Generation and characterization of midbrain-like organoids (MLOs) from healthy human (h) iPSCs (WT-75.1 hiPSCs).

(A) A schematic overview of the procedures for generating MLOs from healthy WT-75.1 hiPSCs. (B) Representative confocal images showing architectural structure of week (Wk) 8 MLOs containing new-born neurons (Tuj1/NeuN), astrocytes (GFAP), dopaminergic neurons (FOXA2/TH) and neural progenitor cells (SOX2/Ki67). Merge images show the distribution of those cell markers. FOXG1 expression in MLO and cerebral organoid (CO). Transcription factor FOXG1 (forebrain marker) was enriched in CO at Wk8 of differentiation but absent in MLO. Pan-neurons (NeuN) were both present in MLO and CO, as shown by NeuN immunostaining (neuronal marker). (C) Quantitative analysis of cell type specific genes expression for midbrain/dopaminergic neuron (FOXA2/ASCL1/LXM1A/PLZF/TH), glial cells (GLAST/S100B) and multipotent stem cells (SOX2/NANOG/OCT4) in Wk3 and Wk8 MLOs by qRT-PCR. (D) Immunoblot of Sox2, Tuj1, MAP2, TH, GFAP and S100B in WT-75.1 hiPSCs and its derived MLO (Wk8) lysate. β-Actin was used as a loading control.

GCase deficiency drives glycosphingolipids accumulation and transcriptomic alteration in GD MLOs.

The GD MLOs were generated as in Figure 1A. (A) Reduced GCase protein in GD MLO (GD2-1260). Wk 8 MLOs (n=3) were pooled as a biological sample. β-Actin was used as loading control. (B) GCase activity in hiPSCs and MLOs. Data was normalized to WT-75.1 control. (C) Representative images of WT-75.1 and GD2-1260 MLOs at Wks 4, 8 and 15 of differentiation. (D) MLO size was measured based on area of MLO spheres and normalized to WT-75.1 control at each indicated time points. N ≥ 10 MLOs were quantified per group. (E, F) Measurement of total glucosylceramide (GluCer) and GluCer species in Wk 15 MLO. (G) Glucosylsphingosine (GluSph) levels in Wk15 and Wk28 MLOs. GluCer and GluSph levels in the organoids were measured by LC-MS/MS and normalized by corresponding total protein of MLO tissue lysate. (H) 3D Principal Component Analysis (PCA) of bulk RNA sequencing (RNA-seq) data. The Euclidean distance of the normalized gene expression among healthy control (WT-75.1) and GD (GD2-1260) MLOs was used for sample clustering. Ellipsoids around each group indicate the distribution and spread of the samples within the sample group. Wk 8 MLOs (n=3) were pooled as one biological sample, and three samples were profiled in each group. (I) MA plot showing the distinct genes differentially expressed in GD MLOs. Statistically significant differentially expressed genes (DEGs; |fold change|≥ 1, p-adj ≤ 0.05 and base mean ≥ 50) were highlighted in red. Number of DEGs downregulated and upregulated in GD2-1260 MLO against WT-75.1 MLO were shown. FC, fold-change. (J, K) Dysregulated pathway in GD MLOs analyzed by GO (gene ontology) (J) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (K) enrichment of DEGs. Both gene counts and level of significance (-log10 of p value) were shown as stacked columns for each category. (L-O) Heat maps of dysregulated pathways or biological functions in GD MLO. Specifically, aberrant expressions of genes involved in WNT signaling (L), anterior-posterior brain specification (M), neuronal function (N) and lysosome-phagosome (O) were shown.

Skewed specification of midbrain patterning and dopaminergic neuron differentiation in GD MLOs.

(A) Gene expression of FOXP1, FOXG1, and PAX6 in week 8 WT-75.1 and GD2-1260 MLOs. Data were plotted using RNA sequencing counts. ***p < 0.001, unpaired Student’s t test. (B, C) Aberrant expression of FOXP1/FOXG1 transcription machinery for forebrain/midbrain patterning in GD MLOs. Representative confocal images (B) and quantification of Wk8 WT-75.1 and GD2-1260 MLOs, immunostained for FOXP1 (red) and FOXG1 (green), with DAPI (blue) labeling nuclei. Yellow arrows indicate FOXP1+FOXG1+ cells. Scale bar, 50 µm. *p < 0.05, **p < 0.01, unpaired Student’s t test. (D) Confocal images of Wk6 MLOs, immunostained for midbrain patterning markers FOXA2 (green) or TH (red), with DAPI (blue) labeling nuclei. Scale bar, 100 µm. (E) Representative images of differentiating DA neurons in MLOs derived from WT-75.1 and GD2-1260 hiPSCs. TH (red), FOXA2 (green) were co-stained, with DAPI (blue) labeling nuclei. Yellow arrows indicate TH+FOXA2+ cells. Scale bar, 50 µm. (F) Quantification of midbrain progenitor markers ASCL1, TH, LMX1A, and PLZF expression in WT-75.1 and GD2-1260 MLOs at Wk3 and Wk8, measured by qRT-PCR and normalized to WT-75.1 hiPSC cells. Data are presented as mean ± SEM (n = 3-4 per group). *p < 0.05, **p < 0.01. (G) Immunoblot analysis of midbrain/dopaminergic neuron markers TH, FOXA2, and MAP2 in Wk16 MLOs. β-Actin was used as a loading control. (H) Relative protein levels of TH, FOXA2, and MAP2 in Wk8 GD2-1260 MLOs compared to WT-75.1. *p < 0.05, **p < 0.01, unpaired Student’s t test. (I) Dopamine levels in MLO culture medium assay by ELISA. Culture medium from 4 GD2-1260 MLOs or WT-75.1 MLOs at Wk12 cultured in 3 mL BGM medium for 72 hours was assayed. Data are presented as mean ± SEM (n = 5 per group). ***p < 0.001, unpaired Student’s t test.

Mutation correction significantly rescued disease phenotypes in GD MLOs.

(A) Schematic overview of CRISPR/Cas9-mediated mutation correction of the GBA1 L444P mutation in GD2-1260 hiPSCs, converting the L444P (L444P/P415R) mutation (Proline, P to Leucine, L) to the wild-type sequence (WT-P415R), generating isogenic iso-GD2-1260 hiPSCs. The mutated base C in amino acid code ‘CCG’ for proline (P), was corrected to T to decode leucine (L, CTG), which was confirmed by genome sequencing of GBA1 locus. (B, C) Immunoblot analysis of GCase protein and quantification in week 8 MLOs derived from WT-75.1, GD2-1260, and iso-GD2-1260 hiPSCs. β-Actin was used as a loading control. Data are presented as mean ± SEM (n = 3 biological replicates per group). ***p < 0.001. (D) Relative GCase activity in GD2-1260 and iso-GD2-1260 hiPSCs and Wk8 MLOs, normalized to WT-75.1 controls. Data are presented as mean ± SEM (2 MLOs pooled, n = 3 per group). ***p < 0.001. (E) Measurement of GluSph levels in WT-75.1, GD2-1260, and iso-GD2-1260 MLOs at Wk15 and Wk28 and their culture medium at Wk 15, quantified by LC-MS/MS and normalized to total protein of tissue lysate. Data are presented as mean ± SEM (3 MLOs pooled, n = 3 per group for MLO and 4 MLOs pooled, n = 3 per group for medium). **p < 0.01; ns, not significant. (F) Representative bright-field images of GD2-1260 and iso-GD2-1260 MLOs at Wk15 of differentiation. Scale bar, 1 mm. (G) MLO size quantification for GD2-1260 and iso-GD2-1260 MLOs at Wks 4, 8, and 15, measured as relative area of MLO spheres and normalized to WT-75.1 controls at indicated time point. N ≥ 10 MLOs were quantified per group. Data are presented as mean ± SEM. *p < 0.05; ns, not significant. (H, I) Immunoblot analysis of midbrain/dopaminergic neuron markers TH and FOXA2 (H) and their relative quantification (I) in Wk8 MLOs. GAPDH was used as a loading control. (J) Dopamine levels in the culture medium of Wk12 MLOs derived from GD2-1260 and iso-GD2-1260 hiPSCs, measured after 72 hours in BGM medium (n = 4 MLOs). Data are presented as mean ± SEM (n = 5∼7 per group). **p < 0.01. (K, L) Immunoblot analysis of autophagy-lysosomal pathway markers LAMP1 and Cathepsin D (K) and quantification (L) in Wk16 MLOs. GAPDH was used as a loading control. Data are presented as mean ± SEM. (M, N) Immunoblot analysis of LC3-I and LC3-II (M) and quantification (N) in Wk16 MLOs. GAPDH was used as a loading control. (O) Immunoblot analysis of mTOR signaling pathway components [4E-BP1, P-4E-BP1(THR37/46), S6, and P-S6 (Ser235/236)] in Wk16 MLOs. β-Actin was used as a loading control. (P) Quantification of protein levels of mTOR signaling pathway components. Data are normalized to WT-75.1 and presented as mean ± SEM. Immunoblot analysis for panel H and I and K-P was performed using the lysate from 3 MLOs pooled per group, 3 repeated experiments. ***p < 0.001; ns, not significant. One-way ANOVA test with Tukey’s test.

Delivery of GCase to MLOs via SapC-DOPS nanoparticles corrects GD phenotypes.

(A) Schematic illustration of SapC-DOPS nanoparticle-mediated delivery of recombinant GCase (fGCase) to MLOs. SapC-DOPS nanoparticles carrying fGCase or fluorescent label CVM were cocultured with MLOs, followed by short-term (48 hours) or 2-week treatment period before analysis. (B) Confocal images of untreated and SapC-DOPS-CVM-treated MLOs, showing uptake of CVM (magenta) with DAPI (blue) labeling nuclei. Scale bars: 200 µm (left panel), 50 µm (right panels, magnified regions a and b). (C) GCase activity in WT-75.1 and GD2-1260 MLOs following a 48-hour treatment with SapC-DOPS-fGCase. Data are presented as mean ± SEM (3 MLOs pooled, n = 3 per group). ***p < 0.001. (D) Confocal images of WT-75.1, GD2-1260, and GD2-10-257 MLOs treated with SapC-DOPS or SapC-DOPS-fGCase for 2 weeks, immunostained for GCase (green) with DAPI (blue) labeling nuclei. Scale bar, 200 µm. (E-G) GCase activity and protein in WT-75.1 and GD (GD2-1260, GD2-10-257) MLOs treated with SapC-DOPS or SapC-DOPS-fGCase for 2 weeks, measured by enzymatic assay and immunoblot. Data are presented as mean ± SEM (3 MLOs pooled, n = 3∼4 per group). ***p < 0.001; ns, not significant. (H, I) GluSph levels in WT-75.1 and GD (GD2-1260, GD2-10-257) MLOs treated with SapC-DOPS or SapC-DOPS-fGCase for 2 weeks, quantified by LC-MS/MS and normalized to total protein. Data are presented as mean ± SEM (3 MLOs pooled, n = 3-4 per group). ***p < 0.001; **p < 0.01; ns, not significant. (J) Immunoblot analysis of autophagy-lysosomal and mTOR pathway proteins in SapC-DOPS or SapC-DOPS-fGCase treated GD2-1260 MLOs. GAPDH was used as a loading control.

AAV9-GBA1 gene therapy mitigates disease phenotypes in GD MLOs.

(A) Schematic illustration of AAV9-GBA1 gene therapy delivery to MLOs using a nanoliter injector. AAV9 vectors carrying the GBA1 gene (AAV9-GBA1) are administered to Wk13 MLOs. The samples were analyzed after 3 weeks of treatment. (B) GCase activity in WT-75.1, GD2-1260, and GD2-10-257 MLOs and AAV9-GBA1 treated MLOs were measured by enzymatic assay. Data are presented as mean ± SEM (3 MLOs pooled, n = 3 to 6 per group). ***p < 0.001. (C) GluSph levels in AAV9-GBA1 treated GD and control MLOs were quantified by LC-MS/MS and normalized to total protein. Data are presented as mean ± SEM (3 MLOs pooled, n ≥ 3 per group). ***p < 0.001; ns, not significant. (D) Immunoblot analysis of LAMP1 and TH in WT-75.1 and in GD2-1260 MLOs untreated or treated with AAV9-GBA1. Protein levels are normalized to WT-75.1 untreated controls (set to 1.0). (E) Transgene expressions (yellow arrows and enlarged insert) in neurons (NeuN), DA neurons (TH) and astrocytes (GFAP) of AAV9-GBA1 treated GD2-1260 MLOs. Scale bar = 50 µm.

Substrate Reduction Therapy with GZ452 reduces lipid accumulation and improves autophagic and lysosomal abnormalities in GD MLOs.

(A) Assessment of GZ452 tolerated dose in healthy MLO and the effect of GZ452 on organoid growth in WT-75.1 MLOs over 6 weeks. (B, C) Total GluCer levels (B) and distribution of GluCer species (C) in WT-75.1 MLOs with various doses of GZ452 at Wk6. (D) Schematic of the experimental timeline for short-term (2 weeks) GZ452 treatment of GD MLOs. (E, F) GluCer (E) and GluSph (F) levels in WT-75.1 and GD2-1260 MLOs at Wk15 under short-term GZ452 treatment. Data were normalized to protein mass. (G) Schematic of the experimental timeline for long-term (28 weeks) GZ452 treatment in GD MLOs. (H) GluSph levels in MLOs at Wk15 under long-term GZ452 treatment. Data were normalized to protein mass. (I) Immunoblot analysis of LAMP1 and LC3-I/II in SRT treated GD2-1260 MLOs for 28 weeks, with β-actin as loading control. (J) Quantification of LAMP1 and LC3-II/I in MLOs. Protein levels are normalized to WT-75.1 untreated controls (set to 1.0). All data presented as mean ± SEM (n = 3∼4). *p < 0.05; **p < 0.01; ns, not significant. One-way ANOVA with Tukey’s test.

Correct L444P mutation in nGD iPSC by CRISPR-Cas9.

(A) Single stranded oligonucleotide design. Silent mutations in upper case. Phosphorothioate modified bases (*). (B) Genomic sequence of mutant, wild type and corrected GBA1. Mutated codon highlighted in red. T insertion for point mutation correction highlighted in yellow. PAM highlighted in grey. sgRNA target sequence underlined. (C) DNA electrophoresis gels showing genome editing and clone screening for iso-GD2-1260 (clone #9). (D) Karyotyping of GD2-1260 and CRISPR/Cas9 corrected iso-GD2-1260 hiPSCs. Normal karyotype was observed in both hiPSC lines. (E) Relative mRNA expression of genes (OCT4, NANOG and SOX2) required for generating and maintaining hiPSCs pluripotency by quantitative RT-PCR. ns, not significant by One-way ANOVA analysis.

Neural rosette formation during MLO maturation were not affected by GBA1 mutation.

(A) Cryosections of Wk 6 MLOs derived from WT-75.1, GD2-1260 and isogenic control iso-GD2-1260 hiPSC cells. Sections were stained with antibodies against Ki67 and Sox2 and nuclei were costained with DAPI. Scale bar, 100 µm. (B) Quantification of the Sox2+, Ki67+ and Sox2+/Ki67+ cells (mean ± SEM; n = 3). ns, not significant by One-way ANOVA analysis.

Restoration of GCase Expression in dopaminergic neuron and astrocytes in SapC-DOPS-fGCase treated nGD MLOs.

(A, B) Representative confocal images of GD2-1260 (A) and GD2-10-257 MLOs (B) treated with SapC-DOPS-fGCase for 2 weeks, immunostained for GCase (green) and dopaminergic neuron (TH, red) with DAPI (blue) labeling nuclei. (C, D) Representative immunostaining images for GCase (green) and astrocytes (GFAP, red) in GD2-1260 (C) and GD2-10-257 MLOs (D) treated with SapC-DOPS-fGCase. Scale bar, 50 µm. Yellow arrows indicate colocalized GCase in TH+ cells.

Restoration of GCase expression in lysosomal and autophagosomal compartments in SapC-DOPS-fGCase treated nGD MLOs.

(A, B) Representative confocal images of GD2-1260 (A) and GD2-10-257 MLOs (B) treated with SapC-DOPS-fGCase for 2 weeks, immunostained for GCase (green) and lysosomal marker LAMP1 (red) with DAPI (blue) labeling nuclei. Yellow arrows indicate colocalized GCase in LAMP1+ compartments. Scale bar, 50 µm. (C, D) Representative confocal images of GD2-1260 (C) and GD2-10-257 MLOs (D) treated with SapC-DOPS-fGCase for 2 weeks, immunostained for GCase (green) and autophagosomal marker LC3B (red) with DAPI (blue) labeling nuclei. Yellow arrows indicate colocalized GCase in LC3B+ compartments. Scale bar, 50 µm.

Influence of SRT drug GZ452 on DA neuron differentiation in WT-75.1 MLOs.

Relative mRNA expression of midbrain markers ASCL1, TH, and PLZF in WT-75.1 at Wk6 in untreated or treated MLOs with indicated concentrations of GZ452. Relative gene expression is normalized to untreated control WT-75.1 MLOs (set to 1.0).