Skin biopsies from six age- and gender-matched subjects were obtained through the Catalan MSA Registry (CMSAR) in Hospital Clínic de Barcelona. Five of the subjects had pathologically confirmed MSA, three with the parkinsonian variant and two with the cerebellar variant. The sixth case was diagnosed as clinically established MSA-C. All cases were graded for histopathological severity according to the Jellinger and Seppi classification, which assesses olivopontocerebellar atrophy (OPCA) and striatonigral degeneration (SND) on a scale from I (mild) to III (severe). Demographic and clinical details are provided in Table 1. The derivation of human adult dermal fibroblasts (HDF-ad) from a skin biopsy was initiated by washing the collected piece of tissue with Dulbecco’s phosphate buffered saline (DPBS; ThermoFisher Scientific) and cutting it into small pieces. These pieces were transferred into gelatin-coated culture flasks with fibroblast medium, containing 88% Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), 0.1 mM non-essential aminoacids solution (NEAA) and 55 µM β-mercaptoethanol (BME; all from ThermoFisher Scientific). During the following 5–8 days, medium changes were performed every other day to maintain optimal conditions and cell growth was monitored. When reaching confluence, fibroblasts were trypsinized and resuspended in cryopreservation medium (50% culture media, 40% FBS and 10% DMSO), this was referred to as passage 0 (P = 0).

Patients-derived skin fibroblasts were thawed and expanded in fibroblast medium. Fibroblasts at low passage (P = 1 or 2) were reprogrammed with the Cytotune-iPS 2.0 Sendai Reprogramming kit (ThermoFishe Scientific), following the manufacturer’s protocol. The kit provides the non-integrative and zero-footprint Sendai virus (SeV) expressing Klf4, Oct3/4, Sox2 and c-Myc transcription factors. Briefly, one well of fibroblasts was used to count cells and perform viral calculations, taking into account the target MOI and the viral titer. Then, the SeV mix was prepared in fibroblast medium and added to a new well of cultured fibroblasts. The following day, the viral medium was replaced by fresh fibroblast medium. On day 7, reprogrammed fibroblasts were plated on irradiated mouse embryonic fibroblasts (iMEFs) using fibroblast medium. On day 8, fibroblast medium was replaced by iPSC medium, containing 78% Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12) supplemented with 20% KnockOut™ serum replacement (KSR), 1% P/S, 0.1 mM NEAA, 55 µM BME and 4 ng/ml basic fibroblast growth factor (bFGF) (all from ThermoFisher Scientific). Three to four weeks after transduction, iPSC colonies had the appropriate size for transfer. They were selected based on their morphology, as they needed to fulfill the following requirements: compact colonies with well-defined and refractive edges, and comprised of cells with high nucleus:cytoplasm ratio. Around 8 passages were performed for each iPSC clone to completely remove the SeV vectors. At first, the iPSC colonies were manually split by cutting them into small pieces in a grid-like pattern and transferring them on iMEFs plates with iPSC medium. Then, iPSC colonies were progressively adapted to 0.5 mM ethylenediaminetetraacetic acid (EDTA, ThermoFisher Scientific) treatment for dissociating them into small clumps and to feeder-free conditions using plates coated with Matrigel (Corning) at a concentration of 8.3 µg/cm² and with mTeSR™ medium (STEMCELL Technologies).

Chromosome harvest was performed when iPSC colonies reached about 60% of confluency. The mitotic spindle inhibitor Colcemid™ (ThermoFisher Scientific) was added in the culture media at a concentration of 0.1 µg/ml during 2 hours. Then, the supernatant was collected. iPSCs were dissociated into single cells using TrypLE Express, and the cell suspension was combined with the supernatant. The mixture was centrifuged and the pellet was incubated with an hypotonic solution of 0.075 M potassium chloride (KCl; Sigma-Aldrich) at 37 °C for 20 min. Cold Carnoy’s fixative solution I (3 parts of methanol and 1 part of glacial acetic acid, both Sigma-Aldrich) was added at a ratio of 5:1 (hypotonic:fixative), and was incubated for 15 min at RT. The chromosome suspension was centrifuged and underwent some additional fixative incubations: (i) cold Carnoy’s fixative solution I for 10 min at RT, two repetitions; and (ii) cold Carnoy’s fixative solution II (2 parts of methanol and 1 part of glacial acetic acid) overnight at 4 °C. A small amount of the chromosome suspension was dropped onto slides. Cytogenetics was conducted according to the standard methods used in Laboratori de Citogenètica at Hospital del Mar (Barcelona). Slides were stained using Wright solution (G-banding). A minimum of 20 metaphase were automated screened with Cytoinsight GSL imaging (Leica Biosystems), evaluated with Cytovysion software and informed according to the International System for Human Cytogenetic Nomenclature (ISCN 2024).

The Copy Number Variation (CNV) assay was designed based on Assou et al., 2020 publication (21). iPSC at passage 8 were harvest and stored as a dry pellet at -20 °C. Genomic DNA was extracted using the column-based PureLink™ Genomic DNA Kit (ThermoFisher Scientific). DNA concentration and purity were determined with the NanoDrop spectrophotometer (ThermoFisher Scientific), by measuring the absorbance at 260 nm and calculating the 260/280 and 260/230 nm absorbance ratios, respectively. The digital PCR mix was prepared by combining 100 ng of genomic DNA, 10 units of the restriction enzyme HindIII (New England Biolabs), the Absolute Q™ DNA digital PCR master mix 1x (ThermoFisher Scientific), the target TaqMan™ copy number assay primer labeled with FAM fluorophore 1x (ThermoFisher Scientific, Table 2), and the reference TaqMan™ copy number assay primer labeled with VIC fluorophore 1x (ThermoFisher Scientific, Table 2). The genomic DNA was digested during the reaction set-up period. This digestion was required for the separation of duplications within genomic proximity, enabling their correct identification. The restriction enzyme was inactivated during the first PCR denaturation step. The PCR reactions were loaded in the microfluidic array plate (MAP, ThermoFisher Scientific), and the Absolute Q™ isolation buffer (ThermoFisher) was added on top of each well to prevent contamination and evaporation. The plate was loaded into the QuantStudio Absolute Q™ Digital PCR System (ThermoFisher Scientific) and the amplification conditions were as follows: 96 °C for 10 min; 40 cycles of 96 °C for 5 sec and 60 °C for 15 sec; and hold at 4 °C. Copy number was assessed using the QuantStudio software. Different quality control parameters were checked to make sure that the digital PCR fulfilled within the performance expectations: (i) the total number of analyzed micro-chambers per sample was > 20,000; (ii) ROX, the reference dye, was uniformly distributed and showed a coefficient of variation (CV) < 11%; and (iii) it was possible to define a clear threshold to separate the positive and the negative population and, thus, the “rain” effect was minimum.

Short Tandem Repeat Analysis (STR) for cell line authentication was conducted by Plataforma de Genomica Translacional at Institut d’Investigacio Germans Trias i Pujol (IGTP, Badalona). AmpFLSTR™ Identifiler™ Plus PCR Amplification Kit (ThermoFisher Scientific) was used to determine the number of allele repeat units in microsatellite regions of both the generated iPSC and their parental fibroblast lines. In a first step, a multiplexed PCR of 16 human loci was performed to amplify the amelogenin gender-determining marker and the following tetranucleotide STR markers: D21S11, CSF1PO, vWA, D8S1179, TH01, D18S51, D5S818, D16S539, D3S1358, D2S1338, TPOX, FGA, D7S820, D13S317, D19S433. The amplification conditions were: 95 °C for 11 min; 28 cycles of 94 °C for 20 sec and 59 °C for 3 min; 60 °C for 10 min and hold at 4 °C. Prior to electrophoresis, samples were prepared by adding the PCR product or the allelic ladder to a Hi-Di formamide - GeneScan™ 500 LIZ™ dye size standard (ThermoFisher Scientific) solution. This mixture was denatured at 95 °C for 3 min and snap-cooled. Samples were injected at 3 kV for 10 sec and electrophoresed at 15 kV for 1500 sec in Performance Optimized Polymer-7 (POP-7 polymer; ThermoFisher Scientific) with a run temperature of 60 °C. The PCR products and the allelic ladder were separated and detected by high-resolution capillary electrophoresis with the ABI PRISM 3130xl genetic analyzer (ThermoFisher Scientific). The resulting electropherograms were analyzed using the GeneMapper ID software v3.2 (ThermoFisher Scientific).

An aliquot of the supernatant, after 48 h culture, was taken. It was incubated at 95 °C for 5 min and centrifuged at 10,000 xg for 30 sec. The PCR reaction was prepared by combining 2 µl of the supernatant, the DreamTaq Green PCR Master Mix 1x (ThermoFisher Scientific), the mycoplasma primers, and the internal control (Qiagen). The primers target the highly conserved 16S rRNA sequences to detect M. pneumoniae, M. hominis, M. fermentans, U. urealyticum, M. pulmonis, M. arthritidis, M. neurolyticum, M. muris and M. collis, which are common human and rodent mycoplasmal species (22). Primer sequences are provided in Table 3. A positive mycoplasma control (InvivoGen) was run alongside the iPSC samples. The PCR conditions were as follows: 95 °C for 3 min; 35 cycles of 95 °C for 30 sec and 55 °C for 30 sec and 72 °C for 1 min; 72 °C for 5 min and hold at 4 °C. The PCR products were separated by agarose gel electrophoresis.

The clones were analyzed by real time RT-PCR to check that they were viral-free iPSCs. 7 days post-transduction fibroblast cells (positive control) and iPSCs at passage 8 (interrogated material for Sendai removal) were harvest (n = 3) and stored as a dry pellet at -20 °C. Total RNA was extracted using the MagMAX−96 Total RNA Isolation Kit (ThermoFisher Scientific). RNA concentration and purity were determined with the NanoDrop spectrophotometer (ThermoFisher Scientific), by measuring the absorbance at 260 nm and calculating the 260/280 and 260/230 nm absorbance ratios, respectively. 1 µg of RNA was treated with the ezDNase enzyme (ThermoFisher Scientific) for genomic DNA removal and reverse-transcribed with the Superscript IV VILO reverse transcriptase (ThermoFisher Scientific). Real time quantitative PCR was performed on a ViiA 7 Real-Time PCR System with OptiFlex Optics System (ThermoFisher Scientific) using PowerUp SYBR Green PCR kit (ThermoFisher Scientific). The PCR reactions were conducted using the following parameters: 95 °C for 2 min; and 40 cycles of 95 °C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. Primer sequences are provided in Table 3. Gene expression levels relative to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene were calculated by the 2^−ΔΔCt method, the Livak method (23). For each patient-derived cell line, the 7 days post-transduction fibroblast population was used as the reference sample. The expression value of the interrogated sample (iPSC population at passage 8) was normalized to this reference sample, giving an expression value relative to 1. The log10 of the relative normalized expression was calculated, and the data was displayed using the Box and Whisker plots.

iPSCs were differentiated to oligodendrocytes as previously described in Douvaras et al., 2015 protocol (24), with some modifications. iPSCs were dissociated with Accutase (ThemoFisher Scientific) as single cells and plated on Matrigel-coated 6-well plates with mTeSR and CEPT at a density between 100,000 - 400,000 cells/well depending on the cell line. This cell density was adjusted for each iPSC line to ensure that in 1–2 days the colonies could reach a diameter of 100 - 250 µm. mTeSR medium was replaced by Neural Induction Media, NIM [DMEM/F12 containing 10 μM SB431542 (Stemgent), 250 nM LDN193189 (Stemgent) and 100 nM all-trans retinoic acid (RA, Sigma-Aldrich)]. After 8 days of culture, NIM medium was switched to N2 medium [DMEM/F12 containing 1x N2 supplement, 100 nM RA and 1 μM smoothened agonist (SAG, Sigma-Aldrich)]. NIM and N2 medium promote the specification of the progenitor motor neuron (pMN) domain. On day 12, cells were overconfluent and piled up generating 3D structures. At this point, cells were mechanically dissociated with a cell scraper (VWR) to obtain spheres in suspension, which were cultured (1:2) in ultra-low attachment 6-well plates with N2B27 medium [DMEM/F12 containing 1x B27 without vitamin A supplement, 1x N2 supplement, 100 nM RA and 1 μM SAG]. Only oligodendrocyte lineage transcription factor 2-positive (OLIG2+) cells are able to form aggregates and, thus, OLIG2- cells are eliminated gradually during medium changes. On day 20, N2B27 media was switched to PDGF media, based on DMEM/F12 supplemented with 1x B27 without vitamin A supplement, 1x N2 supplement, 10 ng/ml platelet-derived growth factor AA (PDGFAA; R&D systems), 10 ng/ml insulin-like growth factor 1 (IGF-I; R&D systems), 5 ng/ml hepatocyte growth factor (HGF; R&D systems), 10 ng/ml neurotrophin 3 (NT3; Sigma-Aldrich), 60 ng/ml 3,3,5-Triiodo-L-thyronine (T3; Sigma-Aldrich), 100 ng/ml biotin (Sigma-Aldrich), 1 µM N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (cAMP analog; Sigma-Aldrich), and 25 µg/ml insulin solution (Sigma-Adrich). These recombinant proteins and chemical compounds drive oligodendrocyte survival and differentiation. On day 30, spheres with round shape, brown color and a diameter of 300 - 800 µm were manually picked using a microscope under sterile conditions and a p200 pipette with a wide-bore tip. The selected spheres were plated on poly-L-ornithine/laminin-coated 6-well plates with PDGF medium, at a concentration of 2 spheres per cm². Cells migrated out of the spheres, first neurons and astrocytes, and around day 50 oligodendrocytes. From day 75 to 90, cells were maintained with glial medium [DMEM/F12 containing 1x B27 without vitamin A supplement, 1x N2 supplement, 10 mM HEPES (Sigma-Aldrich), 60 ng/ml T3, 100 ng/ml biotin, 1 µM cAMP, 25 µg/ml insulin solution, and 20 µg/ml AA] to induce the terminal differentiation of OPCs to more mature OLCs.

At day 75, the immunofluorescence and cell sorting of OPCs with the oligodendrocyte-specific O4 antibody was done with live cells. For immunoflurescence, the O4 primary antibody (R&D systems, 1:50) and 5% goat serum were added into the culture medium. Cells were incubated at 37 °C for 45 min. A wash step with DMEM/F12 was performed. Cells were fixed with 4% PFA for 10 min and the protocol was continued as previously described. A goat anti-mouse secondary antibody conjugated with Alexa Fluor 488 (ThermoFisher, 1:500) was used. For FACS, cells were dissociated with Accutase as single cells. The spheres remained intact after the Accutase treatment, but we were only interested in obtaining the cells that had migrated out of these spheres. Cells were centrifuged and incubated with DMEM/F12 medium containing the O4 primary antibody and 5% goat serum for 45 min on ice. A wash step with DMEM/F12 was performed. Cells were centrifuged and resuspended with DMEM/F12 medium containing the goat anti-mouse secondary antibody conjugated with Alexa Fluor 488 for 25 min on ice. Cells were washed, resuspended with cold PDGF medium and filtered with a 70 µM strainer. During this protocol, the cell suspension was pipetted up and down very gently a maximum of 2–3 times/step to avoid high cell death. Cells were sorted using a BD FACSAria II or a BD Influx (BD Biosciences).

At day 90, samples were fixed and stained as previously described in the «Pluripotency markers validation. Immunofluorescence and confocal microscopy analysis» section. The primary antibodies were the following: for neurons, microtubule-associated protein 2 (MAP2; Abcam); for astrocytes, glial fibrillary acidic protein (GFAP; Dako); for OLCs, myelin basic protein (MBP; Sigma-Aldrich). The corresponding secondary antibodies were: Alexa Fluor 647 goat anti-chicken (ThermoFisher Scientific, 1:500), Alexa Fluor 488 donkey anti-rabbit (ThermoFisher Scientific, 1:1000) and Alexa Fluor 568 goat anti-rat (ThermoFisher Scientific, 1:1000) respectively. Images were captured with an LSM 980 confocal microscope running Zen Black software (Carl-Zeiss) and analyzed with FIJI (ImageJ) software version 2.14.0/1.54f. All reagents and antibodies used in this article are listed in Table 4.

Six MSA cases were selected from the CMSAR in Hospital Clínic de Barcelona to generate a sex-balanced iPSC collection representing the two disease variants (cerebellar and parkinsonian). For five out of six cases, both the skin biopsy and the brain were available and we could confirm a definitive MSA diagnosis as defined by the postmortem identification of aggregated and misfolded aSyn in OLs forming GCIs. Four cases also presented other comorbid neuropathology. The MSA06 case did not donate the brain but fulfills both Gilman’s 2008 criteria for probable MSA (25) and the Movement Disorder Society (MDS) 2022 criteria for clinically established MSA (26) (Table 1).

From the skin biopsies, fibroblast cells were isolated, expanded for two passages, and validated to be mycoplasma-free. HDF-ad appeared spindle-shaped and refractile, with a doubling time of approximately 24 h (Figure 1A). HDF-ad were plated to reach 30 - 60% confluency after 48 h, this seeding density was optimized for each patient’s cell line. HDF-ad were reprogrammed using the non-integrative SeV for delivering the transcription factors Oct3/4, Sox2, c-Myc and Klf4 (OSKM, referred to as the Yamanaka factors). After seven days, transduced cells were plated on irradiated mouse embryonic fibroblasts (iMEFs) and cultured with iPSC medium. At this time point, some cells were already experimenting an evident and distinctive morphology change: from the initial elongated morphology (fibroblast-like) to a compact polygonal morphology (epithelial cell-like) (Figures 1B, C). Approximately between 3- and 4-week after reprogramming, tightly packed colonies were visible, and were characterized by having well-defined edges and containing cells with a high nucleus-cytoplasm ratio, features also shown in human embryonic stem (ES) cell colonies (Figure 1D). Single colonies were picked and clonally expanded, a process during which iPS cells were progressively adapted to mTeSR medium and Matrigel coating.

Our objective was to obtain three independent validated iPSC clones per patient, which met all the pre-defined quality standards (pluripotency expression, genome integrity, and differentiation potential). Some clones could fail in the evaluation process and, thus, a backup repository of nine independent iPSC clones per patient was generated. Then, three clones were selected randomly to evaluate whether they pass the before mentioned quality tests. In this manuscript, we only show the complete dataset of only one validated iPSC clone per patient.

The pluripotency of the newly generated iPSC lines was assessed by studying the expression of a set of signature surface and intracellular pluripotency proteins, SSEA4, NANOG and LIN28A. A combination of quantitative and qualitative data was obtained. The flow cytometry analysis demonstrated that > 99% of the cells were SSEA4+ in all the six iPSC lines (Figure 1E), being the acceptance criteria > 70% (27). The image-based analysis of NANOG and LIN28A revealed that these two proteins were expressed simultaneously and homogenously in the in vitro culture of all the six iPSC lines (Figure 1F).

Reprogrammed MSA hiPSC fully retained their donor-specific pattern of sequence repeats, as confirmed by STR profiling (Figure 2A), and thus cell lines identity could be verified with high accuracy. iPSC maintained their normal diploid karyotype after reprogramming and prolonged cell culture (minimum 10 passages), as determined by a 20-metaphase G-banding analysis (Figure 2B). G-banding offers whole-genome scans of chromosomal structure and size, but with a limited resolution at the level of 5–10 megabases (28). However, human iPSC and ES tend to accumulate recurrent copy number variations smaller than 5 megabases at specific genome locations (hotspots) (21). They include gains at 20q11.2 (encompassing ID1, BCL2L1, DNMT3 and HM13 genes) (29–31); 12p13.31 (NANOG and GDF3 genes) (32, 33); and 17q25.3 (BIRC5 gene) (34). Still under discussion, the amplification of these candidate genes could confer to the abnormal cells an enhanced probability of self-renewal, proliferation and/or survival, rapidly overtaking the culture as an adaptation process (33, 35). For this reason, the genomic DNA of the iPSC cultures at passage 8 was screened by digital PCR, targeting six of the previously identified and described recurrent abnormalities (approximately 60% of cumulated coverage) (21). The iPSC clones had 2 copies of the following genes: ID1 (20q11 locus), NCAPD2 (12p13 locus), STS (Xp22 locus), RPS6KB1 (17q23 locus), SOAT1 (1q25 locus) and PITX1 (5q31 locus), relative to the reference gene RPP30 (10q23 locus) (Figure 2C). This quality control approach should be routinely repeated when cells are cultured for long-term periods.

The selected reprogramming method was the negative-sense single-stranded RNA SeV since it replicates cytosolically in the host cells and it does not have a DNA phase throughout its life cycle, eliminating the risk of genomic integration of OSKM (36). Real Time RT-PCR using SeV-specific primers was performed to check that the reprogramming transgenes were lost during host cell division. It is a passage-dependent decrease (37), which can be accelerated when iPSC are passaged as a single-colony instead of a bulk-population. Consequently, during the firsts 3 passages, the hiPSC expansion was performed as follows: manually selecting a single colony, cutting it in a grid-like pattern and transferring it into a feeder layer- or matrigel- coated plate. For the rest of the passages, EDTA was used to perform a pooled-clone expansion. MSA hiPSC clones were analyzed at passage 8 with quantitative Real Time RT-PCR, and 7-day transduced cells were used as positive controls. The absence of exogenous viral sequences was confirmed for all cases (Figure 2D).

The differentiation potential of the six MSA hiPSC lines was assessed by performing two independent and complementary protocols. Cells were differentiated into the three germ layers (ectoderm, endoderm and mesoderm) by recapitulating early or late stages of the human embryonic development in a dish. First, iPSC were plated as single cells in an adherent monolayer culture and were treated with a commercial, defined and lineage-specific media for 5 or 7 days (STEMdiff™ Trilineage Differentiation Kit from STEMCELL Technologies) (Figure 3A). It is a rapid and reproducible method, based on the chemical induction of the early stages of the trilineage differentiation. On the other hand, embryoid bodies (EBs) were generated and plated in gelatin-coated plates for ectoderm and matrigel-coated plates for endoderm and mesoderm. Following this, the EBs were treated with a homemade, defined and lineage-specific media for 4 weeks (Figure 3B). EBs are three-dimensional cell clusters that can physically recapitulate embryogenesis. Thus, this protocol allows to better observe the morphogenesis of the ectoderm, endoderm and mesoderm and the prolonged culture time enables the emergence of late stages of the trilineage differentiation. The expression of the three germ-layer proteins was monitored through immunofluorescence in both cases. For the ectoderm, neural tube-like rosettes were clearly recognizable in both early- and late- stage differentiations. The transcription factor PAX6 (38) was expressed uniformly by columnar cells. Also, the cytoskeletal protein NES (39) showed that cells developed processes such as axon-like domains. PAX6 and NES are the predominant markers of neural progenitor cells (Figure 3C is exemplary shown for MSA01, Figure 3F; Supplementary Figure 1). The presence of TUBB3-positive cells (40) also confirmed the neuronal fate commitment (Figure 3I). As the differentiation proceeds, this tubulin is expressed by postmitotic immature neurons and, like nestin, is involved in the cytoskeleton remodeling required for neural plasticity. The endoderm formation was demonstrated by the earlier expression of FOXA2 and SOX17 transcription factors that act as epithelial to mesenchymal (EMT) suppressors and as epithelial gatekeepers (41) (Figure 3D is exemplary shown for MSA01, Figure 3G; Supplementary Figure 2). The later expression and localization of the secreted serum protein AFP (42) confirmed the organization of a developing epithelium, characterized by a single layered cup-shaped sheet of cells (Figure 3J). For the mesoderm, cells expressed the adhesion molecule NCAM1 and the transcription factor TBXT, both implicated in cell migration and the commitment to the mesodermal lineage, as it is formed via EMT (43) (Figure 3E is exemplary shown for MSA01, Figure 3H; Supplementary Figure 3). After the EB differentiation, a mesenchymal tissue was identified. Specifically, the expression of the actin protein ACTA2 revealed the presence of stress fibers from the derived stromal cells (Figure 3K).

OLs have been identified as the cell type primarily affected in MSA disease. Their cytoplasm accumulates aggregates of pathological fibrillary forms of aSyn in GCIs that propagate throughout the brain as the disease progresses (44). To validate the use of MSA hiPSCs for disease modeling and drug screening, they were differentiated to OLs following the protocol from Douvaras & Fossati 2015 (24). This protocol recapitulates spinal cord embryonic development and consists of four main stages (Figure 4A): (1) the specification of the pMN domain in monolayer culture via dual-SMAD inhibition and all-trans RA and sonic hedgehog (SHH) signaling; (2) the enrichment of the pMN progenitors (OLIG2+ cells) by generating cell aggregates in suspension; (3) the differentiation to OPCs (O4+ cells) by treating the plated cell aggregates with key factors as PDGF, NT3, T3, IGF-1 and HGF; and (4) the terminal maturation to OLs (O4+MBP+ cells) by the withdrawal of the previously mentioned factors. Due to budget constraints, we were only able to differentiate four MSA hiPSC lines: MSA01, MSA03, MSA04, and MSA06. We excluded MSA02 because this case presented the most extensive and severe co-pathologies, including high-grade chronic traumatic encephalopathy (CTE) related to repeated head trauma, Alzheimer’s disease with an intermediate Amyloid-Braak-CERAD (ABC) score, and cerebral small vessel disease (SVD). Also, MSA05 was not included as no brain donation was available for this case.

The OL differentiation status was monitored at various timepoints (Figures 4B, C; Supplementary Figure 4A). First, we had to adjust the initial plating density of each patient-derived iPSC line to obtain an overconfluent culture at day 12, characterized by cells piling up and forming 3D structures. At day 30, we could observe that the cell clusters in suspension were homogeneous and characterized by being round (spheres), brown with a darker core, and 300 - 800 µm in diameter (Figure 4B). When these spheres were plated into laminin/poly-L-ornithine coated wells, cells started to migrate out. Around day 45, we could confirm the presence of neurons, clearly distinguishable by their long processes that extend out from the cell soma (Figure 4C). From day 55 and onwards, we detected the emergence of many highly branched cells, which were positive for the oligodendrocyte marker O4 (Figures 4D, E). All the MSA hiPSC lines differentiated efficiently to OPCs, as evidenced by the flow cytometry results at day 75 showing O4+ cell percentages ranging from 62% to 81% (Figure 4F; Supplementary Figure 4B). The terminal differentiation of OPCs to more mature OLs from day 75 to day 90 was demonstrated by the presence of cells co-expressing O4 and myelin basic protein, MBP (Figure 4G) (representative image of one MSA hiPSC line). We found glial fibrillary acidic protein (GFAP, astroglial marker) (Figure 4H) and microtubule-associated protein 2 (MAP2, neuronal marker) (Figure 4I) expressing cells in our in vitro culture (representative image of one MSA hiPSC line). These immunofluorescence images evidenced that the protocol also generated astrocytes and neurons (Supplementary Figure 4C), which are essential for supporting the survival and growth of oligodendrocytes.