Pre-motor Mesenchymal stromal Cell Dysfunction Drives Immune Dysregulation in Parkinson’s Disease

Pre-motor Mesenchymal stromal Cell Dysfunction Drives Immune Dysregulation in Parkinson’s Disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Pre-motor Mesenchymal stromal Cell Dysfunction Drives Immune Dysregulation in Parkinson’s Disease Rituparna Ghanty, Kallolika Mondal, Nitish Kamble, Ravi Yadav, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7957744/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Parkinson's disease (PD) is characterized by chronic neuroinflammation and peripheral immune dysfunction, yet the mechanisms underlying systemic-immunomodulatory failure remain unclear. Here we demonstrate that mesenchymal stromal cell (MSC) dysfunction represents an early pathological feature of PD, occurring during the pre-motor phase. Using MPTP-induced rat model, we show that bone-marrow MSC impairment emerges at week 1 post-treatment, coinciding with dopaminergic neurodegeneration but preceding motor-symptoms, with reduced proliferation, impaired migration, elevated oxidative stress, and diminished immunomodulatory capacity. Patient-derived induced pluripotent stem cell-derived MSCs (iMSCs) from sporadic-PD patients recapitulated these dysfunctions and exhibited severely compromised ability to suppress peripheral-blood mononuclear-cell proliferation and PD patient PBMCs in immunomodulation assays. Transplantation studies in MPTP-induced rats revealed that healthy-control iMSCs provided superior neuroprotection, reduced inflammation, promoted neurogenesis, and improved motor-function versus PD-iMSCs. These findings identify MSC immunomodulatory-dysfunction as an upstream contributor to PD pathogenesis and provide rationale for allogeneic over autologous MSC therapeutic strategies in PD treatment. Biological sciences/Neuroscience/Cellular neuroscience Health sciences/Diseases/Neurological disorders/Parkinson's disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Parkinson's disease (PD) represents the second-most prevalent age-related progressive neurodegenerative disorder, affecting not only the central nervous system but also the peripheral nervous system, gastrointestinal tract, and adaptive immune system, justifying its classification as a multisystem disorder (1,2). The classical motor symptoms of PD primarily result from the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) in the midbrain. This pathological process begins decades before clinical manifestation, with approximately 70% of these neurons already lost by the time motor symptoms appear (3). It is now understood that central to PD pathogenesis is the establishment of a self-perpetuating cycle of chronic neuroinflammation. While the etiology of this remains multifactorial, involving genetic predisposition, environmental toxins, and aging (4), transient initiation factors such as chemical insults, infections, particulate matter, and pesticides are known to trigger sustained microglial activation. This results in the increased production of chemokines, cytokines, reactive oxygen/nitrogen species and adhesion molecules, further promoting dopaminergic neuronal death (5,6). While the causal relationship between neuroinflammation and neurodegeneration remains putative, extensive preclinical and clinical evidence demonstrates that chronic inflammation is integral to disease pathogenesis in both sporadic and familial forms of PD. The inflammatory milieu in PD extends beyond the central nervous system to encompass systemic immune dysfunction. Neurodegeneration is accompanied by microglial activation and T lymphocyte infiltration into the SNpc (7–9). Elevated levels of pro-inflammatory cytokines including TGF-β1, IL-6 and IL-1β have been consistently demonstrated in both brain tissue and cerebrospinal fluid of PD patients, supporting a neurotoxic environment involving inflammasome activation across microglia, neurons, astroglia, CNS-associated macrophages, and infiltrating peripheral immune cells (10). Peripheral immune alterations in PD patients are equally complex and heterogeneous. Multiple studies have documented increased neutrophil-to-lymphocyte ratios (NLR), elevated levels of effector and inflammatory T cells, altered monocyte protein expression profiles, and B cell populations skewed toward pro-inflammatory phenotypes with reduced regulatory subsets (11–16). However, these changes exhibit considerable individual variation; for instance, the elevated NLR observed has been attributed to increased neutrophil counts or reduced lymphocyte levels, depending on the study (17–20). Moreover, specific PD clinical subtypes (e.g., akinetic-rigid vs. tremor-dominant) and stages (e.g., PD with mild cognitive impairment) display distinct immune signatures (20–23). Studies also report divergent findings on monocyte subtypes: while some show no difference (14,23–27), others indicate an increase in pro-inflammatory classical monocytes in early PD (28–30). T cell alterations, particularly reduced CD4 + and CD8 + counts, have also been observed, particularly in late-stage disease (31–33). This heterogeneity in immune signatures suggests that therapeutic strategies targeting individual immune cell populations may prove ineffective as a therapeutic strategy. Instead, the observed systemic immunomodulatory failure directs the attention toward upstream regulatory mechanisms. Bone marrow mesenchymal stromal cells (BMMSCs) represent ideal candidates for further investigation, given their master regulatory role in immune homeostasis. Through their extensive secretory profile, BMMSCs modulate both innate and adaptive immunity by controlling cytotoxic and Th17 T cell generation, inhibiting dendritic cell maturation, promoting M1-to-M2 macrophage polarization, and inducing protective regulatory T cells and dendritic cells (34–40). Beyond immunomodulation, MSCs also provide cellular protection by secreting growth factors, transferring mitochondria, and regulating reactive oxygen species (41,42). MSCs possess unique homing properties, allowing them to localize to sites of inflammation or injury—even crossing the blood-brain barrier (BBB) under pathological conditions (43–46). At such sites, MSCs modulate the inflammatory environment, reduce oxidative stress, and enhance cell survival via paracrine signalling, mitochondrial transfer, and trophic factor secretion. This multifactorial regulatory capacity uniquely positions BMMSCs as potential endogenous modulators of the chronic inflammatory cycle (47), characteristic of PD pathogenesis. Some clinical observations further indirectly support MSC involvement in PD pathology. Osteoporosis and osteopenia affect up to 91% of women and 61% of men with PD (48), often manifesting in early disease stages. As BMMSCs serve as progenitors for osteocytes and regulate the balance between adipocyte and osteocyte formation, these bone abnormalities suggest underlying MSC dysfunction (49). Additionally, impaired nerve conduction velocity and pain symptoms in PD patients may relate to MSC-dependent regulation of Schwann cell function and myelination. The temporal dynamics of peripheral immune alterations in PD provide additional insight into disease progression. NLR increases start occurring years before PD diagnosis and correlate with disease duration and severity, while intermediate monocyte elevation and T cell deviations are associated with disease advancement. These findings suggest that immune dysfunction may precede motor symptom onset by years, highlighting the importance of characterizing the timeline of potential MSC impairment. Paradoxically, past clinical trials of autologous MSC transplantation in PD have yielded disappointing results, with some studies reporting worsening of symptoms (50,51). This therapeutic failure further raises critical questions about the functional integrity of endogenous MSCs in PD patients. Precedents exist for MSC impairment in other chronic inflammatory conditions: functional deficits have been documented in MSCs from patients with rheumatoid arthritis and systemic lupus erythematosus, including diminished proliferative, angiogenic, and differentiation capacities (52–54). Similarly, MSCs from progressive supranuclear palsy patients have been shown to exhibit significant mitochondrial dysfunction and reduced differentiation potential compared to healthy controls (55). Current knowledge gaps limit our understanding of MSC involvement in PD pathogenesis. While chronic neuroinflammation is well-established and peripheral immune dysfunction documented, the functional status of endogenous MSCs throughout disease progression remains unmapped. The temporal relationship between MSC impairment and symptom onset is unknown, as is the extent to which MSC immunomodulatory capacity is compromised in PD patients. These uncertainties limit our understanding of endogenous BMMSC dysfunction in PD and impede the development of more effective cell-based therapeutic strategies. Tracking the early phase of PD in human patients presents significant challenges, as clinical diagnosis typically occurs only after motor symptoms have already appeared. The invasive methods required for BMMSC isolation from living subjects present additional barriers to research. However, the advent of patient-derived induced pluripotent stem cells (iPSCs) offers a powerful alternative approach. iPSCs derived from both familial and idiopathic PD patients recapitulate disease-relevant phenotypes in neurons, astrocytes, microglia, and macrophages (56–60). Particularly noteworthy is that differentiated dopaminergic neurons from idiopathic PD patient-derived iPSCs retain age-associated dysfunctions such as impaired chaperone-mediated autophagy (61–64). While iPSC-derived MSCs (iMSCs) have been successfully generated in other research contexts, their immunomodulatory capacity specifically in PD remains entirely unexplored. This study therefore aims to comprehensively characterize the temporal and functional impairment of bone marrow mesenchymal stromal cells in PD through three complementary approaches: (1) assessment of functional integrity of endogenous BMMSCs with regard to neuroinflammation and peripheral inflammation using chronic PD rat models, (2) determination of the precise timing of MSC impairment onset relative to motor dysfunction development, and (3) evaluation of immunomodulatory capacity of hiPSC-derived MSCs (iMSCs) from PD patients versus healthy controls. These investigations will help establish a mechanistic basis for autologous BMMSC dysfunction and inform the development of improved cell-based therapeutic strategies in PD. Methodology Ethics Approval and study design. The protocols and procedures were ethically reviewed and approved by the Institutional Animal Ethics Committee (IAEC) for the use of male Wistar rats (AEC/77/506/B.P/2023-02), the Institutional Biosafety Committee (IBSC) for employing MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) (NIMHANS-IBSC/25 th Nov.2022/01) as an inducing agent to establish a sporadic Parkinson's disease model in rats, and the Institutional Committee of Stem Cell Research (IC-SCR) for utilizing induced pluripotent stem cell (iPSC) lines (SEC/07/035/BP) derived from healthy controls and sporadic PD patients. These approvals were obtained at National Institute of Mental Health and Neurosciences (NIMHANS), India. The sample size for each experiment was determined according to established experimental standards. For all in vivo animal studies and rat primary cultures, a minimum of 5–7 biological replicates (n ≥ 5–7) was maintained per group. For iPSC cultures, six independent clones from individual lines were used to ensure at least 4-5 biological replicates (n ≥ 4-5). These included two sporadic PD lines [NIMHi002-A (referred to as PD02) and NIMHi003-A (referred to as PD03)] and two age- and sex-matched healthy control lines [NIMHAi006-A (referred to as HC03) and NIMHAi005-A (referred to as HC02)], all of which have been previously reported in (65–68). For phase-contrast and confocal imaging, 2–3 independent fields were analysed per replicate, with images acquired 100 µm apart to avoid resampling. Animal model: This study was conducted in compliance with the ARRIVE 2.0 guidelines. Male Albino Wistar rats, 2.5 months old and weighing 280–300 g, were used for all in vivo experiments. The animals were bred, maintained, and obtained from the Central Animal Research Facility (CARF), NIMHANS, in accordance with the Institutional Animal Ethics Committee (IAEC), NIMHANS, Bengaluru, and the regulations of the Committee for the Control and Supervision of Experiments on Animals (CCSEA), India. Rats were housed under controlled laboratory conditions (22 ± 1 °C, 12-h light/dark cycle, 55–60% humidity) with free access to standard laboratory rat chow and water. Age- and weight-matched rats received a single bilateral intranasal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine at 0.1 mg per nostril, while control animals received an equivalent volume of saline (69). The animals were divided into the following groups: 1. Control (Bi-lateral intra-nasal saline administration) (3.5 Months old) 2. 1 st week post MPTP treatment (Wk1 PMT) 3. 2 nd week post MPTP treatment (Wk2 PMT) 4. 3 rd week post MPTP treatment (Wk3 PMT) The number of animals used for each experiment specified in the corresponding figure legends as biological replicates. No experimental units were excluded. Rats were anesthetized with ketamine and xylazine (80:5 mg/kg, intraperitoneally) for experimental procedures and euthanized by isoflurane overdose followed by decapitation for terminal sample collection. Tissues collected included bone marrow for mesenchymal stromal cell isolation, blood serum, and brain samples for analyses such as midbrain neurotransmitter quantification, TNF-α measurement, and immunohistochemistry. Blood for serum isolation was obtained via cardiac puncture, while brain samples for immunohistochemical analysis were collected following transcardial perfusion of anesthetized rats. Behavioural assessments were performed to determine the onset of non-motor and motor symptoms. All animal experiments were conducted by multiple investigators. Random numbers were generated by the first investigator (ID), who was the only one aware of group allocations. Experimental procedures and data analyses were performed by RG and KM. All protocols were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of NIMHANS, Bengaluru, India. Behavioural studies: Olfactory Discrimination: The olfactory discrimination ability of rats was assessed following the method described in (70). Briefly, each rat was placed in a two-compartment cage (30 × 30 × 20 cm) connected by an open doorway, allowing free movement between an unfamiliar compartment containing fresh husk and a familiar compartment with the husk collected from the rat’s home cage over the preceding 48 hours. Each trial lasted 5 minutes. The time spent in the familiar compartment (in minutes) was recorded and presented as mean ± SD for n = 7. Nerve Conduction Velocity: Nerve conduction velocity (NCV) was measured non-invasively as previously described (71). Recordings were obtained and analyzed using the iWorx data acquisition system and LabScribe software (iWorx Systems, Inc., USA). Rats were anesthetized with ketamine: xylazine (80 mg/kg:5 mg/kg, i.p.) and placed on a heated platform to maintain body temperature. The sciatic nerve was stimulated at the sciatic notch with a supramaximal stimulus of 8 V at 20 Hz, and recordings were made from the first interosseous muscle of the hind paw. Latency was determined as the time from the stimulus artifact to the onset of the negative M-wave deflection. NCV was calculated using the formula: NCV (mm/ms) = distance (mm) / proximal latency (t1) – distal latency (t2). where the distance represents the length between the stimulation site and the recording electrodes. Values were expressed as mean ± SD for n = 7. Rotarod: For this study, each rat underwent training with three 30-minute trials per day at a speed of 15 rpm for two consecutive days, followed by 25 rpm on the third day for acclimatization. On the fourth day, during the test session, rats were placed on the rotarod (Rotamex-5 system, Columbus Instruments), and their performance time was recorded for 15 mins (900 sec) (72). Rotarod performance time (s) was expressed as mean ± SD for n = 6-7. IR actimeter: The digital IR actimeter was used to test the locomotor activity of PD rats in which a continuous beam of light falls on photoelectric cells. The apparatus comprises of a frame provided with 16 IR source on X axis and 16 IR source on Y axis creating a 16x16 grid. The instrument control panel displays the number of beam brakes by the animal on all axis and total of all in the actimeter. Any interruption in the continuity of light by the animal was recorded on a digital counter in the form of counts which corresponds to the locomotor activity. The animals from each group were individually placed in the apparatus and allowed to move freely for 5 mins while the beam breaks were recorded automatically and report generated digitally. Measurements were plotted as mean ± SD for n=7. Rat Bone marrow mesenchymal stromal cell isolation and culture: The BMMSC isolation protocol was adapted from our previously published report (71) with slight modifications. Rats from each experimental group were euthanized and hind limb bones were dissected and washed in 1X PBS with 1% anti-anti. To collect bone marrow, a femur and a tibia were placed knee-end down into a 2 ml centrifuge tube with a small hole at the bottom. This 2 ml tube was then nested inside a 15 ml centrifuge tube. The setup was centrifuged at 4000 rpm for 10 min. Upon centrifugation, the 2 ml tube holding the bones was removed with the help of sterile forceps. The pellet was resuspended in equal volume of 1x Phosphate Buffered Saline (PBS), layered onto HiSep™ LSM 1084 in the ratio 1:1, and centrifuged at 500 g for 20 min in brake-free setting. The resultant buffy coat of mononuclear cells was collected, washed once in complete MSC media consisting of Knockout Dulbecco's Modified Eagle's Medium (KO-DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1% Anti-Anti and 1% GlutamaX. After wash cells were resuspend and plated in a T75 culture flask containing same MSC media at 37 ◦C with 5% CO2 in a humidified incubator. After 48h, non-adherent cells were removed and the adherent cells were cultured till P1 with subsequent media changes on every 72h. BM-MSCs were identified by their plastic-adherence property and spindle-shaped morphology using a phase contrast microscope (Olympus CKX41) and characterized by CD73 and CD90 marker expression by immunophenotyping and immunostaining. iPSC culture and differentiation into Mesenchymal Stromal Cells and Dopaminergic neurons: For this study, six clones of previously reported iPSC lines were utilized. The iPSCs were cultured on mitomycin-treated mouse embryonic fibroblast (iMEF) feeder layer in complete iPSC media. The media consisted of 20% KnockOut Serum Replacement (KOSR), 1% Penicillin-Streptomycin (Pen/Strep), 1% GlutaMAX, 1% Non-Essential Amino Acids (NEAA), 0.2% β-ME, and DMEM/F12 as the basal medium supplemented with 10 ng/mL human FGF2 (hFGF2). The culture media was changed daily. For iMSC (iPSC derived MSC) differentiation, iPSCs were used between P10 to P15. iMSCs were differentiated by specific factors in a single step differentiation method followed by selection and maintenance. On day -1 undifferentiated iPSC colonies were manually picked up on to Matrigel coated 35mm dish in complete stemflex medium consisting of stemflex supplement, stemflex basal, 1% Pen/strep and 1% Glutamax. On day 0 , Stemflex media was replaced by MSC differentiation media consisting of DMEM F12 basal medium supplemented with 1% B27(1X) ,1% Penicillin-streptomycin, 1% Glutamax, 10ng/ml FGF2, 2ng/ml Activin A, and 10ng/ml PDGFBB (Platelet derived growth factor -BB). Media was changed daily till day 4. At days 5, when over 80% of the culture plate's surface area was covered with spindle-shaped cells, differentiation media was withdrawn and cells were shifted to MSC maintenance media consist of KO-DMEM as basal media, supplemented with 10% FBS, 1% Pen/strep, 1% GlutamaX, 1% NEAA and 10ng/ml FGF2 and maintained till day 7. At this stage cells were passaged using 0.25% trypsin-EDTA into1:5 ratio in non-coated tissue culture plastic plates and iMSCs were allowed to adhere for 10-12 h. As MSCs have inherent plastic adherent property, non-adherent cells were removed by giving media change and adhered fibroblastic cells were expanded. For validation of successful differentiation, differentiated cells were characterized as per ISCT (International Society for Cell & Gene therapy) guidelines for mesenchymal stromal cell characterization (73). For all the experiments, P3-P4 cells were used. Dopaminergic neuron differentiation protocol is previously reported from our group (67). Briefly, the iPSC colonies were picked manually and plated on Geltrex coated 35mm plates in a Stemflex medium at a density of 2.5 × 10 5 −3.0× 10 5 cells/well and switched to Neural Induction Media (NIM) to differentiated to NPs. For floor plate cell (FPC) generation, NPs were treated with 25 ng/mL FGF-8 in NEM for 7 days. Final differentiation to DA neuron was induced using neurobasal/Adv. DMEM-F12 media supplemented with SHH (10 ng/mL), FGF-8 (25 ng/mL), BDNF (20 ng/mL), GDNF (10 ng/mL), N2 (1X), B27 (1X), ascorbic acid (0.2 Mm) , ITS (1X) , and db-CAMP (50μM), with media changes on alternate days. On day 6, cells were switched to maturation media with SHH reduced to 1 ng/mL. DA neurons were differentiated from HC03, PD02 and PD03 iPSC lines. Trilineage differentiation of iMSCs: To assess the multipotent nature of iMSCs, their osteogenic, adipogenic, and chondrogenic properties were evaluated. For osteogenic differentiation of iMSCs, the cells were plated in 24-well plates with a cell density of 3,000 cells/cm 2 . Upon 90% confluency, they were induced with the osteogenic differentiation medium consisting of MSC maintenance media supplemented with 5 mM glutamine, 10 –8 M dexamethasone, 50 mg/ml ascorbic acid and 10 mM b-glycerophosphate. Media was changed every alternative day. After 21 days of induction, the calcium mineralization was assessed by Alizarin red S staining (74). For adipogenic induction, Stempro™ adipogenic differentiation kit was used. Cultures were checked on regular basis for change in cell shape and the appearance of lipid droplets. The presence of lipid droplets was then confirmed by Oil red O staining after 15days of induction. For chondrogenic differentiation of iMSCs, the cells were plated in 24-well plates with a cell density of 3,000 cells/cm 2 in chondrogenic differentiation medium containing KO-DMEM as basal media supplemented with 1% pen/strep, 1% GlutamaX, 10% FBS and 10ng/ml TGFβ1 in a rocker inside incubator at 37ºC, 5% CO 2 to form micro mass for 21 days. The presence of cartilaginous matter was confirmed by toluidine blue staining after 3 weeks of induction (75). Uninduced iMSCs were used as control for this experimental setup. BODIPY staining of adipocytes: On the 21st day of adipogenic induction, cells were trypsinized and pelleted, then incubated with 2 µM BODIPY 493/503 in 1× DPBS for 30 minutes at 37°C. Following incubation, excess dye was removed by washing twice with 1× DPBS. The resulting single-cell suspension of cells was analysed by flow cytometry, with 10,000 events recorded per sample (76) Flowcytometry: For flowcytometry assay, direct and indirect staining methods were used for fluorophore tagged or untagged primary antibodies respectively. For indirect staining method, the rat BMMSCs, iMSCs, and Osteocytes were dissociated with 0.25% Trypsin EDTA to achieve single cell suspension and fixed in 2% PFA prepared with 1X DPBS for 45min at Room temperature (RT). After fixing, PFA was removed by centrifuging cells at 1800 rpm for 5mins. Cell pellet was then resuspended in 1X DPBS containing 0.01% sodium azide (NaN3). 1×10 5 cells were used per reaction. For intracellular marker (RUNX2) cells were permeabilized using 0.01% Triton X-100 for 30 min at room temperature in the dark followed by blocking with 3% bovine serum albumin (BSA) for 45 min at RT to avoid non-specific binding. For cell surface markers (CD90/THY-1, CD73, CD105, CD11b, CXCR4) permeabilization step was not performed. After blocking, cells were incubated overnight (~16h) with specific primary antibodies at 4ºC followed by incubation with respective secondary antibodies tagged with fluorophore-FITC for 90 mins at RT in dark. Each step is succeeded by a washing step in 1X DPBS, containing 0.01% sodium azide at 10,000 rpm for 10 mins. For direct staining method of cell surface markers (HLA-DR, CD45, CD80, CD86, CD56 and CD35) cells were fixed in PFA and treated with blocking reagent as mentioned before. After blocking, cells were incubated with fluorophore tagged (FITC or APC) antibodies for 45 mins at 4ºC in dark followed by two PBS washes. For flowcytometry acquisition, after the final wash, cells were resuspended in 500 µl of sheath fluid. FACS Verse (BD Biosciences) instrument was used for sample acquisition and 10,000 events were recorded per sample. Events with very low FSC and SSC were excluded by gating the cell population (P1). Isotype control (cells stained with only secondary antibody) was used to gate (P2) the non-specific staining which was overlaid in the represented histogram plots. Mean ± SD of percentage immunopositive population is represented. Immunocytochemistry: Rat BMMSCs and iMSCs were cultured on 12-mm glass coverslips until they reached approximately 85% confluency. The cells were then fixed with 4% PFA for 45 minutes at room temperature. For intracellular marker staining, the cells were permeabilized with 0.1% Triton X-100 in DPBS for 30 minutes. To prevent nonspecific staining, a blocking step with 3% BSA was performed for 45 minutes. Primary antibodies were added and incubated overnight in a humidified chamber at 4°C. Following three washes with DPBS, the cells were incubated with secondary antibodies for 90 minutes at room temperature in a dark, humidified chamber. Coverslips were washed three more times with DPBS, and the nuclei were stained with 300 nM DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) for 2 minutes in the dark. Finally, the coverslips were mounted onto glass slides using 1,4-diazabicyclo [2.2.2] octane (DABCO) for imaging. Confocal images were taken for visualization and analysis. Labelling of iMSCs and rat BMMSCs with PKH-26 dye and transplantation: P2 iMSCs and P0 control rat BMMSCs were trypsinized, counted, and labeled with PKH26 using the MINI26 PKH26 Red Fluorescent Cell Linker Mini Kit (Sigma), following the manufacturer's protocol. Briefly, 2 × 10⁶ cells, pre-washed in serum-free media, were resuspended in 100 µL of Diluent C. A 2x (4 × 10⁻⁶ M) dye solution was prepared separately by mixing 0.4 µL of dye with 100 µL of Diluent C, then added to the cell suspension. The mixture was incubated in dark at room temperature for 5 minutes, achieving a final dye concentration of 2 × 10⁻⁶ M per 2 × 10⁶ cells. The reaction was neutralized by adding an equal volume (200 µL) of FBS. Subsequently, the cells were washed two times with complete media and one final wash with DPBS. For transplantation 1 × 10⁶ PKH-labelled cells were resuspended in 0.5 mL PBS to be injected into one rat. The cells were injected intramuscularly (IM) into the right hind limb of anesthetized rats on day 3 post MPTP treatment. A volume of 0.5 mL DPBS (vehicle) was injected into rats of the control group. Immunohistochemistry: For immunohistochemistry, rats were anesthetized and perfused transcardially with 300 ml of 0.9% saline to remove blood from the circulatory system, followed by 300 ml of 4% paraformaldehyde (PFA) prepared in 1X DPBS. After perfusion, the rats were decapitated and brain tissue was isolated and stored in 4% PFA at 4ºC for 7 days to allow for tissue hardening. For coronal sections, 50 µm thick slices of the midbrain were prepared using a Leica 1200S vibratome. Immunohistochemistry (IHC) was performed on free-floating sections using a 24-well plate. For heat mediated antigen retrieval, the sections were incubated at 65ºC in a water bath for 2.5 hours, immersed in 200 µl of antigen retrieval buffer, consisting of an equal mix of saline-sodium citrate (SSC) buffer and formamide. Permeabilization and blocking were carried out using a solution containing 5% bovine serum albumin (BSA), 5% goat serum, and 0.1% Triton-X for 2 hours at room temperature. After each step, the sections were washed twice for 5 minutes with 1X PBS on a rocker. Following blocking, the brain sections were incubated overnight at 4ºC in primary antibody solution prepared in 5% BSA with 5% goat serum. The next day, after two PBS washes, the sections were incubated with a goat-derived secondary antibody for 6 hours at RT in dark, followed by a counterstaining with 5 µg/ml DAPI for 1 minute. The immunostained brain slices were then mounted on glass coverslips using DABCO as the mounting reagent. Confocal images were taken for visualization and analysis. Confocal microscopy: ICC and IHC slides were visualized using the ZEISS Axio Observer.Z1/7 and LSM 980 confocal microscope, equipped with a Plan-Apochromat 40X/1.3 Oil DIC (UV) VIS-IR M27 objective. Image acquisition was performed using ZEISS ZEN 3.7 software (RRID: SCR_013672), employing a fluorescence contrast method. Detector gain was set to 650V, and the pinhole was adjusted as follows: 1 AU/30 μm for DAPI, 1 AU/36 μm for FITC, 1 AU/45 μm for AF647, and 1 AU/45 μm for PKH26. The excitation/emission settings were 405/495 for DAPI, 488/517 for FITC, 650/665 for AF647, and 551/567 for PKH26. Images were captured at a resolution of 2791 x 2791 pixels with an 8-bit depth and an effective NA of 1.3, then analysed using ImageJ. ELISA: For DA ELISA of rat midbrain samples, tissue lysate was prepared . Briefly, rats were sacrificed and midbrain was dissected out, weighed and homogenized in 1x PBS at 4°C. Homogenized samples was then centrifuged and supernatant was carefully collected and stored in -80°C. To maintain the uniformity of the tissue homogenate, the concentration of the tissue lysate was kept constant at 200mg/ml . For serum sample preparation, 2.5-3ml blood was collected from each rat by cardiac puncture and centrifuged at 3000rpm for 20 min at 4ºC. samples were stored in -80°C until use. For detection of paracrine factors, P0 rat BMMSCs were incubated in serum free media for 24h and cell supernatant was collected and centrifuged for 15 min at 3000 rpm to remove cell debris. For iMSC conditioned media, cells were cultured till 90% confluency and then stimulated with TNF-α (25 ng/ml) for 24h. Then they were washed three times with DPBS, and serum free maintenance media was added and incubated for 24h. The supernatant was collected after removing cell debris and stored at -80°C until use. For DA ELISA, iPSC-derived mature DA neurons were incubated in complete HBSS buffer (pH 7.4) for 5 min, and the supernatant was collected to measure basal vesicular dopamine release. Cells were then stimulated with 56 mM KCl in HBSS, and the supernatant was collected. Samples were centrifuged at 3000 rpm for 15 minutes to remove cellular debris. Cell counts for BMMSCs, iMSCs, and iPSC-derived DA neurons were obtained after conditioned media collection. All ELISA was performed as per manufactures protocol. Readings for standard and samples were taken using the spectrophotometer (SPARK, TECAN, Switzerland). Data is represented as Mean ± SD. Flow cytometry of rat brain samples: Rats were euthanized using an overdose of isoflurane, followed by transcardial perfusion with 350 mL of 0.9% isotonic saline to eliminate blood. The midbrain was carefully dissected and minced into small fragments using a sterile surgical blade. The tissue was then incubated with 2 mL of 0.25% trypsin for 15 minutes at 37°C to facilitate enzymatic digestion. Following digestion, 6 mL of 1× PBS was added to neutralize the trypsin. The tissue was gently triturated by repeated pipetting to obtain a single-cell suspension. The cell suspension was centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The resulting pellet was resuspended in 1× PBS and passed through a 70 µm cell strainer to remove any remaining tissue debris. The filtered single-cell suspension was centrifuged again and fixed in 2% paraformaldehyde (PFA) overnight at 4°C. After fixation, the cells were stored in PBS containing 0.01% sodium azide (NaN₃) until further analysis. For the flowcytometry analysis of PKH+ cells in rat brain, 2 lakh cells were resuspended in sheath fluid, and 10,000 events were acquired per sample in BDFACSLyric instrument. Mean ± SD of percentage immunopositive population is represented. Colony forming unit fibroblast (CFU-f): To check the number of colony-forming units present in the bone marrow, bone marrow was isolated from individual rats and processed as previously described. After isolation all mononuclear cells of the bone marrow was plated into 100mm cell culture plates. Non-adherent cells were removed by giving media change and adhered cells were cultured for 8 days and then fixed in 4% PFA followed by staining in 0.1% toluidine blue prepared in 1% PFA overnight at 4ºC. After staining images were taken and number of colonies were counted in ImageJ software using multi-point tool. Annexin-PI cell apoptosis assay: Apoptosis was measured in rat BMMSCs and iMSCs using FITC Annexin V/Dead cell apoptosis kit as reported before (71). Briefly, 1 × 10 5 cells were incubated in 100μl Annexin binding buffer containing 0.5 μl of Annexin V and 0.1 μl propidium iodide (PI) for 30 min at RT. After one PBS wash, cells were resuspended in 500µl of sheath buffer. Flow cytometric analysis was carried out to determine the number of apoptotic cells using FACSVerse™ (BD Biosciences, USA) using quadrant density plot. Unstained cells were used as experimental control. Mean ± SD of percentage of apoptotic population is represented. Reactive oxygen species assay: ROS generation in rat BMMSCs and iMSCs was estimated as previously reported (71). Cells were washed twice with PBS, counted, and incubated for 15 min with 10 µM H 2 DCF. DA (Invitrogen). Cell number was maintained similar for all groups. After incubation, the fluorescence intensity of DCF on oxidation of H 2 DCF is measured at ex/em-485/535 nm using the spectrophotometer (SPARK, TECAN, Switzerland). In-vitro Migration assay: The transwell migration of rat BM-MSCs and iMSCs was evaluated using a Boyden chamber. A total of 1 × 10⁵ rat BM-MSCs or 2 × 10⁵ iMSCs were seeded in the upper chamber of 8 μm transwell inserts (Corning, USA) in 200 μL of complete media without growth factors and allowed to adhere for 24 hours. Following adherence, migration was induced by adding 25 ng/mL of rat recombinant TNFα for BM-MSCs or hTNFα for iMSCs to 600 μL of basal media in the lower chamber. Cells were allowed to migrate toward the lower chamber for 24 hours. After the incubation period, non-migrated cells were gently removed using a cotton swab. The inserts were then washed with PBS, fixed in 4% PFA, and stained overnight with 0.1% toluidine blue in 1% PFA at room temperature. Images were captured using the EVOS M5000 imaging system (Invitrogen) at 10× magnification. The number of migrated cells per field was quantified using ImageJ software. Ki67 proliferation assay: The protocol was adapted from our previously published report (71). Rat BM-MSCs and iMSCs were harvested using 0.25% trypsin-Ethylenediaminetetraacetic acid (EDTA), fixed with cold 100% methanol for 45 min at RT, and resuspended in wash buffer containing 0.01% sodium azide in PBS. Permeabilization and blocking was performed as mentioned before. The cells were then labeled with proliferation marker Ki67 primary antibody overnight. Further, incubated with secondary antibody conjugated with Alexa Fluor 488 for 90 min at RT. Flow cytometry analysis was performed using FACSVerse™ (BD Biosciences). Cells were identified by light scatter for 10,000 gated events and analysed using BD FACSuite software. Cells stained with only the secondary antibody were used as isotype control and overlaid in the histograms and was used for gating. Mean ± SD of percentage Ki67 positive population for n = 6 is represented. For immunocytochemistry, cells were seeded onto glass coverslips and, after adhering, were fixed using 100% methanol. Immunostaining for ki67 was performed as mentioned before and Confocal images were acquired for further analysis. Mixed Lymphocyte Reaction assay: 5ml of Blood was collected aseptically in K2EDTA treated vacutainer tubes from 3 unrelated healthy individuals and processed after 30min of collection in order to attain room temperature. PBMCs were isolated by gradient centrifugation using Hisep LSM1077 as previously reported (66). After isolation, cells from 3 donors were resuspended separately in complete PBMC media and counted using haemocytometer and plated at 1x10 6 cells/ml concentration in 6 well plates. [Composition of complete PBMCs media was RPMI basal media, 10% FBS, 1% Pen/strep, 1% Glutamax]. After culturing cells for 48h, PBMCs from all 3 donors were pooled in a 15 ml centrifuge tube and mixed properly, followed by centrifugation step at 260g for 5 mins. Cells were resuspended and plated in PBMC media with 1% Phytohemagglutinin-M a positive stimulator, for 72h in optimum sterile culture condition (5% CO2, 37ºC) before setting up for co-culture assay with iMSCs. PHA-M treatment induces a hyper-immunogenic reaction. Pooling donor PBMCs combined with PHA-M stimulation will lead to rapid proliferation and sphere formation. After 72h of PHA-M treatment all PBMCs were collected and centrifuged at 200g for 10 mins. The supernatant was discarded, and the cells were washed twice with PBMC media to remove PHA-M. The cells were then resuspended in complete PBMC media, counted, and plated with mit C-treated iMSCs at a ratio of 1:10 (iMSCs:PBMCs) in a 24-well plate. PBMCs were also plated separately without iMSCs as negative control. Co-culture was set for 24h. After incubation, PBMCs were collected, centrifuged, and fixed in methanol. Methanol-fixed PBMCs were subsequently assessed for Ki67 proliferation marker expression using flow cytometry. Mean ± SD of percentage Ki67 positive population for is represented. Immunomodulation assay using patient PBMCs: Healthy control and PD-patient PBMCs were thawed and centrifuged at 250g for 5 minutes. The cells were resuspended and cultured in PBMC media containing 10% FBS, 1% penicillin-streptomycin, 1% GlutaMAX, and RPMI basal media for 24 hours. Following this, the PBMCs were incubated with 1% Phytohemagglutinin-M (PHA-M) for 72 hours under optimal sterile culture conditions (5% CO2, 37ºC). After 72 hours, the PHA-M-treated PBMCs were collected and centrifuged at 200g for 10 minutes. The supernatant was discarded, and the cells were washed twice with PBMC media to remove any remaining PHA-M. The cells were then resuspended in complete PBMC media, counted, and co-cultured with mitomycin C-treated healthy control and PD iMSCs in a 1:10 ratio (iMSCs) in a 24-well plate. The experimental conditions included co-culturing patient PBMCs with both patient-derived and healthy control iMSCs, and healthy control PBMCs with healthy control iMSCs. Additionally, PHA treated PBMCs of each group were cultured alone as a negative control. After 24 hours of co-culture, PBMCs were collected, centrifuged, and fixed in methanol. The methanol-fixed PBMCs were subsequently analysed for Ki67 proliferation marker expression using flow cytometry. Statistical analysis: Statistical analyses was performed using R software version 3.4.1 (R Foundation; R Project for Statistical Computing, RRID:SCR_001905). One-way ANOVA followed by pairwise comparison (Bonferroni) analysis was used as required (specified in figure legends). In all analyses, statistical significance was set at p < 0.05. F is expressed as F (degrees of freedom, degree of freedom error) = x. For statistical significance, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001. Graphs were prepared using GraphPad Prism 6 (GraphPad Software; GraphPad Prism, RRID:SCR_002798). All error bars in graphs depict standard deviation (SD). Results The degeneration of midbrain dopaminergic (DA) neurons coincides with the initiation of neuroinflammatory processes during pre-motor stage: To track the timeline of neurodegeneration in our MPTP rat model, the status of DA neurons in the SNpc and corresponding non-motor and motor behavioural parameters were assessed. Confocal IHC images showed a significant reduction in the number of TH + cells in the SNpc from week 1 post MPTP treatment (PMT) onwards (Figure 1A & C). In parallel, a gradual increase in phospho a-synuclein serine 129 (p-syn) expression was observed between week 2 and week 3 PMT (Figure 1B). Furthermore, we observed a significant reduction in dopamine levels starting from week 1 PMT, with a further decline at later time points (weeks 2 & 3 PMT) (Figure 1D). At week 1 PMT, the MPTP group exhibited a marked reduction in the time spent in the familiar compartment compared to controls, indicating impaired olfactory discrimination—a hallmark non-motor symptom of PD. This deficit persisted through weeks 1 to 3 PMT (Figure 1E). Additionally, a significant decline in Nerve Conduction Velocity (NCV) was observed from week 1 PMT in the MPTP group, which was retained across all subsequent time points (Figure 1F). Motor coordination assessed by rotarod performance time showed a mild decrease at week 1 PMT, with significant differences emerging from week 2 onward (Figure 1G). In the actimeter-based assessment of locomotor activity, a notable reduction in the number of squares traversed was observed only from week 2 PMT, which persisted through week 3 (Figure 1H). Collectively, these behavioural assessments, along with dopamine ELISA and IHC analysis of TH + neurons in the midbrain, and in alignment with our earlier study (77), indicate that while neurodegeneration and non-motor impairments begin as early as week 1 PMT, motor deficits become apparent only from week 2 PMT onward. Based on these observations, the week 1 PMT is hereafter designated the pre-motor stage in this model, while the onset of motor symptoms from week 2 PMT marks the transition to the motor stage of the disease. The neuroinflammatory status was evaluated based on microgliosis, astrogliosis, and proinflammatory cytokine TNF-α and NLRP3 expression in the SNpc region. Confocal IHC images showed a progressive increase in IBA1 + cell numbers and their interaction with TH + cells from week 1 PMT (insets) (Figure 2A). This was further confirmed by the display of IBA1 + cells showing immunopositive co-localization with TNF-α (yellow puncta) (Figure 2B), with Pearson’s co-localization coefficient significantly elevated in the week 2 PMT group compared to both control and week 1 PMT (Figure 5H). Consistently, TNF-α levels in midbrain lysates showed a significant increase from week 1 PMT onwards (Figure 2I). Post-mortem IHC studies have previously reported infiltration of peripheral immune cells into the PD patient brain (78,79). In line with this, our IHC and flow cytometry analyses of PD rat brain demonstrated a significant increase in CD4⁺ cells at the early pre-motor stage, with a further increase subsequently, while CD8⁺ cells showed a marked increase starting from week 2 PMT (motor stage) (Figure 2E, F, K, L & Supplementary Figure S8 E & G). Also, TNF-α levels in peripheral blood serum showed a significant increase at the pre-motor stage, with further enhancement throughout the motor stages (Figure 2J), indicating the presence of systemic inflammation from the pre-motor stage of the disease. Previous preclinical studies and PD patient data have demonstrated heightened NLRP3 expression with DA neurons (80). Our study also detected an increase in NLRP3 expression in the brains of PD rats that increased as the disease progressed (Figure 2D and M), further supporting the presence of a chronic inflammatory microenvironment from an early stage. The degeneration of midbrain DA neurons and the initiation of neuroinflammatory processes thus appear to occur concurrently during the pre-motor stage of disease progression. The impairment of physiological and functional parameters in PD-BMMSCs correlates with the onset of systemic inflammation and neurodegeneration: BMMSCs isolated from control rats were characterized using immunofluorescence and immunophenotyping to confirm the presence of mesenchymal stromal cell markers. Phase-contrast images (Supplementary Figure S1A) of BMMSC culture on day 1, Day 6, and Day 8 post-isolation demonstrated plastic adherence and stromal cell morphology. Confocal imaging (Supplementary Figure S1B) confirmed the expression of CD90 and CD105 surface markers. Additionally, flow cytometry analysis revealed that over 94% of the isolated BMMSCs were immunopositive for both CD90 (94.85±0.63%) and CD73 (97.30±0.96%) (Supplementary Figure S1C). Next, we proceeded to isolate BMMSCs from MPTP-treated PD rats. BMMSCs (P0) isolated from all three PD groups exhibited an enlarged, thin, and flattened morphology, contrasting with the elongated spindle shape observed in age-matched control BMMSCs (Figure 3A & B). A significant decline in the total number of Ki67 positive proliferative cells was observed from the second week onward, with a further reduction seen in the Week 3 PMT group (Figure 3C & Supplementary Figure S1 D). To assess the self-renewal capacity of MSCs, we compared the number of stromal clonogenic cells in the bone marrow of MPTP-treated groups with age-matched healthy controls, as colony numbers directly indicate self-renewal potential (36). There was a significant reduction in the number of colony-forming units in the bone marrow of MPTP-treated PD rats with time, compared to controls (Figure 3D & E). Furthermore, a substantial increase in the number of apoptotic cells was noted in the Week 2 and Week 3 PMT groups, indicating reduced cell survival. However, no significant difference was observed between the control group and the Week 1 PMT group (Figure 3F & Supplementary Figure S1 E). Morphological changes in MPTP-treated rat BMMSCs at P0 (Figure 3B) suggest cellular aging, while increased apoptosis of BMMSCs for MPTP-treated rats corelates with the decline in the population and proliferation of self-renewing cells in their bone marrow. As homing and migration to inflammatory sites is known to be an essential function of bone marrow MSCs for exerting immunosuppressive effects under chronic inflammatory conditions (81), we next assessed the migratory ability of PD-BMMSCs in response to an inflammatory cue using a transwell migration assay. A significant reduction in the number of migrated cells per field toward TNF-α was observed from week 1 PMT, which further declined in the week 2 and week 3 PMT groups (Figure 3G & H). The migration of BMMSCs is influenced by factors such as CXCR4 surface expression, matrix metalloproteinase secretion, and intracellular ROS levels (82–85). We analysed these factors in PD-BMMSCs and found a significant increase in basal ROS levels from week 2 PMT compared to both control and week 1 PMT (Figure 3J). Additionally, MMP3 concentration in the PD-BMMSC secretome showed a marked reduction from week 1 PMT compared to control (Figure 3K). However, the number of CXCR4-positive cells increased at week 2 PMT but significantly declined at week 3 PMT (Figure 3I). Next, we evaluated the secretion levels of four key immunomodulatory paracrine factors—IDO, PGE2, TGF-β, and IL-10—in the BMMSC secretome. A significant decline in IDO, TGF-β and IL-10 and secretion was observed at week 1 PMT and week 2 PMT (Figure 3L, M & N). However, no significant differences were detected in the secretion levels of PGE2 (Supplementary Figure S2 A). The reduced migration of PD-BMMSCs in vivo may be influenced by elevated intracellular ROS levels, and additionally by a further decrease in MMP3. The observed increase in the CXCR4 + population in the week 2 PMT group may be attributed to elevated levels of inflammatory cytokines in systemic circulation in PD rats. However, despite this increase in CXCR4 expression, migration was not enhanced in BMMSCs of the week 2 PMT group. Therefore, our observations suggest that the onset of BMMSC functional impairment occurs during the pre-motor stage (week 1 PMT) and temporally correlates with the emergence of systemic (rise in blood TNF-a) inflammation, neuroinflammation, and midbrain DA neuron degeneration. The onset of systemic inflammation and neuroinflammation also suggest that this progressive dysfunction of BMMSCs may contribute to the compromised immunomodulatory support during early PD pathogenesis. Impaired differentiation ability of PD-iMSCs: iMSCs were differentiated from healthy control iPSC lines NIMHAi006-A (HC03) and NIMHAi005-A (HC02) as well as from sporadic PD iPSC lines NIMHi002-A (PD02) and NIMHi003-A (PD03). Morphological changes became apparent as early as day 2 of differentiation (Supplementary Figure S3B). Upon switching from differentiation media to maintenance media on day 5, the differentiated cells started to proliferate rapidly. Passaging these cells and plating them onto non-coated, tissue culture-treated plates resulted in a homogeneous population of differentiated iMSCs. iMSCs differentiated from HC lines at different passages (P3, P5 and P10) is shown in Supplementary Figure S3C. To confirm the disease-relevant phenotype, dopaminergic neurons differentiated from the two PD-iPSC lines were characterized (Supplementary Figure S4A-K) and demonstrated distinct pathological features compared to healthy control. These PD-iPSC-derived dopaminergic neurons exhibited impaired vesicular dopamine release (Supplementary Figure S4L) and elevated expression of phosphorylated α-synuclein at serine 129, validating the disease-associated cellular dysfunction (Supplementary Figure S4F & I). iMSCs from all the groups were characterized according to ISCT guidelines for MSC characterization at P2. PD iMSCs exhibited an increase in cell size, with PD03 iMSCs showing a significantly larger size than HC iMSCs. Moreover, PD03 iMSCs displayed a more flattened morphology compared to PD02 iMSCs. (Figure 4B & C). Confocal imaging confirmed the expression of MSC-specific surface markers CD73, CD105, and CD90 in both the HC (Figure 4D, Supplementary Figure 5A) and PD groups (Figure 4E & F). Flow cytometry analysis revealed that over 95% of the cell population was positive for MSC-specific CD markers (Figure 4G, H, I, & Supplementary Figure 5B i-iii), while the expression of co-stimulatory molecules (CD80, CD86) and hematopoietic markers (HLA-DR, CD45, CD56, CD34) was negligible across all three groups (Supplementary Figure 5B iv-ix & Supplementary Figure S6A-C). Mesenchymal stromal cells have characteristic trilineage differentiation ability into adipogenic, osteogenic and chondrogenic lineages. iMSCs derived from both HC and PD groups successfully differentiated into all three lineages, as confirmed by Oil Red O staining for lipid droplets (Figure 5A-C), Alizarin Red S staining for calcium deposition (Figure 5D-E), and Toluidine Blue staining for cartilage formation (Figure 5G-I, Supplementary Figure S5C). However, the adipogenic and osteogenic differentiation capacity of PD-iMSCs showed impairment (Figure 5A-F). Flow cytometry analysis of BODIPY-stained adipocytes revealed a significant reduction in MFI in PD-adipocytes compared to HC (Figure 5J). Additionally, the percentage of Runx2 + cells was lower in the PD-osteocyte population than in HC, with a more pronounced reduction observed in the PD03 group compared to PD02 (Figure 5K). So, while the immunophenotypic expression of MSC and hematopoietic and co-stimulatory markers was similar across HC and PD iMSCs, PD iMSCs showed compromised differentiation capability for osteocytes and adipocytes. PD-iMSCs exhibited reduced proliferation, survival, and migration, along with elevated intercellular basal ROS level: BMMSCs isolated from MPTP induced PD rats had demonstrated increased apoptosis alongside reduced proliferation and migration capabilities. These findings were corroborated in iMSCs derived from sporadic PD patients when maintained under optimal culture conditions. At P3, PD-derived iMSCs exhibited significantly diminished proliferation rates. Flow cytometric analysis revealed a reduced Ki67-immunopositive cell population (Figure 7C & Supplementary Figure S 3D), with PD03-iMSCs showing the most pronounced decrease. Confocal microscopy of Ki67-immunostained cells provided additional validation (Figure 7A), and the calculated Ki67: DAPI ratio (Figure 7B) confirmed these proliferation deficits. Despite optimal culture conditions, PD-derived iMSCs demonstrated increased cell death. Annexin-PI staining revealed significantly higher numbers of apoptotic cells in both PD groups compared to healthy control cells. Notably, PD03-iMSCs exhibited a greater proportion of apoptotic cells than PD02-iMSCs, possibly indicating a severity-related response (Figure 6D Supplementary Figure S3 E). The migratory response to the proinflammatory cytokine TNF-α was also significantly impaired in PD-iMSC groups. The number of migrated cells was substantially lower than HC, mirroring the pattern observed in PD-affected rat BMMSCs (Figure 6E & F). To validate these in vitro observations, PKH-labeled iMSCs were transplanted intramuscularly into PD rat models. Flow cytometric analysis of brain tissue revealed a lower percentage of PKH-positive cells in rats receiving PD-iMSCs compared to those transplanted with HC-iMSCs (Figures 6G & Supplementary Figure S8 C), further reinforcing the reduced viability and migration capacity observed in vitro . Intracellular basal ROS levels were significantly elevated in PD-iMSCs compared to HC03 iMSCs. The PD03-iMSC group exhibited the highest ROS levels (Figure 6H), suggesting a correlation between oxidative stress burden and cellular dysfunction severity. Impaired immunomodulatory function of PD-iMSCs : To evaluate the immunomodulatory capacity of iMSCs, we quantified immunomodulatory paracrine factors in culture media following 48-hour TNF-α treatment. Four key factors were analyzed: transforming growth factor-β (TGF-β), interleukin-10 (IL-10), indoleamine 2,3-dioxygenase (IDO), and prostaglandin E2 (PGE2). Significant reductions in IDO and PGE2 secretion within the PD-iMSC secretome (Figures 6I and 6J) was observed, while TGF-β and IL-10 levels remained unchanged (Supplementary Figure S2B, C). To further characterize the immunosuppressive potential of iMSCs, we employed a mixed lymphocyte reaction assay. This assay exploits the principle that T cells from one donor proliferate when exposed to antigen-presenting cells (APCs) from a genetically distinct donor due to HLA incompatibility, thereby triggering an immune response (Figure 7A & B). Comparing PBMC proliferation in the presence versus absence of iMSCs allows us to evaluate their immunomodulatory potential (Figure 7C & D). Flow cytometric analysis of activated PBMCs with iMSCs demonstrated distinct immunomodulatory patterns, when compared to negative control (defined as PBMC only without the presence of iMSCs). HC iMSCs significantly suppressed PBMC proliferation compared to negative control, confirming their expected immunomodulatory function. In contrast, PD iMSCs failed to significantly reduce PBMC proliferation relative to negative controls (Figure 7E & Supplementary Figure S2 D), indicating compromised immunomodulatory properties. To validate these findings using autologous cell systems, we performed immunomodulation assays with patient-matched PBMCs and iMSCs. This experimental design included HC03-PBMCs, PD02-PBMCs, and PD03-PBMCs (Figure 8A), each activated separately with PHA for 72h (Figure 8B) and then co-cultured with its corresponding iMSCs for 24h (Figure 8E & F). PD patient PBMCs exhibited significantly elevated (~16–21% higher in PD02 and ~12–14% higher in PD03) baseline proliferation compared to HC PBMCs, suggesting an inherently activated immune state. When PD-derived PBMCs were co-cultured with their corresponding PD-iMSCs, proliferation rates remained significantly higher than those observed in co-cultures for HC iMSCs (Figure 8G & Supplementary Figure S2 E). This finding further substantiates the impaired immunoregulatory capacity of PD-derived iMSCs. The combined evidence of significant reduction in IDO and PGE2 secretion, coupled with the inability of PD-iMSCs to effectively suppress PBMC proliferation in both heterologous MLR and autologous co-culture systems, together establish a clear functional deficit in the immunomodulatory capacity of PD-derived iMSCs compared to healthy controls. Transplantation of healthy MSCs in PD rats prevented neurodegeneration, promoted neurogenesis and lowered systemic inflammation more effectively than PD-iMSCs: The compromised immunomodulatory capacity of PD-derived iMSCs may significantly limit their therapeutic effectiveness when transplanted under PD conditions. To assess their disease-modifying and immunomodulatory potential compared to HC iMSCs, MPTP-induced PD rats received transplantations of either HC or PD-derived iMSCs on day 3 of MPTP treatment. Rats transplanted with healthy rat bone marrow-derived MSCs (BMMSCs) served as positive controls. By two weeks post-transplantation, both the rat BMMSC and HC-iMSC transplanted groups demonstrated a significant reduction in IBA1 + microglial cells compared to the disease control group. Notably, this microglial reduction was substantially more pronounced in rats receiving HC-iMSCs than in those transplanted with PD-iMSCs (Figure 9A, E). Moreover, microglia in the PD-iMSC group maintained their activated morphology (as shown in insets), while cells in both the HC-iMSC and rat BMMSC groups exhibited morphology resembling that observed in control animals (Figure 9A, C & Supplementary Figure S7B). Colocalization analysis revealed TNF-α expression in IBA1 + cells within the PD-iMSC group (highlighted in insets), a pattern that was absent in the HC-iMSC transplanted groups (Figure 9C & Supplementary Figure S7B). Consistent with these findings, TNF-α levels were significantly elevated in both brain tissue and peripheral blood of PD-iMSC transplanted rats compared to HC-iMSC recipients (Figure 9F & G). Furthermore, enhanced NLRP3 expression, which colocalized with TH + neurons, was observed exclusively in the PD-iMSC group and was notably absent in HC-iMSC recipients (Figure 9D & Supplementary Figure S7C). Transplantation of HC-iMSCs significantly reduced CD4 + cell infiltration compared to PD-iMSC transplantation (Supplementary Figure S8D). For CD8 + cells, HC-iMSC transplantation maintained infiltration levels similar to 2 WK PMT, while PD-iMSC transplantation resulted in increased infiltration (Supplementary Figure S8F). These findings suggest that PD-iMSCs are less capable of modulating neuroinflammation in PD due to their compromised immunoregulatory functions and altered physiological state. Recent studies using pre-clinical models and postmortem human brain tissue have demonstrated that neurogenesis can occur in the SNpc following treatment with neuroprotective factors (86). Moreover, we have recently reported neurogenesis in the SNpc following transplantation of sEVs derived from human dental pulp stem cells (77). In addition to their immunomodulatory properties, MSCs exhibit strong cytoprotective effects, and thus it is credible that modulating the hostile neuroinflammatory microenvironment may help initiate neurogenesis. To investigate this, we examined the expression of the cell proliferation marker Ki67 through IHC studies, and assessed the floor plate cell marker FOXA2 in the SNpc region of the rat midbrain following transplantation. IHC analysis revealed a significantly higher number of FOXA2 + cells in the SNpc of rats transplanted with healthy MSCs, compared to those receiving PD-MSCs, which showed FOXA2 + levels similar to the disease control group (WK2 PMT) (Figure 10A i – F i, G & Supplementary Figure S7D) Additionally, nuclear localization of Ki67 was prominent in the healthy MSC-transplanted group, indicating active cell proliferation. This was absent in both the control and WK2 PMT groups. Ki67 expression was markedly lower in PD-iMSC transplanted animals compared to those receiving healthy MSCs (Figure 10A ii–F ii & Supplementary Figure S7E). Also, the ratio of TH + Ki67 + cells to total TH + neurons per field was significantly higher in the healthy MSC transplanted group. Notably, there was no significant increase over control in proliferative cells in the PD iMSC transplanted group, suggesting a lack of neurogenesis (Figure 10 H). Consistent with these findings, the total number of TH + neurons in the SNpc region was significantly higher in the healthy MSC-transplanted group compared to the disease control, whereas the PD-MSC transplanted group showed no significant difference from the disease control (Figure 10 C iii–F iii, I & Supplementary Figure S7A). Moreover, midbrain dopamine content was also significantly elevated in the healthy MSC-transplanted group relative to the PD-iMSC group (Figure 10 J). To determine whether the increased dopaminergic neuron survival and possible neurogenesis in the transplanted groups translated into functional recovery, motor coordination was assessed using the rotarod test. The rotarod performance of the HC-iMSC-transplanted group was comparable to that of the control group, whereas the PD-iMSC transplanted group showed significantly impaired performance compared to both control and the HC-iMSC transplanted group (Figure 10K). This finding provides definitive in vivo confirmation that the cytoprotective and immunomodulatory capacity of PD-iMSCs is significantly compromised, as demonstrated by their failure to control neuroinflammation, ultimately leading to failed neurogenesis and poor motor functional recovery. Additionally, these findings reveal that unlike PD-derived iMSCs, healthy MSCs demonstrate robust therapeutic capacity through effective neuroinflammation modulation and enhanced neurogenesis promotion, supporting their clinical potential for PD intervention. Discussion While chronic neuroinflammation and peripheral immune dysfunction are now well-documented features of PD, the functional status of endogenous MSCs throughout disease progression remains uninvestigated. This represents a critical knowledge gap, as MSCs serve as master regulators of immunomodulation in peripheral immune cells and therefore may play a pivotal upstream role in the vicious cycle between neuroinflammation and systemic inflammation that characterizes the disease. Our findings present the first comprehensive evidence that endogenous MSC dysfunction occurs during the early pre-motor stages of PD, fundamentally altering the current understanding of systemic cellular alterations in PD pathogenesis. In our MPTP-induced rat model, we demonstrate the manifestation of BMMSC impairment as early as week 1 post-MPTP treatment (PMT), coinciding precisely with the onset of dopaminergic neurodegeneration and neuroinflammation, and preceding the emergence of motor symptoms. Our temporal analysis demonstrates progressive loss of SNpc TH + dopaminergic neurons with time PMT, which correlates directly with declining midbrain dopamine levels and deteriorating motor coordination and locomotion. An increase in the pathological marker, phospho α-synuclein serine 129 (p-Syn), is also noted with time PMT. This escalating neurodegeneration is accompanied by a corresponding increase in IBA1 + microglial cells, particularly expressing TNF-α, consistent with established evidence that microglia serve as the primary source of TNF-α during neuroinflammation (87). This clustering pattern of activated microglia, previously reported in postmortem brains from MPTP-exposed individuals (88), reflects the status of heightened neuroinflammation accompanying progressive neurodegeneration. This pattern also aligns with postmortem and PET studies demonstrating increased microglial activation in relevant brain structures of PD patients, as well as in atypical parkinsonian disorders including multiple system atrophy, progressive supranuclear palsy, and corticobasal degeneration (89–91). The concomitant elevation of midbrain TNF-α levels and NLRP3 immunopositive cells further supports the neuroinflammatory environment, as NLRP3 is known to be predominantly expressed by activated microglia (92–94). Concurrently, a progressive increase in blood TNF-α levels, as well as the presence of infiltrating CD4 + and CD8 + T cells in the midbrain, demonstrate a failure of systemic and peripheral immunomodulation with disease advancement. These findings align with clinical reports showing elevated serum TNF-α levels in advanced-stage PD patients (Hoehn Yahr Scale stages 3-5) compared to early-stage patients (stages 1-2) (95), and studies that demonstrate elevated plasma high-sensitivity C-reactive protein levels correlating with motor prognosis in PD patients (96). Similarly, clinical and pre-clinical studies have shown increases in CD4 + and CD8 + T cells in the brain with duration post MPTP administration (78,79). Our study reveals multifaceted BMMSC dysfunction in PD, encompassing cellular morphology, proliferation, survival, migration, and crucially, immunomodulatory capacity. PD-BMMSCs exhibited enlarged, flattened morphology characteristic of cellular senescence (83,97), accompanied by significantly reduced proliferation (decreased Ki67 + cells) and increased apoptosis from week 2 PMT. The progressive decline in colony-forming units directly reflects impaired self-renewal capacity, which is essential for maintaining the MSC pool within the bone marrow niche and is crucial to their ability to support tissue homeostasis and participate in immunomodulation (98,99). BMMSCs are also known to provide signals in the specialized bone marrow niche to support the self-renewal, quiescence, and maintenance of HSCs (100,101). The migration deficits observed in PD-BMMSCs represent a critical functional impairment, as MSC homing to inflammatory sites is fundamental to their therapeutic efficacy. Despite increased CXCR4 expression at week 2 PMT—likely a compensatory response to elevated systemic inflammatory cytokines—migration toward TNF-α remained significantly impaired. This paradox can be attributed to elevated intracellular ROS levels and reduced MMP3 secretion, both of which negatively impact MSC mobility and tissue infiltration capacity. In our previous study of diabetic neuropathy, we observed similar features of BMMSC migration impairment (71). This suggests that while these cells may retain the ability to detect inflammatory signals, their downstream migratory response is compromised. This uncoupling of chemokine receptor expression from functional migration underscores the multifaceted nature of MSC dysfunction in PD. To validate these findings in human disease, we differentiated both dopaminergic (DA) neurons and MSCs from patient-derived iPSCs generated from sporadic PD-patient PBMCs. This approach was chosen because PBMCs not only reflect stage-specific inflammatory and reactive signatures in idiopathic PD and Alzheimer's disease (106–108), but also exhibit key pathogenic milestones of PD, including accumulation of pathological α-synuclein species (109,110). Consequently, iPSCs generated from these somatic cells retain the disease state memory of the individual. Furthermore, such iPSCs provide a continuous and stable cellular resource, whereas directly isolating DA neurons and MSCs from patients would require invasive procedures yielding limited cell numbers suitable for only a few experiments. Importantly, modeling sporadic disorders is critical for wider clinical translation, given that the vast majority of patients suffer from sporadic rather than inherited forms of these conditions. DA neurons differentiated from these iPSCs successfully replicated disease pathology and demonstrated impaired vesicular dopamine release, which correlated with patient UPDRS scores and fluorodopa PET results (66). This aligns with previous studies demonstrating that iPSCs from sporadic movement disorders can recapitulate disease pathology in neuronal and glial cells (61–64,102), and similar replication of disease pathology has been shown in other cell types for various sporadic conditions (103–105). Crucially, these sporadic PD-iMSCs recapitulated the major dysfunction patterns observed earlier in rat PD-BMMSCs, including altered morphology, reduced proliferation and survival, impaired migration, elevated basal ROS levels, and—most importantly—compromised immunomodulatory capacity. A previous study conducted on BMMSCs from patients with progressive supranuclear palsy (PSP), a rare neurodegenerative movement disorder, has further reported significant mitochondrial dysfunction, elevated ROS levels, and compromised differentiation potential (55). A particularly interesting observation from our study is the impaired differentiation potential of PD-iMSCs into adipogenic and osteogenic lineages. This aligns with clinical observations where PD patients often present with early-onset symptoms of osteoporosis/osteopenia and unexplained weight loss (48,111). These systemic manifestations may be mechanistically linked to the compromised adipogenic and osteogenic capabilities of MSCs in PD, and merit further investigation in subsequent studies. Most significantly, our study demonstrates profound impairment in iMSC immunomodulatory function. PD-iMSCs exhibited a notable reduction in the secretion of key immunomodulatory paracrine factors, mirroring the deficits observed in PD rat-BMMSCs. Further evaluation by mixed lymphocyte reaction (MLR) assays provided strong evidence for the clinical relevance of these findings. A growing consensus supports the critical role of immunomodulation in governing MSC therapeutic potential (112,113). The ability of MSCs to suppress PBMC proliferation in vitro serves as a reliable indicator of their capacity to modulate inflammatory responses in vivo . Our data support the hypothesis that MSC immunomodulatory dysfunction is intrinsic to the PD disease state. The robust immunosuppressive activity of HC-iMSCs aligns with established MSC functions (114,115), whereas the inability of PD-iMSCs to effectively modulate immune responses suggests disease-related alterations in their regulatory mechanisms. This fundamental impairment in immunomodulatory capacity has critical implications for the design and expected efficacy of MSC-based therapeutic strategies in PD. Patient-specific immunomodulation experiments further corroborate this, revealing a hyperactivated state of PBMCs from PD patients and a reduced ability of their own MSCs to modulate immune responses, compared to MSCs from healthy controls. This constitutive hyperactivated immune state may contribute to the chronic inflammatory milieu characteristic of PD, and aligns with clinical observations of altered immune profiles in PD patients' peripheral blood, including elevated proinflammatory cytokines (116,117). Critically, our patient-matched experimental design revealed that PD-iMSCs exhibit compromised immunosuppressive capacity even when regulating their own immune cells. While PD-iMSCs retained some immunomodulatory function, their suppressive effect on autologous PBMCs was significantly weaker than that achieved by HC-iMSCs on their corresponding cells. The superior ability of HC-iMSCs to suppress PD patient PBMCs compared to the patients' own MSCs further underscores the therapeutic potential of healthy donor-derived MSCs in PD treatment. No previous studies have examined the immunomodulatory capacity of MSCs isolated from the bone marrow of PD patients, in part due to the invasive nature of bone marrow procurement and reduced cell yields resulting from impaired proliferation and self-renewal capacity. Our findings therefore suggest that MSC dysfunction may represent a primary upstream event contributing to the well-documented peripheral immune abnormalities in PD patients, including elevated effector and inflammatory T cells, altered monocyte protein expression profiles, and B cell populations skewed toward proinflammatory phenotypes (32,118,119). These findings align with the failure of recent clinical studies attempting autologous MSC transplantation in PD (51,120), and strongly support the rationale for allogeneic MSC therapeutic approaches using healthy donor sources, which may offer substantially greater therapeutic benefit. To validate these findings in vivo and directly test the therapeutic implications of MSC dysfunction, we transplanted PD-iMSCs in MPTP-treated PD rats and compared the effects against both healthy rat BMMSCs and MSCs derived from iPSCs of healthy individuals (HC-iMSCs). Both healthy rat BMMSCs and HC-iMSCs demonstrated superior neuroprotective and neurorestorative effects compared to PD-iMSCs, as assessed by more effective reduction of neuroinflammation and peripheral inflammation, enhanced dopaminergic neuron survival, and improved motor function. These in vivo findings further validate the impaired immunomodulatory capacity of PD-iMSCs observed in our in vitro studies, confirming their reduced ability to mitigate both neuroinflammation and peripheral inflammation. Beyond modulating the inflammatory environment, we found that healthy iMSC transplantation also promoted neurogenesis in the substantia nigra pars compacta (SNpc), characterized by increased FOXA2 and Ki67 expression, while PD-iMSCs failed to induce such regenerative responses. This finding aligns with our recent study demonstrating that neurogenesis occurs following the administration of small extracellular vesicles (sEVs) derived from human dental pulp stem cells (DPSCs) in the SNpc of MPTP-induced PD rats (77). Additionally, previous research has shown that the microneurotrophin BNN-20 can promote neurogenesis within this region (86). Human studies also support the potential for SNpc neurogenesis. Post-mortem analysis of PD patient brains revealed that adult human neural progenitors can be isolated from the substantia nigra and cultured in vitro under specific growth conditions (121). This study found that multipotent neural stem/progenitor cells reside within the substantia nigra, but lack essential factors required for neural differentiation in PD conditions when cultured independently. However, when co-cultured with human embryonic stem cell-derived neural progenitors, they successfully differentiated into both neurons and glia. By two weeks PMT (1week post-MSC transplantation), we observed significantly increased colocalization of the proliferative marker Ki67 with TH + neurons in the rat-BMMSC and HC-iMSC groups compared to PD-iMSCs. This corresponded with enhanced TH immunopositivity and improved motor function. The failure of PD-iMSC transplanted groups to suppress neuroinflammation likely accounts for the reduced neurogenesis and decreased TH + dopaminergic neurons, ultimately manifesting as lack of motor improvement. The relationship between inflammation and neurogenesis is well-established. Increased NLRP3 activation and TNF-α are individually associated with reduced neurogenesis (122–126), and chronic neuroinflammation negatively affects hippocampal neurogenesis and cognitive processes across the lifespan (127–129). Conversely, anti-inflammatory mediators (130,131), environmental enrichment, and exercise serve as positive modulators of adult hippocampal neurogenesis and associated cognitive function (132–134). Consistent with the neuroinflammation profile, HC-iMSC groups showed reduced CD4 + T-cell infiltration compared to PD-iMSCs, suggesting peripheral immune system modulation as reflected by decreased TNF-α levels in blood serum. Notably, serum TNF-α levels were reduced more dramatically than midbrain TNF-α levels, indicating that systemically administered iMSCs (through the intramuscular route) modulated peripheral immune cells earlier and more effectively than neuroinflammatory cells. Nevertheless, this data indicates that neuroinflammation and neurogenesis can indeed be modulated through systemic iMSC administration without requiring invasive direct brain delivery procedures. The reduced migration of PD-iMSCs to the brain following transplantation, indicated by lower PKH-positive cell levels in flow cytometry analysis, provides a mechanistic explanation for their diminished therapeutic efficacy, demonstrating that the impaired migration capacity of PD-iMSCs observed in transwell migration assays in vitro translates to reduced homing capacity to the SNpc region in vivo , further limiting their therapeutic potential. Taken together, these results demonstrate that the containment of both peripheral inflammation and neuroinflammation by healthy control iMSCs aids the reversal of behavioural impairments, promotes neurogenesis, and highlights their therapeutic superiority. This provides strong evidence that allogeneic iMSC sources may critically enhance the therapeutic efficacy in PD. Beyond therapeutic implications, the correlation between MSC dysfunction and PD progression also suggests several important mechanistic insights. The early appearance of MSC impairments in the pre-motor stage implies that yet unknown systemic factors associated with PD pathogenesis may directly affect MSC function, resulting in impairment of immunomodulation of peripheral immune cells, which in turn leads to failure in containing neuroinflammation. This hypothesis is supported by the elevated NLRP3 expression and sustained microglial activation observed in animals receiving PD-iMSCs compared to those receiving healthy iMSCs. The increase in dopaminergic neurons and corresponding improvement in motor function upon administration of healthy MSCs in the pre-motor stage suggest that the impairment in function of MSCs is not only an early pathological event but also may be critically involved in the advancement of the disease process. Impairment of MSCs is thus not simply a downstream consequence of advanced neurodegeneration, and compromised MSC function may directly contribute to the failure of endogenous neuroprotective mechanisms during the critical pre-motor phase of the disease. These findings fundamentally reframe our understanding of PD pathogenesis. Overall, these findings open several interesting avenues for the future clinical translation of MSC therapy in PD: Firstly, they suggest that autologous MSC therapy using cells from PD patients may have limited efficacy due to intrinsic cellular dysfunction. This challenges current clinical trial designs that predominantly use autologous approaches, and offers a possible explanation for several recent trial failures. Secondly, the mechanistic insights gained from this study suggest potential therapeutic targets for enhancing MSC function in PD. Strategies aimed at reducing oxidative stress, enhancing migration capacity and restoring immunomodulatory function in PD-MSCs could potentially improve their therapeutic efficacy. Thirdly, the immunomodulation assay using PD patient PBMCs and MSCs demonstrates a possible simple test to evaluate the effectiveness of MSCs in promoting immunomodulation under PD conditions. Finally, this study underscores a critical advancement in the emerging field of iPSC-derived cell therapy. By suggesting that HLA-matched allogeneic iMSCs might offer superior therapeutic benefits compared to autologous MSCs derived from PD patients, our findings provide compelling evidence supporting the shift toward allogeneic cell sources. This has potentially significant implications for improving the efficacy and consistency of stem cell-based treatments for neurodegenerative diseases. While this study provides comprehensive evidence for MSC dysfunction in PD, some limitations must be acknowledged. The relatively small number of human iPSC lines examined—two healthy and two sporadic PD lines—limits the generalizability of our findings across the heterogeneous PD patient population. Given the clinical and genetic diversity observed in PD, validation in larger cohorts representing different disease subtypes, stages, and demographic characteristics will be essential to confirm the universal nature of MSC dysfunction in PD. Additionally, while our study demonstrates clear functional impairments in PD-MSCs and their reduced therapeutic efficacy, the specific upstream molecular mechanisms underlying MSC dysfunction in PD require further elucidation. Understanding whether MSC impairment results from direct exposure to PD-associated pathological factors (such as α-synuclein species or inflammatory mediators) or represents an intrinsic cellular defect linked to the disease state will be crucial for developing targeted interventions. Future research should prioritize several key areas. First, identifying the molecular pathways and signalling cascades responsible for MSC impairment in PD will be essential for developing mechanism-based therapeutic strategies. Second, investigating approaches to enhance or restore PD-MSC function—such as preconditioning protocols, genetic modifications, or pharmacological interventions—could potentially salvage autologous cell therapy approaches. Third, it would be particularly valuable to examine whether PD-related genetic variants affect MSC physiology and function, thereby determining if MSC dysfunction is a shared feature across both sporadic and familial forms of PD. Such studies could also reveal whether genetic susceptibility factors influence MSC biology and contribute to disease heterogeneity. Finally, longitudinal studies tracking MSC function throughout disease progression, from pre-motor stages through advanced PD, could provide insights into the temporal relationship between MSC dysfunction and clinical deterioration, potentially identifying windows of opportunity for intervention. Conclusion This study establishes MSC dysfunction as both an early and comprehensive pathological feature of PD, occurring during the critical pre-motor phase when interventions may be most effective. Our findings reveal that MSC impairment encompasses multiple functional domains—including proliferation, survival, migration, differentiation, and most critically, immunomodulatory capacity—suggesting a fundamental alteration in MSC biology rather than isolated functional defects. The profound impairment of MSC immunomodulatory capacity represents a previously unrecognized contributor to PD pathogenesis and suggests that failure of endogenous MSC-mediated immunomodulation and neuroprotection may facilitate disease progression through perpetuation of chronic neuroinflammation and peripheral immune dysfunction. This mechanistic insight positions MSC dysfunction as a potential upstream event in the pathological cascade, rather than merely a consequence of advanced neurodegeneration. From a translational perspective, these findings provide a strong scientific rationale for allogeneic MSC therapeutic strategies using healthy donor sources, while raising important concerns about the efficacy of patient-derived autologous MSC approaches in PD treatment. The superior therapeutic efficacy demonstrated by healthy MSCs in both reducing inflammation and promoting neuroregeneration supports a paradigm shift toward allogeneic cell therapy sources for optimal clinical outcomes. Beyond their immediate therapeutic implications, this work advances the fundamental understanding of PD pathophysiology by identifying a novel cellular dysfunction that bridges the gap between peripheral immune abnormalities and central neuroinflammation. It also opens new avenues for both biomarker development, as MSC functional assays could potentially serve as disease monitoring tools, and therapeutic innovation in regenerative medicine for neurodegenerative diseases. Declarations Data availability - All data generated or analysed during this study are included in this published article [and its supplementary information files]. Competing Interests - All authors declare no financial or non-financial competing interests. Acknowledgements - We acknowledge Dr. Manjunath, Department of Neurovirology, NIMHANS for access to Advanced Flow Cytometer facility. Author Contribution Statement - Conceptualization: I.D; methodology: R.G and I.D; formal analysis and investigation: R.G, I.D, K.M, R.Y, P.K.P, V.H and N.K; writing - original draft preparation: R.G and I.D; writing-review and editing: I.D, R.G; R.Y, P.K.P, V.H and N.K; funding acquisition: I.D, R.Y, and P.K.P, V.H and N.K; resources: I.D, R.Y, P.K.P, V.H and N.K; supervision: I.D. All authors read and approved the final manuscript. Funding - This work is supported by a grant obtained from DBT-BIRAC Biomanufacturing for Precision Biotherapeutics Government of India, New Delhi; contract grant No. 59080 by I.D and R.Y. R.G is supported by CSIR fellowship and K.M by DHR-ICMR YSS fellowship. References Costa HN, Esteves AR, Empadinhas N, Cardoso SM. Parkinson’s Disease: A Multisystem Disorder. Neurosci Bull. 2022 Aug 22;39(1):113–24. Alexander GE. 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Tables Table 1: Table for materials PRODUCT COMPANY CATALOG NUMBER MPTP Sigma Aldrich 5063820001 DPBS Thermo Fisher Scientific (Gibco) 21600010 Tri-sodium citrate Himedia TC249 Formamide Himedia MB012 Nacl Himedia MB023 Anti-Anti Thermo Fisher Scientific (Gibco) 15240062 GlutamaX Thermo Fisher Scientific (Gibco) 35050061 Penstrep Thermo Fisher Scientific (Gibco) 15140122 NEAA Thermo Fisher Scientific (Gibco) 11140050 Insulin-Transferrin-Selenium Thermo Fisher Scientific (Gibco) 51300044 2-Mercaptoethanol Thermo Fisher Scientific (Gibco) 21985023 Phytohemagglutinin, M Thermo Fisher Scientific (Gibco) 10576015 KnockOut™ DMEM Thermo Fisher Scientific (Gibco) 10829018 Neurobasal™ Plus Medium Thermo Fisher Scientific (Gibco) A3582901 Advanced DMEM/F-12 Thermo Fisher Scientific (Gibco) 12634028 DMEM/F-12 Thermo Fisher Scientific (Gibco) 21331020 RPMI 1640 Medium Thermo Fisher Scientific (Gibco) 11875093 Trypsin-EDTA (0.25%) Thermo Fisher Scientific (Gibco) 25200072 StemPro™ Accutase™ Thermo Fisher Scientific (Gibco) A1110501 FBS Thermo Fisher Scientific (Gibco) A5670701 KnockOut™ Serum Replacement Thermo Fisher Scientific (Gibco) 10828028 Normal Goat Serum Abcam ab7481 MitC Sigma Aldrich M4287 Matrigel Corning 356237 Geltrex™ Thermo Fisher Scientific (Gibco) A1413302 B-27™ Plus Supplement Thermo Fisher Scientific (Gibco) A3582801 N-2 Supplement Thermo Fisher Scientific (Gibco) 17502048 StemFlex™ Medium Thermo Fisher Scientific (Gibco) A3349401 PSC Neural Induction Medium Thermo Fisher Scientific (Gibco) A1647801 StemPro™ Adipogenesis Differentiation Kit Thermo Fisher Scientific (Gibco) A1007001 Dead Cell Apoptosis Kits with Annexin V for Flow Cytometry Invitrogen V13242 FGF2 Immunotools 11343627 Activin A Immunotools 11344963 PDGFBB Immunotools 11343673 FGF8 Immunotools 11344834 SHH Immunotools 11344074 GDNF Immunotools 11343795 BDNF Immunotools 11343373 TGF β Immunotools 11343160 Rat TNF alpha Recombinant Protein PeproTech® 400-14-20UG Human TNF-alpha Recombinant Protein Immunotools 11343013 Dibutyryl cyclic-AMP Sigma Aldrich D0260 L-Ascorbic Acid Sigma Aldrich A4544 L-Glutamine Sigma Aldrich G8540 Dexamethasone Himedia TCL211 Β-glycerophosphate Himedia TC463 Hisep LSM 1084 Himedia Hisep LSM 1077 Himedia Alizarin red S Himedia GRM894 Oil red O Himedia TC256 Toluidine Blue Himedia TC257 BODIPY™ 493/503 Invitrogen D3922 PKH26 Sigma Aldrich MINI26 H2DCFDA (H2-DCF, DCF) Invitrogen D399 DAPI Invitrogen D21490 Sheath Fluid BD FACSFlow™ 342003 Sodium azide Himedia TC704 Paraformaldehyde Sigma Aldrich 158127 TritonX Sigma Aldrich T8787 Bovine Serum Albumin Himedia MB083 Rat Dopamine ELISA Sunlong Biotech SL0243Ra Rat TNF alpha ELISA Sunlong Biotech SL0700Ra Rat PGE2 ELISA Krishgen Biosystems KLR0540 Rat TGF beta ELISA Krishgen Biosystems KLR0778 Rat IL-10 ELISA Sunlong Biotech SL0415Ra Rat IDO ELISA Sunlong Biotech SL1203Ra Rat MMP3 ELISA BT Lab E0316Ra Human TGF beta ELISA Sunlong Biotech SL1736Hu Human PGE2 ELISA Sunlong Biotech SL1463Hu Human IDO ELISA Sunlong Biotech SL0922Hu Human IL-10 ELISA Sunlong Biotech SL0967H Human Dopamine ELISA Immunotag™ IT11997 Table 2: Table for Antibodies (RRID) Sl No. Antibody Name Host Concerned titration Catalogue No. & RRID TH Mouse 1:100 cat# T1299 RRID: AB_477560 TH Rabbit 1:100 cat# PAB438Ra01 RRID: AB_3665786 p-Syn (S129) Rabbit 1:100 cat# PA1-4686 RRID: AB_2192960 IBA-1 Mouse 1:100 cat# MA5-27726 RRID: AB_2735228 TNF-Alpha Mouse 1:100 cat# 7124-MSM12-P1 RRID: AB_3714899 GFAP Rabbit 1:100 cat# PAA068Ra01 RRID: AB_3714900 NLRP3 Rabbit 1:100 cat# PA5-79740 AB_2746855 CD4 Mouse 1:100 cat# 14-0040-82 RRID: AB_953584 CD8a Mouse 1:100 cat# 14-0084-82 RRID: AB_1210523 Ki67 Rabbit 1:100 cat# MA5-14520 RRID: AB_10979488 CXCR4 Rabbit 1:100 cat# PAA940Ra01 RRID: AB_3714901 CD90 Mouse 1:100 cat# MAB404Hu21 RRID: AB_3714902 CD105 Rabbit 1:100 cat# PAA980Hu01 RRID: AB_3714903 CD73 Rabbit 1:100 cat# PAB250Hu01 RRID: AB_3714904 Runx2 Rabbit 1:100 cat# PA5-82787 RRID: AB_2789943 FOXA2 Rabbit 1:100 cat# PA5-35097 RRID: AB_2552407 CD90 Mouse 1:100 cat# 554895 RRID: AB_395586 CD73 Mouse 1:100 cat# 551123 RRID: AB_394057 EN1 Rabbit 1:100 cat# PA5-14149 RRID: AB_2231168 MAP2ab Mouse 1:100 cat# M2320 RRID: AB_609904 β-Tub III Rabbit 1:100 cat# ab18207 RRID: AB_444319 Nurr1 Rabbit 1:100 cat# PA5-13416 RRID: AB_2153896 VMAT2 Rabbit 1:100 cat# PA5-22864 RRID: AB_11154073 GIRK2 Rabbit 1:100 cat# ab259909 RRID: AB_3714905 APC-Cy™7 Mouse Anti-Human HLA-DR Mouse 1:100 cat# 335796 RRID: AB_399974 PE Mouse Anti-Human CD56 Mouse 1:100 cat# 347747 RRID: AB_400346 FITC Mouse Anti-Human CD45 Mouse 1:100 cat# 555482 RRID: AB_395874 PE Mouse Anti-Human CD34 Mouse 1:100 cat# 550761 RRID: AB_393871 FITC Mouse Anti-Human CD80 Mouse 1:100 cat# 557226 RRID: AB_396605 FITC Mouse Anti-Human CD86 Mouse 1:100 cat# 555657 RRID: AB_396012 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) Goat 1:200 cat# ab150113 RRID: AB_2576208 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) Goat 1:200 cat# ab150077 RRID: AB_2630356 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) Goat 1:200 cat# ab150115 RRID: AB_2687948 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) Goat 1:200 cat# ab150079, RRID: AB_2722623 Additional Declarations There is NO Competing Interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7957744","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":536152125,"identity":"0e827248-916f-439b-9fba-a42abd754c4f","order_by":0,"name":"Rituparna Ghanty","email":"","orcid":"","institution":"National Institute of Mental Health and Neurosciences","correspondingAuthor":false,"prefix":"","firstName":"Rituparna","middleName":"","lastName":"Ghanty","suffix":""},{"id":536152128,"identity":"74e3c114-aa55-4dc2-9d2a-704426e07b18","order_by":1,"name":"Kallolika Mondal","email":"","orcid":"","institution":"National Institute of Mental Health and Neurosciences","correspondingAuthor":false,"prefix":"","firstName":"Kallolika","middleName":"","lastName":"Mondal","suffix":""},{"id":536152129,"identity":"fa93757b-d338-4d6f-9c05-db6b0bae994e","order_by":2,"name":"Nitish Kamble","email":"","orcid":"","institution":"National Institute of Mental Health \u0026 Neurosciences","correspondingAuthor":false,"prefix":"","firstName":"Nitish","middleName":"","lastName":"Kamble","suffix":""},{"id":536152130,"identity":"8939672a-fc21-47fe-b34c-3f3ad7c0dc11","order_by":3,"name":"Ravi Yadav","email":"","orcid":"","institution":"National Institute of Mental Health \u0026 Neurosciences","correspondingAuthor":false,"prefix":"","firstName":"Ravi","middleName":"","lastName":"Yadav","suffix":""},{"id":536152131,"identity":"12c1ad82-d74e-40eb-ae7c-8e4a5acfbe83","order_by":4,"name":"Vikram Holla","email":"","orcid":"","institution":"National Institute of Mental Health and Neurosciences","correspondingAuthor":false,"prefix":"","firstName":"Vikram","middleName":"","lastName":"Holla","suffix":""},{"id":536152132,"identity":"23a85f0c-eb8e-4f6c-8d88-e05f09cd5e14","order_by":5,"name":"Pramod Pal","email":"","orcid":"","institution":"National Institute of Mental Health \u0026 Neurosciences","correspondingAuthor":false,"prefix":"","firstName":"Pramod","middleName":"","lastName":"Pal","suffix":""},{"id":536152124,"identity":"edd3a73a-44c2-4b60-b685-cc4d80dd425a","order_by":6,"name":"Indrani Datta","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8864-473X","institution":"National Institute Of Mental Health And Neurosciences","correspondingAuthor":true,"prefix":"","firstName":"Indrani","middleName":"","lastName":"Datta","suffix":""}],"badges":[],"createdAt":"2025-10-27 12:15:58","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7957744/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7957744/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94846352,"identity":"c24ff6a0-c6c6-49d2-a432-f245d609374a","added_by":"auto","created_at":"2025-10-31 10:13:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":588744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Representative confocal IHC images of the SNpc region in rat brain slices immunostained for TH⁺ neurons (green) and counterstained with 4’,6-diamidino-2-phenylindole (DAPI, blue). Images acquired at 10× magnification, Scale bar =100 µm; insets show cell bodies of TH⁺ dopaminergic neurons. \u003cstrong\u003eB\u003c/strong\u003e. Confocal IHC images representing phosphorylated P-syn (Ser 129) expression (Red) in SNpc region of rat midbrain, counterstained for TH+ neuron (green) and DAPI (Blue). Insets show magnified regions of the respective images.\u0026nbsp; Magnification 40X. Scale bar 24 µm. \u003cstrong\u003eC\u003c/strong\u003e. Representing number of TH+ neurons per field. Cell numbers were counted \u003cdel\u003emanually\u003c/del\u003e using multi-point tool in Image J. \u003cstrong\u003eD\u003c/strong\u003e. Showing dopamine levels in rat midbrain lysates measured by ELISA for control and PMT groups at different time points (n=6 biological replicates). Longitudinal measurement of olfactory discrimination \u003cstrong\u003e(E)\u003c/strong\u003e, nerve conduction velocity (NCV) \u003cstrong\u003e(F)\u003c/strong\u003e, rotarod performance \u003cstrong\u003e(G)\u003c/strong\u003e, and locomotor activity through actimeter \u003cstrong\u003e(H)\u003c/strong\u003e for control, MPTP treated groups. Statistical significance between Control versus WK1 PMT or WK2 PMT or WK3 PMT is denoted by *(* P\u0026lt;0.05, **P\u0026lt;0.005, ***P\u0026lt;0.001). \u003csup\u003e#\u003c/sup\u003e Represents significant difference between WK1 PMT versus WK2 PMT or WK3 PMT (\u003csup\u003e#\u003c/sup\u003e P\u0026lt;0.05, \u003csup\u003e## \u003c/sup\u003eP\u0026lt;0.005, \u003csup\u003e### \u003c/sup\u003eP\u0026lt;0.001). and ^ represents statistical significance between WK2 PMT versus WK3 PMT (^ P\u0026lt;0.05, ^^P\u0026lt;0.005, ^^^P\u0026lt;0.001) IHC studies were conducted with n = 6 biological replicates, and behavioural studies with n = 7 biological replicates. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA. F values for all the graphs: \u003cstrong\u003eC. \u003c/strong\u003eF (3,20) =54.29, \u003cstrong\u003eD.\u003c/strong\u003e F (3,20) =65.84, \u003cstrong\u003eE. \u003c/strong\u003eF (3,24) =29.68, \u003cstrong\u003eF. \u003c/strong\u003eF (3,24) =402.6, \u003cstrong\u003eG. \u003c/strong\u003eF (3,24) =59.03,\u003cstrong\u003e H.\u003c/strong\u003e F (3,24) =7.855.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7957744/v1/958bd0c780ca19bb0aac9596.png"},{"id":94985195,"identity":"350e1c2b-e315-4aca-8ff0-fcf1e7cd123f","added_by":"auto","created_at":"2025-11-03 06:57:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":901780,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative confocal IHC images of the rat midbrain substantia nigra pars compacta (SNpc) region showing: \u003cstrong\u003e(A)\u003c/strong\u003e IBA-1 (Alexa Fluor 647, red) with TH (green); \u003cstrong\u003e(B)\u003c/strong\u003e TNF-α (red) with IBA-1 (green); \u003cstrong\u003e(C)\u003c/strong\u003e GFAP (red) with TH (green); \u003cstrong\u003e(D)\u003c/strong\u003e NLRP3 (red) with TH (green); \u003cstrong\u003e(E)\u003c/strong\u003e CD4 (green); and \u003cstrong\u003e(F)\u003c/strong\u003e CD8 (green). All sections were counterstained with DAPI (blue). Scale bar =24µm.Insets show magnified regions of the respective images. \u003cstrong\u003eG.\u003c/strong\u003e Quantification of IBA-1⁺ cells per field in control and PMT groups. \u003cstrong\u003eH.\u003c/strong\u003e Pearson’s colocalization coefficient for TNF-α and IBA-1. \u003cstrong\u003eI. \u003c/strong\u003eTNF-α levels in rat midbrain lysates (ELISA). \u003cstrong\u003eJ.\u003c/strong\u003e TNF-α levels in peripheral blood serum (ELISA). \u003cstrong\u003eK, L.\u003c/strong\u003e Percentages of CD4⁺ and CD8⁺ cells, respectively, in the rat midbrain measured by flow cytometry. \u003cstrong\u003eM.\u003c/strong\u003e Quantification of NLRP3⁺ cells per field. Cell numbers were counted \u003cdel\u003emanually\u003c/del\u003e using multi-point tool in Image J.\u0026nbsp; Statistical significance was assessed between Control vs. WK1 PMT, WK2 PMT, or WK3 PMT (*P \u0026lt; 0.05, **P \u0026lt; 0.005, ***P \u0026lt; 0.001); WK1 PMT vs. WK2 PMT or WK3 PMT (\u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.005, \u003csup\u003e###\u003c/sup\u003eP \u0026lt; 0.001); and WK2 PMT vs. WK3 PMT (^P \u0026lt; 0.05, ^^P \u0026lt; 0.005, ^^^P \u0026lt; 0.001). Data represented as mean ± SD from n = 6 (biological replicates) for all graphs. Statistical significance was determined using one-way ANOVA. F values for all the graphs: \u003cstrong\u003eG. \u003c/strong\u003eF (3,20) =67.23, \u003cstrong\u003eH.\u003c/strong\u003e F (3,20) =19.67, \u003cstrong\u003eI. \u003c/strong\u003eF (3,20) =15.16, \u003cstrong\u003eJ. \u003c/strong\u003eF (3,20) =101.8, \u003cstrong\u003eK. \u003c/strong\u003eF (3,20) =356.7,\u003cstrong\u003e L.\u003c/strong\u003e F (3,20) =96.24, \u003cstrong\u003eM. \u003c/strong\u003eF (3,20) =64.61.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7957744/v1/9eaf6279e67608f4825f5120.png"},{"id":94846357,"identity":"9a0dc655-99fb-48dc-9716-66197744b29a","added_by":"auto","created_at":"2025-10-31 10:13:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":570392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Phase-contrast images of rat BMMSCs at Day 8 post-isolation from control and PMT groups (10× magnification; scale bar = 60 µm). \u003cstrong\u003eB. \u003c/strong\u003eQuantification of BMMSC cell area in pixels (3 pixel= 1µm) from control and PMT groups (n \u0026gt; 50 cells from six independent isolations per group). \u003cstrong\u003eC.\u003c/strong\u003e Represents Percentage of Ki67⁺ BMMSCs measured by flow cytometry in control and PMT groups (n= 5 Biological replicates). \u003cstrong\u003eD.\u003c/strong\u003e Representative images of toluidine blue–stained BMMSC colonies in 100 mm tissue culture dishes at P0 Day 7. \u003cstrong\u003eE.\u003c/strong\u003e Graphical Representation of number Colony-forming units (CFUs) per rat, counted manually using the multi-point tool in ImageJ. \u003cstrong\u003eF.\u003c/strong\u003e Flow cytometry analysis of total apoptotic BMMSCs (Annexin V⁺ + PI⁺) in control and PMT groups. \u003cstrong\u003eG.\u003c/strong\u003e Toluidine blue–stained BMMSCs that migrated through transwell membranes in response to TNF-α (25 ng/mL) (10× magnification; scale bar = 300 µm). \u003cstrong\u003eH.\u003c/strong\u003e Quantification of migrated BMMSCs per field. \u003cstrong\u003eI.\u003c/strong\u003e Percentage of CXCR4⁺ BMMSCs in control and PMT groups, assessed by flow cytometry. \u003cstrong\u003eJ.\u003c/strong\u003e Normalized DCF fluorescence intensity in 30k BMMSCs from control and PMT groups. \u003cstrong\u003eK-M.\u003c/strong\u003e Concentration of Matrix Metalloproteinase-3 (MMP3), Indoleamine 2,3-Dioxygenase (IDO), IL-10 and PGE2 in BMMSC conditioned media measured through ELISA. Statistical significance between Control group versus WK1 PMT or WK2 PMT or WK3 PMT is denoted by *(* P\u0026lt;0.05, **P\u0026lt;0.005, ***P\u0026lt;0.001). \u003csup\u003e# \u003c/sup\u003eRepresents significant difference between WK1 PMT versus WK2 PMT or WK3 PMT (\u003csup\u003e# \u003c/sup\u003eP\u0026lt;0.05,\u003csup\u003e ##\u003c/sup\u003e P\u0026lt;0.005, \u003csup\u003e###\u003c/sup\u003e P\u0026lt;0.001). and ^ represents statistical significance between WK2 PMT versus WK3 PMT (^ P\u0026lt;0.05, ^^P\u0026lt;0.005, ^^^P\u0026lt;0.001) n=6 (Biological replicates). For all graphs, Data represented as mean ± SD. Statistical significance was determined using one-way ANOVA. F values for all the graphs: \u003cstrong\u003eB. \u003c/strong\u003eF (3,214) =49.87, \u003cstrong\u003eC.\u003c/strong\u003e F (3,26) =398.7, \u003cstrong\u003eE. \u003c/strong\u003eF (3,20) =24.2, \u003cstrong\u003eF. \u003c/strong\u003eF (3,20) =206.6, \u003cstrong\u003eH. \u003c/strong\u003eF (3,20) =141.6,\u003cstrong\u003e I.\u003c/strong\u003e F (3,20) =99.54, \u003cstrong\u003eJ. \u003c/strong\u003eF (3,20) =494.5, \u003cstrong\u003eK. \u003c/strong\u003eF (3,20) =143.4, \u003cstrong\u003eL. \u003c/strong\u003eF (3,20) =11.66, \u003cstrong\u003eM. \u003c/strong\u003eF (3,20) =7.222, \u003cstrong\u003eN. \u003c/strong\u003eF (3,20) =155.5.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7957744/v1/62a289dccf4c881772494047.png"},{"id":94846353,"identity":"46a76e63-07cb-4f0d-92ff-a39aa316977b","added_by":"auto","created_at":"2025-10-31 10:13:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":690055,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Schematic representation of the iPSC-to-iMSC differentiation protocol. \u003cstrong\u003eB. \u003c/strong\u003eRepresentative phase-contrast images of P2 iMSCs from healthy control (HC) and PD groups (10× magnification; scale bar = 60 µm). \u003cstrong\u003eC.\u003c/strong\u003e Quantification of iMSC cell area (pixels; 3 pixels = 1 µm, n \u0026gt; 30 cells) in HC and PD groups. F (2,85) = 44.09. \u003cstrong\u003eD-F. \u003c/strong\u003eRepresentative confocal images of HC and PD iMSCs immunostained for CD73, CD105, and CD90 (Green) counterstained with DAPI (Blue) (40× magnification; scale bar = 24 µm). \u003cstrong\u003eG-I\u003c/strong\u003e. Flow cytometry histograms showing the percentage (mean ± SD) of CD73⁺, CD105⁺, and CD90⁺ cell populations (P2) in iMSCs. Statistical significance between HC03 iMSCs versus PD02 iMSCs or PD03 iMSCs is denoted by *(* P\u0026lt;0.05, **P\u0026lt;0.005, ***P\u0026lt;0.001). # Represents significant difference between PD02 iMSCs versus PD03 iMSCs (\u003csup\u003e#\u003c/sup\u003e P\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e P\u0026lt;0.005, \u003csup\u003e### \u003c/sup\u003eP\u0026lt;0.001). For each experiment, iMSCs differentiated from six independent iPSC clones were used as biological replicates (n=6) for each cell line (HC03, HC02, PD02, PD03). All data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7957744/v1/7e5edb0a8640036723d2a46a.png"},{"id":94985451,"identity":"0831ea77-4058-43f6-aa8f-0db1f3d2eb40","added_by":"auto","created_at":"2025-11-03 06:58:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":490838,"visible":true,"origin":"","legend":"\u003cp\u003e(A–C) Representative Oil Red O staining of uninduced and induced iMSCs demonstrating adipogenic differentiation (40× magnification, Scale bar 75 µm), (D–F) Alizarin Red S staining indicating osteogenic differentiation (10× magnification, Scale bar 300 µm), and (G–I) Toluidine Blue staining showing chondrogenic differentiation (10× magnification, Scale bar 300 µm) in HC03, PD02, and PD03 iMSCs (from top to bottom).\u003cstrong\u003e (J) \u003c/strong\u003eMedian fluorescence intensity (MFI) of BODIPY-stained adipocytes measured by flow cytometry.\u003cstrong\u003e (K) \u003c/strong\u003eQuantification of Runx2⁺ cells after osteogenic induction by flow cytometry. Statistical significance: HC03 iMSCs vs. PD02/PD03 iMSCs (*P\u0026lt;0.05, **P\u0026lt;0.005, *P\u0026lt;0.001); PD02 vs. PD03 iMSCs (\u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.005, \u003csup\u003e###\u003c/sup\u003eP\u0026lt;0.001). n = 6 biological replicates. All data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA. F values for the graphs: \u003cstrong\u003eJ. \u003c/strong\u003eF (2,15) =420.1, \u003cstrong\u003eK.\u003c/strong\u003e F (2,15) =56.7.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7957744/v1/46e7551208c437fc9130dbfc.png"},{"id":94985650,"identity":"d8d79d9d-c0c8-445e-910f-517f2cc65e5d","added_by":"auto","created_at":"2025-11-03 06:58:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":508980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eConfocal images of iMSCs immunostained for Ki67 (Green) and counterstained with DAPI (Blue). Scale bar = 24µm \u003cstrong\u003eB. \u003c/strong\u003equantitative representation of the ratio of Ki67/DAPI per field (8 fields). \u003cstrong\u003eC. \u003c/strong\u003eGraphical representation of of ki67+ iMSC population (Flowcytometry).\u0026nbsp; \u003cstrong\u003eD. \u003c/strong\u003ePercentage of total apoptotic cells (Annexin V\u003csup\u003e+ \u003c/sup\u003e+ PI\u003csup\u003e+\u003c/sup\u003e) (Flowcytometry). \u003cstrong\u003eE.\u003c/strong\u003e Toluidine Blue staining of iMSCs migrated through transwell membranes in response to hTNF-α (25 ng/mL) (10× magnification; scale bar = 300 µm). \u003cstrong\u003eF.\u003c/strong\u003e Quantification of migrated cells per field. \u003cstrong\u003eG. \u003c/strong\u003e\u0026nbsp;Percentage of PKH positive population in rat midbrain (Flowcytometry). \u003cstrong\u003eH. \u003c/strong\u003eRepresents\u003cstrong\u003e \u003c/strong\u003enormalized DCF fluorescence intensity in iMSCs. \u003cstrong\u003eI-J. \u003c/strong\u003eConcentration of IDO and prostaglandin E2 (PGE2) in iMSC conditioned media (ELISA). Statistical significance between HC03 iMSCs versus PD02 iMSCs or PD03 iMSCs is denoted by *(* P\u0026lt;0.05, **P\u0026lt;0.005, ***P\u0026lt;0.001). # Represents significant difference between PD02 iMSCs versus PD03 iMSCs \u003csup\u003e#\u003c/sup\u003e(\u003csup\u003e#\u003c/sup\u003e P\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e P\u0026lt;0.005, \u003csup\u003e### \u003c/sup\u003eP\u0026lt;0.001). In graph G, statistical significance is indicated as follows: WK2 PMT+HC03 iMSC versus WK2 PMT+HC02/PD02/PD03 iMSC is denoted by * (*P \u0026lt; 0.05, **P \u0026lt; 0.005, *P \u0026lt; 0.001); WK2 PMT+HC02 iMSC versus WK2 PMT+PD02/PD03 iMSC is denoted by ^ (^P \u0026lt; 0.05, ^^P \u0026lt; 0.005, ^^^P \u0026lt; 0.001); and WK2 PMT+PD02 iMSC versus WK2 PMT+PD03 iMSC is denoted by\u003csup\u003e #\u003c/sup\u003e (\u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.005, \u003csup\u003e###\u003c/sup\u003eP \u0026lt; 0.001). n=6 (Biological replicates). All data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA. F values for all the graphs: \u003cstrong\u003eB. \u003c/strong\u003eF (2,21) =262.9, \u003cstrong\u003eC.\u003c/strong\u003e F (2,15) =572, \u003cstrong\u003eD. \u003c/strong\u003eF (2,15) =2382, \u003cstrong\u003eF. \u003c/strong\u003eF (2,15) =555.3, \u003cstrong\u003eG. \u003c/strong\u003eF (3,20) =15.58 \u003cstrong\u003eH. \u003c/strong\u003eF (2,15) =398.4,\u003cstrong\u003e I.\u003c/strong\u003e F (2,15) =20.96, \u003cstrong\u003eJ. \u003c/strong\u003eF (2,15) =7.412.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7957744/v1/43f26e8255049850d5218019.png"},{"id":94846355,"identity":"c99b6376-3f86-499a-830a-864c9f80fad3","added_by":"auto","created_at":"2025-10-31 10:13:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":740158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA-D.\u003c/strong\u003ePhase contrast images of \u003cstrong\u003e(A)\u003c/strong\u003e PBMCs, 48h post isolation. (\u003cstrong\u003eB) \u003c/strong\u003eMixed culture of PBMCs pooled from three donors at 24 h, 48 h, and 72 h following PHA treatment. (\u003cstrong\u003eC) \u003c/strong\u003ePBMCs cultured with or without iMSCs at Day 0 and \u003cstrong\u003e(D) \u003c/strong\u003eDay 1. Magnification 10x, Scale bar 60µm. \u003cstrong\u003eE. \u003c/strong\u003eFlow cytometry analysis showing the percentage of Ki67⁺ PBMCs. Statistical significance between PBMC only versus HC03 iMSCs or PD02 iMSCs or PD03 iMSCs is denoted by *(* P\u0026lt;0.05, **P\u0026lt;0.005, ***P\u0026lt;0.001). n=6 (Biological replicates). Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA. F value for graph \u003cstrong\u003eE. \u003c/strong\u003eF (3,20) =127.7.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7957744/v1/1cf26263cb5760ad2403a972.png"},{"id":94846361,"identity":"0b919072-3ba4-4720-95dd-c06e6fb82165","added_by":"auto","created_at":"2025-10-31 10:13:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":635560,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Phase contrast images of PBMCs isolated from healthy controls and PD patients, from whom iPSC lines were generated. \u003cstrong\u003eB.\u003c/strong\u003e PBMCs at 72 h post-PHA induction. \u003cstrong\u003eC–D\u003c/strong\u003e. Phase contrast images of PBMCs cultured without iMSCs at Day 0 \u003cstrong\u003e(C) \u003c/strong\u003eand 24 h post-PHA treatment \u003cstrong\u003e(D)\u003c/strong\u003e. \u003cstrong\u003eE–F.\u003c/strong\u003e Phase contrast images of PBMC+iMSC co-cultures: HC03 PBMCs+HC03 iMSCs, PD02 PBMCs+PD02 iMSCs, PD02 PBMCs+HC03 iMSCs, PD03 PBMCs+PD03 iMSCs, and PD03 PBMCs+HC03 iMSCs (from left to right) at Day 0 \u003cstrong\u003e(E) \u003c/strong\u003eand Day 1 \u003cstrong\u003e(F)\u003c/strong\u003e post-PHA treatment. All images at 10× magnification; scale bar = 60 µm. (G) Flow cytometry analysis of Ki67⁺ PBMCs. Statistical significance between HC03 PBMC only vs PD02 PBMC only or PD03 PBMC only or HC03 iMSCs+HC03 PBMCs is denoted by *(* P\u0026lt;0.05, **P\u0026lt;0.005, ***P\u0026lt;0.001). PD02 PBMC Only vs. PD02 iMSCs+PD02 PBMCs is denoted by ^(^P\u0026lt;0.05, ^^ P\u0026lt;0.005, ^^^P\u0026lt;0.001).\u0026nbsp; PD02 PBMC Only vs. HC03 iMSCs+PD02 PBMCs is denoted by \u003csup\u003e# \u003c/sup\u003e(\u003csup\u003e#\u003c/sup\u003e P\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e P\u0026lt;0.005, \u003csup\u003e###\u003c/sup\u003e P\u0026lt;0.001), PD03 PBMC only vs. HC03 iMSCs+PD03 PBMCs is denoted by \u003csup\u003e$\u003c/sup\u003e(\u003csup\u003e$\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e$$\u003c/sup\u003e P\u0026lt;0.005, \u003csup\u003e$$$\u003c/sup\u003eP\u0026lt;0.001). PD03 PBMC Only vs. PD03 iMSC+PD03 PBMCs is denoted by \u003csup\u003e@ \u003c/sup\u003e(\u003csup\u003e@\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e@@\u003c/sup\u003e P\u0026lt;0.005, \u003csup\u003e@@@\u003c/sup\u003eP\u0026lt;0.001). HC03 iMSCs+PD02 PBMCs vs. PD02 iMSCs+PD02 PBMCs is denoted by ~ (~P\u0026lt;0.05, ~ ~ P\u0026lt;0.005, ~~~P\u0026lt;0.001). HC03 iMSCs+PD02 PBMCs vs. PD03 iMSCs+PD03 PBMCs is denoted by \u003csup\u003e€ \u003c/sup\u003e(\u003csup\u003e€\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e€€\u003c/sup\u003eP\u0026lt;0.005, \u003csup\u003e€€€\u003c/sup\u003e P\u0026lt;0.001). n=4 (biological replicates). All data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA.F value for graph \u003cstrong\u003eG\u003c/strong\u003e is F(7,24)=144.7.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7957744/v1/eef023652c59c7b1a76e5f86.png"},{"id":94846362,"identity":"ddf37b39-fd57-4655-b230-a6b48d29645d","added_by":"auto","created_at":"2025-10-31 10:13:27","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":769859,"visible":true,"origin":"","legend":"\u003cp\u003e(A–D) Representative confocal IHC images of rat midbrain sections showing (A) IBA-1⁺ microglia (red) with TH⁺ neurons (green), (B) GFAP⁺ astrocytes (red) with TH⁺ neurons (green), (C) TNF-α (red) with IBA-1⁺ cells (green), and (D) NLRP3 (red) with TH⁺ neurons (green). All sections were counterstained with DAPI (blue). Images are shown for control, WK2 PMT, healthy rBMMSC-transplanted, and HC03-, PD02-, or PD03-iMSC-transplanted groups at Day 14 PMT (from left to right). Magnification: 40×; scale bar = 24 µm. (E) Quantification of IBA-1⁺ cells per field. (F) TNF-α levels in rat midbrain lysates and (G) in peripheral blood serum of control, WK2 PMT, healthy rBMMSC-transplanted, and HC03-, PD02-, or PD03-iMSC-transplanted groups measured by ELISA. Statistical significance: Control vs. Wk2 PMT or rBMMSC/HC03/HC02/PD02/PD03 iMSC-transplanted groups (*P\u0026lt;0.05, **P\u0026lt;0.005, *P\u0026lt;0.001); Wk2 PMT vs. rBMMSC/HC03/HC02/PD02/PD03 iMSC-transplanted groups (\u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.005, \u003csup\u003e###\u003c/sup\u003eP\u0026lt;0.001); rBMMSC vs. HC03/HC02/PD02/PD03 iMSC-transplanted groups (^P\u0026lt;0.05, ^^P\u0026lt;0.005, ^^^P\u0026lt;0.001); HC03 iMSC vs. HC02/PD02/PD03 iMSC-transplanted groups (\u003csup\u003e@\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e@@\u003c/sup\u003eP\u0026lt;0.005, \u003csup\u003e@@@\u003c/sup\u003eP\u0026lt;0.001). HC02 iMSC vs. PD02/PD03 iMSC-transplanted groups (\u003csup\u003e$\u003c/sup\u003eP\u0026lt;0.05,\u003csup\u003e $$\u003c/sup\u003eP\u0026lt;0.005, \u003csup\u003e$$$\u003c/sup\u003eP\u0026lt;0.001). No significant differences were observed between PD02- and PD03-iMSC-transplanted groups. n=6 (Biological replicates). All data are presented as mean ± SD. F values for all the graphs: \u003cstrong\u003eE. \u003c/strong\u003eF (6,35) =28.84, \u003cstrong\u003eF.\u003c/strong\u003e F (6,35) =36.3, \u003cstrong\u003eG. \u003c/strong\u003eF (6,35) =185.7.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7957744/v1/92d2322348ac9a08080abd8d.png"},{"id":94846359,"identity":"a31ea6de-1c30-41ce-98b1-2a80a9694746","added_by":"auto","created_at":"2025-10-31 10:13:27","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":844522,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA-i to F-i.\u0026nbsp; \u003c/strong\u003eRepresentative confocal IHC images of rat midbrain showing FOXA2 expression (red) in TH⁺ neurons (green) in (A) Control, (B) Wk2 PMT, (C) healthy rat BMMSC-transplanted, and (D) HC03-, (E) PD02-, and (F) PD03 iMSCs -transplanted groups at the D14 PMT timepoint. \u003cstrong\u003eA-ii to F-ii. \u003c/strong\u003econfocal IHC images showing Ki67 (Red) expression in TH+ neurons (Green) across the same group at same timepoint. All sections were counterstained with DAPI (blue). 40x magnification. Scale bar 24µm. Insets showing magnified regions of the respective images. \u003cstrong\u003eC-iii to F-iii. \u003c/strong\u003eConfocal images of SNpc region of rat midbrain section showing expression of TH+ neurons (Green) at 10x magnification, Scale bar =100µm. \u003cstrong\u003eG. \u003c/strong\u003eNumber of FOXA2+ cells per field. \u003cstrong\u003eH. \u003c/strong\u003eGraph representing ratio the of TH+Ki67+ Cells with total TH + cells per field\u003cstrong\u003e. I. \u003c/strong\u003eQuantification of total number of TH+ neuron per field (10x magnification). \u003cstrong\u003eJ. \u003c/strong\u003eConcentration of Dopamine in rat midbrain lysate measured through ELISA.\u003cstrong\u003e K. \u003c/strong\u003eGraphical representation of the measurement of Rotarod Performance of Control, wk2 PMT, and HC03/HC02/PD02/PD03 iMSC transplanted rats. Statistical significance: Control vs. Wk2 PMT or rBMMSC/HC03/HC02/ PD02/PD03 iMSC-transplanted groups (*P\u0026lt;0.05, **P\u0026lt;0.005, *P\u0026lt;0.001); Wk2 PMT vs. rBMMSC/HC03/HC02/PD02/PD03 iMSC-transplanted groups (\u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.005, \u003csup\u003e###\u003c/sup\u003eP\u0026lt;0.001); rBMMSC vs. HC03/HC02/PD02/PD03 iMSC-transplanted groups (^P\u0026lt;0.05, ^^P\u0026lt;0.005, ^^^P\u0026lt;0.001); HC03 iMSC vs. PD02/HC02/PD03 iMSC-transplanted groups (\u003csup\u003e@\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e@@\u003c/sup\u003eP\u0026lt;0.005, \u003csup\u003e@@@\u003c/sup\u003eP\u0026lt;0.001); HC02 iMSC vs. PD02/PD03 iMSC-transplanted groups (\u003csup\u003e$\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e$$\u003c/sup\u003eP\u0026lt;0.005, \u003csup\u003e$$$\u003c/sup\u003eP\u0026lt;0.001) n=6 (Biological replicates). Statistical significance was determined using one-way ANOVA. All data are presented as mean ± SD. F values for all the graphs: \u003cstrong\u003eG. \u003c/strong\u003eF (6,35) =63.53, \u003cstrong\u003eH.\u003c/strong\u003e F (6,35) =63.53, \u003cstrong\u003eI. \u003c/strong\u003eF (5,30) =71.53, \u003cstrong\u003eJ. \u003c/strong\u003eF (6,35) =29.12, \u003cstrong\u003eK. \u003c/strong\u003eF (5,30) =128.5.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-7957744/v1/3e858ad06e631130b4a4da31.png"},{"id":95798028,"identity":"e9189f34-fda2-4431-b97b-c55aab199f2a","added_by":"auto","created_at":"2025-11-13 08:14:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7884036,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7957744/v1/fca2692e-f6b7-4a3a-b8c3-629ed215cb7a.pdf"},{"id":94984965,"identity":"cc51a020-8c40-414a-95f9-4ceb0ba846c6","added_by":"auto","created_at":"2025-11-03 06:57:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9951344,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7957744/v1/3e70b0681c36bf24b8f22793.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Pre-motor Mesenchymal stromal Cell Dysfunction Drives Immune Dysregulation in Parkinson’s Disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026apos;s disease (PD) represents the second-most prevalent age-related progressive neurodegenerative disorder, affecting not only the central nervous system but also the peripheral nervous system, gastrointestinal tract, and adaptive immune system, justifying its classification as a multisystem disorder (1,2). The classical motor symptoms of PD primarily result from the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) in the midbrain. This pathological process begins decades before clinical manifestation, with approximately 70% of these neurons already lost by the time motor symptoms appear (3).\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is now understood that central to PD pathogenesis is the establishment of a self-perpetuating cycle of chronic neuroinflammation. While the etiology of this remains multifactorial, involving genetic predisposition, environmental toxins, and aging (4), transient initiation factors such as chemical insults, infections, particulate matter, and pesticides are known to trigger sustained microglial activation. This results in the increased production of chemokines, cytokines, reactive oxygen/nitrogen species and adhesion molecules, further promoting dopaminergic neuronal death (5,6). While the causal relationship between neuroinflammation and neurodegeneration remains putative, extensive preclinical and clinical evidence demonstrates that chronic inflammation is integral to disease pathogenesis in both sporadic and familial forms of PD.\u003c/p\u003e\n\u003cp\u003eThe inflammatory milieu in PD extends beyond the central nervous system to encompass systemic immune dysfunction. Neurodegeneration is accompanied by microglial activation and T lymphocyte infiltration into the SNpc (7\u0026ndash;9). Elevated levels of pro-inflammatory cytokines including TGF-\u0026beta;1, IL-6 and IL-1\u0026beta; have been consistently demonstrated in both brain tissue and cerebrospinal fluid of PD patients, supporting a neurotoxic environment involving inflammasome activation across microglia, neurons, astroglia, CNS-associated macrophages, and infiltrating peripheral immune cells (10).\u003c/p\u003e\n\u003cp\u003ePeripheral immune alterations in PD patients are equally complex and heterogeneous. Multiple studies have documented increased neutrophil-to-lymphocyte ratios (NLR), elevated levels of effector and inflammatory T cells, altered monocyte protein expression profiles, and B cell populations skewed toward pro-inflammatory phenotypes with reduced regulatory subsets (11\u0026ndash;16). However, these changes exhibit considerable individual variation; for instance, the elevated NLR observed has been attributed to increased neutrophil counts or reduced lymphocyte levels, depending on the study (17\u0026ndash;20). Moreover, specific PD clinical subtypes (e.g., akinetic-rigid vs. tremor-dominant) and stages (e.g., PD with mild cognitive impairment) display distinct immune signatures (20\u0026ndash;23). Studies also report divergent findings on monocyte subtypes: while some show no difference (14,23\u0026ndash;27), others indicate an increase in pro-inflammatory classical monocytes in early PD (28\u0026ndash;30). T cell alterations, particularly reduced CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e counts, have also been observed, particularly in late-stage disease (31\u0026ndash;33).\u003c/p\u003e\n\u003cp\u003eThis heterogeneity in immune signatures\u0026nbsp;suggests that therapeutic strategies targeting individual immune cell populations may prove ineffective as a therapeutic strategy. Instead, the observed systemic immunomodulatory failure directs the attention toward upstream regulatory mechanisms. Bone marrow mesenchymal stromal cells (BMMSCs) represent ideal candidates for further investigation, given their master regulatory role in immune homeostasis. Through their extensive secretory profile, BMMSCs modulate both innate and adaptive immunity by controlling cytotoxic and Th17 T cell generation, inhibiting dendritic cell maturation, promoting M1-to-M2 macrophage polarization, and inducing protective regulatory T cells and dendritic cells\u0026nbsp;(34\u0026ndash;40). Beyond immunomodulation, MSCs also provide cellular protection by secreting growth factors, transferring mitochondria, and regulating reactive oxygen species\u0026nbsp;(41,42).\u003c/p\u003e\n\u003cp\u003eMSCs possess unique homing properties, allowing them to localize to sites of inflammation or injury\u0026mdash;even crossing the blood-brain barrier (BBB) under pathological conditions (43\u0026ndash;46). At such sites, MSCs modulate the inflammatory environment, reduce oxidative stress, and enhance cell survival via paracrine signalling, mitochondrial transfer, and trophic factor secretion. This multifactorial regulatory capacity uniquely positions BMMSCs as potential endogenous modulators of the chronic inflammatory cycle (47), characteristic of PD pathogenesis.\u003c/p\u003e\n\u003cp\u003eSome clinical observations further indirectly support MSC involvement in PD pathology. Osteoporosis and osteopenia affect up to 91% of women and 61% of men with PD (48), often manifesting in early disease stages. As BMMSCs serve as progenitors for osteocytes and regulate the balance between adipocyte and osteocyte formation, these bone abnormalities suggest underlying MSC dysfunction (49). Additionally, impaired nerve conduction velocity and pain symptoms in PD patients may relate to MSC-dependent regulation of Schwann cell function and myelination.\u003c/p\u003e\n\u003cp\u003eThe temporal dynamics of peripheral immune alterations in PD provide additional insight into disease progression. NLR increases start occurring years before PD diagnosis and correlate with disease duration and severity, while intermediate monocyte elevation and T cell deviations are associated with disease advancement. These findings suggest that immune dysfunction may precede motor symptom onset by years, highlighting the importance of characterizing the timeline of potential MSC impairment.\u003c/p\u003e\n\u003cp\u003eParadoxically, past clinical trials of \u003cem\u003eautologous\u003c/em\u003e MSC transplantation in PD have yielded disappointing results, with some studies reporting worsening of symptoms (50,51). This therapeutic failure further raises critical questions about the functional integrity of endogenous MSCs in PD patients. Precedents exist for MSC impairment in other chronic inflammatory conditions: functional deficits have been documented in MSCs from patients with rheumatoid arthritis and systemic lupus erythematosus, including diminished proliferative, angiogenic, and differentiation capacities (52\u0026ndash;54). Similarly, MSCs from progressive supranuclear palsy patients have been shown to exhibit significant mitochondrial dysfunction and reduced differentiation potential compared to healthy controls (55).\u003c/p\u003e\n\u003cp\u003eCurrent knowledge gaps limit our understanding of MSC involvement in PD pathogenesis. While chronic neuroinflammation is well-established and peripheral immune dysfunction documented, the functional status of endogenous MSCs throughout disease progression remains unmapped. The temporal relationship between MSC impairment and symptom onset is unknown, as is the extent to which MSC immunomodulatory capacity is compromised in PD patients. These uncertainties limit our understanding of endogenous BMMSC dysfunction in PD and impede the development of more effective cell-based therapeutic strategies.\u003c/p\u003e\n\u003cp\u003eTracking the early phase of PD in human patients presents significant challenges, as clinical diagnosis typically occurs only after motor symptoms have already appeared. The invasive methods required for BMMSC isolation from living subjects present additional barriers to research. However, the advent of patient-derived induced pluripotent stem cells (iPSCs) offers a powerful alternative approach. iPSCs derived from both familial and idiopathic PD patients recapitulate disease-relevant phenotypes in neurons, astrocytes, microglia, and macrophages (56\u0026ndash;60). Particularly noteworthy is that differentiated dopaminergic neurons from idiopathic PD patient-derived iPSCs retain age-associated dysfunctions such as impaired chaperone-mediated autophagy (61\u0026ndash;64). While iPSC-derived MSCs (iMSCs) have been successfully generated in other research contexts, their immunomodulatory capacity specifically in PD remains entirely unexplored.\u003c/p\u003e\n\u003cp\u003eThis study therefore aims to comprehensively characterize the temporal and functional impairment of bone marrow mesenchymal stromal cells in PD through three complementary approaches: (1) assessment of functional integrity of endogenous BMMSCs with regard to neuroinflammation and peripheral inflammation using chronic PD rat models, (2) determination of the precise timing of MSC impairment onset relative to motor dysfunction development, and (3) evaluation of immunomodulatory capacity of hiPSC-derived MSCs (iMSCs) from PD patients versus healthy controls. These investigations will help establish a mechanistic basis for autologous BMMSC dysfunction and inform the development of improved cell-based therapeutic strategies in PD.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval and study design.\u0026nbsp;\u003c/strong\u003eThe protocols and procedures were ethically reviewed and approved by the Institutional Animal Ethics Committee (IAEC) for the use of male Wistar rats (AEC/77/506/B.P/2023-02), the Institutional Biosafety Committee (IBSC) for employing MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) (NIMHANS-IBSC/25\u003csup\u003eth\u003c/sup\u003e Nov.2022/01) as an inducing agent to establish a sporadic Parkinson\u0026apos;s disease model in rats, and the Institutional Committee of Stem Cell Research (IC-SCR) for utilizing induced pluripotent stem cell (iPSC) lines (SEC/07/035/BP) derived from healthy controls and sporadic PD patients. These approvals were obtained at National Institute of Mental Health and Neurosciences (NIMHANS), India.\u003c/p\u003e\n\u003cp\u003eThe sample size for each experiment was determined according to established experimental standards. For all in vivo animal studies and rat primary cultures, a minimum of 5\u0026ndash;7 biological replicates (n \u0026ge; 5\u0026ndash;7) was maintained per group. For iPSC cultures, six independent clones from individual lines were used to ensure at least 4-5 biological replicates (n \u0026ge; 4-5). These included two sporadic PD lines [NIMHi002-A (referred to as PD02) and NIMHi003-A (referred to as PD03)] and two age- and sex-matched healthy control lines [NIMHAi006-A (referred to as HC03) and NIMHAi005-A (referred to as HC02)], all of which have been previously reported in (65\u0026ndash;68). For phase-contrast and confocal imaging, 2\u0026ndash;3 independent fields were analysed per replicate, with images acquired 100 \u0026micro;m apart to avoid resampling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal model:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in compliance with the ARRIVE 2.0 guidelines. Male Albino Wistar rats, 2.5 months old and weighing 280\u0026ndash;300 g, were used for all in vivo experiments. The animals were bred, maintained, and obtained from the Central Animal Research Facility (CARF), NIMHANS, in accordance with the Institutional Animal Ethics Committee (IAEC), NIMHANS, Bengaluru, and the regulations of the Committee for the Control and Supervision of Experiments on Animals (CCSEA), India. Rats were housed under controlled laboratory conditions (22 \u0026plusmn; 1 \u0026deg;C, 12-h light/dark cycle, 55\u0026ndash;60% humidity) with free access to standard laboratory rat chow and water. Age- and weight-matched rats received a single bilateral intranasal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine at 0.1 mg per nostril, while control animals received an equivalent volume of saline (69). The animals were divided into the following groups:\u003c/p\u003e\n\u003cp\u003e1. Control (Bi-lateral intra-nasal saline administration) (3.5 Months old)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2. 1\u003csup\u003est\u003c/sup\u003e week post MPTP treatment (Wk1 PMT)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3. 2\u003csup\u003end\u003c/sup\u003e week post MPTP treatment (Wk2 PMT)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4. 3\u003csup\u003erd\u003c/sup\u003e week post MPTP treatment (Wk3 PMT)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe number of animals used for each experiment specified in the corresponding figure legends as biological replicates. No experimental units were excluded. Rats were anesthetized with ketamine and xylazine (80:5 mg/kg, intraperitoneally) for experimental procedures and euthanized by isoflurane overdose followed by decapitation for terminal sample collection. Tissues collected included bone marrow for mesenchymal stromal cell isolation, blood serum, and brain samples for analyses such as midbrain neurotransmitter quantification, TNF-\u0026alpha; measurement, and immunohistochemistry. Blood for serum isolation was obtained via cardiac puncture, while brain samples for immunohistochemical analysis were collected following transcardial perfusion of anesthetized rats. Behavioural assessments were performed to determine the onset of non-motor and motor symptoms. \u0026nbsp;All animal experiments were conducted by multiple investigators. Random numbers were generated by the first investigator (ID), who was the only one aware of group allocations. Experimental procedures and data analyses were performed by RG and KM. All protocols were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of NIMHANS, Bengaluru, India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavioural studies:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOlfactory Discrimination:\u0026nbsp;\u003c/strong\u003eThe olfactory discrimination ability of rats was assessed following the method described in (70). Briefly, each rat was placed in a two-compartment cage (30 \u0026times; 30 \u0026times; 20 cm) connected by an open doorway, allowing free movement between an unfamiliar compartment containing fresh husk and a familiar compartment with the husk collected from the rat\u0026rsquo;s home cage over the preceding 48 hours. Each trial lasted 5 minutes. The time spent in the familiar compartment (in minutes) was recorded and presented as mean \u0026plusmn; SD for n = 7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNerve Conduction Velocity:\u0026nbsp;\u003c/strong\u003eNerve conduction velocity (NCV) was measured non-invasively as previously described (71). Recordings were obtained and analyzed using the iWorx data acquisition system and LabScribe software (iWorx Systems, Inc., USA). Rats were anesthetized with ketamine: xylazine (80 mg/kg:5 mg/kg, i.p.) and placed on a heated platform to maintain body temperature. The sciatic nerve was stimulated at the sciatic notch with a supramaximal stimulus of 8 V at 20 Hz, and recordings were made from the first interosseous muscle of the hind paw. Latency was determined as the time from the stimulus artifact to the onset of the negative M-wave deflection. NCV was calculated using the formula: NCV\u0026nbsp;(mm/ms) = distance\u0026nbsp;(mm) / proximal\u0026nbsp;latency\u0026nbsp;(t1)\u0026nbsp;\u0026ndash;\u0026nbsp;distal\u0026nbsp;latency\u0026nbsp;(t2). where the distance represents the length between the stimulation site and the recording electrodes. Values were expressed as mean \u0026plusmn; SD for n = 7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRotarod:\u0026nbsp;\u003c/strong\u003eFor this study,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eeach rat underwent training with three 30-minute trials per day at a speed of 15 rpm for two consecutive days, followed by 25 rpm on the third day for acclimatization. On the fourth day, during the test session, rats were placed on the rotarod (Rotamex-5 system, Columbus Instruments), and their performance time was recorded for 15 mins (900 sec) (72). Rotarod performance time (s) was expressed as mean \u0026plusmn; SD for n = 6-7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIR actimeter:\u003c/strong\u003e The digital IR actimeter was used to test the locomotor activity of PD rats in which a continuous beam of light falls on photoelectric cells. The apparatus comprises of a frame provided with 16 IR source on X axis and 16 IR source on Y axis creating a 16x16 grid. The instrument control panel displays the number of beam brakes by the animal on all axis and total of all in the actimeter. Any interruption in the continuity of light by the animal was recorded on a digital counter in the form of counts which corresponds to the locomotor activity. The animals from each group were individually placed in the apparatus and allowed to move freely for 5 mins while the beam breaks were recorded automatically and report generated digitally. Measurements were plotted as mean \u0026plusmn; SD for n=7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRat Bone marrow mesenchymal stromal cell isolation and culture:\u0026nbsp;\u003c/strong\u003eThe BMMSC isolation protocol was adapted from our previously published report (71) with slight modifications.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eRats from each experimental group were euthanized and hind limb bones were dissected and washed in 1X PBS with 1% anti-anti. To collect bone marrow, a femur and a tibia were placed knee-end down into a 2 ml centrifuge tube with a small hole at the bottom. This 2 ml tube was then nested inside a 15 ml centrifuge tube. The setup was centrifuged at 4000 rpm for 10 min. Upon centrifugation, the 2 ml tube holding the bones was removed with the help of sterile forceps. The pellet was resuspended in equal volume of 1x Phosphate Buffered Saline (PBS), layered onto HiSep\u0026trade; LSM 1084 in the ratio 1:1, and centrifuged at 500\u003cem\u003eg\u0026nbsp;\u003c/em\u003efor 20 min in brake-free setting. The resultant buffy coat of mononuclear cells was collected, washed once in complete MSC media consisting of Knockout Dulbecco\u0026apos;s Modified Eagle\u0026apos;s Medium (KO-DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1% Anti-Anti and 1% GlutamaX. After wash cells were resuspend and plated in a T75 culture flask containing same MSC media at 37 ◦C with 5% CO2 in a humidified incubator. After 48h, non-adherent cells were removed and the adherent cells were cultured till P1 with subsequent media changes on every 72h.\u0026nbsp;BM-MSCs were identified by their plastic-adherence property and spindle-shaped morphology using a phase contrast microscope (Olympus CKX41) and characterized by CD73 and CD90 marker expression by immunophenotyping and immunostaining.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eiPSC culture and differentiation into Mesenchymal Stromal Cells and Dopaminergic neurons:\u0026nbsp;\u003c/strong\u003eFor this study, six clones of previously reported iPSC lines were utilized. The iPSCs were cultured on mitomycin-treated mouse embryonic fibroblast (iMEF) feeder layer in complete iPSC media. The media consisted of 20% KnockOut Serum Replacement (KOSR), 1% Penicillin-Streptomycin (Pen/Strep), 1% GlutaMAX, 1% Non-Essential Amino Acids (NEAA), 0.2% \u0026beta;-ME, and DMEM/F12 as the basal medium supplemented with 10 ng/mL human FGF2 (hFGF2). The culture media was changed daily.\u003c/p\u003e\n\u003cp\u003eFor iMSC (iPSC derived MSC) differentiation, iPSCs were used between P10 to P15. \u0026nbsp;iMSCs were differentiated by specific factors in a single step differentiation method followed by selection and maintenance. On day -1 undifferentiated iPSC colonies were manually picked up on to Matrigel coated 35mm dish in complete stemflex medium consisting of stemflex supplement, stemflex basal, 1% Pen/strep and 1% Glutamax. On day 0\u003cstrong\u003e,\u003c/strong\u003e Stemflex media was replaced by MSC differentiation media consisting of DMEM F12 basal medium supplemented with 1% B27(1X) ,1% Penicillin-streptomycin, 1% Glutamax, 10ng/ml FGF2, 2ng/ml Activin A, and 10ng/ml PDGFBB (Platelet derived growth factor -BB). Media was changed daily till day 4. At days 5, when over 80% of the culture plate\u0026apos;s surface area was covered with spindle-shaped cells, differentiation media was withdrawn and cells were shifted to MSC maintenance media consist of KO-DMEM as basal media, supplemented with 10% FBS, 1% Pen/strep, 1% GlutamaX, 1% NEAA and 10ng/ml FGF2 and maintained till day 7. At this stage cells were passaged using 0.25% trypsin-EDTA into1:5 ratio in non-coated tissue culture plastic plates and iMSCs were allowed to adhere for 10-12 h. As MSCs have inherent plastic adherent property, non-adherent cells were removed by giving media change and adhered fibroblastic cells were expanded. For validation of successful differentiation, differentiated cells were characterized as per ISCT (International Society for Cell \u0026amp; Gene therapy) guidelines for mesenchymal stromal cell characterization\u0026nbsp;(73). For all the experiments, P3-P4 cells were used.\u003c/p\u003e\n\u003cp\u003eDopaminergic neuron differentiation protocol is previously reported from our group (67). Briefly, the iPSC colonies were picked manually and plated on Geltrex coated 35mm plates in a Stemflex medium at a density of 2.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e\u0026minus;3.0\u0026times; 10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ecells/well and switched to Neural Induction Media (NIM) to differentiated to NPs. For floor plate cell (FPC) generation, NPs were treated with 25 ng/mL FGF-8 in NEM for 7 days. Final differentiation to DA neuron was induced using neurobasal/Adv. DMEM-F12 media supplemented with SHH (10 ng/mL), FGF-8 (25 ng/mL), BDNF (20 ng/mL), GDNF (10 ng/mL), N2 (1X), B27 (1X), ascorbic acid (0.2 Mm) , ITS (1X) , and db-CAMP (50\u0026mu;M), with media changes on alternate days. On day 6, cells were switched to maturation media with SHH reduced to 1 ng/mL. DA neurons were differentiated from HC03, PD02 and PD03 iPSC lines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrilineage differentiation of iMSCs:\u0026nbsp;\u003c/strong\u003eTo assess the multipotent nature of iMSCs, their osteogenic, adipogenic, and chondrogenic properties were evaluated. For osteogenic differentiation of iMSCs, the cells were plated in 24-well plates with a cell density of 3,000 cells/cm\u003csup\u003e2\u003c/sup\u003e. Upon 90% confluency, they were induced with the osteogenic differentiation medium consisting of MSC maintenance media supplemented with 5 mM glutamine, 10\u003csup\u003e\u0026ndash;8\u003c/sup\u003e M dexamethasone, 50 mg/ml ascorbic acid and 10 mM b-glycerophosphate. Media was changed every alternative day. After 21 days of induction, the calcium mineralization was assessed by Alizarin red S staining\u0026nbsp;(74). For adipogenic induction, Stempro\u0026trade; adipogenic differentiation kit was used. Cultures were checked on regular basis for change in cell shape and the appearance of lipid droplets. The presence of lipid droplets was then confirmed by Oil red O staining after 15days of induction. For chondrogenic differentiation of iMSCs, the cells were plated in 24-well plates with a cell density of 3,000 cells/cm\u003csup\u003e2\u003c/sup\u003e in chondrogenic differentiation medium containing KO-DMEM as basal media supplemented with 1% pen/strep, 1% GlutamaX, 10% FBS and 10ng/ml TGF\u0026beta;1 in a rocker inside incubator at 37\u0026ordm;C, 5% CO\u003csub\u003e2\u003c/sub\u003e to form micro mass for 21 days. The presence of cartilaginous matter was confirmed by toluidine blue staining after 3 weeks of induction (75). Uninduced iMSCs were used as control for this experimental setup.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBODIPY staining of adipocytes:\u0026nbsp;\u003c/strong\u003eOn the 21st day of adipogenic induction, cells were trypsinized and pelleted, then incubated with 2 \u0026micro;M BODIPY 493/503 in 1\u0026times; DPBS for 30 minutes at 37\u0026deg;C. Following incubation, excess dye was removed by washing twice with 1\u0026times; DPBS. The resulting single-cell suspension of cells was analysed by flow cytometry, with 10,000 events recorded per sample (76)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlowcytometry:\u0026nbsp;\u003c/strong\u003eFor flowcytometry assay, direct and indirect staining methods were used for fluorophore tagged or untagged primary antibodies respectively. For indirect staining method,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethe rat BMMSCs, iMSCs, and Osteocytes were dissociated with 0.25% Trypsin EDTA to achieve single cell suspension and fixed in 2% PFA prepared with 1X DPBS for 45min at Room temperature (RT). After fixing, PFA was removed by centrifuging cells at 1800 rpm for 5mins. Cell pellet was then resuspended in 1X DPBS containing 0.01% sodium azide (NaN3). 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were used per reaction. For intracellular marker (RUNX2) cells were permeabilized using 0.01% Triton X-100 for 30 min at room temperature in the dark followed by blocking with 3% bovine serum albumin (BSA) for 45 min at RT to avoid non-specific binding. For cell surface markers (CD90/THY-1, CD73, CD105, CD11b, CXCR4) permeabilization step was not performed. After blocking, cells were incubated overnight (~16h) with specific primary antibodies at 4\u0026ordm;C followed by incubation with respective secondary antibodies tagged with fluorophore-FITC for 90 mins at RT in dark. Each step is succeeded by a washing step in 1X DPBS, containing 0.01% sodium azide at 10,000 rpm for 10 mins. For direct staining method of cell surface markers (HLA-DR, CD45, CD80, CD86, CD56 and CD35) cells were fixed in PFA and treated with blocking reagent as mentioned before. After blocking, cells were incubated with fluorophore tagged (FITC or APC) antibodies for 45 mins at 4\u0026ordm;C in dark followed by two PBS washes. For flowcytometry acquisition, after the final wash, cells were resuspended in 500 \u0026micro;l of sheath fluid. FACS Verse (BD Biosciences) instrument was used for sample acquisition and 10,000 events were recorded per sample. Events with very low FSC and SSC were excluded by gating the cell population (P1). Isotype control (cells stained with only secondary antibody) was used to gate (P2) the non-specific staining which was overlaid in the represented histogram plots. Mean \u0026plusmn; SD of percentage immunopositive population is represented.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunocytochemistry:\u0026nbsp;\u003c/strong\u003eRat BMMSCs and iMSCs were cultured on 12-mm glass coverslips until they reached approximately 85% confluency. The cells were then fixed with 4% PFA for 45 minutes at room temperature. For intracellular marker staining, the cells were permeabilized with 0.1% Triton X-100 in DPBS for 30 minutes. To prevent nonspecific staining, a blocking step with 3% BSA was performed for 45 minutes. Primary antibodies were added and incubated overnight in a humidified chamber at 4\u0026deg;C. Following three washes with DPBS, the cells were incubated with secondary antibodies for 90 minutes at room temperature in a dark, humidified chamber. Coverslips were washed three more times with DPBS, and the nuclei were stained with 300 nM DAPI (4\u0026prime;,6-diamidino-2-phenylindole dihydrochloride) for 2 minutes in the dark. Finally, the coverslips were mounted onto glass slides using 1,4-diazabicyclo [2.2.2] octane (DABCO) for imaging. Confocal images were taken for visualization and analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLabelling of iMSCs and rat BMMSCs with PKH-26 dye and transplantation:\u0026nbsp;\u003c/strong\u003eP2 iMSCs and P0 control rat BMMSCs were trypsinized, counted, and labeled with PKH26 using the MINI26 PKH26 Red Fluorescent Cell Linker Mini Kit (Sigma), following the manufacturer\u0026apos;s protocol. Briefly, 2 \u0026times; 10⁶ cells, pre-washed in serum-free media, were resuspended in 100 \u0026micro;L of Diluent C. A 2x (4 \u0026times; 10⁻⁶ M) dye solution was prepared separately by mixing 0.4 \u0026micro;L of dye with 100 \u0026micro;L of Diluent C, then added to the cell suspension. The mixture was incubated in dark at room temperature for 5 minutes, achieving a final dye concentration of 2 \u0026times; 10⁻⁶ M per 2 \u0026times; 10⁶ cells. The reaction was neutralized by adding an equal volume (200 \u0026micro;L) of FBS. Subsequently, the cells were washed two times with complete media and one final wash with DPBS. For transplantation 1 \u0026times; 10⁶ PKH-labelled cells were resuspended in 0.5 mL PBS to be injected into one rat. The cells were injected intramuscularly (IM) into the right hind limb of anesthetized rats on day 3 post MPTP treatment. A volume of 0.5 mL DPBS (vehicle) was injected into rats of the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry:\u0026nbsp;\u003c/strong\u003eFor immunohistochemistry, rats were anesthetized and perfused transcardially with 300 ml of 0.9% saline to remove blood from the circulatory system, followed by 300 ml of 4% paraformaldehyde (PFA) prepared in 1X DPBS. After perfusion, the rats were decapitated and brain tissue was isolated and stored in 4% PFA at 4\u0026ordm;C for 7 days to allow for tissue hardening. For coronal sections, 50 \u0026micro;m thick slices of the midbrain were prepared using a Leica 1200S vibratome. Immunohistochemistry (IHC) was performed on free-floating sections using a 24-well plate. \u0026nbsp;For heat mediated antigen retrieval, the sections were incubated at 65\u0026ordm;C in a water bath for 2.5 hours, immersed in 200 \u0026micro;l of antigen retrieval buffer, consisting of an equal mix of saline-sodium citrate (SSC) buffer and formamide. Permeabilization and blocking were carried out using a solution containing 5% bovine serum albumin (BSA), 5% goat serum, and 0.1% Triton-X for 2 hours at room temperature. After each step, the sections were washed twice for 5 minutes with 1X PBS on a rocker. Following blocking, the brain sections were incubated overnight at 4\u0026ordm;C in primary antibody solution prepared in 5% BSA with 5% goat serum. The next day, after two PBS washes, the sections were incubated with a goat-derived secondary antibody for 6 hours at RT in dark, followed by a counterstaining with 5 \u0026micro;g/ml DAPI for 1 minute. The immunostained brain slices were then mounted on glass coverslips using DABCO as the mounting reagent. Confocal images were taken for visualization and analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfocal microscopy:\u003c/strong\u003e ICC and IHC slides were\u0026nbsp;visualized using the ZEISS Axio Observer.Z1/7 and LSM 980 confocal microscope, equipped with a Plan-Apochromat 40X/1.3 Oil DIC (UV) VIS-IR M27 objective. Image acquisition was performed using ZEISS ZEN 3.7 software (RRID: SCR_013672), employing a fluorescence contrast method. Detector gain was set to 650V, and the pinhole was adjusted as follows: 1 AU/30 \u0026mu;m for DAPI, 1 AU/36 \u0026mu;m for FITC, 1 AU/45 \u0026mu;m for AF647, and 1 AU/45 \u0026mu;m for PKH26. The excitation/emission settings were 405/495 for DAPI, 488/517 for FITC, 650/665 for AF647, and 551/567 for PKH26. Images were captured at a resolution of 2791 x 2791 pixels with an 8-bit depth and an effective NA of 1.3, then analysed using ImageJ.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA:\u0026nbsp;\u003c/strong\u003eFor DA ELISA of rat midbrain samples, tissue lysate was prepared\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eBriefly,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003erats were sacrificed and midbrain was dissected out, weighed and homogenized in 1x PBS at 4\u0026deg;C. Homogenized samples was then centrifuged and supernatant was carefully collected and stored in -80\u0026deg;C. To maintain the uniformity of the tissue homogenate, the concentration of the tissue lysate was kept constant at 200mg/ml\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor serum sample preparation, 2.5-3ml blood was collected from each rat by cardiac puncture and centrifuged at 3000rpm for 20 min at 4\u0026ordm;C. samples were stored in -80\u0026deg;C until use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor detection of paracrine factors, P0 rat BMMSCs were incubated in serum free media for 24h and cell supernatant was collected and centrifuged for 15 min at 3000 rpm to remove cell debris. For iMSC conditioned media, cells were cultured till 90% confluency and then stimulated with TNF-\u0026alpha; (25 ng/ml) for 24h. Then they were washed three times with DPBS, and serum free maintenance media was added and incubated for 24h. The supernatant was collected after removing cell debris and stored at -80\u0026deg;C until use. For DA ELISA, iPSC-derived mature DA neurons were incubated in complete HBSS buffer (pH 7.4) for 5 min, and the supernatant was collected to measure basal vesicular dopamine release. Cells were then stimulated with 56 mM KCl in HBSS, and the supernatant was collected. Samples were centrifuged at 3000 rpm for 15 minutes to remove cellular debris. Cell counts for BMMSCs, iMSCs, and iPSC-derived DA neurons were obtained after conditioned media collection. All ELISA was performed as per manufactures protocol. Readings for standard and samples were taken using the spectrophotometer (SPARK, TECAN, Switzerland). Data is represented as Mean \u0026plusmn; SD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry of rat brain samples:\u003c/strong\u003e Rats were euthanized using an overdose of isoflurane, followed by transcardial perfusion with 350 mL of 0.9% isotonic saline to eliminate blood. The midbrain was carefully dissected and minced into small fragments using a sterile surgical blade. The tissue was then incubated with 2 mL of 0.25% trypsin for 15 minutes at 37\u0026deg;C to facilitate enzymatic digestion. Following digestion, 6 mL of 1\u0026times; PBS was added to neutralize the trypsin. The tissue was gently triturated by repeated pipetting to obtain a single-cell suspension. The cell suspension was centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The resulting pellet was resuspended in 1\u0026times; PBS and passed through a 70 \u0026micro;m cell strainer to remove any remaining tissue debris. The filtered single-cell suspension was centrifuged again and fixed in 2% paraformaldehyde (PFA) overnight at 4\u0026deg;C. After fixation, the cells were stored in PBS containing 0.01% sodium azide (NaN₃) until further analysis. For the flowcytometry analysis of PKH+ cells in rat brain, 2 lakh cells were resuspended in sheath fluid, and 10,000 events were acquired per sample in BDFACSLyric instrument. \u0026nbsp;Mean \u0026plusmn; SD of percentage immunopositive population is represented.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony forming unit fibroblast (CFU-f):\u0026nbsp;\u003c/strong\u003eTo check the number of colony-forming units present in the bone marrow, bone marrow was isolated from individual rats and processed as previously described. After isolation all mononuclear cells of the bone marrow was plated into 100mm cell culture plates. Non-adherent cells were removed by giving media change and adhered cells were cultured for 8 days and then fixed in 4% PFA followed by staining in 0.1% toluidine blue prepared in 1% PFA overnight at 4\u0026ordm;C. After staining images were taken and number of colonies were counted in ImageJ software using multi-point tool.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnnexin-PI cell apoptosis assay:\u0026nbsp;\u003c/strong\u003eApoptosis was measured in rat BMMSCs and iMSCs using FITC Annexin V/Dead cell apoptosis kit as reported before\u0026nbsp;(71). Briefly, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were incubated in 100\u0026mu;l Annexin binding buffer containing 0.5 \u0026mu;l of Annexin V and 0.1 \u0026mu;l propidium iodide (PI) for 30 min at RT. After one PBS wash, cells were resuspended in 500\u0026micro;l of sheath buffer. Flow cytometric analysis was carried out to determine the number of apoptotic cells using FACSVerse\u0026trade; (BD Biosciences, USA) using quadrant density plot. Unstained cells were used as experimental control. Mean \u0026plusmn; SD of percentage of apoptotic population is represented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReactive oxygen species assay:\u0026nbsp;\u003c/strong\u003eROS generation in rat BMMSCs and iMSCs was estimated as previously reported (71). Cells were washed twice with PBS, counted, and incubated for 15 min with 10 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eDCF. DA (Invitrogen). Cell number was maintained similar for all groups. After incubation, the fluorescence intensity of DCF on oxidation of H\u003csub\u003e2\u003c/sub\u003eDCF is measured at ex/em-485/535 nm using the spectrophotometer (SPARK, TECAN, Switzerland).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn-vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Migration assay:\u0026nbsp;\u003c/strong\u003eThe transwell migration of rat BM-MSCs and iMSCs was evaluated using a Boyden chamber. A total of 1 \u0026times; 10⁵ rat BM-MSCs or 2 \u0026times; 10⁵ iMSCs were seeded in the upper chamber of 8 \u0026mu;m transwell inserts (Corning, USA) in 200 \u0026mu;L of complete media without growth factors and allowed to adhere for 24 hours. Following adherence, migration was induced by adding 25 ng/mL of rat recombinant TNF\u0026alpha; for BM-MSCs or hTNF\u0026alpha; for iMSCs to 600 \u0026mu;L of basal media in the lower chamber. Cells were allowed to migrate toward the lower chamber for 24 hours. After the incubation period, non-migrated cells were gently removed using a cotton swab. The inserts were then washed with PBS, fixed in 4% PFA, and stained overnight with 0.1% toluidine blue in 1% PFA at room temperature. Images were captured using the EVOS M5000 imaging system (Invitrogen) at 10\u0026times; magnification. The number of migrated cells per field was quantified using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKi67 proliferation assay:\u003c/strong\u003e \u0026nbsp;The protocol was adapted from our previously published report\u0026nbsp;(71). Rat BM-MSCs and iMSCs were harvested using 0.25% trypsin-Ethylenediaminetetraacetic acid (EDTA), fixed with cold 100% methanol for 45 min at RT, and resuspended in wash buffer containing 0.01% sodium azide in PBS. Permeabilization and blocking was performed as mentioned before. The cells were then labeled with proliferation marker Ki67 primary antibody overnight. Further, incubated with secondary antibody conjugated with Alexa Fluor 488 for 90 min at RT. Flow cytometry analysis was performed using FACSVerse\u0026trade; (BD Biosciences). Cells were identified by light scatter for 10,000 gated events and analysed using BD FACSuite software. Cells stained with only the secondary antibody were used as isotype control and overlaid in the histograms and was used for gating. Mean \u0026plusmn; SD of percentage Ki67 positive population for n = 6 is represented. For immunocytochemistry, cells were seeded onto glass coverslips and, after adhering, were fixed using 100% methanol. Immunostaining for ki67 was performed as mentioned before and Confocal images were acquired for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMixed Lymphocyte Reaction assay:\u0026nbsp;\u003c/strong\u003e5ml of Blood was collected aseptically in K2EDTA treated vacutainer tubes from 3 unrelated healthy individuals and processed after 30min of collection in order to attain room temperature. PBMCs were isolated by gradient centrifugation using Hisep LSM1077 as previously reported\u0026nbsp;(66). After isolation, cells from 3 donors were resuspended separately in complete PBMC media and counted using haemocytometer and plated at 1x10\u003csup\u003e6\u003c/sup\u003e cells/ml concentration in 6 well plates. [Composition of complete PBMCs media was RPMI basal media, 10% FBS, 1% Pen/strep, 1% Glutamax]. After culturing cells for 48h, PBMCs from all 3 donors were pooled in a 15 ml centrifuge tube and mixed properly, followed by centrifugation step at 260g for 5 mins. Cells were resuspended and plated in PBMC media with 1% Phytohemagglutinin-M a positive stimulator, for 72h in optimum sterile culture condition (5% CO2, 37\u0026ordm;C) before setting up for co-culture assay with iMSCs. PHA-M treatment induces a hyper-immunogenic reaction. Pooling donor PBMCs combined with PHA-M stimulation will lead to rapid proliferation and sphere formation. After 72h of PHA-M treatment all PBMCs were collected and centrifuged at 200g for 10 mins. The supernatant was discarded, and the cells were washed twice with PBMC media to remove PHA-M. The cells were then resuspended in complete PBMC media, counted, and plated with mit C-treated iMSCs at a ratio of 1:10 (iMSCs:PBMCs) in a 24-well plate. PBMCs were also plated separately without iMSCs as negative control. Co-culture was set for 24h. After incubation, PBMCs were collected, centrifuged, and fixed in methanol. Methanol-fixed PBMCs were subsequently assessed for Ki67 proliferation marker expression using flow cytometry. Mean \u0026plusmn; SD of percentage Ki67 positive population for is represented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunomodulation assay using patient PBMCs:\u003c/strong\u003e Healthy control and PD-patient PBMCs were thawed and centrifuged at 250g for 5 minutes. The cells were resuspended and cultured in PBMC media containing 10% FBS, 1% penicillin-streptomycin, 1% GlutaMAX, and RPMI basal media for 24 hours. Following this, the PBMCs were incubated with 1% Phytohemagglutinin-M (PHA-M) for 72 hours under optimal sterile culture conditions (5% CO2, 37\u0026ordm;C). After 72 hours, the PHA-M-treated PBMCs were collected and centrifuged at 200g for 10 minutes. The supernatant was discarded, and the cells were washed twice with PBMC media to remove any remaining PHA-M. The cells were then resuspended in complete PBMC media, counted, and co-cultured with mitomycin C-treated healthy control and PD iMSCs in a 1:10 ratio (iMSCs) in a 24-well plate. The experimental conditions included co-culturing patient PBMCs with both patient-derived and healthy control iMSCs, and healthy control PBMCs with healthy control iMSCs. Additionally, PHA treated PBMCs of each group were cultured alone as a negative control. After 24 hours of co-culture, PBMCs were collected, centrifuged, and fixed in methanol. The methanol-fixed PBMCs were subsequently analysed for Ki67 proliferation marker expression using flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis:\u0026nbsp;\u003c/strong\u003eStatistical analyses was performed using R software version 3.4.1 (R Foundation; R Project for Statistical Computing, RRID:SCR_001905). One-way ANOVA followed by pairwise comparison (Bonferroni) analysis was used as required (specified in figure legends). In all analyses, statistical significance was set at p \u0026lt; 0.05. F is expressed as F (degrees of freedom, degree of freedom error) = x. For statistical significance, * represents \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** represents \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and *** represents \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. Graphs were prepared using GraphPad Prism 6 (GraphPad Software; GraphPad Prism, RRID:SCR_002798). All error bars in graphs depict standard deviation (SD).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe degeneration of midbrain dopaminergic (DA) neurons coincides with the initiation of neuroinflammatory processes during pre-motor stage:\u0026nbsp;\u003c/strong\u003eTo track the timeline of neurodegeneration in our MPTP rat model, the status of DA neurons in the SNpc and corresponding non-motor and motor behavioural parameters were assessed. Confocal IHC images showed a significant reduction in the number of TH\u003csup\u003e+\u003c/sup\u003e cells in the SNpc from week 1 post MPTP treatment (PMT) onwards (Figure 1A \u0026amp; C). In parallel, a gradual increase in phospho a-synuclein serine 129 (p-syn) expression was observed between week 2 and week 3 PMT (Figure 1B). \u0026nbsp;Furthermore, we observed a significant reduction in dopamine levels starting from week 1 PMT, with a further decline at later time points (weeks 2 \u0026amp; 3 PMT) (Figure 1D). \u0026nbsp;At week 1 PMT, the MPTP group exhibited a marked reduction in the time spent in the familiar compartment compared to controls, indicating impaired olfactory discrimination\u0026mdash;a hallmark non-motor symptom of PD. This deficit persisted through weeks 1 to 3 PMT (Figure 1E). \u0026nbsp;Additionally, a significant decline in Nerve Conduction Velocity (NCV) was observed from week 1 PMT in the MPTP group, which was retained across all subsequent time points (Figure 1F). Motor coordination assessed by rotarod performance time showed a mild decrease at week 1 PMT, with significant differences emerging from week 2 onward (Figure 1G). In the actimeter-based assessment of locomotor activity, a notable reduction in the number of squares traversed was observed only from week 2 PMT, which persisted through week 3 (Figure 1H). Collectively, these behavioural assessments, along with dopamine ELISA and IHC analysis of TH\u003csup\u003e+\u003c/sup\u003e neurons in the midbrain, and in alignment with our earlier study (77), indicate that while neurodegeneration and non-motor impairments begin as early as week 1 PMT, motor deficits become apparent only from week 2 PMT onward. Based on these observations, the week 1 PMT is hereafter designated the pre-motor stage in this model, while the onset of motor symptoms from week 2 PMT marks the transition to the motor stage of the disease.\u003c/p\u003e\n\u003cp\u003eThe neuroinflammatory status was evaluated based on microgliosis, astrogliosis, and proinflammatory cytokine TNF-\u0026alpha; and NLRP3 expression in the SNpc region. Confocal IHC images showed a progressive increase in IBA1\u003csup\u003e+\u003c/sup\u003e cell numbers and their interaction with TH\u003csup\u003e+\u003c/sup\u003e cells from week 1 PMT (insets) (Figure 2A). This was further confirmed by the display of IBA1\u003csup\u003e+\u003c/sup\u003e cells showing immunopositive co-localization with TNF-\u0026alpha; (yellow puncta) (Figure 2B), with Pearson\u0026rsquo;s co-localization coefficient significantly elevated in the week 2 PMT group compared to both control and week 1 PMT (Figure 5H). Consistently, TNF-\u0026alpha; levels in midbrain lysates showed a significant increase from week 1 PMT onwards (Figure 2I). Post-mortem IHC studies have previously reported infiltration of peripheral immune cells into the PD patient brain (78,79). In line with this, our IHC and flow cytometry analyses of PD rat brain demonstrated a significant increase in CD4⁺ cells at the early pre-motor stage, with a further increase subsequently, while CD8⁺ cells showed a marked increase starting from week 2 PMT (motor stage) (Figure 2E, F, K, L \u0026amp; Supplementary Figure S8 E \u0026amp; G). \u0026nbsp;Also, TNF-\u0026alpha; levels in peripheral blood serum showed a significant increase at the pre-motor stage, with further enhancement throughout the motor stages (Figure 2J), indicating the presence of systemic inflammation from the pre-motor stage of the disease. Previous preclinical studies and PD patient data have demonstrated heightened NLRP3 expression with DA neurons (80). Our study also detected an increase in NLRP3 expression in the brains of PD rats that increased as the disease progressed (Figure 2D and M), further supporting the presence of a chronic inflammatory microenvironment from an early stage. The degeneration of midbrain DA neurons and the initiation of neuroinflammatory processes thus appear to occur concurrently during the pre-motor stage of disease progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe impairment of physiological and functional parameters in PD-BMMSCs correlates with the onset of systemic inflammation and neurodegeneration:\u003c/strong\u003e BMMSCs isolated from control rats were characterized using immunofluorescence and immunophenotyping to confirm the presence of mesenchymal stromal cell markers. Phase-contrast images (Supplementary Figure S1A) of BMMSC culture on day 1, Day 6, and Day 8 post-isolation demonstrated plastic adherence and stromal cell morphology. Confocal imaging (Supplementary Figure S1B) confirmed the expression of CD90 and CD105 surface markers. Additionally, flow cytometry analysis revealed that over 94% of the isolated BMMSCs were immunopositive for both CD90 (94.85\u0026plusmn;0.63%) and CD73 (97.30\u0026plusmn;0.96%) (Supplementary Figure S1C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we proceeded to isolate BMMSCs from MPTP-treated PD rats.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eBMMSCs (P0) isolated from all three PD groups exhibited an enlarged, thin, and flattened morphology, contrasting with the elongated spindle shape observed in age-matched control BMMSCs (Figure 3A \u0026amp; B). A significant decline in the total number of Ki67 positive proliferative cells was observed from the second week onward, with a further reduction seen in the Week 3 PMT group (Figure 3C \u0026amp; Supplementary Figure S1 D). To assess the self-renewal capacity of MSCs, we compared the number of stromal clonogenic cells in the bone marrow of MPTP-treated groups with age-matched healthy controls, as colony numbers directly indicate self-renewal potential (36). There was a significant reduction in the number of colony-forming units in the bone marrow of MPTP-treated PD rats with time, compared to controls (Figure 3D \u0026amp; E). Furthermore, a substantial increase in the number of apoptotic cells was noted in the Week 2 and Week 3 PMT groups, indicating reduced cell survival. However, no significant difference was observed between the control group and the Week 1 PMT group (Figure 3F \u0026amp; Supplementary Figure S1 E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMorphological changes in MPTP-treated rat BMMSCs at P0 (Figure 3B) suggest cellular aging, while increased apoptosis of BMMSCs for MPTP-treated rats corelates with the decline in the population and proliferation of self-renewing cells in their bone marrow.\u003c/p\u003e\n\u003cp\u003eAs homing and migration to inflammatory sites is known to be an essential function of bone marrow MSCs for exerting immunosuppressive effects under chronic inflammatory conditions (81), we next assessed the migratory ability of PD-BMMSCs in response to an inflammatory cue using a transwell migration assay. A significant reduction in the number of migrated cells per field toward TNF-\u0026alpha; was observed from week 1 PMT, which further declined in the week 2 and week 3 PMT groups (Figure 3G \u0026amp; H). The migration of BMMSCs is influenced by factors such as CXCR4 surface expression, matrix metalloproteinase secretion, and intracellular ROS levels (82\u0026ndash;85). We analysed these factors in PD-BMMSCs and found a significant increase in basal ROS levels from week 2 PMT compared to both control and week 1 PMT (Figure 3J). Additionally, MMP3 concentration in the PD-BMMSC secretome showed a marked reduction from week 1 PMT compared to control (Figure 3K). However, the number of CXCR4-positive cells increased at week 2 PMT but significantly declined at week 3 PMT (Figure 3I). Next, we evaluated the secretion levels of four key immunomodulatory paracrine factors\u0026mdash;IDO, PGE2, TGF-\u0026beta;, and IL-10\u0026mdash;in the BMMSC secretome. A significant decline in IDO, TGF-\u0026beta; and IL-10 and secretion was observed at week 1 PMT and week 2 PMT (Figure 3L, M \u0026amp; N). However, no significant differences were detected in the secretion levels of PGE2 (Supplementary Figure S2 A). The reduced migration of PD-BMMSCs \u003cem\u003ein vivo\u003c/em\u003e may be influenced by elevated intracellular ROS levels, and additionally by a further decrease in MMP3. The observed increase in the CXCR4\u003csup\u003e+\u003c/sup\u003e population in the week 2 PMT group may be attributed to elevated levels of inflammatory cytokines in systemic circulation in PD rats. However, despite this increase in CXCR4 expression, migration was not enhanced in BMMSCs of the week 2 PMT group. Therefore, our observations suggest that the onset of BMMSC functional impairment occurs during the pre-motor stage (week 1 PMT) and temporally correlates with the emergence of systemic (rise in blood TNF-a) inflammation, neuroinflammation, and midbrain DA neuron degeneration. The onset of systemic inflammation and neuroinflammation also suggest that this progressive dysfunction of BMMSCs may contribute to the compromised immunomodulatory support during early PD pathogenesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpaired differentiation ability of PD-iMSCs:\u0026nbsp;\u003c/strong\u003eiMSCs were differentiated from healthy control iPSC lines NIMHAi006-A (HC03) and NIMHAi005-A (HC02) as well as from sporadic PD iPSC lines NIMHi002-A (PD02) and NIMHi003-A (PD03). Morphological changes became apparent as early as day 2 of differentiation (Supplementary Figure S3B). Upon switching from differentiation media to maintenance media on day 5, the differentiated cells started to proliferate rapidly. Passaging these cells and plating them onto non-coated, tissue culture-treated plates resulted in a homogeneous population of differentiated iMSCs. iMSCs differentiated from HC lines at different passages (P3, P5 and P10) is shown in Supplementary Figure S3C. To confirm the disease-relevant phenotype, dopaminergic neurons differentiated from the two PD-iPSC lines were characterized (Supplementary Figure S4A-K) and demonstrated distinct pathological features compared to healthy control. These PD-iPSC-derived dopaminergic neurons exhibited impaired vesicular dopamine release (Supplementary Figure S4L) and elevated expression of phosphorylated \u0026alpha;-synuclein at serine 129, validating the disease-associated cellular dysfunction (Supplementary Figure S4F \u0026amp; I).\u003c/p\u003e\n\u003cp\u003eiMSCs from all the groups were characterized according to ISCT guidelines for MSC characterization at P2. PD iMSCs exhibited an increase in cell size, with PD03 iMSCs showing a significantly larger size than HC iMSCs. Moreover, PD03 iMSCs displayed a more flattened morphology compared to PD02 iMSCs. (Figure 4B \u0026amp; C). Confocal imaging confirmed the expression of MSC-specific surface markers CD73, CD105, and CD90 in both the HC (Figure 4D, Supplementary Figure 5A) and PD groups (Figure 4E \u0026amp; F). Flow cytometry analysis revealed that over 95% of the cell population was positive for MSC-specific CD markers (Figure 4G, H, I, \u0026amp; Supplementary Figure 5B i-iii), while the expression of co-stimulatory molecules (CD80, CD86) and hematopoietic markers (HLA-DR, CD45, CD56, CD34) was negligible across all three groups (Supplementary Figure 5B iv-ix \u0026amp; Supplementary Figure S6A-C).\u003c/p\u003e\n\u003cp\u003eMesenchymal stromal cells have characteristic trilineage differentiation ability into adipogenic, osteogenic and chondrogenic lineages. iMSCs derived from both HC and PD groups successfully differentiated into all three lineages, as confirmed by Oil Red O staining for lipid droplets (Figure 5A-C), Alizarin Red S staining for calcium deposition (Figure 5D-E), and Toluidine Blue staining for cartilage formation (Figure 5G-I, Supplementary Figure S5C). However, the adipogenic and osteogenic differentiation capacity of PD-iMSCs showed impairment (Figure 5A-F). Flow cytometry analysis of BODIPY-stained adipocytes revealed a significant reduction in MFI in PD-adipocytes compared to HC (Figure 5J). Additionally, the percentage of Runx2\u003csup\u003e+\u003c/sup\u003e cells was lower in the PD-osteocyte population than in HC, with a more pronounced reduction observed in the PD03 group compared to PD02 (Figure 5K). So, while the immunophenotypic expression of MSC and hematopoietic and co-stimulatory markers was similar across HC and PD iMSCs, PD iMSCs showed compromised differentiation capability for osteocytes and adipocytes. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePD-iMSCs exhibited reduced proliferation, survival, and migration, along with elevated intercellular basal ROS level:\u0026nbsp;\u003c/strong\u003eBMMSCs isolated from MPTP induced PD rats had demonstrated increased apoptosis alongside reduced proliferation and migration capabilities. These findings were corroborated in iMSCs derived from sporadic PD patients when maintained under optimal culture conditions. At P3, PD-derived iMSCs exhibited significantly diminished proliferation rates. Flow cytometric analysis revealed a reduced Ki67-immunopositive cell population (Figure 7C \u0026amp; Supplementary Figure S 3D), with PD03-iMSCs showing the most pronounced decrease. Confocal microscopy of Ki67-immunostained cells provided additional validation (Figure 7A), and the calculated Ki67: DAPI ratio (Figure 7B) confirmed these proliferation deficits. Despite optimal culture conditions, PD-derived iMSCs demonstrated increased cell death. Annexin-PI staining revealed significantly higher numbers of apoptotic cells in both PD groups compared to healthy control cells. Notably, PD03-iMSCs exhibited a greater proportion of apoptotic cells than PD02-iMSCs, possibly indicating a severity-related response (Figure 6D Supplementary Figure S3 E). The migratory response to the proinflammatory cytokine TNF-\u0026alpha; was also significantly impaired in PD-iMSC groups. The number of migrated cells was substantially lower than HC, mirroring the pattern observed in PD-affected rat BMMSCs (Figure 6E \u0026amp; F). To validate these \u003cem\u003ein vitro\u003c/em\u003e observations, PKH-labeled iMSCs were transplanted intramuscularly into PD rat models. Flow cytometric analysis of brain tissue revealed a lower percentage of PKH-positive cells in rats receiving PD-iMSCs compared to those transplanted with HC-iMSCs (Figures 6G \u0026amp; Supplementary Figure S8 C), further reinforcing the reduced viability and migration capacity observed \u003cem\u003ein vitro\u003c/em\u003e. Intracellular basal ROS levels were significantly elevated in PD-iMSCs compared to HC03 iMSCs. The PD03-iMSC group exhibited the highest ROS levels (Figure 6H), suggesting a correlation between oxidative stress burden and cellular dysfunction severity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpaired immunomodulatory function of PD-iMSCs\u003c/strong\u003e: \u0026nbsp;To evaluate the immunomodulatory capacity of iMSCs, we quantified immunomodulatory paracrine factors in culture media following 48-hour TNF-\u0026alpha; treatment. Four key factors were analyzed: transforming growth factor-\u0026beta; (TGF-\u0026beta;), interleukin-10 (IL-10), indoleamine 2,3-dioxygenase (IDO), and prostaglandin E2 (PGE2). Significant reductions in IDO and PGE2 secretion within the PD-iMSC secretome (Figures 6I and 6J) was observed, while TGF-\u0026beta; and IL-10 levels remained unchanged (Supplementary Figure S2B, C). To further characterize the immunosuppressive potential of iMSCs, we employed a mixed lymphocyte reaction assay. This assay exploits the principle that T cells from one donor proliferate when exposed to antigen-presenting cells (APCs) from a genetically distinct donor due to HLA incompatibility, thereby triggering an immune response (Figure 7A \u0026amp; B). Comparing PBMC proliferation in the presence versus absence of iMSCs allows us to evaluate their immunomodulatory potential (Figure 7C \u0026amp; D). Flow cytometric analysis of activated PBMCs with iMSCs demonstrated distinct immunomodulatory patterns, when compared to negative control (defined as PBMC only without the presence of iMSCs). HC iMSCs significantly suppressed PBMC proliferation compared to negative control, confirming their expected immunomodulatory function. In contrast, PD iMSCs failed to significantly reduce PBMC proliferation relative to negative controls (Figure 7E \u0026amp; Supplementary Figure S2 D), indicating compromised immunomodulatory properties. To validate these findings using autologous cell systems, we performed immunomodulation assays with patient-matched PBMCs and iMSCs. This experimental design included HC03-PBMCs, PD02-PBMCs, and PD03-PBMCs (Figure 8A), each activated separately with PHA for 72h (Figure 8B) and then co-cultured with its corresponding iMSCs for 24h (Figure 8E \u0026amp; F). PD patient PBMCs exhibited significantly elevated (~16\u0026ndash;21% higher in PD02 and ~12\u0026ndash;14% higher in PD03) baseline proliferation compared to HC PBMCs, suggesting an inherently activated immune state. When PD-derived PBMCs were co-cultured with their corresponding PD-iMSCs, proliferation rates remained significantly higher than those observed in co-cultures for HC iMSCs (Figure 8G \u0026amp; Supplementary Figure S2 E). This finding further substantiates the impaired immunoregulatory capacity of PD-derived iMSCs. The combined evidence of significant reduction in IDO and PGE2 secretion, coupled with the inability of PD-iMSCs to effectively suppress PBMC proliferation in both heterologous MLR and autologous co-culture systems, together establish a clear functional deficit in the immunomodulatory capacity of PD-derived iMSCs compared to healthy controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransplantation of healthy MSCs in PD rats prevented neurodegeneration, promoted neurogenesis and lowered systemic inflammation more effectively than PD-iMSCs:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe compromised immunomodulatory capacity of PD-derived iMSCs may significantly limit their therapeutic effectiveness when transplanted under PD conditions. To assess their disease-modifying and immunomodulatory potential compared to HC iMSCs, MPTP-induced PD rats received transplantations of either HC or PD-derived iMSCs on day 3 of MPTP treatment. Rats transplanted with healthy rat bone marrow-derived MSCs (BMMSCs) served as positive controls.\u003c/p\u003e\n\u003cp\u003eBy two weeks post-transplantation, both the rat BMMSC and HC-iMSC transplanted groups demonstrated a significant reduction in IBA1\u003csup\u003e+\u003c/sup\u003e microglial cells compared to the disease control group. Notably, this microglial reduction was substantially more pronounced in rats receiving HC-iMSCs than in those transplanted with PD-iMSCs (Figure 9A, E). Moreover, microglia in the PD-iMSC group maintained their activated morphology (as shown in insets), while cells in both the HC-iMSC and rat BMMSC groups exhibited morphology resembling that observed in control animals (Figure 9A, C \u0026amp; Supplementary Figure S7B).\u003c/p\u003e\n\u003cp\u003eColocalization analysis revealed TNF-\u0026alpha; expression in IBA1\u003csup\u003e+\u003c/sup\u003e cells within the PD-iMSC group (highlighted in insets), a pattern that was absent in the HC-iMSC transplanted groups (Figure 9C \u0026amp; Supplementary Figure S7B). Consistent with these findings, TNF-\u0026alpha; levels were significantly elevated in both brain tissue and peripheral blood of PD-iMSC transplanted rats compared to HC-iMSC recipients (Figure 9F \u0026amp; G). Furthermore, enhanced NLRP3 expression, which colocalized with TH\u003csup\u003e+\u003c/sup\u003e neurons, was observed exclusively in the PD-iMSC group and was notably absent in HC-iMSC recipients (Figure 9D \u0026amp; Supplementary Figure S7C). Transplantation of HC-iMSCs significantly reduced CD4\u003csup\u003e+\u003c/sup\u003e cell infiltration compared to PD-iMSC transplantation (Supplementary Figure S8D). For CD8\u003csup\u003e+\u003c/sup\u003e cells, HC-iMSC transplantation maintained infiltration levels similar to 2 WK PMT, while PD-iMSC transplantation resulted in increased infiltration (Supplementary Figure S8F). These findings suggest that PD-iMSCs are less capable of modulating neuroinflammation in PD due to their compromised immunoregulatory functions and altered physiological state.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRecent studies using pre-clinical models and postmortem human brain tissue have demonstrated that neurogenesis can occur in the SNpc following treatment with neuroprotective factors (86). Moreover, we have recently reported neurogenesis in the SNpc following transplantation of sEVs derived from human dental pulp stem cells (77). In addition to their immunomodulatory properties, MSCs exhibit strong cytoprotective effects, and thus it is credible that modulating the hostile neuroinflammatory microenvironment may help initiate neurogenesis.\u003c/p\u003e\n\u003cp\u003eTo investigate this, we examined the expression of the cell proliferation marker Ki67 through IHC studies, and assessed the floor plate cell marker FOXA2 in the SNpc region of the rat midbrain following transplantation. IHC analysis revealed a significantly higher number of FOXA2\u003csup\u003e+\u003c/sup\u003e cells in the SNpc of rats transplanted with healthy MSCs, compared to those receiving PD-MSCs, which showed FOXA2\u003csup\u003e+\u003c/sup\u003e levels similar to the disease control group (WK2 PMT) (Figure 10A i \u0026ndash; F i, G \u0026amp; Supplementary Figure S7D)\u003c/p\u003e\n\u003cp\u003eAdditionally, nuclear localization of Ki67 was prominent in the healthy MSC-transplanted group, indicating active cell proliferation. This was absent in both the control and WK2 PMT groups. Ki67 expression was markedly lower in PD-iMSC transplanted animals compared to those receiving healthy MSCs (Figure 10A ii\u0026ndash;F ii \u0026amp; Supplementary Figure S7E). Also, the ratio of TH\u003csup\u003e+\u003c/sup\u003e Ki67\u003csup\u003e+\u003c/sup\u003e cells to total TH\u003csup\u003e+\u003c/sup\u003e neurons per field was significantly higher in the healthy MSC transplanted group. Notably, there was no significant increase over control in proliferative cells in the PD iMSC transplanted group, suggesting a lack of neurogenesis (Figure 10 H). Consistent with these findings, the total number of TH\u003csup\u003e+\u003c/sup\u003e neurons in the SNpc region was significantly higher in the healthy MSC-transplanted group compared to the disease control, whereas the PD-MSC transplanted group showed no significant difference from the disease control (Figure 10 C iii\u0026ndash;F iii, I \u0026amp; Supplementary Figure S7A). Moreover, midbrain dopamine content was also significantly elevated in the healthy MSC-transplanted group relative to the PD-iMSC group (Figure 10 J). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine whether the increased dopaminergic neuron survival and possible neurogenesis in the transplanted groups translated into functional recovery, motor coordination was assessed using the rotarod test. The rotarod performance of the HC-iMSC-transplanted group was comparable to that of the control group, whereas the PD-iMSC transplanted group showed significantly impaired performance compared to both control and the HC-iMSC transplanted group (Figure 10K). This finding provides definitive \u003cem\u003ein vivo\u003c/em\u003e confirmation that the cytoprotective and immunomodulatory capacity of PD-iMSCs is significantly compromised, as demonstrated by their failure to control neuroinflammation, ultimately leading to failed neurogenesis and poor motor functional recovery. Additionally, these findings reveal that unlike PD-derived iMSCs, healthy MSCs demonstrate robust therapeutic capacity through effective neuroinflammation modulation and enhanced neurogenesis promotion, supporting their clinical potential for PD intervention.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWhile chronic neuroinflammation and peripheral immune dysfunction are now well-documented features of PD, the functional status of endogenous MSCs throughout disease progression remains uninvestigated. This represents a critical knowledge gap, as MSCs serve as master regulators of immunomodulation in peripheral immune cells and therefore may play a pivotal upstream role in the vicious cycle between neuroinflammation and systemic inflammation that characterizes the disease.\u003c/p\u003e\n\u003cp\u003eOur findings present the first comprehensive evidence that endogenous MSC dysfunction occurs during the early pre-motor stages of PD, fundamentally altering the current understanding of systemic cellular alterations in PD pathogenesis. In our MPTP-induced rat model, we demonstrate the manifestation of BMMSC impairment as early as week 1 post-MPTP treatment (PMT), coinciding precisely with the onset of dopaminergic neurodegeneration and neuroinflammation, and \u003cu\u003epreceding\u003c/u\u003e the emergence of motor symptoms.\u003c/p\u003e\n\u003cp\u003eOur temporal analysis demonstrates progressive loss of SNpc TH\u003csup\u003e+\u003c/sup\u003e dopaminergic neurons with time PMT, which correlates directly with declining midbrain dopamine levels and deteriorating motor coordination and locomotion. An increase in the pathological marker, phospho \u0026alpha;-synuclein serine 129 (p-Syn), is also noted with time PMT. This escalating neurodegeneration is accompanied by a corresponding increase in IBA1\u003csup\u003e+\u003c/sup\u003e microglial cells, particularly expressing TNF-\u0026alpha;, consistent with established evidence that microglia serve as the primary source of TNF-\u0026alpha; during neuroinflammation (87). This clustering pattern of activated microglia, previously reported in postmortem brains from MPTP-exposed individuals (88), reflects the status of heightened neuroinflammation accompanying progressive neurodegeneration. This pattern also aligns with postmortem and PET studies demonstrating increased microglial activation in relevant brain structures of PD patients, as well as in atypical parkinsonian disorders including multiple system atrophy, progressive supranuclear palsy, and corticobasal degeneration (89\u0026ndash;91). The concomitant elevation of midbrain TNF-\u0026alpha; levels and NLRP3 immunopositive cells further supports the neuroinflammatory environment, as NLRP3 is known to be predominantly expressed by activated microglia \u0026nbsp;(92\u0026ndash;94).\u003c/p\u003e\n\u003cp\u003eConcurrently, a progressive increase in blood TNF-\u0026alpha; levels, as well as the presence of infiltrating CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in the midbrain, demonstrate a failure of systemic and peripheral immunomodulation with disease advancement. These findings align with clinical reports showing elevated serum TNF-\u0026alpha; levels in advanced-stage PD patients (Hoehn Yahr Scale stages 3-5) compared to early-stage patients (stages 1-2) (95), and studies that demonstrate elevated plasma high-sensitivity C-reactive protein levels correlating with motor prognosis in PD patients (96). Similarly, clinical and pre-clinical studies have shown increases in CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in the brain with duration post MPTP administration (78,79).\u003c/p\u003e\n\u003cp\u003eOur study reveals multifaceted BMMSC dysfunction in PD, encompassing cellular morphology, proliferation, survival, migration, and crucially, immunomodulatory capacity. PD-BMMSCs exhibited enlarged, flattened morphology characteristic of cellular senescence (83,97), accompanied by significantly reduced proliferation (decreased Ki67\u003csup\u003e+\u003c/sup\u003e cells) and increased apoptosis from week 2 PMT. The progressive decline in colony-forming units directly reflects impaired self-renewal capacity, which is essential for maintaining the MSC pool within the bone marrow niche and is crucial to their ability to support tissue homeostasis and participate in immunomodulation (98,99). BMMSCs are also known to provide signals in the specialized bone marrow niche to support the self-renewal, quiescence, and maintenance of HSCs (100,101).\u003c/p\u003e\n\u003cp\u003eThe migration deficits observed in PD-BMMSCs represent a critical functional impairment, as MSC homing to inflammatory sites is fundamental to their therapeutic efficacy. Despite increased CXCR4 expression at week 2 PMT\u0026mdash;likely a compensatory response to elevated systemic inflammatory cytokines\u0026mdash;migration toward TNF-\u0026alpha; remained significantly impaired. This paradox can be attributed to elevated intracellular ROS levels and reduced MMP3 secretion, both of which negatively impact MSC mobility and tissue infiltration capacity. In our previous study of diabetic neuropathy, we observed similar features of BMMSC migration impairment (71). This suggests that while these cells may retain the ability to detect inflammatory signals, their downstream migratory response is compromised. This uncoupling of chemokine receptor expression from functional migration underscores the multifaceted nature of MSC dysfunction in PD.\u003c/p\u003e\n\u003cp\u003eTo validate these findings in human disease, we differentiated both dopaminergic (DA) neurons and MSCs from patient-derived iPSCs generated from sporadic PD-patient PBMCs. This approach was chosen because PBMCs not only reflect stage-specific inflammatory and reactive signatures in idiopathic PD and Alzheimer\u0026apos;s disease (106\u0026ndash;108), but also exhibit key pathogenic milestones of PD, including accumulation of pathological \u0026alpha;-synuclein species (109,110). Consequently, iPSCs generated from these somatic cells retain the disease state memory of the individual. Furthermore, such iPSCs provide a continuous and stable cellular resource, whereas directly isolating DA neurons and MSCs from patients would require invasive procedures yielding limited cell numbers suitable for only a few experiments. Importantly, modeling sporadic disorders is critical for wider clinical translation, given that the vast majority of patients suffer from sporadic rather than inherited forms of these conditions. DA neurons differentiated from these iPSCs successfully replicated disease pathology and demonstrated impaired vesicular dopamine release, which correlated with patient UPDRS scores and fluorodopa PET results (66). This aligns with previous studies demonstrating that iPSCs from sporadic movement disorders can recapitulate disease pathology in neuronal and glial cells (61\u0026ndash;64,102), and similar replication of disease pathology has been shown in other cell types for various sporadic conditions (103\u0026ndash;105).\u003c/p\u003e\n\u003cp\u003eCrucially, these sporadic PD-iMSCs recapitulated the major dysfunction patterns observed earlier in rat PD-BMMSCs, including altered morphology, reduced proliferation and survival, impaired migration, elevated basal ROS levels, and\u0026mdash;most importantly\u0026mdash;compromised immunomodulatory capacity. A previous study conducted on BMMSCs from patients with progressive supranuclear palsy (PSP), a rare neurodegenerative movement disorder, has further reported significant mitochondrial dysfunction, elevated ROS levels, and compromised differentiation potential (55).\u003c/p\u003e\n\u003cp\u003eA particularly interesting observation from our study is the impaired differentiation potential of PD-iMSCs into adipogenic and osteogenic lineages. This aligns with clinical observations where PD patients often present with early-onset symptoms of osteoporosis/osteopenia and unexplained weight loss (48,111). These systemic manifestations may be mechanistically linked to the compromised adipogenic and osteogenic capabilities of MSCs in PD, and merit further investigation in subsequent studies.\u003c/p\u003e\n\u003cp\u003eMost significantly, our study demonstrates profound impairment in iMSC immunomodulatory function. PD-iMSCs exhibited a notable reduction in the secretion of key immunomodulatory paracrine factors, mirroring the deficits observed in PD rat-BMMSCs. Further evaluation by mixed lymphocyte reaction (MLR) assays provided strong evidence for the clinical relevance of these findings. A growing consensus supports the critical role of immunomodulation in governing MSC therapeutic potential (112,113). The ability of MSCs to suppress PBMC proliferation \u003cem\u003ein vitro\u003c/em\u003e serves as a reliable indicator of their capacity to modulate inflammatory responses \u003cem\u003ein vivo\u003c/em\u003e. Our data support the hypothesis that MSC immunomodulatory dysfunction is intrinsic to the PD disease state. The robust immunosuppressive activity of HC-iMSCs aligns with established MSC functions (114,115), whereas the inability of PD-iMSCs to effectively modulate immune responses suggests disease-related alterations in their regulatory mechanisms. \u0026nbsp;This fundamental impairment in immunomodulatory capacity has critical implications for the design and expected efficacy of MSC-based therapeutic strategies in PD.\u003c/p\u003e\n\u003cp\u003ePatient-specific immunomodulation experiments further corroborate this, revealing a hyperactivated state of PBMCs from PD patients and a reduced ability of their own MSCs to modulate immune responses, compared to MSCs from healthy controls. This constitutive hyperactivated immune state may contribute to the chronic inflammatory milieu characteristic of PD, and aligns with clinical observations of altered immune profiles in PD patients\u0026apos; peripheral blood, including elevated proinflammatory cytokines (116,117).\u003c/p\u003e\n\u003cp\u003eCritically, our patient-matched experimental design revealed that PD-iMSCs exhibit compromised immunosuppressive capacity even when regulating their own immune cells. While PD-iMSCs retained some immunomodulatory function, their suppressive effect on autologous PBMCs was significantly weaker than that achieved by HC-iMSCs on their corresponding cells. The superior ability of HC-iMSCs to suppress PD patient PBMCs compared to the patients\u0026apos; own MSCs further underscores the therapeutic potential of healthy donor-derived MSCs in PD treatment. No previous studies have examined the immunomodulatory capacity of MSCs isolated from the bone marrow of PD patients, in part due to the invasive nature of bone marrow procurement and reduced cell yields resulting from impaired proliferation and self-renewal capacity. Our findings therefore suggest that MSC dysfunction may represent a primary upstream event contributing to the well-documented peripheral immune abnormalities in PD patients, including elevated effector and inflammatory T cells, altered monocyte protein expression profiles, and B cell populations skewed toward proinflammatory phenotypes (32,118,119). These findings align with the failure of recent clinical studies attempting autologous MSC transplantation in PD (51,120), and strongly support the rationale for allogeneic MSC therapeutic approaches using healthy donor sources, which may offer substantially greater therapeutic benefit.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo validate these findings \u003cem\u003ein vivo\u003c/em\u003e and directly test the therapeutic implications of MSC dysfunction, we transplanted PD-iMSCs in MPTP-treated PD rats and compared the effects against both healthy rat BMMSCs and MSCs derived from iPSCs of healthy individuals (HC-iMSCs). Both healthy rat BMMSCs and HC-iMSCs demonstrated superior neuroprotective and neurorestorative effects compared to PD-iMSCs, as assessed by more effective reduction of neuroinflammation and peripheral inflammation, enhanced dopaminergic neuron survival, and improved motor function. These \u003cem\u003ein vivo\u003c/em\u003e findings further validate the impaired immunomodulatory capacity of PD-iMSCs observed in our \u003cem\u003ein vitro\u003c/em\u003e studies, confirming their reduced ability to mitigate both neuroinflammation and peripheral inflammation.\u003c/p\u003e\n\u003cp\u003eBeyond modulating the inflammatory environment, we found that healthy iMSC transplantation also promoted neurogenesis in the substantia nigra pars compacta (SNpc), characterized by increased FOXA2 and Ki67 expression, while PD-iMSCs failed to induce such regenerative responses. This finding aligns with our recent study demonstrating that neurogenesis occurs following the administration of small extracellular vesicles (sEVs) derived from human dental pulp stem cells (DPSCs) in the SNpc of MPTP-induced PD rats (77). Additionally, previous research has shown that the microneurotrophin BNN-20 can promote neurogenesis within this region (86).\u003c/p\u003e\n\u003cp\u003eHuman studies also support the potential for SNpc neurogenesis. Post-mortem analysis of PD patient brains revealed that adult human neural progenitors can be isolated from the substantia nigra and cultured \u003cem\u003ein vitro\u003c/em\u003e under specific growth conditions (121). This study found that multipotent neural stem/progenitor cells reside within the substantia nigra, but lack essential factors required for neural differentiation in PD conditions when cultured independently. However, when co-cultured with human embryonic stem cell-derived neural progenitors, they successfully differentiated into both neurons and glia.\u003c/p\u003e\n\u003cp\u003eBy two weeks PMT (1week post-MSC transplantation), we observed significantly increased colocalization of the proliferative marker Ki67 with TH\u003csup\u003e+\u003c/sup\u003e neurons in the rat-BMMSC and HC-iMSC groups compared to PD-iMSCs. This corresponded with enhanced TH immunopositivity and improved motor function. The failure of PD-iMSC transplanted groups to suppress neuroinflammation likely accounts for the reduced neurogenesis and decreased TH\u003csup\u003e+\u003c/sup\u003e dopaminergic neurons, ultimately manifesting as lack of motor improvement.\u003c/p\u003e\n\u003cp\u003eThe relationship between inflammation and neurogenesis is well-established. Increased NLRP3 activation and TNF-\u0026alpha; are individually associated with reduced neurogenesis (122\u0026ndash;126), and chronic neuroinflammation negatively affects hippocampal neurogenesis and cognitive processes across the lifespan (127\u0026ndash;129). Conversely, anti-inflammatory mediators (130,131), environmental enrichment, and exercise serve as positive modulators of adult hippocampal neurogenesis and associated cognitive function (132\u0026ndash;134).\u003c/p\u003e\n\u003cp\u003eConsistent with the neuroinflammation profile, HC-iMSC groups showed reduced CD4\u003csup\u003e+\u003c/sup\u003e T-cell infiltration compared to PD-iMSCs, suggesting peripheral immune system modulation as reflected by decreased TNF-\u0026alpha; levels in blood serum. Notably, serum TNF-\u0026alpha; levels were reduced more dramatically than midbrain TNF-\u0026alpha; levels, indicating that systemically administered iMSCs (through the intramuscular route) modulated peripheral immune cells earlier and more effectively than neuroinflammatory cells. Nevertheless, this data indicates that neuroinflammation and neurogenesis can indeed be modulated through systemic iMSC administration without requiring invasive direct brain delivery procedures. The reduced migration of PD-iMSCs to the brain following transplantation, indicated by lower PKH-positive cell levels in flow cytometry analysis, provides a mechanistic explanation for their diminished therapeutic efficacy, demonstrating that the impaired migration capacity of PD-iMSCs observed in transwell migration assays \u003cem\u003ein vitro\u003c/em\u003e translates to reduced homing capacity to the SNpc region \u003cem\u003ein vivo\u003c/em\u003e, further limiting their therapeutic potential.\u003c/p\u003e\n\u003cp\u003eTaken together, these results demonstrate that the containment of both peripheral inflammation and neuroinflammation by healthy control iMSCs aids the reversal of behavioural impairments, promotes neurogenesis, and highlights their therapeutic superiority. This provides strong evidence that allogeneic iMSC sources may critically enhance the therapeutic efficacy in PD.\u003c/p\u003e\n\u003cp\u003eBeyond therapeutic implications, the correlation between MSC dysfunction and PD progression also suggests several important mechanistic insights. The early appearance of MSC impairments in the pre-motor stage implies that yet unknown systemic factors associated with PD pathogenesis may directly affect MSC function, resulting in impairment of immunomodulation of peripheral immune cells, which in turn leads to failure in containing neuroinflammation. This hypothesis is supported by the elevated NLRP3 expression and sustained microglial activation observed in animals receiving PD-iMSCs compared to those receiving healthy iMSCs. The increase in dopaminergic neurons and corresponding improvement in motor function upon administration of healthy MSCs in the pre-motor stage suggest that the impairment in function of MSCs is not only an early pathological event but also may be critically involved in the advancement of the disease process. Impairment of MSCs is thus not simply a downstream consequence of advanced neurodegeneration, and compromised MSC function may \u003cu\u003edirectly\u003c/u\u003e contribute to the failure of endogenous neuroprotective mechanisms during the critical pre-motor phase of the disease. These findings fundamentally reframe our understanding of PD pathogenesis.\u003c/p\u003e\n\u003cp\u003eOverall, these findings open several interesting avenues for the future clinical translation of MSC therapy in PD:\u003c/p\u003e\n\u003cp\u003eFirstly, they suggest that autologous MSC therapy using cells from PD patients may have limited efficacy due to intrinsic cellular dysfunction. This challenges current clinical trial designs that predominantly use autologous approaches, and offers a possible explanation for several recent trial failures.\u003c/p\u003e\n\u003cp\u003eSecondly, the mechanistic insights gained from this study suggest potential therapeutic targets for enhancing MSC function in PD. Strategies aimed at reducing oxidative stress, enhancing migration capacity and restoring immunomodulatory function in PD-MSCs could potentially improve their therapeutic efficacy.\u003c/p\u003e\n\u003cp\u003eThirdly, the immunomodulation assay using PD patient PBMCs and MSCs demonstrates a possible simple test to evaluate the effectiveness of MSCs in promoting immunomodulation under PD conditions.\u003c/p\u003e\n\u003cp\u003eFinally, this study underscores a critical advancement in the emerging field of iPSC-derived cell therapy. By suggesting that HLA-matched allogeneic iMSCs might offer superior therapeutic benefits compared to autologous MSCs derived from PD patients, our findings provide compelling evidence supporting the shift toward allogeneic cell sources. This has potentially significant implications for improving the efficacy and consistency of stem cell-based treatments for neurodegenerative diseases.\u003c/p\u003e\n\u003cp\u003eWhile this study provides comprehensive evidence for MSC dysfunction in PD, some limitations must be acknowledged. The relatively small number of human iPSC lines examined\u0026mdash;two healthy and two sporadic PD lines\u0026mdash;limits the generalizability of our findings across the heterogeneous PD patient population. Given the clinical and genetic diversity observed in PD, validation in larger cohorts representing different disease subtypes, stages, and demographic characteristics will be essential to confirm the universal nature of MSC dysfunction in PD.\u003c/p\u003e\n\u003cp\u003eAdditionally, while our study demonstrates clear functional impairments in PD-MSCs and their reduced therapeutic efficacy, the specific upstream molecular mechanisms underlying MSC dysfunction in PD require further elucidation. Understanding whether MSC impairment results from direct exposure to PD-associated pathological factors (such as \u0026alpha;-synuclein species or inflammatory mediators) or represents an intrinsic cellular defect linked to the disease state will be crucial for developing targeted interventions.\u003c/p\u003e\n\u003cp\u003eFuture research should prioritize several key areas. First, identifying the molecular pathways and signalling cascades responsible for MSC impairment in PD will be essential for developing mechanism-based therapeutic strategies. Second, investigating approaches to enhance or restore PD-MSC function\u0026mdash;such as preconditioning protocols, genetic modifications, or pharmacological interventions\u0026mdash;could potentially salvage autologous cell therapy approaches.\u003c/p\u003e\n\u003cp\u003eThird, it would be particularly valuable to examine whether PD-related genetic variants affect MSC physiology and function, thereby determining if MSC dysfunction is a shared feature across both sporadic and familial forms of PD. Such studies could also reveal whether genetic susceptibility factors influence MSC biology and contribute to disease heterogeneity.\u003c/p\u003e\n\u003cp\u003eFinally, longitudinal studies tracking MSC function throughout disease progression, from pre-motor stages through advanced PD, could provide insights into the temporal relationship between MSC dysfunction and clinical deterioration, potentially identifying windows of opportunity for intervention.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study establishes MSC dysfunction as both an early and comprehensive pathological feature of PD, occurring during the critical pre-motor phase when interventions may be most effective. Our findings reveal that MSC impairment encompasses multiple functional domains\u0026mdash;including proliferation, survival, migration, differentiation, and most critically, immunomodulatory capacity\u0026mdash;suggesting a fundamental alteration in MSC biology rather than isolated functional defects.\u003c/p\u003e\n\u003cp\u003eThe profound impairment of MSC immunomodulatory capacity represents a previously unrecognized contributor to PD pathogenesis and suggests that failure of endogenous MSC-mediated immunomodulation and neuroprotection may facilitate disease progression through perpetuation of chronic neuroinflammation and peripheral immune dysfunction. This mechanistic insight positions MSC dysfunction as a potential upstream event in the pathological cascade, rather than merely a consequence of advanced neurodegeneration.\u003c/p\u003e\n\u003cp\u003eFrom a translational perspective, these findings provide a strong scientific rationale for allogeneic MSC therapeutic strategies using healthy donor sources, while raising important concerns about the efficacy of patient-derived autologous MSC approaches in PD treatment. The superior therapeutic efficacy demonstrated by healthy MSCs in both reducing inflammation and promoting neuroregeneration supports a paradigm shift toward allogeneic cell therapy sources for optimal clinical outcomes.\u003c/p\u003e\n\u003cp\u003eBeyond their immediate therapeutic implications, this work advances the fundamental understanding of PD pathophysiology by identifying a novel cellular dysfunction that bridges the gap between peripheral immune abnormalities and central neuroinflammation. It also opens new avenues for both biomarker development, as MSC functional assays could potentially serve as disease monitoring tools, and therapeutic innovation in regenerative medicine for neurodegenerative diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e - All data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e- All authors declare no financial or non-financial competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e - We acknowledge Dr. Manjunath, Department of Neurovirology, NIMHANS for access to Advanced Flow Cytometer facility.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e - Conceptualization: I.D; methodology: R.G and I.D; formal analysis and investigation: R.G, I.D, K.M, R.Y, P.K.P, V.H and N.K; writing - original draft preparation: R.G and I.D; writing-review and editing: I.D, R.G; R.Y, P.K.P, V.H and N.K; funding acquisition: I.D, R.Y, and P.K.P, V.H and N.K; resources: I.D, R.Y, P.K.P, V.H and N.K; supervision: I.D. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e - This work is supported by a grant obtained from DBT-BIRAC Biomanufacturing for Precision Biotherapeutics\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eGovernment of India, New Delhi; contract grant No. 59080 by I.D and R.Y. R.G is supported by CSIR fellowship and K.M by DHR-ICMR YSS fellowship. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCosta HN, Esteves AR, Empadinhas N, Cardoso SM. Parkinson\u0026rsquo;s Disease: A Multisystem Disorder. Neurosci Bull. 2022 Aug 22;39(1):113\u0026ndash;24. \u003c/li\u003e\n\u003cli\u003eAlexander GE. Biology of Parkinson\u0026rsquo;s disease: pathogenesis and pathophysiology of a multisystem neurodegenerative disorder. Dialogues Clin Neurosci. 2004 Sept 30;6(3):259\u0026ndash;80. \u003c/li\u003e\n\u003cli\u003eFearnley JM, Lees AJ. 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Effects of environmental enrichment and voluntary exercise on neurogenesis, learning and memory, and pattern separation: BDNF as a critical variable? Semin Cell Dev Biol. 2011 July;22(5):536\u0026ndash;42. \u003c/li\u003e\n\u003cli\u003eGould E, Beylin A, Tanapat P, Reeves A, Shors TJ. Learning enhances adult neurogenesis in the hippocampal formation. Nat Neurosci. 1999 Mar;2(3):260\u0026ndash;5. \u003c/li\u003e\n\u003cli\u003eKempermann G, Kuhn HG, Gage FH. More hippocampal neurons in adult mice living in an enriched environment. Nature. 1997 Apr;386(6624):493\u0026ndash;5. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1: Table for materials\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePRODUCT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCOMPANY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCATALOG NUMBER\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMPTP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSigma Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e5063820001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDPBS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e21600010\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTri-sodium citrate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eHimedia\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eTC249\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFormamide\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eHimedia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eMB012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNacl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eHimedia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eMB023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnti-Anti\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e15240062\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGlutamaX\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e35050061\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePenstrep\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e15140122\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNEAA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e11140050\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInsulin-Transferrin-Selenium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e51300044\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2-Mercaptoethanol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e21985023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhytohemagglutinin, M\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e10576015\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKnockOut\u0026trade; DMEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e10829018\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNeurobasal\u0026trade; Plus Medium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eA3582901\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAdvanced DMEM/F-12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e12634028\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDMEM/F-12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e21331020\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRPMI 1640 Medium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e11875093\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTrypsin-EDTA (0.25%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e25200072\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStemPro\u0026trade; Accutase\u0026trade;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eA1110501\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFBS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eA5670701\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKnockOut\u0026trade; Serum Replacement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e10828028\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormal Goat Serum\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eAbcam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eab7481\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMitC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSigma Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eM4287\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMatrigel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eCorning\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e356237\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGeltrex\u0026trade;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eA1413302\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eB-27\u0026trade; Plus Supplement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eA3582801\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN-2 Supplement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e17502048\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStemFlex\u0026trade; Medium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eA3349401\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePSC Neural Induction Medium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eA1647801\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStemPro\u0026trade; Adipogenesis Differentiation Kit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eThermo Fisher Scientific (Gibco)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eA1007001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDead Cell Apoptosis Kits with Annexin V for Flow Cytometry\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eInvitrogen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eV13242\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFGF2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eImmunotools\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e11343627\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eActivin A\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eImmunotools\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e11344963\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePDGFBB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eImmunotools\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e11343673\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFGF8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eImmunotools\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e11344834\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSHH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eImmunotools\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e11344074\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGDNF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eImmunotools\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e11343795\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBDNF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eImmunotools\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e11343373\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTGF \u0026beta;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eImmunotools\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e11343160\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRat TNF alpha\u003c/strong\u003e \u003cstrong\u003eRecombinant Protein\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003ePeproTech\u0026reg;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e400-14-20UG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHuman TNF-alpha Recombinant Protein\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eImmunotools\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e11343013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDibutyryl cyclic-AMP\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSigma Aldrich\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.sigmaaldrich.com/IN/en/product/sigma/d0260\"\u003eD0260\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eL-Ascorbic Acid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSigma Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eA4544\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eL-Glutamine\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSigma Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eG8540\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDexamethasone\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eHimedia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eTCL211\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026Beta;-glycerophosphate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eHimedia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eTC463\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHisep LSM 1084\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eHimedia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHisep LSM 1077\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eHimedia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlizarin red S\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eHimedia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eGRM894\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOil red O\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eHimedia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eTC256\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eToluidine Blue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eHimedia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eTC257\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBODIPY\u0026trade; 493/503\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eInvitrogen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eD3922\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePKH26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSigma Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eMINI26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eH2DCFDA (H2-DCF, DCF)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eInvitrogen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eD399\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDAPI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eInvitrogen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eD21490\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSheath Fluid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eBD FACSFlow\u0026trade;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e342003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSodium azide\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eHimedia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eTC704\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParaformaldehyde\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSigma Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003e158127\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTritonX\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSigma Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eT8787\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBovine Serum Albumin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eHimedia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eMB083\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRat Dopamine ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSunlong Biotech\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eSL0243Ra\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRat TNF alpha ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSunlong Biotech\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eSL0700Ra\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRat PGE2 ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eKrishgen Biosystems\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eKLR0540\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRat TGF beta ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eKrishgen Biosystems\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eKLR0778\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRat IL-10 ELISA\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSunlong Biotech\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eSL0415Ra\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRat IDO ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSunlong Biotech\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eSL1203Ra\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRat MMP3 ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eBT Lab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eE0316Ra\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHuman TGF beta ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSunlong Biotech\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eSL1736Hu\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHuman PGE2 ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSunlong Biotech\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eSL1463Hu\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHuman IDO ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSunlong Biotech\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eSL0922Hu\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHuman IL-10 ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eSunlong Biotech\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eSL0967H\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHuman Dopamine ELISA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 37.4376%;\"\u003e\n \u003cp\u003eImmunotag\u0026trade;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.2845%;\"\u003e\n \u003cp\u003eIT11997\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Table for Antibodies (RRID)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSl No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibody Name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHost\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcerned titration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCatalogue No. \u0026amp; RRID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eTH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# T1299\u003c/p\u003e\n \u003cp\u003eRRID: AB_477560\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"2\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eTH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# PAB438Ra01\u003c/p\u003e\n \u003cp\u003eRRID: AB_3665786\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"3\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003ep-Syn (S129)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# PA1-4686\u003c/p\u003e\n \u003cp\u003eRRID: AB_2192960\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"4\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eIBA-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# MA5-27726\u003c/p\u003e\n \u003cp\u003eRRID: AB_2735228\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"5\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eTNF-Alpha\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# 7124-MSM12-P1\u003c/p\u003e\n \u003cp\u003eRRID: AB_3714899\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"6\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eGFAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# PAA068Ra01\u003c/p\u003e\n \u003cp\u003eRRID: AB_3714900\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"7\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eNLRP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# PA5-79740\u003c/p\u003e\n \u003cp\u003eAB_2746855\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"8\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eCD4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# 14-0040-82\u003c/p\u003e\n \u003cp\u003eRRID: AB_953584\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"9\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eCD8a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# 14-0084-82\u003c/p\u003e\n \u003cp\u003eRRID: AB_1210523\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"10\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eKi67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# MA5-14520\u003c/p\u003e\n \u003cp\u003eRRID: AB_10979488\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"11\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eCXCR4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# PAA940Ra01\u003c/p\u003e\n \u003cp\u003eRRID: AB_3714901\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"12\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eCD90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# MAB404Hu21\u003c/p\u003e\n \u003cp\u003eRRID: AB_3714902\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"13\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eCD105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# PAA980Hu01\u003c/p\u003e\n \u003cp\u003eRRID: AB_3714903\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"14\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eCD73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# PAB250Hu01\u003c/p\u003e\n \u003cp\u003eRRID: AB_3714904\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"15\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eRunx2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# PA5-82787\u003c/p\u003e\n \u003cp\u003eRRID: AB_2789943\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"16\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eFOXA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# PA5-35097\u003c/p\u003e\n \u003cp\u003eRRID: AB_2552407\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"17\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eCD90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# 554895\u003c/p\u003e\n \u003cp\u003eRRID: AB_395586\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"18\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eCD73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# 551123\u003c/p\u003e\n \u003cp\u003eRRID: AB_394057\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"19\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eEN1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# PA5-14149\u003c/p\u003e\n \u003cp\u003eRRID: AB_2231168\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"20\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eMAP2ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# M2320\u003c/p\u003e\n \u003cp\u003eRRID: AB_609904\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"21\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003e\u0026beta;-Tub III\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# ab18207\u003c/p\u003e\n \u003cp\u003eRRID: AB_444319\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"22\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eNurr1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# PA5-13416\u003c/p\u003e\n \u003cp\u003eRRID: AB_2153896\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"23\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eVMAT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# PA5-22864\u003c/p\u003e\n \u003cp\u003eRRID: AB_11154073\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"24\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eGIRK2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# ab259909\u003c/p\u003e\n \u003cp\u003eRRID: AB_3714905\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"25\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eAPC-Cy\u0026trade;7 Mouse Anti-Human HLA-DR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# 335796\u003c/p\u003e\n \u003cp\u003eRRID: AB_399974\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"26\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003ePE Mouse Anti-Human CD56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# 347747\u003c/p\u003e\n \u003cp\u003eRRID: AB_400346\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"27\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eFITC Mouse Anti-Human CD45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# 555482\u003c/p\u003e\n \u003cp\u003eRRID: AB_395874\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"28\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003ePE Mouse Anti-Human CD34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# 550761\u003c/p\u003e\n \u003cp\u003eRRID: AB_393871\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"29\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eFITC Mouse Anti-Human CD80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# 557226\u003c/p\u003e\n \u003cp\u003eRRID: AB_396605\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"30\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eFITC Mouse Anti-Human CD86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# 555657\u003c/p\u003e\n \u003cp\u003eRRID: AB_396012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"31\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eGoat Anti-Mouse IgG H\u0026amp;L (Alexa Fluor\u0026reg; 488)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eGoat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# ab150113\u003c/p\u003e\n \u003cp\u003eRRID:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eAB_2576208\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"32\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eGoat Anti-Rabbit IgG H\u0026amp;L (Alexa Fluor\u0026reg; 488)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eGoat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# ab150077\u003c/p\u003e\n \u003cp\u003eRRID: AB_2630356\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"33\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eGoat Anti-Mouse IgG H\u0026amp;L (Alexa Fluor\u0026reg; 647)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eGoat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# ab150115\u003c/p\u003e\n \u003cp\u003eRRID: AB_2687948\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.34891%;\"\u003e\n \u003col start=\"34\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.0568%;\"\u003e\n \u003cp\u003eGoat Anti-Rabbit IgG H\u0026amp;L (Alexa Fluor\u0026reg; 647)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003eGoat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003e1:200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.8648%;\"\u003e\n \u003cp\u003ecat# ab150079,\u003c/p\u003e\n \u003cp\u003eRRID:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eAB_2722623\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7957744/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7957744/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Parkinson's disease (PD) is characterized by chronic neuroinflammation and peripheral immune dysfunction, yet the mechanisms underlying systemic-immunomodulatory failure remain unclear. Here we demonstrate that mesenchymal stromal cell (MSC) dysfunction represents an early pathological feature of PD, occurring during the pre-motor phase. Using MPTP-induced rat model, we show that bone-marrow MSC impairment emerges at week 1 post-treatment, coinciding with dopaminergic neurodegeneration but preceding motor-symptoms, with reduced proliferation, impaired migration, elevated oxidative stress, and diminished immunomodulatory capacity. Patient-derived induced pluripotent stem cell-derived MSCs (iMSCs) from sporadic-PD patients recapitulated these dysfunctions and exhibited severely compromised ability to suppress peripheral-blood mononuclear-cell proliferation and PD patient PBMCs in immunomodulation assays. Transplantation studies in MPTP-induced rats revealed that healthy-control iMSCs provided superior neuroprotection, reduced inflammation, promoted neurogenesis, and improved motor-function versus PD-iMSCs. These findings identify MSC immunomodulatory-dysfunction as an upstream contributor to PD pathogenesis and provide rationale for allogeneic over autologous MSC therapeutic strategies in PD treatment.","manuscriptTitle":"Pre-motor Mesenchymal stromal Cell Dysfunction Drives Immune Dysregulation in Parkinson’s Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 10:13:22","doi":"10.21203/rs.3.rs-7957744/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9128895d-d0d9-46c9-a676-97382699cc24","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57002107,"name":"Biological sciences/Neuroscience/Cellular neuroscience"},{"id":57002108,"name":"Health sciences/Diseases/Neurological disorders/Parkinson's disease"}],"tags":[],"updatedAt":"2025-11-10T17:11:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-31 10:13:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7957744","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7957744","identity":"rs-7957744","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}