Pentraxin 3 secreted by human adipose‐derived stem cells promotes dopaminergic neuron repair in Parkinson's disease via the inhibition of apoptosis

The FASEB JournalVolume 35, Issue 7 e21748 RESEARCH ARTICLEOpen Access Pentraxin 3 secreted by human adipose-derived stem cells promotes dopaminergic neuron repair in Parkinson's disease via the inhibition of apoptosis Changlin Lian, Changlin Lian orcid.org/0000-0001-8901-2160 Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorQiongzhen Huang, Qiongzhen Huang Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorXiangyang Zhong, Xiangyang Zhong Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorZhenyan He, Zhenyan He Department of Neurosurgery, The Affiliated Tumor Hospital of Zhengzhou University, Zhengzhou, ChinaSearch for more papers by this authorBoyang Liu, Boyang Liu Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorHuijun Zeng, Huijun Zeng Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorNingbo Xu, Ningbo Xu Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorZhao Yang, Zhao Yang Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorChenxin Liao, Chenxin Liao Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorZhao Fu, Zhao Fu Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorHongbo Guo, Corresponding Author Hongbo Guo guohongbo911@126.com Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, China Correspondence Hongbo Guo, Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, 253 Gongye Middle Avenue, Haizhu District, Guangzhou, Guangdong 510280, China. Email: guohongbo911@126.comSearch for more papers by this author Changlin Lian, Changlin Lian orcid.org/0000-0001-8901-2160 Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorQiongzhen Huang, Qiongzhen Huang Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorXiangyang Zhong, Xiangyang Zhong Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorZhenyan He, Zhenyan He Department of Neurosurgery, The Affiliated Tumor Hospital of Zhengzhou University, Zhengzhou, ChinaSearch for more papers by this authorBoyang Liu, Boyang Liu Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorHuijun Zeng, Huijun Zeng Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorNingbo Xu, Ningbo Xu Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorZhao Yang, Zhao Yang Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorChenxin Liao, Chenxin Liao Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorZhao Fu, Zhao Fu Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorHongbo Guo, Corresponding Author Hongbo Guo guohongbo911@126.com Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, China Correspondence Hongbo Guo, Neurosurgery Center, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, 253 Gongye Middle Avenue, Haizhu District, Guangzhou, Guangdong 510280, China. Email: guohongbo911@126.comSearch for more papers by this author First published: 21 June 2021 https://doi.org/10.1096/fj.202100408RRCitations: 1 Changlin Lian and Qiongzhen Huang contributed equally to the experimental work. Funding information This study was supported by the Natural Science Foundation of Guangdong Province-Major Basic Cultivation Project (Grant/Award Numbers: 2017A030308001), the National Natural Science Foundation of China, (Grant/Award Numbers: 81874079, 81672477), Guangdong Province Science and Technology Innovation Strategy Special Fund (2018A030310422), Guangdong Medical Science and Technology Research Fund (A2018542), and Key development and promotion project of Henan province (No. 212102310662) AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Although adipose-derived human mesenchymal stem cell (hADSC) transplantation has recently emerged as a promising therapeutic modality for Parkinson's disease (PD), its underlying mechanism of action has not been fully elucidated. This study evaluated the therapeutic effects of stereotaxic injection of hADSCs in the striatum of the 6-OHDA-induced mouse model. Furthermore, an in vitro PD model was constructed using tissue-organized brain slices. The therapeutic effect was also evaluated using a co-culture of the hADSCs and 6-OHDA-treated brain slice. The analysis of hADSC exocrine proteins using RNA-sequencing, human protein cytokine arrays, and label-free quantitative proteomics identified key extracellular factors in the hADSC secretion environment. The degeneration and apoptosis of the dopaminergic neurons were measured in the PD samples in vivo and in vitro, and the beneficial effects were evaluated using quantitative reverse transcription-polymerase chain reaction, western blotting, Fluoro-Jade C, TUNEL assay, and immunofluorescence analysis. This study found that hADSCs protected the dopaminergic neurons in the in vivo and vitro models. We identified Pentraxin 3 (PTX3) as a key extracellular factor in the hADSC secretion environment. Moreover, we found that human recombinant PTX3 (rhPTX3) treatment could rescue the pathophysiological behavior of the PD mice in vivo, prevent dopaminergic neuronal death, and increase neuronal terminals in the ventral tegmental area + substantia nigra pars compacta and striatum in the PD brain slices in vitro. Furthermore, testing of the pro-apoptotic markers in the PD mouse brain following rhPTX3 treatment revealed that rhPTX3 can prevent apoptosis and degeneration of the dopaminergic neurons. This study discovered that PTX3, a hADSC-secreted protein, potentially protected the dopaminergic neurons against apoptosis and degeneration during PD progression and improved motor performance in PD mice, indicating the possible mechanism of action of hADSC replacement therapy for PD. Thus, our study discovered potential translational implications for the development of PTX3-based therapeutics for PD. Abbreviations 6-OHDA 6-hydroxydopamine 6-OHDA group 6-OHDA-induced brain slices APO rotation apomorphine rotation co-hADSCs group hADSC co-cultured with 6-OHDA-induced brain slices DA dopamine Dil CellTracker CM-Dil hADSC group single hADSCs hADSCs human adipose mesenchymal stem cells LDH lactate dehydrogenase si-PTX3 knocking down PTX3 SNc substantia nigra pars compacta SNr substantia nigra pars reticulate STR striatum TH(+) tyrosine hydroxylase (positive) VTA ventral tegmental area W weeks 1 INTRODUCTION Parkinson's disease (PD) is the second most common neurodegenerative disorder that affects approximately 1% of the population over the age of 60 years.1 It is primarily characterized by a massive loss of dopaminergic neurons in the ventral tegmental area + substantia nigra pars compacta (VTA+SNc) and the consequent deficit in dopamine (DA) release in the striatum (STR).2 Treatment with L-dopa or DA agonists can effect a slight amelioration of the symptoms; however, these symptomatic treatments are accompanied by considerable side effects and their efficacy diminishes with time.2-4 In recent years, mesenchymal stem cell (MSC) transplantation has emerged as a potential therapy for PD.5, 6 Human adipose-derived mesenchymal stem cells (hADSCs) have become the mainstay of regeneration research due to the convenience of handling and abundant sources.7-9 They also possess immune properties and the ability to be differentiated into fat, osteoblasts, and neural-like cells.10, 11 Studies have reported that MSCs can induce local repair mechanisms by releasing paracrine factors and eliciting changes in the microenvironment.12 Some previous studies had found that transplanting undifferentiated MSCs into a 6-hydroxydopamine (6-OHDA)-induced PD mouse model could restore the dopaminergic pathway.13-15 It was recently reported that a hADSC-conditioned medium could restore H2O2-induced toxic SH-SY5Yd cells to normal axonal morphology.16 However, whether the protective effect of hADSCs on dopaminergic neurons entails the secretion of cytokines still requires further exploration. Pentraxin 3 (PTX3) is a typical acute-phase protein belonging to the pentraxin family.17 Studies have reported that PTX3 plays an important role in acute inflammation and can also inhibit cell apoptosis.18, 19 Recent studies reported that PTX3 secreted by bone marrow MSCs could promote wound healing through fibrin remodeling.20 At the same time, other studies showed that PTX3 secreted by human umbilical cord blood MSCs promoted functional recovery, vascular remodeling, and nerve regeneration in stroke rat models.21-23 Both clinical and laboratory studies have found an increased expression of PTX3 in some chronic central nervous system diseases, such as PD and Alzheimer's disease.24, 25 Current evidence indicates that PTX3 is involved in a variety of pathological processes in central nervous system diseases, and its role in clinical practice continues to garner considerable attention, even though its mechanism of action remains to be fully understood. 6-OHDA is a hydroxylated derivative of the neurotransmitter DA, which is injected into the substantia nigra area, STR area, or medial forebrain tract area of the animal to create animal models of PD.26 6-OHDA causes death and degeneration of the dopaminergic neurons through oxidative stress, mitochondrial damage, induction of cell apoptosis, and mediation of inflammation.27 Therefore, 6-OHDA damages the substantia nigra-striatal dopaminergic system to produce symptoms similar to PD, providing the necessary experimental animal model for studying the pathogenesis of PD, drug efficacy evaluation, cell transplantation therapy, gene therapy, etc.26 Apoptosis is one of the processes involved in the pathogenesis of PD.28 Numerous in vivo and in vitro experiments have confirmed the activation of caspase during the apoptosis process induced by 6-OHDA.27 In vitro experiments confirmed that 6-OHDA can be selectively absorbed by the dopaminergic neurons, and activate caspase to cause a cascade reaction and promote cell apoptosis.29, 30 The chief signal transduction system in the process of apoptosis is currently believed to include the membrane receptor and mitochondrial pathways.31 Both pathways activate the caspase cascade and ultimately lead to cell death. Studies have shown that the Fas-associated death domain protein (FADD) is involved in the pathogenesis of dopaminergic neuron apoptosis in PD, and it is speculated that the death receptor signal transduction pathway mediated by FADD is one of the pathomechanisms of PD.32 The current study not only confirmed the protective effect conferred by hADSCs in PD using mice models, but also verified the neuroprotective effects of hADSCs through the in vitro model (comprised of co-culture of the hADSCs and PD organotypic brain slices). Subsequently, we found that PTX3 secreted by hADSCs plays an important role in the protection of dopaminergic neurons using RNA-sequencing (RNA-seq), human protein cytokine arrays, and label-free quantitative proteomics. Moreover, we established that the topical application of PTX3 protected the dopaminergic neurons via the inhibition of the apoptosis pathway. 2 MATERIALS AND METHODS 2.1 hADSC isolation The hADSCs were derived from 5 mL of sterile adipose tissue obtained from the fat of the inner left thigh, which was provided by the Department of Plastic Surgery, Zhujiang Hospital, Southern Medical University. Type I collagenase digestion was used to extract the hADSCs, and the cell layer was resuspended in Dulbecco's Modified Eagle Medium (DMEM)/Nutrient Mixture F12 cell culture medium containing 10% fetal bovine serum (FBS), which was inoculated into a 6-cm culture dish and placed in an incubator for culture. Adipose stem cells (P2, P4, and P8) were observed under the microscope (DMC4500, Leica) after 48 hours. Adipogenic and osteogenic differentiation was induced to identify the differentiation ability of the hADSCs. These experiments were performed as described by a previous study33 (See Figure S1 for details). 2.2 Induction of neural differentiation We used this cell culture system (DMEM/F12 + 2% B27 + 20 ng/mL epidermal growth factor +20 ng/mL basic fibroblast growth factor +100 µ/mL penicillin/streptomycin +2.5 mmol/L NaHCO3) to induce neural differentiation of the hADSCs at a pH of 6.5-7.0. The hADSCs were mounted on slides and flattened with coverslips for 24 hours, followed by washing with phosphate-buffered saline (PBS) two times, and fixation with 4% paraformaldehyde (about 2 mL/well) for 30 minutes, and washing three times with PBS. We ruptured the membrane with 0.25% Triton X-100 (approximately 2 mL/well) for 20 minutes, followed by washing three times with PBS. Blocking was performed with 5% bovine serum albumin (BSA) for 30 minutes for the reduction of nonspecific binding, followed by treatment with (150 µL/well) the primary antibody (1:100 nestin and SOX9, Abcam) diluted in PBS containing 1% BSA and incubation for 2 hours at room temperature, and washing three times with PBS. We diluted the PBS and 1% BSA solution with the anti-fluorescence secondary antibody (150 µL/well) at room temperature for 2 hours, followed by washing three times. 4,6-diamidino-2-phenylindole (DAPI, 1:2000, final concentration 1 µg/mL) was used for the nuclear staining of the cells at 37°C for 3-4 minutes, followed by washing two times with PBS. The neurospheres were observed under a fluorescence or confocal microscope (See Figure S1 for details). 2.3 Flow cytometry After attaining a confluence of 80%-90%, we rinsed the specimen once with PBS, performed cell digestion, created a cell suspension, performed centrifugation at 800 rpm for 5 minutes, and discarded the resulting supernatant. We washed the sample two times with PBS, added 20 µL of primary antibodies CD29-APC(BD), CD44-PE(BD), CD45-FITC(BD), and incubated the sample on ice for 20 minutes in the dark. Finally, we washed it with an appropriate amount of PBS (200 µL microplate, 1 mL test tube), subjected it to centrifugation (1000 rpm, 5 minutes, 4 degrees), and aspirated the supernatant. We added an appropriate amount of PBS to the suspension (500 µL/tube) in the flow cytometer and analyzed the data (See Figure S1 for details). 2.4 Animals All experimental protocols were conducted in accordance with the guidelines issued by the committee on animal research of Zhujiang Hospital, Southern Medical University, and were approved by the institutional ethics committee. C57BL/6 male mice were housed in cages under 12-hour/12-hour light/dark cycles and acclimated to the experimental environment for 1 week before modeling. All procedures were reviewed and approved by the institutional animal care committee. All efforts were made to minimize animal suffering in this study. 2.5 6-Hydroxydopamine hydrobromide lesion induction The PD model was created by inducing the formation of a 6-OHDA lesion.26 Adequate anesthesia was administered and the animals were secured onto a stereotactic frame (RWD, 68001, China). A solution of 6-OHDA (Sigma-Aldrich, 3 µL, 5 mg/mL in sterile saline containing 0.02% ascorbic acid) was injected into the right SNc using a microliter syringe at an infusion rate of 0.5 µL/minute using a Hamilton syringe and back pump (RWD) for a total dose of 15 µg at the following coordinates with the bregma as the reference point: anteroposterior (AP), −3 mm; mediolateral (ML), +1.3 mm; and dorsoventral (DV), −4.7 mm. The needle was withdrawn slowly after a waiting period of 5 minutes. 2.6 Labeling hADSCs with CM-Dil The hADSCs (1 × 105/well) were incubated into 6-well plates for 24 hours, and the cell membrane was stained with Dil (MedChemExpress, USA) for 10 minutes and the hADSCs were transferred to another medium. 2.7 Transplantation of hADSCs The hADSCs (suspended in PBS) were implanted in the STR at the stereotaxic coordinates (AP, +0.9 mm, ML, +2.2 mm, and DV, −2.8 mm). A total of 1 × 105 cells were injected at each point at a volume of 5 µL with a 5 µL Hamilton syringe (1.5 µL/min). 2.8 Open field test The open field activity test was conducted to assess the spontaneous locomotor activity of the mice using the opening experiment video analysis system. All mice were acclimated to the test chamber (100 cm × 100 cm × 40 cm) for 1 hour before the test. Thereafter, they were placed at the center of the chamber and allowed to freely explore the area for 10 minutes. The apparatus was cleaned with ethanol after each animal was exposed to the box. The total running distance, average speed, and resting time of the mice were recorded. 2.9 Rotation behavior analysis Apomorphine hydrochloride (APO; Sigma-Aldrich, Taufkirchen, Germany) was dissolved in sterile saline containing 0.02% ascorbic acid and subcutaneously administered at a dose of 0.5 mg/kg of body weight. The mice were injected with APO (0.5 mg/kg) subcutaneously, placed in square chamber (40 cm2), and the number of contralateral turns within a 30-min period was recorded. Mice with more than seven contralateral turns per minute were used as valid animal models of PD pathology. 2.10 Immunohistochemical staining The animals were euthanized using tribromoethanol (Sigma) and transcardially perfused with 4% paraformaldehyde (YONG JIN BIOTECH) in 0.1 M PBS. The animals' brains were extracted, post-fixed, paraffinized, and sectioned at a thickness of 4 µm (VTA+SNc and STR regions). Six coronal sections were obtained per mouse and were treated with the SP kit (ZSGB-BIO, SP-9001), in accordance with the manufacturer's instructions. The sections were subsequently incubated with the primary antibody anti-tyrosine hydroxylase (TH, 1:500, Abcam-EP1532Y). A bright-field microscope (model no. DM2500; Leica) and ImageJ software (National Institutes of Health) were used to measure the density of the TH+ neurons in the VTA+SNc and STR. 2.11 Immunofluorescence The mice brains were removed, post-fixed in 4% paraformaldehyde for 24 hours at 4°C, and cryoprotected in 30% sucrose solution. Brain sections (6 µm and 100 µm thickness) were prepared, washed two times in PBS, and incubated in 0.2% Triton X-100 for 60 minutes at room temperature. The samples were blocked with 0.5% BSA (Sigma-Aldrich, A7906) for 60 minutes. Subsequently, the sections were rinsed two times with modified PBS and incubated overnight at 4°C with the primary antibody, that is, rabbit anti-TH antibodies (1:200, Millipore-AB152, USA). The sections were rinsed with PBS and treated with a secondary antibody conjugated to a fluorescent dye. The secondary antibodies included donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody (Alexa Fluor 488, Thermo Fisher Scientific, catalog # A-21206, RRID AB_2535792), followed by imaging under a fluorescence microscope (Nikon, Ti2-E). 2.12 Organotypic brain slice cultures The organotypic slice cultures were prepared according to the membrane interface method.34 The mice brains were removed (3-4 weeks). Sagittal nigrostriatal brain slices were obtained in the sagittal plane35 at a thickness of 350 µm thick using a McIlwain tissue chopper (Microslicer DTK-1000N, Japan, Dosaka Company). Subsequently, four slices were plated onto four separate 0.4-µm porous polytetrafluoroethylene (PTFE) membrane inserts (TCS000012, JET Biofil), placed in 6-well plates filled with 1 mL of slice culture media containing 50% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered Eagle's Minimum Essential Medium (MEM) (Gibco), 25% heat-inactivated horse serum (Gibco), 25% Hank's Balanced Salt Solution (HBSS) (Gibco), and 1 mM L-glutamine (Gibco) at a pH of 7.4 and maintained in a cell culture incubator at 37°C with 5% CO2. The media were changed 1 day after preparation and subsequently every 3-4 days. 2.13 PD models-organotypic brain slices co-culture with hADSCs We cultured the organotypic brain slices with 6-OHDA (600 µM; Figure S3) for 1 hour36 and removed the 6-OHDA-containing medium and added 1 mL of serum-free medium [75% HEPES-buffered MEM (Gibco), 25% HBSS (Gibco), and 1 mM L-glutamine (Gibco)]. At the same time, 1 × 105 hADSCs were spread over a 6-well plate, and the membrane insert carrying the 6-OHDA-induced brain slices was placed in a 6-well plate for indirect co-cultivation with hADSCs for 4 days. The serum-free medium was changed every 2 days. 2.14 Lactate dehydrogenase assay Lactate dehydrogenase (LDH) activity in the cell culture medium was determined using a commercial kit, according to the Nanjing Jiancheng Bioengineering Institute's instructions. Briefly, the cell culture medium was collected and treated with the LDH assay. The absorbance was measured with a microplate reader (BioTek, ELX808, USA) at 450 nm. The cell death ratio was calculated using the following formula according to the manufacturer's instructions. 2.15 RNA-seq analysis Oligo(dT)-attached magnetic beads were used to purify the mRNA. The purified mRNA was fragmented into small pieces using a fragment buffer at the appropriate temperature. Subsequently, first-strand cDNA was generated using random hexamer-primed reverse transcription, followed by second-strand cDNA synthesis. Thereafter, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from the previous step were amplified using PCR, the products were purified using AMPure XP Beads, and dissolved in EB solution. The product was validated on the Agilent Technologies 2100 Bioanalyzer for quality control. The double-stranded PCR products obtained from the previous step were subjected to heat denaturation and circularized using the splint oligo sequence to acquire the final library. The single-stranded circular DNA was formatted as the final library. The final library was amplified with phi29 to generate a DNA nanoball (DNB), which had more than 300 copies of each molecule. The DNBs were loaded into the pa