2.1 hNTSCs generation and cell culture

The study using hNTSCs followed the guidelines of the Institutional Review Board of Seoul St. Mary’s Hospital (KC08TISS0341), Catholic University of Korea, including informed consent regulations and the Declaration of Helsinki. Before surgery, the participants provided written informed consent to participate in the study. The tissue was donated by patient who conducted hypertrophied nasal inferior turbinate volume reduction surgery. hNTSCs were isolated from the discarded tissue following partial turbinectomy using previously described methods [30]. First, the tissues were washed with 0.9% saline and phosphate-buffered saline (Thermo Fisher Scientific). The specimens were cut into 1 mm pieces and placed in a culture dish. Finally, a sterilized glass cover slide was placed on top. The tissue was placed in a humidified incubator and maintained at a temperature of 37 °C with 5% CO2. The specimen was cultured in α-minimum essential medium (α-MEM; Thermo Fisher Scientific) which was supplemented with 1% penicillin/streptomycin (Invitrogen, CA, USA), and 10% fetal bovine serum (FBS; Thermo Fisher Scientific). The culture medium was changed every two days for three weeks. The cells were harvested from the tissue by removing the glass cover slide and using 0.25% trypsin and 1 mM EDTA solution. hNTSCs were cultured and expanded for use in experiments.

2.2 hBM-MSC generation and cell culture

Healthy donors provided human bone marrow aspirates from the iliac crest following approval from the Institutional Review Board of Seoul St. Mary’s Hospital (KC10CSSE0651). Bone marrow aspirates were sent to the good manufacturing practice-compliant facility of the Catholic Institute of Cell Therapy (Seoul, Korea, http://www.cic.re.kr) for hBM-MSCs isolation, expansion, and quality control, with written consent from the participants [26]. The hBM-MSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penicillin/streptomycin (Invitrogen) and 20% FBS (Thermo Fisher Scientific). The cells were incubated at 37 °C in a humidified atmosphere containing 5% of CO2.

2.3 SH-SY5Y cell culture and differentiation

SH-SY5Y cells were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in the humidified incubator, which maintained 37 °C and contained 5% CO2, in α-MEM supplemented with 1% penicillin/streptomycin (Invitrogen), and 10% FBS (Thermo Fisher Scientific). SH-SY5Y cells were seeded at a density of 3 × 104 cells in a 6-well-plate, and the differentiation medium was changed according to a previously described protocol [37].

2.4 Trans-well assay

DA-like cells (1 × 106 cells of DA-like cells were cultured in the bottom chamber, treated with 500 mM MPP+ in culture medium overnight, and the following day, the medium was replaced with fresh medium. One day later, 5 × 105 hNTSCs and hBC-MSCs were cultured in the upper chamber, after being placed at the chamber bottom, and incubated overnight.

2.5 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model generation

Seven-week-old C57BL/6N mice were obtained from Orient Bio (Gyeonggi-do, South Korea). Ten mice were randomly assigned to one of four groups: control, MPTP-saline, MPTP_hNTSCs, and MPTP_hBC-MSCs. MPTP was administered at a dose of 25 mg/kg for seven consecutive days. The motor dysfunction of the PD mouse model was validated using the rotarod and open field tests.

2.6 Stereotaxic surgery

hNTSCs and hBM-MSCs were incubated for 5 min with trypsin–EDTA (Thermo Fisher Scientific), centrifuged, and then, the supernatant was removed. Mice were anesthetized, and their heads were fixed in a stereotaxic apparatus. After cleaning the surface of the skull, according to the Allen Brain Atlas, the distance between the two points of the lambda and bregma was identified. The hNTSCs and hBM-MSCs [1 × 105 cells/10 µl N-Acetyl-L-cysteine(NAC)] were injected into the substantia nigra par compacta. The coordinates were as follows: AP = − 3.2, ML = 0.71 mm and DV = 4.6 mm.

2.7 Behavior test

One week after MPTP IP injection, the motor function of the mice was validated by rotarod and open field tests, using a rotarod machine with falling sensors (MED-Associates Inc., FL, USA). In the rotarod test, ten mice for each group were placed side by side in a rotarod machine. The mice were allowed to remain in the machine for 5 min. Each session was performed after habituation. The acceleration of the rotation and rotation speed were set to 0–15 m/min and 0–50 rpm, respectively. Each rail on the machine was turned off as soon as the mouse fell off the platform. The rotarod test was conducted consecutively for four days. The open field test was conducted using SMART software (version 3.0) and an open field box (Panlab, MA, USA). The open field box was a 42 × 42 × 42 cm polyvinyl chloride box, which was monitored and recorded using a camera connected to the SMART 3.0 software. The camera measured the movements of the mice in the peripheral and central zones. Trajectory tracing, total travel distance, and time spent in the zone were measured to analyze the motor function of the animal.

2.8 Flow cytometry

hNTSCs (1 × 105 cells) were collected in a round bottom tube and resuspended to 100 μL of 5% FBS (Thermo Fisher Scientific) diluted in Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher Scientific). Cells were stained with human CD90-PE conjugated (BD Bioscience, NJ, USA) and CD34 (FITC) at RT for 2 h. The cells were then evaluated using a FACSAria III (BD Biosciences) machine.

2.9 Immunofluorescence staining

DA-Like cells were washed three times with DPBS (Thermo Fisher Scientific) and then fixed in cold methanol on ice for 15 min. After fixation, normal goat serum was used as a blocking buffer, for an hour at RT. Primary antibodies were diluted to 1:100 in blocking buffer and added to the cells. The cells were incubated overnight at 4 °C and then washed with DPBS three times. Secondary antibodies were diluted 1:500 in DPBS and incubated in the buffer for an hour. DAPI (Vector Laboratories) was diluted 1:5000 and incubated with the samples for 5 min at RT. The brain of MPTP-induced PD mouse model was fixed in 4% paraformaldehyde for 2 h and incubated in 30% of sucrose until it is crysectioned. The brain was sectioned 7 μm from the bregma to the end. The brain section slide was selected by referring to mouse brain atlas map and the slide was blocked with blocking buffer for an hour at RT. Primary antibody was diluted to 1:100 in blocking buffer and added to the slide. The slide was incubated overnight at 4 °C and washed with DPBS three times. Secondary antibody was diluted 1:500 in DPBS and incubated with the slide for an hour. DAPI was diluted 1:5000 and incubated for 5 min at RT. The fluorescence intensity of the cells and the slide were measured using a Zeiss LSM 800 Confocal Laser Scanning Microscope (Zeiss, Jena, Germany).

2.10 Protein isolation

Brain proteins were extracted using RIPA buffer (LPS Solution, Daejeon, Korea) with EDTA-free Complete Ultra tablets and an EASYpack Protease Inhibitor Cocktail (Roche, Basel, Switzerland). The tissues were homogenized using a tissue grinder with lysis buffer containing proteinase and phosphatase inhibitors. Lysates were then sonicated for 10 secs in an ice bath sonication machine, followed by centrifuging at 14,000 × g for 20 min at 4 °C. The protein concentrations were measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific).

2.11 Immunoblotting

The proteins (15 μg) used for immunoblotting were mixed with 5 μL of NuPAGE™ LDS Sample Buffer (4x; Thermo Fisher Scientific), 2 μL of dithiothreitol (10x; Sigma-Aldrich, MO, USA), and distilled water, added to a final volume of 20 μL. The protein mixture was heated up to 75 °C for 10 min to denature the proteins. The protein mixture then underwent electrophoresis in a 10% SDS-PAGE gel for 40 min, in Invitrogen™ NuPAGE™ MOPS SDS Running Buffer (20x; Invitrogen). Proteins were separated by SDS-PAGE using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The PVDF membranes were blocked with 5% skim milk for 2 h at RT. The membranes were then washed three times with 0.1% tween-20 diluted in tris-buffered saline (0.1% T-TBS) for 5 min, three times, at RT. The transferred PVDF was then incubated with the primary antibody, either eukaryotic translation initiation factor 2 alpha subunit (EIF2α; Cell signaling technology, MA, USA), phosphorylated EIF2α activating transcription factor 4 (ATF4, Cell signaling technology), NEDD4L (Cell signaling), Yes-associated protein/transcription activator with PDZ binding motif (YAP/TAZ; Cell signaling), phosphorylated YAP/TAZ (Cell signaling), Large neutral amino acids transporter small subunit 1(LAT1; Cell signaling), or phosphorylated LAT1(Cell signaling). All the antibodies were diluted to 1:1000 with 0.1% sodium azide (Sigma-Aldrich) and incubated with the membranes at 4 °C overnight. The PVDF membranes were then washed with T-TBS for 10 min, three times, and then incubated with a goat anti-rabbit IgG antibody labeled with peroxidase (Vector Laboratory, Inc., CA, USA), diluted 1:5000, for 10 min at RT. The PVDF membranes were then analyzed using an LAS-3000 (FUJI PHOTO FILM CO., LTD, Tokyo, Japan).

2.12 RNA isolation and cDNA synthesis

Midbrain tissue was homogenized using a tissue grinder and 300 μL of TRIzol® reagent (Invitrogen). Tissue lysates were added to 60 μL of chloroform (DAEJUNG CHEMICALS & METALS Co., Ltd, Gyeonggi-do, Korea) and vortexed for 3 secs, to mix. The lysates were then centrifuged at 12,000 × g for 15 min at 4 °C. Supernatants were transferred to new 1.7 mL tubes, incubated in 0.5 mL of isopropanol (Sigma-Aldrich), and centrifuged at 12,000 × g for 15 min at 4 °C. The resulting RNA pellet was washed with 70% cold ethanol and centrifuged at 12,000 × g for 10 min at 4 °C. The RNA pellet was then dissolved in 30–50 μL of diethyl pyrocarbonate-treated water, according to the size of the pellet. The RNA was then incubated for 10 min at 65 °C. cDNA was synthesized from 1 µg of total RNA using the iScript™ cDNA Synthesis Kit (Bio-RAD).

2.13 Poly-chain reaction (PCR) assay

The mRNA was isolated from the midbrain of each group using a manual protocol. cDNA was synthesized using the iScript™ cDNA Synthesis Kit (Bio-RAD). Then, 1 µg of cDNA was used for gene expression level analysis. CFX386 touch (Bio-RAD) was used to conduct qRT-PCR. The reaction efficiency and number of cycles were determined using innate software.

2.14 Statistics

The Kruskal–Wallis and Mann–Whitney U post-hoc tests were used to compare the results across groups. SPSS version 22 (IBM Corporation, NY, USA) was used to determine statistically significant differences between groups. The results are expressed as mean ± standard deviation (SD). Differences were considered significant at *p < 0.05 versus Control; **p < 0.01 versus Control; ***p < 0.001 versus control; #p < 0.05 versus MPTP-saline and MPP+-saline; ##p < 0.01 versus MPTP-saline and MPP+-saline; ###p < 0.001 versus MPTP-saline and MPP+-saline.