Neural stem cells derived from α-synuclein-knockdown iPS cells alleviate Parkinson’s disease – Cell Death Discovery

Mouse embryonic fibroblast cell isolation and animal model

We obtained C57BL/6J and SNCA A53T transgenic mice from the Jackson Lab (Bar Harbor, ME, USA), whose detailed information can be found at the following links (http://www.jax.org/strain/000664 and http://www.jax.org/strain/008859, respectively). These transgenic mice express human A53T variant SNCA under the promoter of human thymus cell antigen 1. Hind limb paralysis and a resting tremor typically begin to appear around 8 months of age. We isolated primary mouse embryonic fibroblast cells (MEFs) from SNCA A53T transgenic mice at 13.5 days old. During the procedure, the embryos were retrieved by cesarean section, freed from the placenta, and their internal organs, legs, and brains were removed. Trypsin-EDTA (GIBCO BRL, Grand Island, NY, USA) was used to digest the remaining embryo parts. Immediately following digestion, MEFs were cultured in MEF medium (DMEM/high glucose (GIBCO BRL) with 10% heat-inactivated FBS (HyClone, Logan, Utah, USA), penicillin (100 U/ml; GIBCO BRL), streptomycin (100 µg/ml; GIBCO BRL), nonessential amino acids (0.1 mM, GIBCO BRL) and L-glutamine (2 mM, GIBCO BRL)) in a humidified incubator with 5% CO₂. The Institutional Animal Care and Use Committee at China Medical University approved all experimental protocols (CMUIACUC-2017-313).

The generation of iPS cells

On day 0, SNCA A53T MEFs were transduced with lentiviral vectors expressing mouse Oct4, Sox2, Klf4, and c-Myc (Cellexium Biomedica Inc. Taipei, Taiwan). ES-like colonies were found on day 9, and these cells were transferred to feeder cells in an iPS cell culture medium consisting of DMEM/high glucose with 15% heat-inactivated defined FBS (HyClone, Logan, Utah, USA), penicillin (100 U/ml), streptomycin (100 µg/ml), nonessential amino acids (0.1 mM), L-glutamine (2 mM), 2-Mercaptoethanol (0.1 mM) (Merck KGaA, Darmstadt, Germany), and mouse Leukemia Inhibitory factor (LIF, 10³ units/ml; EMD Millipore Corporation, Temecula, California, USA). On day 21, iPS cells (ES cell-like clones) were obtained.

Alkaline phosphatase staining and indirect immunofluorescent staining

The alkaline phosphatase (AP) staining procedure (Vector Laboratories, Cat. No. SK-5100, Newark, California, USA) was followed according to the manufacturer’s instructions. We treated the cells at room temperature with 0.5% Triton X-100 for 15 min after fixing them with 4% paraformaldehyde in PBS for 30 min. After washing with PBS, cells were treated with blocking buffer (0.5% BSA in PBS) for 1 h at room temperature and washed 3 times with PBS. We used the following primary antibodies at 1:1000 dilutions overnight at 4 °C: anti-Nanog (Genetex, Cat. No. GTX100863, Irvine, CA, USA), anti-SSEA1 (Millipore, Cat. No. MAB4301, Burlington, MA, USA), anti-GATA4 (Genetex, Cat. No. GTX113194), anti-SMA (Millipore, Cat. No. CBL171), anti-Tuj-1 (Millipore, Cat. No. MAB1637) and anti-Nestin (Abcam, Cat. No. ab6142, Cambridge, UK). Cells were washed with three cold PBS washes and then incubated with FITC-conjugated anti-mouse IgG or TRITC-conjugated anti-rabbit IgG (Sigma-Aldrich, Cat. No. AP160F and T6778). A confocal fluorescence microscope (TCS-NT, Hilden, Germany) was used to visualize the signals. Cell nuclei were stained with DAPI (Sigma-Aldrich).

Histological analysis of spontaneous differentiation in vitro

The iPS cells were seeded into bacterial culture dishes at a density of 5 × 10⁶ cells/ml in iPS cell culture media without LIF for 72 h, and the aggregated cells (embryoid bodies, EBs) were then plated onto gelatin-coated culture plates for another 3 days. A confocal fluorescence microscope was used to observe the signals of spontaneously differentiated cells stained with anti-GATA4, anti-SMA, and anti-Tuj1.

NSC differentiation from iPS cells

On day 1, we dissociated iPS cells into single cells using accutase (Thermo Fisher Scientific, Waltham, MA, USA), then transferred them to bacterial culture dishes containing EB differentiation media (DMEM supplemented with 20% FBS (Gibco), glutamine (2 mM), nonessential amino acids (0.1 mM), and 2-mercaptoethanol). Upon renewing the culture medium every 2 days, iPS cells differentiated spontaneously into EBs. EBs were replated on day 5 with poly-D-lysine coated plates (Gibco). By switching the medium to ITS-FN medium (DMEM/F12, glutamine (2 mg), 1X ITS-G supplement (Gibco), Fibronectin (5 µg/ml, Gibco), 2-mercaptoethanol (0.1 mM)), neural stem cells were produced. After 4 days, cells were washed with PBS and dissociated with trypsin-EDTA for 5 min. The cell pellets were resuspended in N2 medium (DMEM/F12, glutamine (2 mM), 1X N2 supplement (Gibco), and bFGF (20 ng/ml, Gibco)) and seeded onto Poly-L-ornithine/Fibronectin-coated cultureware following brief centrifugation. NSCs could be maintained for five passages, and the culture medium was replaced every 2 days.

Efficacy of shRNAs in knocking down SNCA genes

We examined the knockdown efficiency of different shRNAs in GBM8901 glioma cells in this study. The cells were cultured in RPMI-1640 medium containing FBS, nonessential amino acids, glutamine, penicillin, and streptomycin. According to the manufacturer’s instructions, shRNA encoded in plasmids was transfected into GBM8901 cells using FuGENE (Promega, Madison, WI, USA). SNCA gene expression levels were determined using real-time PCR using the delta-delta Ct method.

Real-time PCR and Western Blot

RNA was collected from GBM8901 cells transfected with shRNA using Trizol reagent (Thermo Fisher Scientific). We used Fast SYBR TM Green Master Mix (Applied Biosystems, Waltham, MA, USA) and StepOnePlus TM Real-Time PCR Environment (Applied Biosystems) to measure gene expression (i.e., Actin and SNCA). According to the manufacturer’s instructions, 1 mg of total RNA was reverse transcribed-PCR using Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) and oligo(dT) primers in a final volume of 20 ml. Real-time PCR reactions were conducted following the manufacturer’s standard PCR protocol. Gapdh was used as a reference gene for normalizing mRNA expression levels. Negative controls were siRNAs against GFP. GBM8901 cells transfected with shRNA were lysed, and proteins were collected for Western blot analysis. Antibodies against SNCA (Novus Biologicals, Cat. No. NBP2-15365) and β-actin (Genetex, Cat. No. GTX629630) were used in this study.

The production and transduction of shRNA-6 lentiviruses

293T cells were co-transfected with shRNA-6 plasmids, pCMV-DR8.91, and pMD.G using FuGENE. After transfection, 293T cells were cultured for 24 h with FBS, NEAA, L-glutamine, and PS in DMEM/high glucose medium, followed by 24 h in ITS-FN medium. The virus supernatant was collected, filtered through a 0.45 mm filter, and stored at −80 °C until the next step. For transduction, the iPS cells were incubated with virus solution for 24 h (MOI:5), followed by selection with 0.5 mg/ml of puromycin for another 24 h.

Neurological behavioral measurements after transplantation

For the SNCA A53T transgenic mice, six training sessions were conducted over 2 weeks at the age of 5 months. After the last training session, mice were transplanted with 1 × 10⁶ NSC-SNCA or NSC-shSNCA cells, which were counted using the Countess 2 (Thermo Fisher Scientific), into three cortical areas adjacent to the right middle cerebral artery (MCA), positioned 3 to 3.5 mm below the dura after ligation [37]. Normal saline injections were administered as a control group for mice. According to “Methods of behavior analysis in neuroscience” [38], locomotor activity, beam walking, and rotarod were used to assess the therapeutic effects of NSCs and NSC-shSNCA cells. Briefly, the neuronal behaviors of target mice were examined using 3 separate apparatuses: a beam walking was used to monitor balancing ability (width: 80 cm), a rotarod was used to monitor coordination, and a locomotor device was used to monitor overall activity. During this study, mice’s balance ability was measured by recording their time crossing an 80-cm beam and measuring the frequency with which their rear feet slipped. During 3 min, animals were observed to remain stable on a rota. An 8-channel locomotor activity box was used to monitor overall activity for 1 h. The movement of the animals in the chamber, their resting time, and the distance they traveled during the last 30 min were also examined. The behavioral assessments were conducted 1 day before transplantation (week 0) and every 7 days thereafter for 22 weeks. Afterward, the mice were sacrificed, and their brain tissue was collected.

Assessment of the cell numbers of dopaminergic neurons

We prepared frozen sections of brain tissue after the behavioral study. A dopaminergic cell marker, tyrosine hydroxylase (TH; Millipore), was stained in sections of the substantia nigra (SN). The number of TH-positive cells was determined using ImageJ software (National Institutes of Health, USA).

TUNEL assay

An apoptotic cell count was determined using ImageJ software by staining the frozen sections with the TUNEL assay kit (Abcam, Cat. No. ab66108).

Statistical analysis

Data were obtained from at least three independent experiments and compared for statistical significance using the Prism software (version 5.01, San Diego, CA, USA) at a significance level of P < 0.05 (*P < 0.05, **P < 0.01, and ***P < 0.001). One-way analysis of variance, followed by the appropriate multiple comparisons test, was used to investigate statistical significance. Data were expressed as the mean ± SD. Behavioral data were compared by two-way analysis of variance (ANOVA) followed by a post hoc Bonferroni test. Data were expressed as the mean ± SEM. Survival analysis was done using the Kaplan-Meier estimator and the log-rank test for group comparison. Variables with a significant P-value in the univariate analysis were exposed to a multivariate analysis using Cox regression proportional hazard model.

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• Source: https://www.nature.com/articles/s41420-024-02176-z