Efficient generation of brain organoids using magnetized gold nanoparticles

Abstract

Brain organoids, which are three-dimensional cell culture models, have the ability to mimic certain structural and functional aspects of the human brain. However, creating these organoids can be a complicated and difficult process due to various technological hurdles. This study presents a method for effectively generating cerebral organoids from human induced pluripotent stem cells (hiPSCs) using electromagnetic gold nanoparticles (AuNPs). By exposing mature cerebral organoids to magnetized AuNPs, we were able to cultivate them in less than 3 weeks. The initial differentiation and neural induction of the neurosphere occurred within the first week, followed by maturation, including regional patterning and the formation of complex networks, during the subsequent 2 weeks under the influence of magnetized AuNPs. Furthermore, we observed a significant enhancement in neurogenic maturation in the brain organoids, as evidenced by increased histone acetylation in the presence of electromagnetic AuNPs. Consequently, electromagnetic AuNPs offer a promising in vitro system for efficiently generating more advanced human brain organoids that closely resemble the complexity of the human brain.

Figure 1

(A) Schematic presentation of the efficient generation of the brain organoids using electromagnetized AuNPs. The conjugation of RGD-AuNPs in brain organoids coupled with exposure to electromagnetized field can facilitate the proliferation and differentiation of neural stem cells and activate epigenetic histone modification, including H3K9ac. (B) Schematic depicting the main steps for magnetizable RGD (arginine-glycine-aspartic acid)-conjugated AuNPs synthesis. (C) Data showing HPLC chromatogram and MS (ion masses) of the synthetic peptide, CYGRGDS. Analysis of RGD-AuNPs by (D) UV–vis spectroscopy, (E) DLS, (F) Zeta-potential, and (G) FE-TEM analysis.

Figure 2

(A) Schematic depicting the main steps for electromagnetized AuNP-brain organoids production. To generate AuNP-brain organoids, iPSC-derived embryonic bodies, and RGD–AuNPs were gently mixed and then embedded in a Matrigel matrix for the structural organization. The brain organoids were exposed to an electromagnetized field (60 Hz and 1 mT EMF) for 6 h per day for 3 weeks. (B) Immunofluorescence for PAX6 and NESTIN in the control and electromagnetized AuNP organoids at 2 weeks. Scale bar = 50 µm. (C) Immunofluorescence for PAX6 and NESTIN in control organoids at 8 weeks. Scale bar = 50 µm. (D,E) Quantifications of the PAX6 + (D) and NESTIN + (E) cells in control organoids and electromagnetized AuNP organoids. Data represent mean ± SEM. one-way ANOVA, *P < 0.05, **P < 0.01; n = 5 per group. (F) Immunofluorescence for SOX2 and TBR2 in control and electromagnetized AuNP organoids. Scale bar = 20 µm. (G) Quantifications of the TBR2 + cells in control organoids and electromagnetized AuNP organoids. Data represent mean ± SEM. one-way ANOVA, **P < 0.01; n = 5 per group. Control-EMF brain organoid without AuNPs and EMF exposure, Control + EMF EMF-exposed brain organoid without AuNPs, AuNP-EMF AuNP-brain organoid without EMF exposure, AuNP + EMF AuNP-brain organoid with EMF exposure.

Figure 3

(A) Immunofluorescence for DCX+, TUJ1+, MAP2+, and VGLUT1 + neurons in brain organoids with or without electromagnetized AuNPs. Scale bar = 20 µm. (B) Quantification of DCX+, TUJ1+, MAP2+, and VGLUT1+ neurons in each condition at 3 weeks. Data represent mean ± SEM. Student’s t-test, *P < 0.05, **P < 0.01; n = 5 per group. (C,D) qRT-PCR analysis of the specific markers for cholinergic neurons, ChAT (C), and GABAergic neurons, GAD67 (D), at different time points. Data represent mean ± SEM. Student’s t-test, *P < 0.05, **P < 0.01; n = 3 per group. (E) qRT-PCR analysis of neuronal markers including NEFL, MAPT, Synapsin1, and MAP2 in control-EMF, control + EMF, AuNP-EMF, and AuNP + EMF organoids. Data represent mean ± SEM. one-way ANOVA, *P < 0.05, **P < 0.01; n = 3 per group. (F) Representative FACS plots of synapsin-RFP-positive cells from control-EMF, control + EMF, AuNP-EMF, and AuNP + EMF organoids. (G) Quantification of synapsin-RFP-positive cells in each group. Data represent mean ± SEM. one-way ANOVA, **P < 0.01; n = 3 per group. Control-EMF brain organoid without AuNPs and EMF exposure, Control + EMF EMF-exposed brain organoid without AuNPs, AuNP-EMF AuNP-brain organoid without EMF exposure, AuNP + EMF AuNP-brain organoid with EMF exposure.

Figure 4

(A) Gene expression profiling for histone modifiers, including histone methyltransferases, histone demethylases, histone acetyltransferases, and histone deacetylases. The yellow and dark blue represent higher and lower gene expression levels, respectively. (B) Immunofluorescence for MAP2+ and KAT2B + neurons in brain organoids with or without electromagnetized AuNPs. Scale bar = 20 µm. (C) Quantification of MAP2 and KAT2B-positive cells in each condition at 3 weeks. Data represent mean ± SEM. Student’s t-test, **P < 0.01; n = 5 per group. (D) Western blot analysis of KAT2B in brain organoids with or without electromagnetized AuNPs at different time points. Original blots are presented in Supplementary Fig. 4. Data represent mean ± SEM. Student’s t-test, *P < 0.05, **P < 0.01; n = 3 per group. (E) ChIP-PCR analysis showing the occupancy of KAT2B at the SLC2A1 promoter regions in brain organoids with or without electromagnetized AuNPs. Data represent mean ± SEM. one-way ANOVA, **P < 0.01; n = 3 per group. (F) Quantification of the binding of KAT2B at MT3 promoter regions in brain organoids with or without electromagnetized AuNPs. Data represent mean ± SEM. one-way ANOVA, **P < 0.01; n = 3 per group. Control-EMF brain organoid without AuNPs and EMF exposure, Control + EMF EMF-exposed brain organoid without AuNPs-EMF, AuNP AuNP-brain organoid without EMF exposure, AuNP + EMF AuNP-brain organoid with EMF exposure.

Figure 5

(A) Representative images of H3K9ac-positive cells in brain organoids with or without electromagnetized AuNPs. Scale bar = 20 µm. (B) Quantification of H3K9ac-positive cells at 3 weeks. Data represent mean ± SEM. Student’s t-test, **P < 0.01; n = 5 per group. (C) Quantification of the fluorescence intensity of H3K9ac in nuclei using a single confocal section. Data represent mean ± SEM. Student’s t-test, *P < 0.05; n = 15 from three samples per group. (D) Western blot analysis of histone H3K9 acetylation and histone H3K27 and H3K4 trimethylation in control-EMF, control + EMF, AuNP-EMF, and AuNP + EMF organoids. Original blots are presented in Supplementary Fig. 5. (E) The relative intensities of histone H3K9 acetylation and histone H3K27 and H3K4 trimethylation. Data represent mean ± SEM. one-way ANOVA, **P < 0.01; n = 3 per group. (F) Representative images of KI67− and NESTIN-positive cells in electromagnetized AuNP-organoids treated with KAT2B-shRNA. Scale bar = 50 µm. (G,H) Quantification of KI67 + (G) and NESTIN + (H) NSCs in AuNP-EMF and AuNP + EMF organoids treated with KAT2B-shRNA. Data represent mean ± SEM. two-way ANOVA, *P < 0.05; n = 3 per group. (I) Representative images of NESTIN− and SOX2-positive cells in electromagnetized AuNP-organoid treated with KAT2B-shRNA. Scale bar = 50 µm. (J) Representative images of MAP2− and TUJ1-positive cells in electromagnetized AuNP-organoid treated with KAT2B-shRNA. Scale bar = 20 µm. (K) Quantification of NESTIN+, SOX2 + NSCs in AuNP-EMF and AuNP + EMF organoids treated with KAT2B-shRNA. Data represent mean ± SEM. two-way ANOVA, *P < 0.05; n = 3 per group. (L) Quantification of MAP2+, TUJ1 + neurons in AuNP-EMF and AuNP + EMF organoids treated with KAT2B-shRNA. Data represent mean ± SEM. two-way ANOVA, **P < 0.01; n = 3 per group. Control-EMF brain organoid without AuNPs and EMF exposure, Control + EMF EMF-exposed brain organoid without AuNPs, AuNP-EMF AuNP-brain organoid without EMF exposure, AuNP + EMF AuNP-brain organoid with EMF exposure.