Human post-mortem tissue

Frozen putaminal post-mortem tissue of five MSA patients and controls was obtained from the Netherlands Brain Bank (NBB), Netherlands Institute for Neuroscience, Amsterdam (open access: http://www.brainbank.nl). MSA patients were clinically and neuropathologically diagnosed according to current consensus guidelines. Absence of neurological diseases and aSyn pathology was prerequisite for age-and sex-matched controls. For histological analyses, tissue was sectioned in 6 µm cryosections prior to fixation and staining (Suppl. Table 1). Fresh frozen tissue was used for Western blot and reverse transcription quantitative PCR for gene expression analysis.

hiPSC culture and genome-editing

Dermal fibroblasts were obtained from skin biopsies of two healthy donors (C1: male 71 years, C2: female 66 years) and reprogrammed using the Cytotune 2.0 Sendai kit (Thermo Fisher) to generate hiPSCs. All skin biopsy sampling procedures were performed at the Outpatient Center for Movement Disorder at the University Hospital Erlangen in accordance with the local Institutional Review Board approval (No. 259_17B, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany). Written informed consents of donors are on file at the Division of Molecular Neurology, University Hospital Erlangen, Germany. hiPSCs were cultured on Matrigel (growth-factor reduced, Corning) at 37 °C in a humidified 5% CO2 incubator and passaged twice a week using Gentle Cell Dissociation Reagent. The control hiPSC lines UKERiO3H-S1-006 (C1) and UKERi82A-S1-005 (C2) were included in this study.

Stable integration of SOX10, OLIG2, and NKX6.2 (SON) cassette in iPSCs

For the generation of inducible SON-iPSC lines, we adapted an one-in-all vector strategy for genomic integration to a safe genomic harbour as described earlier [15]. Human iPSCs were targeted with two zinc finger nucleases integrating the expression cassette containing the SON transcriptions factors [9] under control of a Tet-On inducible promotor (TRE3G), a reverse tetracycline transactivator (rtTA) under control of the CMV promoter as well as a puromycin resistance for selection, into the adeno-associated-virus-site 1 (AAVS1) locus on chromosome 19 [34, 50]. Surviving single cells were grown to colonies, picked, and analyzed via PCR for the correct and bi-allelic integration of the construct. Primer sequences are summarized in Suppl. Table 2.

Generation of neural precursor cells (NPCs)

NPCs were derived from hiPSCs as previously described [41, 52]. After dual SMAD inhibition, NPCs were cultured in N2B27 medium consisting of 50% DMEM-F12 / GlutaMAX, 50% neurobasal medium, 1X B27 supplement (lacking vitamin A), 1X N2 supplement, and 1% penicillin/streptomycin (PS). N2B27 was further supplemented with 3 µM CHIR, 0.5 µM SAG, and 150 µM ascorbic acid (AA; NPC-medium, NPCM). Cultures were passaged once a week using accutase with medium changes every other day.

Oligodendrocyte differentiation

NPCs were plated at a density of 26,300 cells/cm2 in NPCM supplemented with 3 µg/mL doxycycline (dox). RI Y-27632 dihydrochloride (2 µM, Enzo Life Sciences) was added for 24 h. Cells were transduced with EF-SNCA or EF- green fluorescent protein (GFP) lentiviral vector (generated as described below in “Generation of lentiviral particles”) using a multiplicity of infection of 1. Viral medium was removed after 24 h and replaced by glial induction medium (GIM) consisting of N2B27 (without neurobasal medium), 1 µM SAG, 10 ng/mL PDGF-AA, 10 ng/mL NT-3, 10 ng/ml IGF-1, 10 ng/mL T3, 200 µM AA, 0.1% Trace Elements B, and 3 µg/mL dox. GIM was changed every other day and replaced by differentiation medium (DM) supplemented with 3 µg/mL dox after four days. DM consists of N2B27 (without neurobasal medium), 10 ng/mL NT-3, 10 ng/mL IGF-1, 90 ng/mL T3, 200 µM AA, 0.1% Trace Elements B, and 100 µM db-cAMP. Seven days after glial induction, cells were detached using accutase and replated at a density of 26,300 cells/cm2 in DM supplemented with 3 µg/mL dox. RI (2 µM) was added for 24 h. From day 12 on, dox was withdrawn from the medium and cells were cultured in DM with media changes every other day.

Nanofiber assay

Aligned polycaprolactone (PCL) nanofibers with a diameter of 700 nm were synthesized by electrospinning as previously described [19, 43], fixed onto tissue carriers (13 mm), and coated with Cy5-labeled poly-L-lysine (PLL, Nanocs) at a final concentration of 100 µg/mL at room temperature (RT) overnight followed by Matrigel coating. Differentiating hOLs were replated onto nanofibers in DM supplemented with RI (2 µM) at day 9 of differentiation. 100,000 cells were seeded per tissue carrier.

Human triple-culture

First, Neurogenin 2 (NGN2)-inducible cortical neurons were derived from the hiPSC line BIONi010-C-13 (sample ID: SAMEA103988285), obtained from the European Bank for Induced Pluripotent Stem Cells (EBiSC), and generated as previously described [60]. NGN2 neurons were plated at a density of 10,000 cells/cm2 in neuronal basal medium (NBM) consisting of 50% DMEM-F12/GlutaMAX, 50% neurobasal medium, 0.5X B27 supplement, 0.5X N2 supplement, 0.5X GlutaMAX™ Supplement, 0.5X MEM Non-Essential Amino Acids solution, 0.5 mM sodium pyruvate, 50 µM 2-mercaptoethanol, 0.025% insulin solution human and 1% PS. NBM was further supplemented with 2 µg/mL dox. After 4 days of differentiation, human primary astrocytes (ScienCell, #1800) were plated at a density of 10,000 cells/cm2 onto the pre-differentiated neurons. Co-cultures of neurons and astrocytes were maintained using NBM + 20 ng/ mL GDNF and 20 ng/ mL BDNF for 2 weeks. Differentiating hOLs were replated onto these co-cultures in DM supplemented with 20 ng/mL GDNF, 20 ng/ mL BDNF, 3 µg/mL dox, and RI (2 µM) at day 9 of differentiation. 30,000 cells were seeded per 24-well. From day 12 on, dox was withdrawn from the medium and cells were cultured in DM supplemented with 20 ng/ mL GDNF, 20 ng/mL BDNF with media changes every other day. Human triple-cultures were fixed at day 23 of differentiation.

Immunocytochemistry and imaging

Cells were sequentially fixed with 2% paraformaldehyde (PFA, 8 min, RT) in culture medium and thereafter with 4% PFA in PBS (10 min, RT). As an exception, O4-staining was performed prior to fixation (30 min, 37 °C). After fixation, cells were blocked and permeabilized with PBS/3% donkey serum/0.1% Triton X-100 for 1 h at RT, or with fish skin gelatin buffer (FSGB: tris-buffered saline with 0.4% cold water fish skin gelatin in water, 1% bovine serum albumin (BSA), and 0.1% Triton X-100) for 30 min at RT. For tubulin polymerization promoting protein TPPP/p25α (TPPP) immunostaining, FSGB containing 0.2% TritonX-100 was used. Primary antibodies (Suppl. Table 3) and phalloidin-iFluor 647 (1:500, Abcam) were diluted in blocking solution and applied overnight at 4 °C. Cells were washed with PBS and incubated for 1 h at RT with fluorescently labeled secondary antibodies (1:1,000; donkey or goat Alexa Fluor 488-, 568-, 546-, 647-labeled IgG and Alexa Fluor 488-labeled IgM, from Thermo Fisher; donkey or goat Cy3-, Cy5-labeled IgG, from Dianova) against the corresponding primary antibody species (see Suppl. Table 3). Nuclei were counterstained with DAPI (1:10,000, Sigma-Aldrich) and cells were imaged on an AxioObserver or a laser scanning microscope LSM 780 using Zen blue v. 1.1.2.0 or Zen black software v. 8.1 (Carl Zeiss), respectively. Identical illumination and acquisition conditions were applied for each experiment. Imaging and data analyses were performed blinded to the experimental conditions.

Immunohistochemistry

Human sections were thawed for 1–2 min, rinsed in Tris-buffered saline (TBS, 50 mM Tris/HCl, pH 7.4, 150 mM NaCl) and fixed using 4% PFA for 5 min (all at RT). Prior and after all staining steps, sections were washed 2–3 times in TBS. Incubation with Phalloidin-iFluor 647 Reagent solution (1:500 in TBS) was performed for 20 min using a humid chamber. Finally, sections were stained using DAPI solution (1:10,000 in TBS) for 10 min at RT and mounted using ProLong™ Gold antifade reagent.

Quantifications

Cell differentiation and morphological phenotyping: Quantification of cell populations was performed on 5–10 coverslip regions per experiment and condition using the cell counter plugin of ImageJ 1.51p [45].

Length of myelin basic protein (MBP +) segments: Confocal image stacks of GFP + hOLs plated on nanofibers were processed using ImageJ 1.51p (generation of maximum intensity projections and binary images) [45]. Maximum intensity projections of z-stacks for the PLL and MBP channels were generated and converted into binary images. Co-localized pixels were consequently identified in overlay images and retained for further analysis. The length of MBP + segments along nanofibers was determined using the particle analyzer plugin by applying the following parameters: circularity, 0–0.02; size, 10 µm – infinity. Oligodendroglial processes were counted manually. 3D reconstructions of image stacks were generated with Imaris 7.7.1 © (Bitplane).

Length of MBP + segments in co-cultures: Confocal image stacks of co-cultures were analyzed using ImageJ 1.52 k [45]. A region of interest (ROI) of 950 × 700 pixels around individual hOLs was defined and the length of MBP + neurites was manually measured in overlay images of TUBB3 and MBP channels. The ROI was transferred to the corresponding TUBB3 image and a threshold was set to identify the TUBB3 + area, followed by binarization, inversion, and particle analysis (parameters: size: 0–infinity, circularity: 0.00–1.00). Only hOLs exhibiting ensheathment events and ROIs containing > 10 K TUBB3 + pixels were included in the analysis.

Cell size and phalloidin staining intensity in vitro: GFP + hOLs were outlined in the center of confocal image stacks based on O4 staining using ImageJ 1.51p and MBP staining using Image J2 [44]. The cell contour was chosen as ROI and cell size was calculated based on the ROI area. The signal intensity of phalloidin staining was determined within the ROI as mean gray value after exclusion of the nucleus and background subtraction.

Phalloidin staining intensity in vivo: White matter striae were outlined and assessed as previously described for cell culture approaches, omitting nuclear correction. One putaminal section of human post-mortem tissue with ten randomly chosen WM striae per donor were assessed (total: 10 ROIs per donor). Mean gray values of all chosen white matter striae were averaged per donor prior to statistical analyses.

Reverse transcription quantitative PCR for gene expression analysis

Total RNA was extracted from cell lysates using the RNeasy plus mini kit (Qiagen) according to manufacturer’s instructions. RNA concentration was determined by spectrometry using NanoDrop ND1000 (PeqLab). cDNA was generated using the GoScriptTM Reverse Transcription System (Promega) and reverse transcription quantitative PCR was performed on a Roche Light Cycler 480 system using the SSoFastTMEvaGreen® Supermix (Bio-Rad). Relative expression levels were calculated by the 2−∆∆ct method and normalized to the mean of two housekeeping genes (18S rRNA, and GAPDH). Primer sequences are summarized in Suppl. Table 2.

Flow cytometry analysis

Cells were enzymatically detached using accutase and stained using mouse IgM anti-O4-APC antibody or the corresponding isotype control (Suppl. Table 3) according to manufacturer’s instruction. Antibodies were removed by centrifugation and cells were washed twice with PBS. All centrifugation steps were performed at 300 g for 7 min. Life-dead discrimination was carried out by DAPI staining (1:10,000). Flow cytometry analysis was performed on a BD LSR FortessaTMX-20 (BD) acquiring 10,000 cells. Raw data was processed using FlowJo software. Fluorescence-activated cell sorting (FACS) for RNA isolation and protein extraction was conducted on a MoFlo Astrios EQ (Beckman Coulter).

Western blot analyses

Cells were mechanically detached, centrifuged, and homogenized in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate). Fresh frozen post-mortem tissue was homogenized in RIPA buffer as well. Protein concentration was determined using the Pierce® BCA protein assay kit (Thermo Scientific) according to manufacturer’s instructions. Total protein (6 µg for unsorted hOLs, 12 µg for sorted hOLs and 20 µg for post-mortem tissue samples) was separated on NuPAGETM 4–12% Bis–Tris gels (Invitrogen) and blotted onto an EMD Millipore Immobilon™−FL PVDF Transfer Membrane (Merck Millipore). Membranes were fixed with 4% PFA in TBS (30 min) and blocked with TBS / 1% BSA / 0.1% Tween for 1 h at RT followed by the incubation with respective primary antibodies (Suppl. Table 3) overnight at 4 °C. Membranes were washed with TBS / 0.1% Tween and fluorescently labeled secondary antibodies (1:1,000, donkey or goat Alexa Fluor 488-, Alexa Fluor 647-labeled IgG, from Thermo Fisher; 1:10,000, donkey or goat 680RD-, 800CW-labeled IgG, from LI-COR) against the corresponding primary antibody species (see Suppl. Table 3) were applied for 1 h at RT. Fluorescent signals were captured with the Fusion FX7 (PeqLab), the LI-COR Odyssey M (LI-COR), or the iBright 1500 (Invitrogen) detection system and densitometric analysis was performed using the Bio1D (Vilber Lourmat), the Image Studio 5.5 (LI-COR), or the Empiria Studio 3.3 (LI-COR) software. Relative expression levels were calculated by normalizing arbitrary units (AU) to GAPDH signal.

Generation of lentiviral particles

Third-generation lentiviral particles coding for human aSyn (NM 000345) and/or an internal ribosomal entry site (ires) followed by the coding sequence of GFP under the control of the EF1α-promoter were produced as previously described [10]. Following transfection and virus particle assembly in HEK293T cells, virus-containing supernatant was harvested after 48 h and filtered through a 0.45 µm PVDF membrane. Lentiviral particles were concentrated by ultracentrifugation at 28,000 rpm for 2 h at 4 °C.

Statistical analyses

Data were processed and visualized using OriginPro, Version 2021 and GraphPad Prism® 6.07, respectively. If not stated differently, data were obtained from four hOL lines derived from two individuals. Bar graphs depict the mean + standard deviation (SD) and box plots represent the median and the 25./75. percentile. Corresponding whiskers indicate the 5./95. percentile. The Shapiro–Wilk test was used to assess the normal distribution of the parameters for small sample sizes (n < 20). Additionally, the F-test was applied to test for equality of variances. Statistical significance was determined by one-way analysis of variance (ANOVA), two-way ANOVA, Kruskal–Wallis test, two-tailed student’s t-test, Mann–Whitney U-test, or one sample t-test. The respective tests are indicated in the figure legends. P values < 0.05 were considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).