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. 2024 Jan 12:25:250-263.
doi: 10.1016/j.reth.2023.12.018. eCollection 2024 Mar.

Estradiol enhanced neuronal plasticity and ameliorated astrogliosis in human iPSC-derived neural models

Sopak Supakul  1 , Chisato Oyama  2 , Yuki Hatakeyama  1 , Sumihiro Maeda  1 , Hideyuki Okano  1
Affiliations

Estradiol enhanced neuronal plasticity and ameliorated astrogliosis in human iPSC-derived neural models

Sopak Supakul et al. Regen Ther. .

Abstract

Introduction: 17β-Estradiol (E2) is a sex hormone that has been previously demonstrated to have neurotherapeutic effects on animal models of Alzheimer's disease (AD). However, clinical trials on E2 replacement therapy for preventing AD onset yielded inconsistent results. Therefore, it is imperative to clarify the therapeutic effects of E2 on human cells. In this study, we utilized induced pluripotent stem cells (iPSCs) derived from multiple AD donors to explore the therapeutic effects of E2 on the in vitro model of human cells.

Methods: We conducted a systematic review and meta-analysis using a random-effects model of the previously reported AD clinical trials to summarize the effects of E2 replacement therapy on AD prevention. Subsequently, we induced iPSCs from the donors of the healthy control (1210B2 line (female) and 201B7 line (female)), the familial AD (APP V717L line (female) and APP KM670/671NL line (female)), and the sporadic AD (UCSD-SAD3.7 line (APOE ε3/ε3) (male), UCSD-SAD7D line (APOE ε3/ε4) (male), and TMGH-1 line (APOE ε3/ε3) (female)), then differentiated to neurons. In addition to the mono-culture model of the neurons, we also examined the effects of E2 on the co-culture model of neurons and astrocytes.

Results: The meta-analysis of the clinical trials concluded that E2 replacement therapy reduced the risk of AD onset (OR, 0.69; 95 % confidence interval [CI], 0.53-0.91; I2 = 82 %). Neural models from the iPSCs of AD donors showed an increase in secreted amyloid-beta (Aβ) levels in the mono-culture model and an astrogliosis-like phenotype in the co-culture model. E2 treatment to the neuronal models derived from the iPSCs enhanced neuronal activity and increased neurite complexity. Furthermore, E2 treatment of the co-culture model ameliorated the astrogliosis-like phenotype. However, in contrast to the previous reports using mouse models, E2 treatment did not change AD pathogenesis, including Aβ secretion and phosphorylated tau (pTau) accumulation.

Conclusion: E2 treatment of the human cellular model did not impact Aβ secretion and pTau accumulation, but promoted neuronal plasticity and alleviated the astrogliosis-like phenotype. The limited effects of E2 may give a clue for the mixed results of E2 clinical trials.

Keywords: 17β-estradiol; Alzheimer's disease; Astrocytes; Induced pluripotent stem cells (iPSCs); Neurons.

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Conflict of interest statement

Hideyuki Okano is a founder scientist and a Scientific Advisory Board Member for SanBio Co., Ltd., and K Pharma Inc. The remaining authors declare no conflicts of interest.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Meta-analysis of the clinical trials investigating the effects of estrogen replacement therapy and the prevalence of Alzheimer's disease. A Flowchart of the approach for identifying studies included in the analysis. B Forest plot (random-effects model) of the clinical studies included in the meta-analysis (n = 15).
Fig. 2
Fig. 2
Utilizing induced pluripotent stem cells (iPSCs) as a platform to elucidate the hormonal effects in human cells. A Induction method for the differentiation of neurons from iPSCs using the dual SMAD inhibition. B RT-qPCR analysis of stem cell marker (OCT4), stem cell and neural progenitor cell marker (SOX2), and forebrain markers (FOXG1 and PAX6) compared between the original iPSCs (n = 3) and the induced neural progenitor cells (NPCs) at PID 15 (n = 3). Bars, mean ± SEM. OCT4: ∗∗p = 0.0011; SOX2: non-significant (ns); FOXG1: ∗∗p = 0.0090; PAX6: ∗p = 0.0151 (Unpaired t-test). C Bright-field images of the iPSCs, the induced neural progenitor cells at PID 15, and the induced neurons at PID 45 of the healthy control donor (1210B2 line). Scale bar; 100 μm. D Immunocytochemistry images of iPSC-derived neurons at PID 45 stained with neuronal markers (MAP2, Tau, and NeuN) and synaptic markers (Synapsin I). Scale bar; 50 μm and 20 μm. The relative proportion of markers compared to Hoechst (n = 4). Bars, mean ± SEM.
Fig. 3
Fig. 3
iPSCs-derived neurons from the AD patients expressed AD phenotypes. A Table of iPSC lines comprised of healthy control (1210B2 line (WT1) and 201B7 line (WT2)), familial AD (APP2E26 line (APP V717L) (FAD1) and FA16-601 line (APP KM670/671NL) (FAD2)), and sporadic AD (SAD3 line (APOE ε3/ε3) (SAD1), SAD7 line (APOE ε3/ε4) (SAD2), and TMGH-1 line (APOE ε3/ε3) (SAD3)) used in this study. B Relative Aβ1-42 levels, Aβ1-40 levels, and Aβ1-42/Aβ1-40 ratio of the iPSC-derived neurons at PID 45 (n = 16 each). Bars, mean ± SEM. Aβ1-42: ∗∗∗p = 0.0009; Aβ1-40: ∗p = 0.0116; Aβ1-42/Aβ1-40 ratio: ∗∗∗∗p < 0.0001 (Kruskal-Wallis test). C Aβ1-42 and Aβ1-40 levels (pmol/L) of the iPSC-derived neurons after treatment with 5 μM γ-Secretase Inhibitor (DAPT) for 2 days (n = 8 each). Bars, mean ± SEM. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 (Kruskal-Wallis test).
Fig. 4
Fig. 4
Treatment with 17β-estradiol (E2) elevated neuronal activities and neurite branching of the iPSC-derived neurons after PID 45 (line WT1, FAD1, and SAD3). A Immunocytochemistry images of iPSC-derived neurons at PID 45 stained with anti-estrogen receptor antibodies (ERs) (ERα or ERβ). Scale bar; 50 μm. B Ca2+ oscillations of the iPSC-derived neurons at PID 45 after 15-min 100 nM E2 treatment measured with Ca2+ imaging (n = 45 each). WT1: ∗∗p = 0.0010 & ∗p = 0.0203; FAD1: ∗∗∗∗p < 0.0001 & ∗∗∗∗p < 0.0001; SAD3: ∗p = 0.0142 & ∗∗p = 0.0087 (Mann Whitney test). C Increased neurite branching of iPSC-derived neurons after 4-day 100 nM E2 treatment measured by Sholl analysis (n = 20 each). WT1, FAD1, SAD3: ∗∗∗∗p < 0.0001 (Two-way ANOVA).
Fig. 5
Fig. 5
The effects of E2 treatment on AD pathologies of the iPSC-derived neurons. A Treatment with 100 nM E2 for 2 months to the iPSC-derived neurons at PID 45 did not change the levels of the secreted amyloid-beta (Aβ1-42, Aβ1-40, and Aβ1-42/Aβ1-40 ratio) in every cell line (n = 15 each). ns: non-significant (Kruskal-Wallis test). B Treatment with 100 nM E2 for 2 months to the iPSC-derived neurons at PID 45 did not change the level of phosphorylated Tau proteins (AT180, PHF-1, and CP13) in every cell line (n = 3 each). ns: non-significant (Kruskal-Wallis test).
Fig. 6
Fig. 6
Treatment with E2 alleviated the astrogliosis-like phenotype developed in the fAD iPSC-derived co-culture model. A Induction method for the differentiation of astrocytes from iPSCs. B Bright-field image of human astrocytes induced from iPSCs. Scale bar; 50 μm. C Immunocytochemistry images of iPSC-derived astrocytes after PID 98 stained with markers of estrogen receptors (ERs) (ERα and ERβ). Scale bar; 50 μm. D Immunocytochemistry images of the co-culture model derived from the control (WT2 line) and the AD (FAD1 line) donors at 30 days after co-culturing the iPSC-derived neurons and astrocytes stained with S100β, GFAP, and MAP2. Scale bar; 100 μm. E Treatment with 100 nM E2 for 4 days alleviated the astrogliosis-like phenotype in the co-culture model generated from AD donor (FAD1 line) (n = 30 each). Scale bar; 100 μm. Bars, mean ± SEM. ∗∗∗∗p < 0.0001 (One-way ANOVA).
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