All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Dankook University Medical School (approval number: DKU-23-058) and were conducted in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. Furthermore, we confirm that all procedures are reported in compliance with the ARRIVE guidelines. Animals were housed under a 12-h light/dark cycle with free access to standard food pellets and water.

Primary cortical neurons were prepared from embryonic day 17 (E17) Sprague–Dawley rat fetuses, as previously described with minor modifications (Hong et al., 2023). Pregnant rats were anesthetized with 16.5% urethane, and the fetal cortices were dissected in calcium- and magnesium-free HEPES-buffered Hank’s balanced salt solution (pH 7.45, Gibco, Life Technologies Corporation, NY, United States). Cortical tissues were gently dissociated using a series of flame-polished Pasteur pipettes. The dissociated cells were seeded at a density of 1.6 × 10⁴ cells/well onto 25-mm glass coverslips pre-coated with 0.2 mg/mL Matrigel (Corning, Bedford, MA, United States) for 1 h. Neurons were maintained in Neurobasal medium supplemented with 2% B27 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, United States), 0.25% GlutaMAX-I(Gibco; Life Technologies Corporation), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.025 μg/mL amphotericin B (Sigma-Aldrich, St. Louis, MO, United States) in a humidified incubator (10% CO₂, 90% air, 37 °C, pH 7.4). The medium was replenished every 4 days by replacing 75% of the volume with fresh medium. All experiments were conducted using six independent cultures. On day in vitro (DIV) 13, neurons were exposed to either 10 μM or 100 μM hydrogen peroxide (H₂O₂) for 30 min, after which the medium was completely replaced with fresh culture medium.

This study was approved by the Institutional Review Board of Dankook University Hospital (Approval No. DKUH-2020-05-001), and all procedures were conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all donors prior to the collection of human-derived biological materials (hMSCs). The manuscript does not contain any personally identifiable information of the donors other than age and sex. A 50-year-old female patient who provided informed consent prior to surgery participated in this study. Cell sorting and differentiation were performed by N-BIOTEK (Bucheon-si, Gyeonggi-do, 14,502, Republic of Korea), which is an officially approved stem cell engineering center by the Korean Government. Further information is provided in a previous publication (Chang et al., 2023). hMSCs were isolated from human adipose tissue. The adipose tissue was washed three times with 1X phosphate-buffered saline (PBS) and digested with 0.1% (w/v) type I collagenase (Nordmark, Uetersen, Germany) for 45 min at 37 °C. Undigested tissue and excess oil were filtered out using a screen mesh, followed by centrifugation at 1500 rpm for 5 min. The passage number used in the experiment was passage 4. hMSCs were cultured in α-MEM (Corning) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin at 37 °C in a 5% CO₂ incubator. To confirm the characteristics of the hMSC spheroids, the expression of CD73 (Invitrogen, 41–0200), CD90 (Abcam, ab307736), and CD105 (Invitrogen, MA5-17041) was examined as a positive marker, while CD45 (Abcam, ab10558) was examined as a negative marker. Cells were seeded in 90 mm culture dishes and subcultured when they reached approximately 80–90% confluence. For spheroid formation, hMSCs were seeded in ultra-low attachment U-bottom 96-well plates (Sumitomo Bakelite Co., Ltd., Kobe, Japan) at a density of 3.5 × 10³ cells per well in 100 μL of complete medium. The plates were centrifuged at 1,000 rpm for 3 min to promote cell aggregation and then incubated for 48 h to allow spontaneous spheroid formation. At the time point when primary cortical neurons had established mature neuronal networks and hMSC spheroids had developed a compact and transferable structure, the two cell types were co-cultured. One hMSC spheroid was added to each well containing cortical neurons.

hMSC spheroids were irradiated with either 660 nm (10 mW/cm²) or 850 nm (12 mW/cm²) wavelength light for 10 min prior to their application to cortical neuron cultures. On DIV 13, 30 min after H₂O₂ exposure, the irradiated hMSC spheroids were introduced to the neuronal cultures. PBM exposure was performed in the dark, and a power meter (PD300 and VEGA, Ophir Photonics) was used to confirm light intensity. Neurons were fixed 24 h after H₂O₂ treatment for subsequent analyses. To assess power density-dependent effects of PBM on spheroid-mediated neuroprotection, additional spheroid groups were irradiated at multiple power densities prior to co-culture. For 660 nm PBM, spheroids were exposed at 5, 10, or 20 mW/cm², and for 850 nm PBM at 6, 12, or 24 mW/cm² (10 min), followed by co-culture under H₂O₂ injury conditions and MTT assessment at 24 h (Supplementary Figure 1).

Cortical neurons and hMSC spheroids were fixed with cold methanol (−20 °C) for 8 min. After fixation, cells were permeabilized with 0.3% Triton X-100 for 5 min and blocked with 10% bovine serum albumin (BSA) for 1 h. Neuronal samples were incubated overnight at 4 °C with a primary antibody against mouse anti-MAP2 (Sigma-Aldrich, M9942), followed by incubation with an Alexa Fluor 555-conjugated anti-mouse IgG secondary antibody (ThermoFisher, A21422). For the immunophenotypic characterization of hMSC spheroids, the samples were stained with primary antibodies against CD73 (Invitrogen, 41–0200), CD90 (Abcam, ab307736), CD105 (Invitrogen, MA5-17041), and CD45 (Abcam, ab10558), followed by appropriate Alexa Fluor 488-conjugated secondary antibodies. After staining, all samples were mounted with VECTASHIELD Antifade Medium containing DAPI (Vector Laboratories, H-1200) and imaged using a confocal microscope (FV3000, Olympus, Tokyo, Japan). Excitation/emission wavelengths for Alexa Fluor 488 and 555 were 488/520 nm and 561/568 nm, respectively.

Confocal imaging was performed using a 20 × objective on an FV3000 microscope (Olympus). Z-stacked images were acquired across 10 μm (1 μm step size) and merged into a single maximum intensity projection. For each condition, 3–4 random fields were captured. MAP2-labeled dendritic structures were visualized and analyzed using the Metamorph software (Molecular Devices) to assess dendritic morphology in primary cortical neurons.

To evaluate cell viability, cortical neurons were treated with H₂O₂ and hMSC spheroids. After 24 h, cells were incubated for 4 h with 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich). The resulting formazan crystals were dissolved in dimethyl sulfoxide, and absorbance was measured at 570 nm using a microplate reader. The levels of neurotrophic and angiogenic factors secreted by hMSC spheroids were quantified using enzyme-linked immunosorbent assays (ELISA). Brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF) concentrations in culture supernatants were measured using the following commercially available human ELISA kits: Human BDNF SimpleStep ELISA Kit (ab212166, Abcam, United Kingdom), Human NGF ELISA Kit (ab193760, Abcam, United Kingdom), and Human VEGF ELISA Kit (ab222510, Abcam, United Kingdom), according to the manufacturers’ instructions. Briefly, culture media were collected immediately after the PBM treatment of hMSC spheroids, centrifuged at 2,000 × g for 5 min to remove cellular debris, and stored at −20 °C until analysis. For each assay, standards and samples (50 μL per well) were added to pre-coated 96-well plates, followed by the addition of the corresponding antibody cocktail. The plates were incubated for 1 h at room temperature with gentle shaking. After washing, tetramethylbenzidine (TMB) substrate solution was added, and the reaction was stopped using stop solution. Absorbance was measured at 450 nm using a microplate reader. All samples were analyzed in duplicate, and concentrations were calculated based on standard curves generated for each factor.

The results were analyzed using the Prism software 8.4.3 (GraphPad Software, La Jolla, CA, United States; RRID: SCR_002798). Normality of the data distribution was assessed prior to statistical analysis. Two-tailed Mann–Whitney U tests (non-parametric) and unpaired non-parametric t-tests were performed to compare the hMSC size over time, as these datasets did not meet the assumptions of normality and involved independent group comparisons. One-way repeated measure analysis of variance (ANOVA) followed by Tukey’s multiple comparisons tests was used to compare cell viability, axonal length, and branch number, as these parameters were measured repeatedly across multiple experimental conditions within the same samples and met parametric assumptions. p-values < 0.05 were considered to indicate significance. All statistical comparisons and corresponding p-values for each figure are summarized in Table 1.

To resemble regenerating neurons from various types of neuronal damage, primary cortical neurons were harvested from rat pups and cultured (Figure 1A). With the appropriate application of factors (see methods), the initially heterogeneous cell population gradually became homotypic, consisting of neural cells with axonal network connections (Figure 1B). These axonal connections were identified through epifluorescence analysis of MAP2 antibody expression. hMSCs were aggregated into spheroids (using the cell number of 3.5 × 10³), and these spheroids were structurally stable for over 48 h (Figure 1C). The average size of the hMSC spheroid reached 300 μm 48 h after initial cell seeding for spheroid formation, showing a statistically significant increase compared to 24 h (p < 0.0001; Figure 1D). To verify the characteristics of hMSC spheroids, the expression of representative hMSC markers was examined. Among these, the positive markers CD73, CD90, and CD105 were expressed (Figure 1E). The hMSC spheroids were divided into three groups: a spheroid-only group that received no additional treatment, a 660 nm PBM group irradiated for 10 min, and an 850 nm PBM group irradiated for 10 min. After this short PBM preconditioning, the hMSC spheroids were co-cultivated with primary cortical neuronal networks that had been damaged with varying concentrations of H₂O₂ (10 or 100 μM for 30 min; Figure 1A). A quantitative comparison of secreted neurotrophic and angiogenic factors in hMSC spheroids after PBM treatment was conducted. ELISA analysis revealed that PBM-treated hMSC spheroids exhibited significantly higher levels of the BDNF, NGF, and VEGF compared to non-PBM-treated spheroids (Figure 1F), indicating an enhanced paracrine secretory profile following PBM preconditioning.

H₂O₂ exposure represents a cytotoxic stimulus that can cause damage to neuronal cells. Exposure to 10 μM H₂O₂ resulted in a significant reduction in cell viability to approximately 75% relative to the control (p = 0.0016; Figure 2A). At higher H₂O₂ concentrations, cell viability was drastically decreased to 21% (p < 0.0001; Figure 2B). To investigate the neuronal regenerative potential of hMSCs in an H₂O₂ toxicity model, hMSC spheroids, with or without PBM preconditioning (660 nm or 850 nm), were co-incubated with primary cortical neurons exposed to H₂O₂. Under low H₂O₂ concentration conditions, co-incubation with various hMSC spheroids did not alter cell viability (Figure 2A). In contrast, under high H₂O₂ concentration conditions, co-incubation with hMSC spheroids led to an increase in cell viability. Notably, both 660 nm- and 850 nm-PBM-treated hMSC spheroids significantly improved cell viability compared to the H₂O₂-only group and the spheroid group without PBM (p = 0.0006 for 660 nm vs. H₂O₂ only; p = 0.0013 for 850 nm vs. H₂O₂ only; Figure 2B). To assess whether PBM efficacy depends on irradiation power density, we performed a power titration for each wavelength prior to spheroid application (660 nm: 5/10/20 mW/cm²; 850 nm: 6/12/24 mW/cm²; 10 min). While PBM power titration did not markedly alter viability under low oxidative stress conditions, several power settings improved viability under high H₂O₂ conditions, indicating that PBM effects on hMSC spheroid-mediated neuroprotection are parameter-sensitive (Supplementary Figure 1).

Primary cortical neurons were exposed to cytotoxic H₂O₂ (10 or 100 μM for 30 min) and subsequently co-cultivated with spheroids, with or without PBM. On day 1, samples were collected for cell viability assessment and epifluorescence analysis. After co-cultivation, hMSCs spread out, and some were in contact with primary cortical neurons (Figure 3A). For microscopic analyses, areas not in contact with hMSCs were assessed. (Figure 3A). After H₂O₂ exposure, even at low concentrations, the number of cells observed in light microscopy (Figure 3A) and the number of neuronal cells (MAP2) in epifluorescence analysis (Figure 3B) were significantly decreased. Epifluorescence analysis showed that the number of MAP2-expressing cells after high-concentration H₂O₂ exposure was also significantly reduced (Figure 3C). To evaluate the axonal re-sprouting potential of hMSCs in an H₂O₂ toxicity model, the total outgrowth length and rate of branching were evaluated. Across all H₂O₂ treatment conditions, axonal sprouting was decreased and shortened (Figures 3B,C). Under low-concentration H₂O₂ conditions, sprouting frequency (branches) was reduced to 38.19 ± 4.34% of control levels (p = 0.0008), and total outgrowth length decreased to 45.48 ± 4.35% of control levels (p = 0.0015). Under high H₂O₂ concentration conditions, sprouting frequency (branches) was reduced to 30.67 ± 4.58% of control levels (p = 0.0019), and total outgrowth length decreased to 37.28 ± 5.13% of control levels (p = 0.0023; Figures 3D,E). As shown in representative images after low H₂O₂ exposure, the frequency of axonal branching was relatively higher in hMSC spheroid co-cultures, especially those involving PBM-treated hMSC spheroids (Figure 3B). There was a statistically higher sprouting frequency in the 850 nm PBM-treated hMSC spheroid co-incubation group (83.25 ± 10.49% of control, p = 0.0263) compared to the low-concentration H₂O₂-only group (Figure 3E). Under high H₂O₂ concentration conditions, PBM-treated hMSC spheroids showed restoration of neurite parameters. Specifically, total outgrowth length increased to 94.19 ± 15.79% (660 nm, p = 0.0148) and 101.49 ± 11.71% (850 nm, p = 0.0083) of control levels (Figure 3D), while branching frequency increased to 93.81 ± 16.76% (660 nm, p = 0.0122) and 99.36 ± 13.77% (850 nm, p = 0.0102) of control levels (Figure 3E), compared to the H₂O₂-only group. To distinguish between direct cell–cell interactions and paracrine-mediated effects, we compared a direct co-culture of hMSC spheroids with primary cortical neurons to an indirect co-culture system using 0.4 μm Transwell inserts. The inserts physically prevent cell contact while allowing the diffusion of secreted factors. Quantitative analysis of MAP2-positive neurite outgrowth and branch density revealed that neuronal survival and axonal regeneration were comparably enhanced in both direct and Transwell co-culture conditions (Supplementary Figure 2). These results indicate that the neuroprotective and neuritogenic effects of PBM-treated hMSC spheroids are predominantly mediated by paracrine signaling rather than direct cell–cell contact.