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, 11 (9), e0161969
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Self-Organizing 3D Human Neural Tissue Derived From Induced Pluripotent Stem Cells Recapitulate Alzheimer's Disease Phenotypes

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Self-Organizing 3D Human Neural Tissue Derived From Induced Pluripotent Stem Cells Recapitulate Alzheimer's Disease Phenotypes

Waseem K Raja et al. PLoS One.

Abstract

The dismal success rate of clinical trials for Alzheimer's disease (AD) motivates us to develop model systems of AD pathology that have higher predictive validity. The advent of induced pluripotent stem cells (iPSCs) allows us to model pathology and study disease mechanisms directly in human neural cells from healthy individual as well as AD patients. However, two-dimensional culture systems do not recapitulate the complexity of neural tissue, and phenotypes such as extracellular protein aggregation are difficult to observe. We report brain organoids that use pluripotent stem cells derived from AD patients and recapitulate AD-like pathologies such as amyloid aggregation, hyperphosphorylated tau protein, and endosome abnormalities. These pathologies are observed in an age-dependent manner in organoids derived from multiple familial AD (fAD) patients harboring amyloid precursor protein (APP) duplication or presenilin1 (PSEN1) mutation, compared to controls. The incidence of AD pathology was consistent amongst several fAD lines, which carried different mutations. Although these are complex assemblies of neural tissue, they are also highly amenable to experimental manipulation. We find that treatment of patient-derived organoids with β- and γ-secretase inhibitors significantly reduces amyloid and tau pathology. Moreover, these results show the potential of this model system to greatly increase the translatability of pre-clinical drug discovery in AD.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Organoids created from patient-derived iPSCs exhibit robust Alzheimer’s disease (AD)-like pathology.
(A) Concentration of Aβ1–40 and Aβ1–42 from supernatant of control (Ctrl; CS-0020-01) and familial AD (fAD; APPDp1-1) organoid cultures, measured by ELISA, as well as the ratio of Aβ1–42 to Aβ1–40 concentrations. Unpaired two-tailed t-test with equal variance: *p = 0.047 (Aβ1–40), unpaired two-tailed t-test with Welch’s correction for unequal variance: **p = 0.004 (Aβ1–42), p = 0.48 (Aβ1-42/Aβ1–40). (B) Tissue sections from fAD (APPDp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against amyloid β (Aβ) using two antibodies (D54D2: white, 4G8: green), as well as antibodies against the neuronal marker MAP2 (red) and stained with the nuclear dye Hoechst (blue). Insets demonstrate Aβ immunoreactivity that appears both extracellular (i, arrow) and intracellular (ii, arrowhead) based upon MAP2 co-localization. (C) Z-projection of immunolabeled tissue sections from 90 day old Ctrl and fAD organoids showing immunoreactivity for Aβ (D45D2: white) and MAP2 (red). The edge of the tissue section is visible at the left bottom corner of each example. (D) Quantification of Aβ immunoreactivity in fAD and Ctrl organoids following 60d and 90d culture. Particle Counts: one-way ANOVA with post-hoc Fishers Least Significant Difference (LSD) test for multiple comparisons; F (3,28) = 4.385, ***p = 0 0.0008, R2 = 0.32 (i-60 days); F (5,43) = 3.346, *p = 0 0.012, R2 = 0.28 (90 days). Particle Size: Two-tailed Mann Whitney test for non-normal distributions (normality α < 0.05), **p = 0.006 (60 days), ***p = 0.001 (90 days). (E) Tissue sections from fAD (APPDp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against phosphorylated Tau (pTau, green) at Serine 396 (S396) and MAP2 (red) following 90d culture. Hoechst (blue) labels cell nuclei. (F) Quantification of pTau immunoreactivity for the Ser396 at 60d and 90d, and for the Threonine 181 (Thr181) pTau at 90d. Values are plotted as mean intensity of immunoreactivity as fold change of Ctrl. Unpaired two-tailed t-test with equal variance: p = 0.67 (60 day Ser396), **p = 0.001 (90 day Ser396), *p = 0.03 (90 day Thr181). (G) Sections from fAD (APPDp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against the early endosome antigen 1 (EEA1, green) and MAP2 (red). The dotted white line outlines the region of higher magnification to show EEA1 detail. (H) Quantification of EEA1 immunoreactivity in fAD and Ctrl organoids following 90d culture. EEA1 Particle Counts: one-way ANOVA with post-hoc Fisher’s LSD test for multiple comparisons; F (3,16) = 4.0, *p = 0.026, R2 = 0.43. EEA1 Particle size: unpaired two-tailed t-test with Welch’s correction: *p = 0.041. (I) Organoids from Ctrl and fAD lines were subjected to the transferrin endocytosis assay to label pools of clathrin-coated early endosomes. (Each data point represent one organoid) Quantification of the average size of transferrin-positive particles: unpaired two-tailed t-test with equal variance, **p = 0.005. Average number (count) of transferrin-positive particles, unpaired two-tailed t-test with equal variance, p = 0.64. On charts: *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2
Fig 2. Organoids created from different lines of AD patient iPSCs exhibit AD phenotypes.
(A) Tissue sections from fAD (APPDp2-3, ND34732, AG068840) and control (Ctrl; CS-0020-01, AG09173) organoids were processed for immunoreactivity against Aβ (D45D2, white), MAP2 (red), and pTau (S396, green) and labeled with the nuclear dye Hoechst. (B) Quantification of Aβ immunoreactivity in fAD and Ctrl organoids following 90 days of culture. Values between the two control lines did not significantly differ. Number of Aβ-positive aggregates in two size classes (Particle Counts): one-way ANOVA with post-hoc Tukey’s multiple comparisons test; F (4,21) = 6.15, **p = 0.0019, R2 = 0.5396 (1–3μm); F (4,21) = 7.95, ***p = 0.0005, R2 = 0.6024 (3–6 μm). (C) Quantification of the average intensity of pTau Ser396 immunoreactivity as a fold change of Ctrl in fAD and Ctrl organoids following 90 days of culture. Values between the two control lines did not significantly differ. (Each data point represent one organoid). One-way ANOVA with post-hoc Tukey’s multiple comparisons test; F (4,20) = 9.629, ***p = 0.0002, R2 = 0.6582. On charts: *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3
Fig 3. Organoids created from AD patient iPSCs respond to compound treatment.
(A) Schematic of beta (BACE-1) and gamma (Comp-E) secretase inhibitor treatment (top). At 30 days of culture, fAD (APPDp1-1) organoids were treated with low dose (BACE-1, 1μM and Comp-E, 3nM) or high dose (BACE-1, 5 μM and Comp-E 6nM) combined compounds, or equivalent DMSO vehicle. Following 30 or 60 days of culture and drug treatment, organoids at 60 and 90 days of culture, respectively, were processed for immunohistochemistry (IHC). Tissue sections from fAD (APPDp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against Aβ (D45D2, white), pTau (Ser396, green), and MAP2 (red). Examine images are from 90 day organoids. (B) Quantification of Aβ particle number and size in compound treated and fAD organoids following 30 days of administration. Number of Aβ-positive aggregates in two size classes (Particle Counts): one-way ANOVA with Fishers LSD test for multiple comparisons; F (5,24) = 3.58, *p = 0.014, R2 = 0.4296. Particle size: one-way ANOVA with Kruskal-Wallis test for non-normal distribution (α < 0.05), p = 0.475. (C) Quantification of Aβ particle number and size in treated (high dose) and untreated fAD organoids following 60 days of compound administration. Number of Aβ-positive aggregates in three size classes (Particle Counts): one-way ANOVA with Fishers LSD test for multiple comparisons; F (5,19) = 5.02, **p = 0.004, R2 = 0.5691. Particle size: Mann-Whitney two-tailed test for non-normal distribution (α < 0.05), p = 0.09. (D) Quantification of the average intensity of pTau Ser396 immunoreactivity as a fold change of Ctrl in fAD organoids following 30 and 60 days of compound treatment. 30 day treatment. (Each data point represent one organoid). Unpaired two-tailed t-test with equal variance, p = 0.69. 60 day treatment: one-way ANOVA with Tukey’s multiple comparisons test, F (2,13) = 19.82, ***p = 0.0001, R2 = 0.7530. On charts: *p < 0.05, **p < 0.01, ***p < 0.001.

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