Human umbilical cord mesenchymal stem cells (hUCMSCs)
hUCMSCs were obtained from Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. hUCMSCs were maintained in low serum defined medium, which consisted of 56% low-glucose Dulbecco’s Modified Eagle Medium (DMEM-LG; Invitrogen, CA, USA), 37% MCBD 201 (Sigma, MO, USA), 2% fetal bovine serum (Thermo, Logan, UT), 0.5 mg/mL of AlbuMAX® I (Life technology, NY, USA), 1X Insulin-Transferrin-Selenium-A (Life technology, NY, USA), 10 nM Dexamethasone (Sigma, MO, USA), 10 ng/mL Epidermal growth factor (PeproTech, NJ, USA), 50 nM L-ascorbic acid 2-phosphate (Sigma, MO, USA), and 1 ng/mL of platelet-derived growth factor-BB (PeproTech, NJ, USA). The cells were incubated in a humidified chamber with 5% CO2 at 37 °C. Detaching with HyClone® HyQtase (GE, UT, USA) and replated at a ratio 1:4 when cells reached 70–80% confluence.
Human fibroblast-like synoviocytes: rheumatoid arthritis (HFLS-RA)
HFLS-RA were purchased from Cell Applications, INC., (San Diego, CA, USA), and cultured in Synoviocyte Growth Medium (Cell Applications, CA, USA) according to the general instructions for culturing. The cells were incubated in a humidified chamber with 5% CO2 at 37 °C with the medium exchanged every 3 days. When the cells reached 80–90% confluence, they were detached with HyClone® HyQtase (GE, UT, USA) and replated at a ratio of 1:4. All the experiments used 3rd to 7th passages cells.
MTT cell viability assay
hUCMSCs and HFLS-RA cells were plated in 96-well plates and then starved in a serum-free DMEM-LG medium for 16 h. After starvation, cells were stimulated with 100 ng/mL IL-1β for 24 h or with 25 μM, 50 μM, and 100 μM LFA-1-ICAM-1 inhibitor Lovastatin for an hour. After cytokine and inhibitor treatment, cells were incubated with 1 mg/mL MTT reagent (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, SERVA, Heidelberg, Germany) in DMEM-LG medium for 4 h while protected from light. Aspirating the supernatant carefully, the formazan was dissolved with dimethyl sulfoxide (DMSO) for 2 h at 37 °C. The results were detected with Multimode micro-plate readers (Infinite 200, TECAN) at a wavelength of 545 nm.
Pre-stimulating hUCMSCs or HFLS-RA cells with IL-1β
hUCMSCs or HFLS-RA were cultured in 10 cm petri dish til 80% confluence and then starved in serum-free DMEM-LG medium for 16 h. After starvation, cells were treated with 50 ng/mL, 100 ng/mL, and 200 ng/mL IL-1β for 24 h. Proteins were extracted immediately. The procedures are described in the Western blot passage.
Adhesion assay
HFLS-RA were seeded in 96-well black plates. After forming a confluent monolayer, cells were starved in serum-free DMEM-LG medium for 16 h and stimulated with or without 100 ng/mL IL-1β for 24 h. At the same time, hUCMSCs were also cultured in 10 cm dishes with serum-free DMEM-LG medium for 16 h and with the same cytokine-stimulated condition of HFLS-RA. Before co-culturing, hUCMSCs were incubated with 2.5 μM Calcein AM (Tocris, UK) in serum-free DMEM-LG medium for 30 min at 37 °C protected from light, then washed with PBS 2 times and resuspended in DMEM-LG medium. hUCMSCs (5 × 103/100 μL) were added into HFLS-RA wells with or without 50 μM Lovastatin for an hour adhesion at 37 °C protected from light. The non-adhesion cells in each well were washed out using PBS with Ca2+/Mg2+ 3 times. The results were detected by Multimode microplate readers (Infinite 200, TECAN) with a wavelength of 480 nm for excitation and 520 nm for emission.
Fluorescence study of cell adhesion assay
HFLS-RA were seeded on 12 mm microscope cover glasses in 24-well culture plates. Then, the cells were starved in serum-free DMEM-Low Glucose medium (Gibco, NY, USA) for 16 h. After starvation, cells were stimulated with or without 100 ng/mL IL-1β for 24 h. Meanwhile, hUCMSCs were also starved in serum-free DMEM-LG medium for 16 h and with the same cytokine-stimulated condition of HFLS-RA. Before co-culturing, hUCMSCs were incubated with 6 μM Calcein AM (Tocris, UK) in serum-free DMEM-LG medium for 30 min at 37 °C protected from light. After incubation, cells were centrifuged and washed with PBS 2 times, then resuspended in DMEM-LG medium. hUCMSCs (5 × 104/900 μL) were added into each HFLS-RA containing wells with or without 50 μM Lovastatin for an hour adhesion at 37 °C protected from light. After adhesion, the non-adhesion cells in each well were washed out using PBS with Ca2+/Mg2+ 3 times. Cells were fixed in 4% paraformaldehyde (Ferak, Berlin, Germany) at 4 °C overnight and stained with Hoechst 33258 (Sigma, MO, USA) at 1:5000 dilution after washing three times with PBS. Last of all, cells were mounted with Fluorescence Mounting Medium (Ibidi, Planegg, Germany). Images of cells were taken using Fluorescent Microscope (Leica DM6000B, Wetzlar, Germany) and the statistical results were counted with cell numbers in five random fields of view (FOV) at 10 × magnification.
Western blot
Cells were washed with PBS and lysed by M-PER Mammalian Protein Extraction Reagent (Thermo, IL, USA) with 1% Halt Protease Inhibitor Cocktail (Thermo, IL, USA). The extraction was gently shaken for 3 min and centrifuged at 14,000g for 10 min, 4 °C. To isolate the cytosol proteins and membrane proteins, cells were lysed by Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo, IL, USA). Cells were washed with PBS and detached by HyClone® HyQtase (GE, UT, USA), and then centrifuged at 1000 rpm for 3 min to collect cell pellets. Cell pellet was washed with Cell Wash Solution and incubated with Permeabilization Buffer for 30 min at 4 °C on shaker, and then centrifuged at 16,000g for 15 min at 4 °C to collect the supernatant containing cytosol proteins. Solubilization Buffer was added to the pellet and mixed in by pipetting. The Mixture liquid was incubated for an hour at 4 °C and centrifuged at 16,000g for 15 min at 4 °C to collect the membrane proteins. Protein concentration was determined by Bio-Rad Protein Assay Dye Reagent (BIO-RAD, CA, USA) and multimode microplate readers (Infinite 200, TECAN). Protein samples were resolved using 10 or 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (BIO-RAD, CA, USA). After transferring, membranes were blocked with 5% Fish Gelatin Blocking Buffer (AMRESCO, OH, USA) in tris-buffered saline with tween-20 (TBST) for an hour at room temperature. After blocking, membranes were incubated with primary antibody LFA-1(GeneTex, CA, USA) with 1:1000 dilution, pan-cadherin antibody (Abcam, Cambridge, UK) diluted at 1:2000, TRAIL R1 (Novus, St. Charles, USA) with 1:2000 dilution, DR5 antibody (Abcam, Cambridge, UK) with 1:1000 dilution, ICAM-1 antibody (Abcam, Cambridge, UK) with 1:2000 dilution, TRAIL antibody (Abcam, Cambridge, UK) with 1:2000 dilution or beta-actin antibody (GeneTex, CA, USA) with 1:5000 dilution in 5% Fish Gelatin Blocking Buffer TBST at 4 °C overnight. The membranes were washed with TBST three times for 5 min each, then incubated with horseradish peroxidase-conjugated (HRP) Mouse or Rabbit IgG secondary 20 antibodies (GeneTex, CA, USA) for an hour at room temperature. The membranes were washed with TBST three times for 5 min each. The proteins were visualized using the LAS-4000 Luminescence Imaging System (GE, CT, USA) with enhanced chemiluminescence substrate (ECL) (PerkinElmer, MA, USA).
Cell immunofluorescence and images
hUCMSCs and HFLS-RA were seeded on 12 mm microscope cover glasses in 24-well culture plates. Then, the cells were starved in serum-free DMEM-LG for 16 h. After starvation, cells were stimulated with IL-1β in different concentrations or different durations. For different concentrations of IL-1β, cells were treated with 50 ng/mL, 100 ng/mL, 200 ng/mL IL-1β for 24 h. For difference in duration, cells were treated with 100 ng/mL IL-1β for 6, 16, 24, or 48 h. After cytokine stimulation, cells were fixed in 4% paraformaldehyde (Ferak, Berlin, Germany) at 4 °C overnight, and were permeabilized with 0.1% Triton X-100 (Sigma, MO, USA) in PBS for 10 min. Next, cells were blocked with 2% bovine serum albumin (BSA) (Sigma, MO, USA) for 30 min, and then incubated with primary antibody TRAIL R1 (Novus, St. Charles, USA) with 1:200 dilution, DR5 antibody (Abcam, Cambridge, UK) with 1:100 dilution, ICAM-1 antibody (Abcam, Cambridge, UK) with 1:200 dilution, or TRAIL antibody (Abcam, Cambridge, UK) with 1:200 dilution at 4 °C overnight. After washing three times with PBS, cells were incubated for an hour at room temperature with Alexa Fluor 488-conjugated AffiniPure Rabbit Anti-Mouse or Goat Anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, PA, USA) for a dilution of 1:100 or 1:200 depending on each primary antibody. Then, cells were stained with Hoechst 33258 (Sigma, MO, USA) at 1:5000 dilution after washing three times with PBS. Last of all, cells were mounted with Fluorescence Mounting Medium (Ibidi, Planegg, Germany). Images of cells were captured using Fluorescent Microscope (Leica DM6000B, Wetzlar, Germany) or Laser Confocal Microscope (LSM880, Oberkochen, Germany).
LFA-1 siRNA oligonucleotides and negative control siRNA transfection
LFA-1 Silencer Select predesigned siRNA (s534808 and s229716, Ambion, Austin, USA) and Silencer Select negative control no. 1 (Ambion, Austin, USA) were used to downregulate LFA-1 expression in cells. When cells were cultured in 10 cm dish until 80% confluence, 5 nM LFA-1-specific siRNA or negative control siRNA and lipofectamine® RNAiMAX transfection reagent dilute in Opti-MEM® Medium (Invitrogen, CA, USA) was prepared following by the manufacturer’s instruction. Then cells were cultured in the 1:1 mixture of dilute siRNA and dilute lipofectamine® RNAiMAX for 24 h.
Direct co-culture of hUCMSCs and HFLS-RA
hUCMSCs were seeded in 6-wells and HFLS-RA were seeded in 6-wells or cultured on 12 mm microscope cover glasses in 24-wells culture plate. Both cells were stimulated with/without 100 ng/mL IL-1β for 24 h. Then 5 × 105 of hUCMSCs were added to 6 wells and 5 × 104 hUCMSCs were added to 24 wells of HFLS-RA containing wells for 24 h. For Western blot, cells were washed with PBS, then the cells were lysed using M-PER Mammalian Protein Extraction Reagent (Thermo, IL, USA) with 1% Halt Protease Inhibitor Cocktail (Thermo, IL, USA). Extractions were vortexed for 1 min and centrifuged at 14,000g for 10 min at 4 °C.
Caspase-8, -9, and -3 detection in directly co-culture HFLS-RA and hUCMSCs
HFLS-RA were cultured in 24-wells and hUCMSCs were cultured in 10 cm dishes. For cytokine stimulation, hUCMSCs and HFLS-RA were stimulated with 100 ng/mL IL-1β for 24 h. After cytokine stimulation, hUCMSCs were labeled with 5 μM CellTracker Orange (Life technology, NY, USA) for 30 min at 37 °C, protected from light. Cells were centrifuged at 1500 rpm for 5 min and washed 3 times with PBS, protected from light. Finishing the label steps, 5 × 104 of hUCMSCs cells were added to HFLS-RA containing 24-wells. After 24 h direct co-culture, cells were fixed in 4% paraformaldehyde (Ferak, Berlin, Germany) at 4 °C overnight, and were permeabilized with 0.1% Triton X-100 (Sigma, MO, USA) in PBS for 10 min. Next, cells were blocked with 2% BSA (Sigma, MO, USA) for 30 min, and then incubated with primary antibody of caspase-8, caspase-9 or caspase-3 (GeneTex, CA, USA) with 1:200 dilution, at 4 °C overnight. After washing three times with PBS, cells were incubated an hour at room temperature with Alexa Fluor 488-conjugated AffiniPure Goat Anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, PA, USA) for a dilution of 1:200. Then, cells were stained with Hoechst 33258 (Sigma, MO, USA) at 1:5000 dilution after washing three times with PBS. Last of all, cells were mounted with Fluorescence Mounting Medium (Ibidi, Planegg, Germany). Images of cells were obtained using Fluorescent Microscope (Leica DM6000B, Wetzlar, Germany).
Annexin V/PI detection by immunofluorescence study
HFLS-RA cells were seeded on 12 mm microscope cover glasses and cultured in a 24-well plate, and hUCMSCs were cultured in 10 cm dishes. For cytokine stimulation, hUCMSCs and HFLS-RA were stimulated with 100 ng/mL IL-1β for 24 h. After cytokine stimulation, hUCMSCs were labeled with 5 μM CellTracker Orange (Life technology, NY, USA) for 30 min at 37 °C, protected from light. After labeling, cells were centrifuged at 1500 rpm for 5 min and washed 3 times with PBS, then centrifuged at 1500 rpm protected from light. Finishing the labeling steps, 5 × 104 of hUCMSCs cells were added to HFLS-RA containing 24-wells. After 24 h of direct co-culture, cells were stained with Annexin V-FITC Apoptosis Detection Kit (Strong Biotech Corporation, Taipei, Taiwan). According to the manual guide, the procedures are briefly described below. 2 μL of Annexin V-FITC was diluted in 100 μL Binding Buffer and 2 μL PI added for each assay as the preparation of staining solution. The cover glasses were removed and 100 μL staining solution was applied to each sample for 15 min incubation at room temperature. After incubation, samples were immediately examined and captured by Fluorescent Microscope (Leica DM6000B, Wetzlar, Germany).
Animal experiment and the induce of collagen-induced arthritis
All experimental were approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang Ming Chiao Tung University (on April 20, 2018) and were conducted in accordance with ethical regulations (Approval no. 1070410). Reporting of animal experiments follows recommendations in the ARRIVE guidelines. The mice were maintained in the Laboratory Animal Center of National Yang Ming Chiao Tung University in a 12 h dark/12 h light cycle with abundant food and water. Eight weeks old DBA/1J mice were used for all experiments in this study (n = 60). If the animal died during the experiment, it was excluded from the collection of data.
All the experiments adhered to the “Protocol for the Successful Induction of Collagen-Induced Arthritis (CIA) in Mice” (Chondrex, Inc.) Eight weeks old DBA/1J mice were injected intradermally (i.d) with 100 μg of bovine type II collagen (CII) (Chondrex, Redmond, WA, USA) emulsified with Complete Freund’s Adjuvant (CFA) (Chondrex, Redmond, WA, USA) at the base of the tail. For a booster injection, Incomplete Freund’s adjuvant (IFA) containing 100 μg of CII was administered on the day 21. Arthritis would develop 28–35 days after the first injection. On day 36, 1 × 106 hUCMSCs in 100 μL PBS were administered via tail vein. The Sham group was injected with the same volume of PBS as the inducing agent and hUCMSCs. Mice were sacrificed by cervical dislocation after isoflurane anesthesia on day 76 after starting CIA induction.
Therapeutic efficacy evaluation of hUCMSCs on CIA mouse model
The severity of RA symptom was evaluated with paw thickness using a digital caliper and body weight was measured with an electronic weigher every 10 days for four periods. According to the “Mouse Arthritis Scoring System” (Chondrex, Inc), clinical scores were judged on the redness and swelling of front and hind paws of each mouse. Three joint types were observed for scoring: the interphalangeal joint, the metacarpophalangeal joint, and the carpal and tarsal joint. The score is defined as follow: score 0 = normal, score 1 = one joint type has redness and swelling, score 2 = two joint types have redness and swelling, score 3 = all three joints have redness and swelling, score 4 = symptoms of the entire paw were maximally severe and the anatomic appearance was hard to distinguish. The total score was obtained from 4 paws, so the maximum score was 16 for each mouse.
Hematoxylin and eosin (H&E) stain
After sacrificing, the front and hind limbs were removed for histopathological examination. The tissues were fixed, decalcified and embedded in paraffin, then sectioned into 5 μm slides. To describe briefly, the slides were deparaffinized and rehydrated before staining with hematoxylin and eosin (H&E). The results were observed and photographed by microscope.
TUNNEL assay
After sacrificing, the hind legs were removed for histopathological examination. The tissues were fixed, decalcified and embedded with paraffin, then sectioned into 5 μm slides. The slides were deparaffinized and rehydrated, then processed following the manufacturer’s instructions of TUNEL Assay Kit—HRP-DAB (Abcam, Cambridge, UK). The results were observed and photographed by microscope.
18F-FDG microPET/MRI imaging
18F-FDG was used to track the inflammatory response. The response was evaluated every 10 days after hUCMSCs administration. The mice were anesthetized using 1–3% isoflurane and intravenously injected with 7.4 MBq/0.2 mL 18F-FDG for an hour. All images were acquired for 10 min by Bruker 7T PET/MR after injection. The regions of interest (ROI) were selected and the accumulation of 18F-FDG was measured using Amide’s Medical Imaging Data Examiner (AMIDE) software. Standardized uptake value (SUV) was calculated from the palm, knee and paw.
Statistical analysis
Quantitation data was analyzed by Student’s t-test and one-way ANOVA. P values < 0.05 were considered as statistically significant. All statistical analysis was performed with Prism 5 software.
Ethics approval and consent to participate
All experimental protocols were approved on April 20, 2018 by the Institutional Animal Care and Use Committee (IACUC) of National Yang Ming Chiao Tung University and were conducted in accordance with ethical regulations (Approval no. 1070410). The title of the approved project is “Effects of Human Umbilical Cord Wharton’s Jelly-derived Mesenchymal Stem Cells on Fibroblast-like Synoviocytes from Rheumatoid Arthritis”.
- SEO Powered Content & PR Distribution. Get Amplified Today.
- PlatoData.Network Vertical Generative Ai. Empower Yourself. Access Here.
- PlatoAiStream. Web3 Intelligence. Knowledge Amplified. Access Here.
- PlatoESG. Automotive / EVs, Carbon, CleanTech, Energy, Environment, Solar, Waste Management. Access Here.
- PlatoHealth. Biotech and Clinical Trials Intelligence. Access Here.
- ChartPrime. Elevate your Trading Game with ChartPrime. Access Here.
- BlockOffsets. Modernizing Environmental Offset Ownership. Access Here.
- Source: https://www.nature.com/articles/s41598-023-42585-1