In this study, a normal iPSC line previously established in our laboratory was used as a control [12], and one iPSC line was generated from the urine of an SCA6 patient. The iPSCs were maintained on dishes coated with 1% Matrigel in mTeSR-1 medium (STEMCELL Technologies, Seoul, Korea), with daily replacement of the culture medium. Passaging was performed as small clumps using ReLeSR (STEMCELL Technologies).

Urinary cells (UCs) were isolated from the patient’s urine as described previously [8]. Briefly, urine samples were washed with a buffer consisting of dPBS (Welgene, Gyeongsan, Korea) supplemented with 1% antibiotic-antimycotic (AA, Gibco; Thermo Fisher Scientific Inc., Seoul, Korea) and centrifuged at 200×g for 10 minutes. After discarding the supernatant, the pellet was resuspended in a primary medium composed of DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% AA, and REGM supplement (Lonza, Basel, Switzerland). The resuspended cells were then seeded onto plates coated with 0.2% gelatin (Sigma, St. Louis, MO, USA).

On the following day, the primary medium was replenished and maintained for 3 days. On day 4, the primary medium was replaced with a proliferation medium, consisting of REGM basal medium (Lonza) supplemented with 1% AA and REGM supplement. Starting the next day, half of the proliferation medium was replaced daily. Once colonies formed and reached 80-90% confluency, subculturing was performed using 0.05% trypsin-EDTA (Gibco). The isolated UCs were cryopreserved and could be passaged 5 to 10 times.

To generate iPSCs, the expanded UCs were reprogrammed using the CytoTune iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific Inc.). The cells were transduced with Sendai viral vectors carrying hKOS, hc-MYC, and hKlf4 at multiplicities of infection of 5, 5, and 3, respectively. Transduced cells were then seeded onto mitomycin C-treated mouse embryonic fibroblast feeder layers and maintained in a reprogramming medium composed of DMEM/F12 supplemented with 20% Knockout Serum Replacement (Gibco), 1% Pen/Strep (Gibco), 1% non-essential amino acids (Gibco), 1% GlutaMax (Gibco), 1X beta-mercaptoethanol (Gibco), 20 ng/mL basic fibroblast growth factor (bFGF; R&D Systems, Minneapolis, MN, USA), and 10 mM nicotinamide (Sigma). The iPSC colonies were mechanically transferred to fresh feeder plates to ensure continued growth and maintenance.

After dissociation of iPSCs with Accutase (Sigma), cells were seeded at a density of 2×10⁴ cells per well in an ultra-low attachment 96-well plate to facilitate embryoid body (EB) formation in mTeSR medium supplemented with 10 μM Y-27632 (Tocris, Bristol, UK). The next day, the medium was replaced with TeSR-E6 medium (STEMCELL Technologies), with half of the medium changed every other day. After 2 weeks of culture, the embryoid bodies were dissociated by gentle pipetting and seeded onto dishes coated with 1:100 Matrigel. The TeSR-E6 medium was replaced by half every 2 days to maintain optimal conditions for cell differentiation and growth.

iPSC-derived cerebellar neurons were generated as described in a published study. iPSCs, dissociated into single cells using Accutase, were seeded in an ultra-low attachment 96-well plate at a density of 6×10³ cells per well to allow self-organization. After the formation of embryoid bodies, the medium was replaced with gfCDM (a 1:1 mixture of Iscove’s modified Dulbecco’s medium (Corning, New York, NY, USA) and Ham’s F12 medium (Corning), supplemented with 1% chemically defined lipid concentrate, 1% Pen/Strep, and 7 μg/mL insulin) supplemented with 50 μg/mL bFGF and 10 μM SB431542 (Tocris), with changes on alternate days. On day 14, the medium was replaced with gfCDM supplemented with 100 ng/mL FGF19 (PeproTech; Thermo Fisher Scientific Inc.). After 1 week, the medium was replaced with neurobasal medium supplemented with 1X GlutaMax, 1X N2 supplement (Gibco), and 1% Pen/Strep; from day 28, 300 ng/mL SDF1 (PeproTech) was added. On day 35, the cerebellar organoids were dissociated into single cells and seeded at a density of 8×10⁴ cells per cm² on a dish coated with 1:100 Matrigel, using BrainPhys Neuronal medium supplemented with NeuroCult SM1 Neuronal supplement (STEMCELL Technologies), N2 supplement-A, 20 ng/mL BDNF (PeproTech), 20 ng/mL GDNF (PeproTech), 1 mM db-cAMP (BioGems, Seoul, Korea), and 200 nM ascorbic acid. The medium was half-changed every other day.

The differentiated organoids and cerebellar neurons were rinsed with 1X PBS and then lysed using the easy-BLUE Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea). RNA isolation was performed manually using chloroform and isopropanol. The resulting RNA pellet was washed with cold 70% ethanol and briefly air-dried. The concentration and quality of the isolated RNA were assessed using a NanoDrop spectrophotometer.

A total of 1,200 ng of RNA was reverse-transcribed into complementary DNA using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. Gene expression levels were analyzed by quantitative polymerase chain reaction (PCR) using Fast SYBR Green PCR Master Mix (Applied Biosystems, Santa Clara, CA, USA) and gene-specific primers for each marker.

hTBP, F- TGA GTT GCT CAT ACC GTG CTG CTA, R- CCC TCA AAC CAA CTT GTC AAC AGC

hOCT4, F- GAG AAG GAT GTG GTC CGA GTG TG, R- CAG AGG AAA GGA CAC TGG TCC C

hSOX2, F- AGA ACC CCA AGA TGC ACA AC, R- ATG TAG GTC TGC GAG CTG GT

hNANOG, F- CAA AGG CAA ACA ACC CAC TT, R- ATT GTT CCA GGT CTG GTT GC

hREX1, F- AAT GCG TCA TAA GGG GTG AG, R- TCA ATG CCA GGT ATT CCT CC

hEN, F- CCG GCG TGG GTC TAC TGT A, R- GGC CGC TTG TCC TCT TTG TT

hOLIG2, F- GAC AAG CTA GGA GGC AGT GG, R- CGG CTC TGT CAT TTG CTT CTT G

hATOH1, F- TGT TAT CCC GTC GTT CAA CAA C, R- TGG GCG TTT GTA GCA GCT C

hNEUROGRANIN, F- TCA AAG TTC CCG AGG AGA GA, R- CTA AAA GGG CAC GGA CTC AG

hPARVALBUMIN, F- TTC TCC CCA GAT GCC AGA GA, R- GAG ATT GGG TGT TCA GGG CA

hALDOC, F- ACT CCA TAC CAC AGC CCT TG, R- GCA ATT TCT TCT GCC CTC AG

hLHX5, F- CAG CAA CGC TGT AGC CAA TTT, R- TCC GGA TCC TCA TCT TTG TC

hL7/PCP2, F- ACC AGG AGG GCT TCT TCA AT, R- CTG TCA CAC GTT GGT CAT CC

hCBLN1, F- TTT GAT TCA GAA CGC AGC AC, R- TTG GAT TAG GAC TCC GTT GC

hCORL2, F- CCA GGT GTT AAA AGG AAA CAC A, R- GCT CCC TTT TCA TCT GAT CCT

hVGLUT1, F- TAC ACG GCT CCT TTT TCT GG, R- CTG AGG GGA TCA GCA TGT TT

hKIRREL, F- GAA TGT CAG GCT ACA CAA GCA GG, R- TTC GCA GGA ACT CCA GCA ACC A

Genomic DNA (gDNA) was extracted using the G-spin Total DNA Extraction Kit (iNtRON Biotechnology) following the manufacturer’s instructions. Briefly, collected cell pellets were lysed using Buffer CL supplemented with RNase A and Proteinase K, followed by vigorous vortexing. Buffer BL was then added to the lysate, mixed thoroughly, and incubated at room temperature for 2 minutes. The mixture was subsequently incubated at 56°C for 10 minutes to enhance protein digestion. After incubation, ethanol was added and the solution was briefly centrifuged. The resulting mixture was transferred to a spin column and centrifuged at 13,000 rpm for 1 minute. After discarding the filtrate, the column was washed twice with Buffer WB, and the membrane was dried by centrifugation for 1 minute. Genomic DNA was eluted from the membrane using Buffer CE.

To amplify the extended CAG repeat in SCA6 patient iPSCs, PCR was performed using the hCACNA1A primer set: (F: GGC CAC ACG TGT CCT ATT CC / R: GAC CCG CCT CTC CAT CCT).

Cerebellar organoids, neurons, and 2-dimensional (2D) cells were fixed in 4% paraformaldehyde (PFA) for 3 hours and 30 minutes at room temperature, then rinsed with 1X PBS. The fixed organoids were immersed in 30% sucrose at 4°C for 1 day for cryoprotection, followed by another rinse with 1X PBS. Organoids were then embedded in OCT compound (Sakura, Torrance, CA, USA) and sectioned into 7 μm slices using a cryostat (LEICA CM1520). The sections were permeabilized for 1 hour with 0.3% Triton X-100 (Sigma) in 0.025% TBS-T. Following permeabilization, the samples were blocked for 1 hour using a solution containing 3% BSA and 0.02% sodium azide in 0.025% TBS-T.

Primary antibody staining was performed overnight at 4°C using the following primary antibodies: MAP2 (R&D Systems; #MAB933), TUJ1 (BioLegend, San Diego, CA, USA; #802001), PCP-2 (Santa Cruz, Starr County, TX, USA; #SC-137064), KIRREL (LS Bio, Lynnwood, WA, USA; #LS-C336220), SOX2 (Seven Hills Bioreagent, Cincinnati, OH, USA; #WRAB1236), Nanog (R&D Systems; #AF1997), SSEA4 (R&D Systems; #MAB1435), OCT4 (Abcam, Cambridge, UK; #ab109183), αSMA (R&D Systems; #MAB1420), Vimentin (Cell Signaling, Danvers, MA, USA; #5731), Nestin (Abcam; #ab6320), GATA4 (Santa Cruz; #SC25310), and NKX2.1 (Abcam; #ab76013). After 3 washes with 1X TBS-T, the samples were incubated at room temperature for 1 hour in dim light with diluted Hoechst (Thermo Fisher Scientific Inc.) for nuclear staining, along with secondary antibodies Alexa Fluor 488 goat anti-mouse IgG (Life Technologies, Carlsbad, CA, USA) and Alexa Fluor 555 donkey anti-rabbit IgG (Life Technologies). The stained sections were examined using a confocal microscope (ZEISS LSM800) to capture high-resolution images and assess protein expression.

Alkaline phosphatase (AP) staining was performed using the ALKALINE PHOSPHATASE kit (Sigma) according to the manufacturer’s instructions. Briefly, iPSC colonies were washed with distilled water (DW) and fixed with 4% PFA for 10 minutes at room temperature. Following fixation, the colonies were washed again with DW to remove any residual fixative. After discarding the wash solution, the colonies were treated with a mixture of sodium nitrite solution, FRV-alkaline solution, and naphthol AS-BI Alkaline solution, and incubated at room temperature for 30 minutes to develop staining. After incubation, the colonies were thoroughly washed with DW to remove excess reagents. The stained colonies were subsequently imaged to assess AP activity.

All experiments were performed independently at least 3 times. Statistical analyses were conducted using GraphPad Prism 9.5.1 software, employing a non-parametric test to evaluate differences between groups. Data are presented as the mean±standard deviation. Statistical significance was defined as follows: p<0.05, p<0.01, p<0.001, p<0.0001. Significant differences were indicated using the corresponding symbols.

To obtain somatic cells from an SCA6 patient using a non-invasive method and generate iPSCs, we collected urine samples and cultured urinary epithelial cells. Colony formation was observed from day 6 onward. When colonies reached approximately 70% to 80% confluency (Fig. 1A), the cells were transferred for reprogramming. Reprogramming was performed using a Sendai virus system. After 3 weeks of Sendai virus treatment, colonies exhibiting pluripotent stem cell (PSC) morphology were observed, and this typical morphology was maintained even after transfer (Fig. 1B).

To confirm pluripotency, AP staining was performed, and AP-positive colonies were selected for further analysis (Fig. 1B). Additionally, to verify the SCA6-specific mutation, increased CAG repeats were confirmed in the patient-derived iPSCs (Fig. 1C). When PCR amplification was performed using a primer set targeting the CAG repeat region in the CACNA1A gene, the normal iPSCs showed a single amplified band. In contrast, the SCA6 patient-derived iPSCs exhibited 2 bands: one corresponding to the normal-sized PCR product and another representing a larger product. This result suggests an increased CAG repeat length on one allele of the patient-derived iPSCs.

To further verify the pluripotency of SCA6 patient-derived iPSCs, we assessed the expression of pluripotency markers through immunostaining. Specifically, we confirmed the protein expression of SRY-box transcription factor 2 (SOX2), Nanog, stage-specific embryonic antigen-4 (SSEA4), and octamer-binding transcription factor 4 (OCT4) in the patient’s UC-derived iPSC colonies (Fig. 2A). In addition, the mRNA expression levels of OCT4, SOX2, NANOG, and REX1 were validated by real-time PCR (Fig. 2B).

To assess the differentiation potential of the UC-derived iPSCs into the 3 germ layers, EBs were formed and subsequently analyzed by immunostaining. The results demonstrated that the UC-derived iPSCs successfully expressed markers for all 3 germ layers: mesoderm (αSMA and vimentin), ectoderm (Nestin and Tuj1), and endoderm (GATA4 and NKX2.1). These findings confirm that the UC-derived iPSCs possess the ability to differentiate into all 3 germ layers, thereby demonstrating their pluripotency (Fig. 2C).

Since the phenotype of SCA6 is closely associated with cerebellar Purkinje cell dysfunction [13-15], we differentiated iPSCs into cerebellar organoids. Phase-contrast imaging of these organoids revealed the formation of characteristic wrinkles (Fig. 3A).

To assess the differentiation status, we analyzed the mRNA expression levels of various cerebellar neuron progenitor markers. Real-time PCR analysis showed that the expression levels of engrailed homeobox (EN), oligodendrocyte transcription factor 2 (OLIG2), atonal bHLH transcription factor 1 (ATOH1), NEUROGRANIN, PARVALBUMIN, aldolase, fructose-bisphosphate C (ALDOC), and LIM homeobox 5 (LHX5) increased significantly on day 20 and then decreased by day 37. In contrast, the mRNA expression levels of cerebellar neuron markers—including Purkinje cell protein 2 (L7/PCP2), cerebellin 1 precursor (CBLN1), cerebellin 2 precursor (CORL2), solute carrier family 17 member 7 (VGLUT1), and kirre-like nephrin family adhesion molecule 1 (KIRREL)—increased significantly on day 37 (Fig. 3B).

Furthermore, immunostaining of cerebellar organoids confirmed the expression of neural markers Tuj1 and MAP2, along with PCP-2, a specific marker of cerebellar neurons, on day 37 (Fig. 3C).

These results demonstrate that the organoids differentiated from human iPSCs successfully generated cerebellar neurons, indicating that our differentiation protocol effectively recapitulated cerebellar neuronal characteristics.

To determine whether the SCA6 phenotype could be reproduced using SCA6 patient-derived iPSCs, we differentiated both SCA6 patient-derived iPSCs and normal control iPSCs into cerebellar neurons. During differentiation, cerebellar organoids derived from the SCA6 group were significantly smaller than those from the normal group. Furthermore, following dissociation and subsequent 2D culture, neurite formation was notably reduced in the SCA6 group compared to controls (Fig. 4A).

To further assess Purkinje cell differentiation, we evaluated the mRNA expression level of KIRREL, a specific marker for Purkinje cells. The results demonstrated a significant decrease in KIRREL expression in SCA6-derived neurons compared to normal controls (Fig. 4B).

Immunostaining analysis revealed distinct differences in KIRREL expression and neuronal morphology between the groups. In the normal group, KIRREL expression was observed in both the soma and neurites, consistent with typical neuronal morphology. In contrast, the SCA6 group exhibited KIRREL expression only in the soma, with no detectable expression in the neurites (Fig. 4C).

These findings indicate that cerebellar neurons differentiated from SCA6 patient-derived iPSCs exhibit reduced differentiation capacity and impaired neurite formation compared to those derived from normal iPSCs, effectively recapitulating the SCA6 phenotype.