Fig 2: Characterization of hiPSC derived hNPCs, neurons and astrocytes.a Immunostainings exemplarily shown for O3H-R1-003. hiPSC pluripotency staining for markers OCT4, NANOG, SOX2. Scale bar=200 µm. hNPC staining for markers SOX1, SOX2, NESTIN, PAX6. Scale bar=100 µm. Neuron staining for markers TUBB3 and DAn marker TH. Scale bar=100 µm. Astrocyte staining for markers GFAP and SLC1A3. Scale bar=100 µm. b Summary of somatic CNVs identified in hNPC clones by chromosomal microarray analysis shown as total number of somatic CNVs detected per analyzed clone and average length of CNVs (in kb; green=copy number gain; orange=copy number loss) per analyzed clone. n = 5 Ctrl and 7 sPD patients. c Circos plot showing the genomic distribution of somatic CNVs in Ctrl (blue) and sPD (red) clones. d Quantification of RBFOX3 (synonym: NeuN) positive as well as TH / RBFOX3 double-positive cells in DAn populations. n = 5 Ctrl and 7 sPD clones, in triplicates. e Characterization of neurite morphologies of DAns. Boxplots show the average number of neurites emerging from TH positive cell bodies, their average number of branch points and their average length. n = 5 Ctrl and 7 sPD clones, in triplicates. Boxplots display the median and range from the 25th to 75th percentile. Whiskers extend from the min to max value. Each dot represents one patient. P-values were determined by two-sided t-test d (right), e; two-sided Mann–Whitney-U test b, d (left). *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

Fig 3: EXSISERS can be programmed with different reporters and effectors.a,b, HaloTag was flanked by intein-CC domains and type I and type II transmembrane domains (TMDs), resulting in a surface functionalization with a HaloTag inserted into exon 10 of MAPT. c, Cells were incubated with chloroalkane-functionalized Alexa Fluor 660 (3.5 µM) for 10 min at 37 °C and washed with HBSS. d, Anti-pan-tau immunoblot showed the typical tau pattern indicating scarless excision. Data shown represent one independent experiment. e, Epifluorescence microscopy image of anti-pan-tau immunocytochemistry of EXSISERSMAPT:10Halo cells after MAPT induction showing filamentous tau staining. Data shown represent 2 independent experiments f, AF660 live-cell-staining after MAPT-induction resulted in a distinct membrane staining. Data shown represent 2 independent experiments. g, scAvidin was used as a high-affinity binding domain for biotinylated compounds. Test constructs for both designs contained mNG-NLS as surrogate exteins, which are expected to be spatially decoupled after the excision of the intein-domains. h,i, Surface localization of scAvidin or HaloTag in transfected HEK293T was confirmed by live-staining with biocytin-AF594 and chloroalkane-AF660, while mNeonGreen-NLS (mNG-NLS) was detected in the nucleus. Data shown represent one experiment from 2 cell culture wells. j, The binding moiety of the membrane anchors was exchanged for NLuc flanked by furin sites (FS), such that the Golgi-resident furin can cleave off NLuc and releases it into the supernatant. k, NLuc activity was measured over time in the supernatant of cells expressing the sheddable NLuc without/with furin sites. Data shown represent the mean values ± s.d. (n = 3 biological replicates). *** denotes p < 0.001 of selected Bonferroni MCT after two-way ANOVA with repeated measures (full statistical results in Supplementary Table 1). l, Intein-flanked mNG was inserted into the mouse Tubb3 gene. m, Immunoblot confirms successful intein splicing of mNG. Data shown represent 7 independent experiments. n, Mouse N2a cell line with insertion of the intein-mNG reporter into Tubb3 shows typical Tubb3 filaments via immunocytochemistry. The fluorescent mNG signal (inset) was observed throughout the cell, indicating successful post-translational splicing. Data shown represent 2 independent experiments. Unprocessed blots and numerical source data are provided in Source Data Extended Data Fig. 8.Source data

Fig 4: Immunofluorescence labeling of two differentiated organoids at day 43. The left panels show sections of organoids with blue nuclei stained with Fluoromount DAPI, scale Bar = 100 µm (10×). The dashed-line square represents the field visualized at higher magnification in the subsequent panels. The upper figures show the presence of neurons TUBB3 (red) and astrocytes GFAP (yellow). The bottom panels show oligodendrocytes O4 (red) and embryonic stem cells SOX2 (yellow). Scale bar 20 µm (40×).

Fig 5: Primary cilia morphology in sPD.a (right) Density plot illustrating the distribution of PC length (in µm) in hiPSCs. (middle) Fraction of ciliated cells. PC length was measured in immunostainings (left) of hiPSCs positive for anti-SOX2 (red) and anti-ARL13B (green). n = 37-79 cilia per clone. b (right) Density plot illustrating the distribution of PC length (in µm) in hNPCs. (middle) Fraction of ciliated cells. PC length was measured in immunostainings (left) of hNPCs positive for anti-NES and anti-ARL13B. n = 74-123 cilia per clone. c (right) Density plot illustrating distribution of PC length (in µm) in neurons. (middle) Fraction of ciliated cells. PC length was measured in immunostainings (left) of neurons positive for anti-TUBB3 (red) and anti-ARL13B (green). n = 131-385 cilia per clone. d (right) Density plot illustrating distribution of PC length (in µm) in astrocytes. (middle) Fraction of ciliated cells. PC length was measured in immunostainings (left) of astrocytes positive for anti-GFAP (red) and anti-ARL13B (green). n = 100 cilia per clone. All experiments were performed in triplicates, n = 5 Ctrl and 7 sPD. Immunostainings are exemplarily shown for G3G-R1-039 with scale bars=10 µm. Boxplots display the median and range from the 25th to 75th percentile. Whiskers extend from the min to max value. Each dot represents one patient. P-values were determined by two-sided t-test a (middle), b (middle), c (middle), d (middle); two-sided Kolmogorov-Smirnov test (ks) + linear mixed effects model (lm) a (right - ks p = 0.15; lm p = 0.48), b (right - ks p = 0.0007; lm p = 0.04), c (right - ks p = 2.2 × 10-16; lm p = 0.009), d (right - ks p = 0.31; lm p = 0.94). #p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.