A detailed list of chemicals and materials used in this study is provided in Supplementary Material 1, Table S1.
Cell culture conditions
Human ASCs, MSCs harvested from normal human adipose tissue (C12977, hMSC-AT), were purchased from PromoCell GmBH and cultured in a mixture of 25% mesenchymal stem cell growth medium (C28009, PromoCell) and 75% Dulbecco’s modified Eagle’s medium high glucose (DMEM-high; 12400-061, Gibco), supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1% penicillin/streptomycin (P/S) (Gibco). Hs68 cells (BCRC No. 60038), a human foreskin fibroblast cell line, were obtained from the Bioresource Collection and Research Center (BCRC) of Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan), and the rat schwannoma cell line RT4 (RT4-D6P2T, ATCC CRL-2768) was acquired from ATCC. Both Hs68 and RT4 cells were cultured in DMEM-high media supplemented with 10% FBS and 1% P/S. All cells were maintained in a humidified incubator with 5% CO2 at 37 °C.
3D sphere induction
For chitosan-induced sphere formation, cells were seeded onto the chitosan-coated surface at a density of 1 × 106 cells in 10 ml growth media on a 9 cm plate to induce spheroid formation for 72 hours as following the established protocols (35, 37). For sphere formation in ultra-low attachment (ULA) microplates (7007, Corning), 3000 cells were seeded per well in 200 μl growth media to assembly the 3D spheres for 72 hours.
Pathway inhibitors and treatments
The epigenetic inhibition compounds were added to the growth media at the same time as sphere induction: 70 μM SNDX-5613 (Revumenib, a KMT2A inhibitor) targeting KMT2A (MLL1) to reduce H3K4me3, 25 nM SUVi (Chateocin, a SUV39H1 inhibitor) to block H3K9me3, 10 μM GSK126 (an EZH2 inhibitor) for inhibiting H3K27me3, and 500 nM LMK235 as a HDAC5 inhibitor. The Food and Drug Administration (FDA)-approved peroxisome proliferator-activated receptor gamma (PPARγ) agonist, Rosiglitazone (RSG), was used to investigate the gain-of-function in PPARγ signaling. The PPARγ antagonist, GW9662, was applied to verify the loss-of-function in the PPARγ pathway. The RSG or GW9662 were added to the growth media during sphere formation at the concentration of 2, 5, 10, and 20 μM.
Mitochondrial staining, observation, and artificial intelligence-based Classification
Prior to sphere formation on chitosan-coated surface, ASCs were incubated with MitoTracker™ Deep Red FM dye at a concentration of 30nM for 45 min at 37°C. After labeling the mitochondria, cells were washed twice with PBS to remove excess dye. Following 3 days of sphere formation, the spheres were rinsed once with PBS and fixed with 4% paraformaldehyde (PFA, EM grade) in PBS at 4°C overnight. To observe the mitochondria with cytoskeleton arrangement, rhodamine-phalloidin dye (R415, Thermo Fisher Scientific) was added and incubated overnight at 4°C. Subsequently, the spheres were rinsed three times with PBS, permeabilized by 0.5% Triton X-100 for 2 hours, and then mounted using ProLong® Diamond Antifade Mountant with DAPI in the dark. Confocal microscopy was utilized to examine mitochondrial morphology using the Olympus FluoView™ FV3000 confocal microscope (Olympus, Japan).
For defining and quantifying MitoTracker-labeled mitochondrial shapes, the "Maximum Extent Inner" feature in the cellSens software was employed. This feature represents the maximum length of a line connecting two boundary points within the object. Subsequently, classification was performed using an artificial intelligence (AI) machine-learning-based mitochondrial neural network within Olympus cellSens Dimension software (Olympus, Japan), categorizing them into three classes based on their inner lengths: Fragmented (0.7-1.2 μm), Tubular (1.21-4.5 μm), and Compact (or Clustered) (4.51 μm and beyond). The quantification of MitoTracker fluorescent intensities in different categorized mitochondria was normalized to cell numbers by nuclei staining using DAPI or Hoechst 33342.
Transmission electron microscopy analysis of the mitochondrial ultrastructure
Collected 3D spheres and adherent ASCs were rinsed with pre-cooled PBS and then immersed in a fixative solution containing 2.5% glutaraldehyde and 3 mM CaCl3 in 0.1M cacodylate buffer for 1 hour at 4°C. The adherent 2D-cultured ASCs were then scraped into a tube. After washing with 0.1M cacodylate buffer containing 3mM CaCl3, cells underwent post-fixation by immersion in 0.1M cacodylate buffer containing 1% osmium tetroxide and 1.5% potassium ferricyanide at 4°C for 40 min. Following the post-fixation step, the samples were washed with distilled water and then gradually dehydrated by immersion in a series of ethanol solutions with increasing concentrations: 70%, 90%, and 95%, with each stage lasting 15 min. Subsequently, the samples were immersed in 100% ethanol three times, with each immersion lasting 30 min. The dehydrated samples were then infiltrated in stages with spur resin–ethanol solutions containing 50%, then 75%, and finally 100% resin, with each stage lasting 1 hour. After overnight incubation in 100% spur resin suppression, the specimens were embedded in fresh resin and polymerized at 70°C for 24 hours. The embedded samples were sectioned into 70 nm ultrathin slices using an ultramicrotome (Ultracut S, Leica Reichart) equipped with a diamond knife, and the sections were collected on nickel grids. These grids were subsequently post-stained with uranyl acetate and lead citrate. TEM analysis was performed using a JEM-1400 transmission electron microscope (JEOL, Ltd., Japan) operating at 120 keV, coupled with a 4k x 4k CCD Camera System 895 (UltraScan 4000, Gatan Inc., USA).
Single-cell RNA sequencing assay
The scRNA-seq data from this study build upon an important aspect of our previous research. The procedures for scRNA-seq libraries preparation, sequencing, and analysis for 2D-cultured ASCs and 3D ASC spheres were detailed in a prior publication (35). Monocle 2 software (38) was used for trajectory analysis to explore cell fate and cluster associations for investigating the cell differentiation trajectories between ASCs and chitosan-induced ASC spheres. The gene expression pattern analysis and directional predictions were conducted using Ingenuity Pathway Analysis software (IPA®, QIAGEN Inc, CA, USA).
Western blotting analysis
The 2D-cultured ASCs or 3D ASC spheres were lysed using a cell lysis buffer (9083; Cell Signaling Tech) supplemented with a protease inhibitor cocktail (7012; Cell Signaling Tech) and PMSF (8553, Cell Signaling Tech). The protein concentration was measured using a Bio-Rad protein assay dye (Bio-Rad Laboratories Inc., USA). Thirty micrograms of total protein were loaded in Western blotting analysis in according to previous study (37, 39). The target proteins were immunoblotted with specific antibodies (detailed in Table S2), and signal visualization was achieved using Trident femto Western HRP Substrate and a UVP EC3 imaging system (UVP Inc., Upland, CA, USA). The quantification of signal intensity was performed using ImageJ software (Image J, NIH).
ATP production detection assay
An ATP luminescence detection assay kit (A22066, Molecular Probes, Inc.; Invitrogen, Ltd., UK) was used to measure the ATP production of 2D-cultured ASCs and 3D ASC spheres, following the experiment procedure outlined in the manufacturer's instructions. In brief, ten micrograms of total protein in a 10 μl cell lysate were mixed with 100 μl of assay buffer containing D-Luciferin, DTT, and firefly luciferase. The luciferase intensity was then measured at 560 nm. The ATP concentration was normalized by total protein.
Real-time assessment of mitochondrial function using the Seahorse system
Mitochondrial oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were assessed using the Seahorse XFe24 Analyzer (Agilent, Santa Clara, CA, USA). The procedures were carried out according to the manufacturer's instruction manual. In brief, a XFe24 cell culture microplate was coated with fibronectin (FN, 20 μg/ml) and incubated at 37°C for 30 min. For 2D-cultured ASCs, 5000 cells were seeded onto the FN-coated plates one day before the experiment. For 3D spheroid formation, spheres were transferred and settled in microplates for an hour before the Seahorse assay.
Mitochondrial respiration was analyzed under basal conditions and in response to sequential injections of Oligomycin (1.5 μM), fluoro-carbonyl cyanide phenylhydrazone (FCCP, 1 μM), and Rotenone/Antimycin (0.5 μM each) using the Seahorse XF Cell MitoStress Test Kit. Real-time measurements for each drug were recorded over 5 cycles of 3 min in the Seahorse XF Cell Mito Stress Test program in Wave Controller 2.4 software (Agilent). The OCR was normalized to total protein content per well. The ATP production rates were determined using the Seahorse XF Real-Time ATP Rate Assay Kit following the recommended protocol. Data sets were analyzed using the Wave software and Excel software.
RNA extraction, reverse transcriptase PCR and real-time PCR analysis
Total RNA was extracted from ASCs or spheres using TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA) and the Direct-zol RNA Miniprep kit (Zymo Research, Inc., CA, USA), following the manufacturer’s protocol. The concentration of total RNA was determined by measuring absorbance at 260 and 280 nm using a nucleic acid spectrometer (Nanodrop ND1000, Thermo Fisher Scientific). Two micrograms of total RNA were reverse-transcribed into cDNA using the SuperScript II Reverse Transcriptase Kit (Invitrogen). The real-time PCR (qPCR) analysis was conducted using the SYBR Green Master Mix (Thermo Scientific) and ABI StepOne PlusTM (Applied Biosystems). The qPCR conditions were as follows: 50°C for 2 min, 95 °C for 2 min, and 45 cycles of 95 °C for 3 seconds (s) and 60 °C for 20 s, followed by routine melting and cooling steps. The primer pairs used for qPCR in current study were listed in Table S3. GAPDH was employed as the reference gene for mRNA levels, and each sample was examined in triplicate. The fold change for target gene expression was calculated using the ∆∆Ct method.
ROS assay
The cellular ROS assay kit (Ab113851, Abcam) was used to detect the occurrence of oxidative stress during 3D sphere formation. In 2D-cutlured ASCs, adherent ASCs were seeded on an 8-well chamber slide and rinsed once with PBS before being stained with 2', 7'-dichlorofluorescin diacetate (DCFDA) for 1 hour. To detect the ROS in spheres, the 3D-assembled spheres were collected in a conical tube by centrifugation and rinsed once in PBS. The spheres were stained by resuspending them in the DCFDA solution and incubating at 37°C for 3 hours. After removing the DCFDA solution, the spheres were rinsed with PBS and resuspended in complete media without phenol red, supplemented with 10% FBS. Nuclei were stained with Hoechst 33342 for 30 min before microscopy examination.
Nuclear/Cytosol fractionation
To separate the nuclear extract from the cytoplasmic fraction of cells, the Nuclear Extraction Kit (No. 10009277, Cayman Chemical Inc., MI, USA) was used. Briefly, adherent 2D-cultured ASCs were washed with PBS containing protease inhibitors and then scraped into the hypotonic buffer. For 3D spheroids, chitosan-induced ASC spheres were transferred to a centrifuge tube and collected by centrifugation at 800 rpm for 5min, after which the medium was discarded. Spheres were then washed with PBS containing protease inhibitors and centrifuged again before being resuspended in the hypotonic buffer. After a 15-min incubation, 200 μl of 10% NP-40 per ml of hypotonic buffer was added, and the mixture was centrifuged at 14,000 g for 30 seconds at 4°C. The supernatant containing the cytosolic fraction was transferred to a new tube. The pellet was washed twice with ice-cold PBS and resuspended in the nuclear extraction buffer. To isolate the nuclear proteins, the suspension was vortexed vigorously for 15 seconds, incubated on ice for 15 min, followed by a 30-second vortex, and another 15-min incubation on ice. Finally, the supernatant containing the nuclear fraction was collected by centrifugation at 14,000 g for 10 min at 4°C. Both the nuclear and cytoplasmic protein fractions were stored at -80°C for further analyses.
PPARγ transcription factor assay
The specific transcription factor DNA binding activity of PPARγ was detected using a PPARγ transcription factor assay kit (Ab133101, Abcam), following the manufacturer's protocol. Briefly, ten micrograms of total protein in a 10 μl cell lysate were mixed with 90 μl Transcription Factor Binding Assay Buffer (TFB) per well of 96-well plate and incubated overnight ar 4°C without agitation. After five PBS washes, PPARγ primary antibody was added and incubated at room temperature for 1 hour. Subsequently, after five PBS washes, the second antibody (goat anti-rabbit HRP conjugate) was added and incubated for 1 hour. To each well, 100 µl of developing solution was added and incubated for 15 min. The colorimetric absorbance was read at 450 nm within five min of adding the stop solution.
Cell inflammation models and cell-free mitochondrial transfer experiments
A. Isolation of enhanced mitochondria from spheres: Mitochondria in 2D-cultured ASCs or chitosan-induced 3D spheres were extracted using Mitochondria Isolation Kit for cultured cells (Abcam, ab110170). Cells and spheres were collected by centrifugation at 800 rpm for 5 min, rinsed once with PBS, and subjected to a freeze-thaw process to disrupt the cell membrane. The samples from different groups were then suspended in Reagent A for 10 min and homogenized with 30 strokes using a Dounce homogenizer. The resultant mixture was centrifuged at 4000 rpm for 10 min. The supernatant containing Reagent A was collected. Next, Reagent B was added to resuspend the cell pellet, followed by a 10-min incubation, cell homogenization, and another centrifugation at 4000 rpm for 10 min. The combined supernatants (Reagent A and B) were centrifuged at 12000 rpm for 15 min. The pellet was washed once with PBS, centrifuged again. After removing the PBS, the mitochondria were resuspended in growth media, and then administered to inflamed cells. Approximately 50 μg of mitochondria, quantified using the Bio-Rad protein assay, can be extracted from spheres formed by 1 × 106 ASC cells in a 9 cm chitosan-coated dish after 72 hours of culture. To visualized the extracted mitochondria, the MitoTracker™ Deep Red FM dye (red fluorescent color) were applied to ASCs or ASC spheres for staining and tracing the mitochondria prior isolation. The extracted mitochondria (exMito) were freshly applied for in vitro cell rescue experiments.
B. Inflamed cell models and treatments: For investigating the anti-inflammation and cell rescue outcome, RT4 cells (2.5 × 105 cells) were seeded in 6 cm culture dishes overnight and then subjected to 20 μg/ml lipopolysaccharide (LPS) or 10 ng/ml Tumor Necrosis Factor-α (TNFα) for 3 hours. The extracted mitochondria (50 μg) from various stem cell groups were added to the inflamed RT4 cells growing in a 10 cm plate and incubated for additional 48 hours. The RT4 cells were harvested for Western blotting to measure the inflammatory protein expressions, and qPCR for cytokine mRNA expressions.
To observe the delivery of exMito and the intracellular mitochondrial dynamics between exMito and endogenous mitochondria, the RT4 cells were first labeled with MitoTracker™ Green FM dye (green fluorescent color) for 45 min at 37°C before inflammatory induction by LPS or TNFα. After applying the exMito from different treatment groups for 48 hours, the inflamed RT4 cells were observed using living cell confocal microscopy (Olympus FluoView™ FV3000, Olympus Corp., Japan). Fluorescent-labeled mitochondria were visualized along with the nuclei stain using Hoechst 33342 (NucBlue Live Ready Probes Reagent, Thermo Fisher Scientific) to investigate the mitochondrial morphologies and interactions between exMito (red) and endogenous mitochondria (green) within the cells.
Statistical analysis
Statistical analysis was conducted utilizing GraphPad Prism 6 software (GraphPad, La Jolla, CA, USA). A significant threshold of p-value <0.05 was applied to determine statistical significance in this study. Specific statistical methods corresponding to each figure were detailed in the respective Figure Legends.