Altered neuroepithelial morphogenesis and migration defects in iPSC-derived cerebral organoids and 2D neural stem cells in familial bipolar disorder

Abstract

Bipolar disorder (BD) is a severe mental illness that can result from neurodevelopmental aberrations, particularly in familial BD, which may include causative genetic variants. In the present study, we derived cortical organoids from BD patients and healthy (control) individuals from a clinically dense family in the Indian population. Our data reveal that the patient organoids show neurodevelopmental anomalies, including organisational, proliferation and migration defects. The BD organoids show a reduction in both the number of neuroepithelial buds/cortical rosettes and the ventricular zone size. Additionally, patient organoids show a lower number of SOX2-positive and EdU-positive cycling progenitors, suggesting a progenitor proliferation defect. Further, the patient neurons show abnormal positioning in the ventricular/intermediate zone of the neuroepithelial bud. Transcriptomic analysis of control and patient organoids supports our cellular topology data and reveals dysregulation of genes crucial for progenitor proliferation and neuronal migration. Lastly, time-lapse imaging of neural stem cells in 2D in vitro cultures reveals abnormal cellular migration in BD samples. Overall, our study pinpoints a cellular and molecular deficit in BD patient-derived organoids and neural stem cell cultures.

Keywords: bipolar disorder patients; cellular migration; cortical organoids; neuroepithelial bud organization.

Graphical Abstract

Figure 1

(A) Pedigree of clinically dense family and (B) table showing the clinical information for BD patients

Figure 2

Qualitative and quantitative representation of cortical organoid structure across healthy controls (HC1 and HC2) and patients (A1 and A2). (A) IHC of organoids with DAPI, SOX2 and DCX to show overall topology of organoids. The arrowheads in DAPI panel show NEBs. (B) Representation of boundaries drawn to quantify the area of the VZ/IZ region in NEBs, where area of VZ/IZ = area of the outer boundary of NEB - area of the lumen (C) box plot showing total number of NEBs found in each organoid across patients and healthy controls, where N = 12 organoids, 6 organoids/subject, p < 0.001. (D) the box plot shows the area of VZ/IZ in μm2 across organoids of healthy control (N = 171 NEBs) and patients (N = 56 NEBs), p < 0.0001.(E) the line graph shows the size distribution and number of NEBs. Scale bar represents 500 μm. Statistically significant differences are shown as asterisks

Figure 3

Fewer progenitors and abnormally positioned neurons in BD organoids (A) immunostaining of NEBs with SOX2, apical progenitor marker and TUJ1(βIII tubulin), neuronal marker (B) representative image showing the area defined as VZ/IZ and PMZ in NEBs for quantification of SOX2 and TUJ1 intensity (C) presence of TUJ1 positive neurons in VZ of patient organoid showing neuronal migration defect when compared to control (D) box plot showing percentage of SOX2 positive cells out of ~200 DAPI stained cells counted across patients and control (p-value <0.001). (E and F) box plot showing quantification of SOX2 intensity in VZ (p < 0.01) and PMZ regions (non-significant). (G, H, I) box plots showing quantification of TUJ1 intensity in VZ/IZ (p < 0.01), PMZ regions (non-significant) and the combined intensities of VZ/IZ and PMZ regions (non-significant). For panels D-I, N = 20–22, for each line of patient and control, 5–6 organoids per subject, and 2 fields per organoid. The scale bar represents 50 μm

Figure 4

Transcriptome of cortical organoids derived from BD patients and healthy controls. (A) Principal component analysis showing clustering of patients and control based on their transcriptome profile. (B) Volcano plot showing the logFC and -log of p values of differentially expressed genes (DEGs). (C) the heatmap shows the normalised expression values for significant DEGs in BD organoids compared to healthy control. (D) GO ontology enrichment analysis for all DEGs in patients (upregulated and downregulated). (E, F) Heatmaps show DEGs for genes involved in progenitor proliferation and neuronal migration. (G) Bar graph showing the number of dysregulated genes present in a cell type-specific manner when compared to Trevino et al., 2021. Refer to Supplementary Table 2 for the full description of abbreviated cell types.(H) relative cell fractions of different cell type-specific clusters in the bulk transcriptome of HC1, A1 and A2 organoids, after deconvolution using CIBERSORTx. Refer to Supplementary Table 2 for the full description of abbreviated cell types. (I) Abundance of late radial glia (cluster 10) across HC1, A1 (p < 0.01), and A2 (p < 0.001) organoids, estimated through deconvolution.(J) DEGs in late radial glia (cluster 10), across control and patient organoids

Figure 5

Cell proliferation assay for organoids and iPSCs lines. (A) Representative IHC images of controls and patient organoids showing SOX2, EdU and Ki67.(B, C) percentage of cells showing the presence of proliferation markers in organoids (N = 12–13 fields) (B) p < 0.001, (C) p < 0.001(D) representative IHC images of controls and patient iPSCs showing SOX2, EdU and Ki67 (N = 12 fields).(E, F) percentage of cells showing the presence of proliferation markers in iPSCs, p is non-significant. Scale bar is 50 μm

Figure 6

2D NSC migration assay (A) phase contrast images of the cell migration assay at 0 hours and 20 hours. The trajectories of NSC migration are superimposed to show the displacement of single cells from their original position. Box plot of (B) total displacement and (C) average speed of the migrating cells after 20 h for n of healthy controls =735 and patients =1080 (p < 0.0001)

Figure 7

Position of data points representing patients A1 and A2 on the boxplots of T1-weighted structural MRI data for five severe mental illnesses, representing centile calculations of total cortical GMV, subcortical GMV, and total cerebral WMV, using maximum likelihood estimation