Fig 2: 3R and 4R tau expression in marmoset brain. (A) RT‐PCR products of marmoset prefrontal cortex. Subjects are three unrelated marmosets spanning an age range of postnatal day 1 (PND1) through 9 years. The PCR products without exon 10 (304 bp) and with exon 10 MAPT (396 bp) isoforms were amplified from the cDNA library of the prefrontal cortex. Lanes are identified as follows: Lane 1, DNA ladder: PND1 (postnatal day 1, female); 13 M (13 months old, female); 9Y (9 years of age, female). The two bands observed in each lane were indicating PCR product with or without exon10. (B) 3R and 4R tau isoform expression in marmoset entorhinal cortex (EC) and (C) hippocampus (Hip) prepared as 1% Sarkosyl soluble lysate in individual outbred wild‐type marmosets (PND1 to 9 years old), and compared to mouse brain (PND 21 and 17 months old, respectively). The PND 21 sample is a positive control for 3R tau and the 17‐month‐old sample is a negative control for 3R tau. Figure 1B and C: recombinant human tau ladder showing six tau isoforms. Row 1 is a short‐time exposure to RD3 immunoblot, and row 2 is a longer exposure to RD3 immunoblot. 3R tau was detected by anti‐3R tau‐specific antibody RD3, 4R tau was detected by anti‐4R tau‐specific antibody RD4. Total tau expression was determined by anti‐human tau‐specific antibody HT7 and anti‐tau antibody Tau5. GAPDH is a loading control. M: male; F: female.

Fig 3: PSP‐RS MOs accumulated hyperphosphorylated tau protein. (A–D) Quantification of pThr231 (Invitrogen, MN1040) (A), pSer396 (Invitrogen, 44752G) (B), pThr181 (Cell Signaling, 12,885) (C), and AT8 (pSer202/Thr205) (Invitrogen, MN1020) (D) protein levels at different time points (day 60, day 90, and day 120) assessed using immunoblot analysis in HC and PSP‐RS MOs. Expression levels of p‐Tau were quantified using OD measurement. Data are presented as mean ± standard error (SEM). Significance was calculated versus relative HC MOs using the t test, *p ≤ 0.05, **p ≤ 0.01. GAPDH was used as loading control. (E) Representative immunofluorescence images of pThr231, pS396, pThr181, and AT8 labeling in HC and PSP‐RS MOs at day 90. DAPI (blue) was used to stain nuclei. Scale bar 50 μm. (F) Immunoblot analysis of 4R tau isoform in HC and PSP‐RS MOs at day 90 detected with a specific antibody (Cell Signaling, 79327S). (G) Quantification of 4R‐tau Western blot. Data are presented as mean ± standard error (SEM). Significance was calculated versus relative HC MOs using the t test, **p ≤ 0.01. GAPDH was used as loading control. HC = healthy control; MOs = midbrain organoids; OD = optical density; PSP‐RS = progressive supranuclear palsy Richardson's syndrome. [Color figure can be viewed at www.annalsofneurology.org]

Fig 4: Genetic background of patients with PSP‐RS and cell proliferation analysis. (A) Genotyping of the MAPT H1/H2 haplotype using end point PCR, with GAPDH as the loading control. (B) The qRT‐PCR analysis of genes associated with PSP neurodegeneration at day 120. Values are shown as fold change relative to healthy control MOs, with statistical analysis performed using a 2‐tailed unpaired t test. **p ≤ 0.01, ***p ≤ 0.001. Data are presented as mean ± SEM. (C) Proliferation assay on iPSCs derived from healthy controls and patients with PSP‐RS. Data are presented as the mean ± SEM of 3 independent experiments, performed in duplicate at each time point. (D) Cell cycle analysis of iPSCs from healthy donors and patients with PSP‐RS, showing the percentage of cells in G1, S, and G2/M phases 48 hours after nocodazole synchronization (12 hours). (E) Contribution of individual cell lines to the mosaic MO was assessed by demultiplexing single‐cell RNA sequencing data (from day 90 mosaic MO) based on genetic variants (SNPs) identified via whole‐genome sequencing (data not shown). iPSCs = induced pluripotent stem cells; MAPT = microtubule‐associated protein tau; MO = midbrain organoid; PCR = polymerase chain reaction; PSP‐RS = progressive supranuclear palsy Richardson's syndrome; qRT‐PCR = quantitative real‐time PCR; SNPs = single nucleotide polymorphisms. [Color figure can be viewed at www.annalsofneurology.org]