6E10 antibody also detected sAPP (~100 kDa), which would represent the amounts of full-length APPs in these cell lines (n = 3 per each sample).

Supplementary Figure 9 Aggregated for of pTau in AD neuron.

Phosphorylated tau (pTau) at Ser202/Thr206 was confirmed by immunofluorescent staining (pTau antibody AT8) in both neuritic and soma bodies of 3D Neu + AC + MG AD (Numberdevice = 5; All experiments were repeated ≥ 3 times, green: GFP, scale bars: 50 μm).

Supplementary Figure 10 Schematic representation for the definition of the recruitment index, R.I.

Microglia recruitment was quantified by comparing the fraction of cells inside the central chamber (CC) to the total number of cells in the corresponding angular chamber device (AC). The R.I. is calculated as R.I. = (CCDay i - CCDay 0) / ACDay i, where Day i is the number of cells in the central chamber and ADDay i is the number of cells in angular chamber. The recruitment index, R.I., is obtained by normalizing with the subtraction at ‘CCDay 0’ cell numbers.

Supplementary Figure 11 CCL2 dependent recruitment of microglial cells.

Microglial cell recruitment significantly reduced in AD neuron/astrocyte co-culture condition with neutralizing Anti-CCL2. Two-way ANOVA revealed significant differences in between 2D Neu + AC + MG AD vs 2D Neu + AC + MG AD + Anti-CCL2 (**p < 0.0020, F (1, 4) = 51.09) and 3D Neu + AC + MG AD vs 3D Neu + AC + MG AD + Anti-CCL2 (***p < 0.0007, F (1, 4) = 86.39), All experiments were repeated ≥ 3 times. Each data point is the average ± SEM. of five different devices.

Supplementary Figure 12 ATP measurement of different brain model.

Extracellular released ATP was measured from conditioned media in different type of brain model at week 0 and week 9. Two-way ANOVA revealed significant differences in between 3D Neu + AC vs 3D Neu + AC AD in week 9 (**p < 0.2389, F (1, 4) = 1.912) and 2D Neu + AC + MG vs 3D Neu + AC + MG AD in week 9 (***p < 0.0001, F (1, 4) = 732.3). (Numberdevice = 5; All experiments were repeated ≥ 3 times, blue: 3D Neu + AC, purple: 2D Neu + AC AD, red: 3D Neu + AC AD).

Supplementary Figure 13 Membrane kit assay for the cytokine measurement.

. 36 cytokine/chemokine were measured with different conditions 3D Neu + AC + MG, 3D Neu + AC AD and 3D Neu + AC + MG AD. (Numberdevice = 5, All experiments were repeated ≥ 3 times,).

Supplementary Figure 14 Axotomy mediated by microglia.

Time-lapse images showed that microglia (red) mediated neurite cleavage (green) in co-culture condition. (Numberdevice = 5; All experiments were repeated ≥ 3 times, Scale bar: 20 μm).

Supplementary Figure 15 Microglial cells are colocalized with 3D Neu + AC + MG AD showed large area disruption of neuron/astrocyte.

(Numberdevice = 5; All experiments were repeated ≥ 3 times, Scale bar: 100 μm).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15

Reporting Summary

Supplementary Video 1

Reconstructed 3D confocal image

Supplementary Video 2

Whole device rotation

Supplementary Video 3

Calcium imaging of AD neuron/astrocyte

Supplementary Video 4

Calcium imaging of AD neuron/astrocyte in week 3

Supplementary Video 5

Calcium imaging of AD neuron/astrocyte in week 9

Supplementary Video 6

Microglial expansion

Supplementary Video 7

Microglia mediated axotomy

Supplementary Video 8

Microglia mediated neurite retraction

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Park, J., Wetzel, I., Marriott, I. et al. A 3D human triculture system modeling neurodegeneration and neuroinflammation in Alzheimer’s disease. Nat Neurosci 21, 941–951 (2018). https://doi.org/10.1038/s41593-018-0175-4

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