Isolation and culture of PL-MSCs

The study was approved by the Human Ethics Committee of Thammasat University (Medicine) [Approval number: 037/2021]. Placental tissues were obtained from pregnant women after normal delivery with written informed consent. The MSCs were isolated from the placenta and characterized as previously described [11]. The tissues were intensively washed with washing buffer [1X phosphate buffered saline (PBS) containing 100 units/ml of penicillin and 100 µg/ml streptomycin (GibcoBRL, USA)] and minced into small pieces. Subsequently, the tissues were digested with 0.5% trypsin–EDTA (Invitrogen, USA) at 37 °C for 2 h with shaking. After washing twice with washing buffer, the cells were cultured with complete DMEM medium [Dulbecco's Modified Eagle’s Medium (DMEM; GibcoBRL, USA) containing 10% fetal bovine serum (FBS; HyClone™, USA), 1% GlutaMAX™ (GibcoBRL, USA), 100 units/ml of penicillin and 100 µg/ml streptomycin (GibcoBRL, USA)] in 25-cm2 culture flasks (Corning costar®, China). The cultures were maintained at 37 °C in a humidified tissue culture incubator with 5% CO2. The culture medium was changed every 3 days. Plastic-adherent cells with approximately 80–90% confluence were subcultured with 0.25% trypsin–EDTA (GibcoBRL, USA), cryopreserved in a freezing medium, and stored in liquid nitrogen for further experiments.

Characterization of PL-MSCsMorphology

The morphology of PL-MSCs kept in an incubator was observed by inverted microscopy (Nikon Eclipse Ts2R-FL, Japan) every day. Adherent cells were sub-passaged when they reached 80–90% confluence.

Immunophenotype

The PL-MSCs in passages 3 to 5 were trypsinized with 0.25% trypsin–EDTA and washed with 1X PBS. After centrifugation, 5 × 105 cells were resuspended in 50 µl of 1X PBS and incubated with 5 µl of fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated antibody against HLA-DR (Bio Legend, USA), cluster of differentiation (CD) 34 (BioLegend, USA), CD45 (BioLegend, USA), CD73 (BioLegend, USA), CD90 (BioLegend, USA) and CD105 (BD Bioscience, USA) at 4 °C for 30 min in the dark. After washing with 1X PBS, the cells were fixed with 1% paraformaldehyde in PBS. The positive cells were identified by comparing with isotype-matched controls [FITC-conjugated mouse immunoglobulin G1 (IgG1) and PE-conjugated mouse immunoglobulin G2a (IgG2a)]. At least twenty thousand labeled cells were acquired and analyzed using flow cytometry (DxFLEX flow cytometer, Beckman Coulter, USA) and CytExpert software (DxFLEX flow cytometer, Beckman Coulter, USA).

Osteogenic differentiation

After trypsinization, PL-MSCs in passages 3–5 were seeded in 6-well plates (Corning costar®, China) at a density of 4.5 × 103 cells/cm2. Cells were cultured with complete DMEM medium at 37° C in a humidified incubator with 5% CO2 until they reached 80% confluence. After washing with 1X PBS, 1.5 ml of osteogenic differentiation medium [complete DMEM medium supplemented with 100 nM dexamethasone (Sigma-Aldrich, USA), 10 mM β-glycerophosphate (Sigma-Aldrich, USA), and 50 µg/ml ascorbic acid (Sigma-Aldrich, USA)] was added. The medium was changed every 3 days. On day 28, the cultured cells were fixed with 10% formaldehyde and stained with 40 mM Alizarin Red S (Sigma-Aldrich, USA) for 20 min at room temperature. The cells were washed with distilled water and observed under an inverted microscope (Nikon Eclipse Ts2R-FL, Japan). Differentiated MSCs were visualized as red cells correlated with calcium deposits in the cells. For the control, cells were cultured in a complete DMEM medium without any osteogenic stimuli, performed in parallel with the experimental group, and stained in the same way.

Adipogenic differentiation

After trypsinization, PL-MSCs in passages 3–5 were cultured with complete DMEM medium in 6-well plates (Corning costar®, China) at a density of 4.5 × 103 cells/cm2. Cells were kept at 37 °C in a humidified incubator with 5% CO2 until they reached 80% confluence. Subsequently, cells were washed with 1X PBS and 1.5 ml of adipogenic differentiation medium [complete DMEM medium supplemented with 2.5 mM glucose (Sigma-Aldrich, USA), 0.5 mM isobutyl methylxanthine (IBMX; Sigma-Aldrich, USA), 1 µM dexamethasone (Sigma-Aldrich, USA), 10 µM insulin (Sigma-Aldrich, USA) and 0.2 mM indomethacin (Sigma-Aldrich, USA)] was added. The medium was changed every 3 days. On day 28, cells were fixed with 37% formalin solution vapor and stained with 0.5% oil red O (Sigma-Aldrich, USA) in 6% isopropanol. The cells were then washed with distilled water and observed by inverted microscopy (Nikon Eclipse Ts2R-FL, Japan). Lipid droplets in differentiated PL-MSCs were marked red, in correlation with lipid accumulation in the cells. For the control group, cells were maintained in the complete medium without adipogenic stimuli, performed in parallel with the experimental group, and stained in the same manner.

Chondrogenic differentiation

After trypsinization, PL-MSCs in passages 3–5 were initially cultured with complete DMEM medium at a density of 3 × 106 cells/cm2 in 96-well U-bottom cell culture plates (Jet Biofil, China) at 37°C with 5% CO2 overnight. The medium was removed and MSCgoTM Chondrogenic XF medium (Sartorius, Germany) was added. On day 21, the spherical mass was fixed with 10% formalin solution for 30 min at room temperature. The mass was then stained with a 1% Alcian Blue Solution (HiMedia, India) in a dark environment at room temperature overnight. The stained sample was examined using inverted microscopy (Nikon Eclipse Ts2R, Japan). For the control, PL-MSCs were cultured in a complete DMEM medium without chondrogenic stimuli, and processed similarly to cells in differentiation medium.

Cytotoxicity assay

To evaluate the effect of metformin on viability of PL-MSCs, 1 × 103 PL-MSCs were cultured with complete DMEM medium in a 96-well plate (SPL Life Science, Korea) at 37 °C in a 5% CO2 humidified incubator. Cells were allowed to adhere to the plate for 24 h. Subsequently, the cells were washed with 1X PBS and treated with 100 µl of complete DMEM medium containing 0.5, 10, 40, 80, 160, 320, and 640 µM metformin. The control was cultured in complete DMEM medium without metformin. The cultures were maintained for 14 days and cell viability was measured every 2 days using an MTT assay (Sigma-Aldrich, USA). Briefly, 2 mg/ml of MTT solution (3- (4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium bromide) in complete DMEM medium was added to each well and incubated at 37°C in a humidified incubator with 5% CO2 for 4 h. Subsequently, the entire solution was removed and the purple formazan crystals were dissolved in 100 µl of dimethyl sulfoxide (DMSO, VWR BDH Chemicals, France). The absorbance was measured at 570 nm using a microplate reader (BioTex, USA). The percentage of cell viability was calculated using the following equation:

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Osteogenic differentiation assay

The osteogenic differentiation potential of PL-MSCs was evaluated using an osteogenic differentiation assay as previously described [22]. The PL-MSCs were seeded in 6-well plates (Corning costar®, China) at a density of 4.5 × 103 cells/cm2 and cultured with complete DMEM medium at 37 °C in a humidified incubator with 5% CO2 until 80% confluence. The cells were then washed with 1X PBS and 1.5 ml of osteogenic differentiation medium containing 10, 40, 80, 160, and 320 µM metformin was added. PL-MSCs cultured with osteogenic differentiation medium without metformin were used as a control. On days 7, 14, 21, and 28, the expression of osteogenic genes, the alkaline phosphatase (ALP) activity assay, and the Alizarin Red S staining were performed as follows:

Expression of osteogenic genes

Total RNA extraction was performed using TRIzol® reagent (Invitrogen, USA). Total RNA was reverse transcribed into cDNA using iScript™ reverse transcription Supermix for RT-qPCR (Bio-Rad, USA) according to the manufacturer's instructions. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure the expression of osteogenic markers (RUNX2, Osterix, Osteocalcin and Collagen I) in metformin-treated PL-MSCs compared to untreated PL-MSCs. Gene expression was quantified using the SYBR Green PCR Master Mix (Applied Biosystems, USA). The cycling program was set as follows: thermal activation at 95 °C for 30 s and 40 cycles of PCR (melting at 95 °C for 15 s, annealing at 60 °C for 60 s). The primer sequences are shown in Table 1. Relative expression was evaluated using the 2ΔΔCt method and normalized by the Ct of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All reactions were performed in at least triplicate and analysis was performed using an Applied Biosystems StepOne™ Plus real-time PCR system and StepOne™ Software version 2.3 (Applied Biosystems; ABI, USA).

Table 1 Sequence of primers used for qRT-PCRAlkaline phosphatase activity assay

Alkaline phosphatase (ALP) activity was measured using the SensoLyte® pNPP Alkaline Phosphatase Assay Kit (Ana Spec, USA). On days 7, 14, 21, and 28 after treatment, the culture medium was removed, the cell monolayer was washed with 1X PBS and cell lysis was performed using 200 µl of lysis buffer (0.1 M glycine, 1% Nonidet P-40, 1 mM MgCl2 and 1 mM ZnCl2, pH 9.6). The cells were then scratched off the plastic surface and incubated on ice for 20 min. After centrifugation at 2500 × g, 4 °C for 10 min, the supernatant was collected and transferred to a new 96-well plate (SPL Life Science, Korea). Then, 50 µl of p-nitrophenyl phosphate (pNPP) was added to each well, mixed by gently shaking the plate for 30 s, and incubated for 30 min at 37°C. The final solution, a yellow-colored product, was measured using a microplate reader (BioTex, USA) at an absorbance of 405 nm. The ALP activity in each sample was calculated by comparing the measured OD values with a standard curve generated from 0 to 200 ng/ml of the standard ALP solution. Each test condition was performed in triplicate and normalized with the concentrations of total cellular proteins measured by the Bradford assay (Bio-Rad, USA).

Alizarin Red staining

The formation of calcium-containing mineral nodules during osteogenic differentiation of metformin-treated PL-MSCs compared to untreated PL-MSCs was determined using Alizarin Red S staining. After washing with 1X PBS, the PL-MSCs were fixed with 10% formaldehyde at room temperature for 15 min. The cells were then washed with distilled water and stained with 40 mM Alizarin Red S (Sigma-Aldrich, USA) for 20 min at room temperature. The cells were washed with distilled water and observed under an inverted microscope (Nikon Eclipse Ts2R-FL, Japan).

For the quantification of Alizarin Red S staining, 400 µl of 10% acetic acid was added to each well and the plate was incubated at room temperature for 30 min. The monolayer was then scratched off the plastic surface, transferred to the microcentrifuge tube, and sealed with parafilm to prevent evaporation. After vortexing for 30 s, the samples were heated at exactly 85 °C for 10 min, and incubated on ice for 5 min. The lysate was centrifuged at 20,000 x g for 15 min and 300 µl of the supernatant was transferred to a new 1.5 ml microcentrifuge tube. Subsequently, 150 µl of 10% ammonium hydroxide was added to neutralize the acid. The samples, a yellow-colored product, were measured using a microplate reader (BioTex, USA) at an absorbance of 405 nm.

AMPK activity assay

To study the relationship between AMPK activation and osteogenic differentiation potential of metformin-treated PL-MSCs, we investigated the levels of AMPK and phosphorylated AMPK in metformin-treated PL-MSCs and compared them to levels in untreated PL-MSCs. PL-MSCs were seeded in 6-well plates (Corning costar®, China) at a density of 4.5 × 103 cells/cm2 and cultured with complete DMEM medium at 37 °C in a humidified incubator with 5% CO2 until 80% confluence. The cells were then washed with 1X PBS and 1.5 ml of osteogenic differentiation medium containing 10, 40, 80, 160, and 320 µM metformin was added. To determine the role of the AMPK signaling pathway in metformin-induced osteogenic differentiation, 10 µM Compound C (6-[4-(2-Piperidin-1-ylethoxy) phenyl]-3-pyridin-4-ylpyrazolo [1,5-a] pyrimidine; the AMPK inhibitor) was added to the culture. PL-MSCs cultured in osteogenic differentiation medium without metformin were used as a control. Western blot and qRT-PCR were performed to examine the expression of AMPK after metformin treatment.

Western blot analysis

The protein was extracted using RIPA reagent (Bio-Rad, USA) containing a protease and phosphatase inhibitor cocktail (Cell Signaling Technology, USA). The protein concentration was determined using a Bradford assay (Bio-Rad, USA). The total protein (20 µg) from each sample was diluted with the 3X reducing SDS loading buffer (Cell Signaling Technology, USA), and a molecular weight marker (Abcam, USA) was then separated by electrophoresis using a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis was carried out at 120 V for 90 min. The proteins on SDS-PAGE gels were transferred to a nitrocellulose membrane (0.45 µm pore-size; Bio-Rad, USA) using a mini trans-blot electrophoretic transfer cell machine (Bio-Rad, USA) at 120 V for 90 min. Following transfer, the membranes were gently stained using 0.1% (w/v) Ponceau S in 5% acetic acid. Next, the membranes were carefully cut to facilitate the incubation process with different primary antibodies. The membranes were then blocked with 5% skim milk in TBS at room temperature for 1 h. Next, the membranes were incubated with primary antibodies, including rabbit anti-human AMPKα (1:1000 dilution; Cell Signaling Technology, USA), rabbit anti-human phospho-AMPKα (Thr172) (1:1000 dilution; Cell Signaling Technology, USA) and rabbit anti-human actin (1:10,000 dilution; Proteintech, USA) at 4 °C, overnight. The membranes were washed with 0.1% Tween-20 in TBS 3 times. Subsequently, the membranes were incubated with peroxidase-conjugated IgG fraction monoclonal mouse anti-rabbit secondary antibody (1:10,000 dilution; Jackson ImmunoResearch, USA) for 1 h at room temperature. The proteins were visualized using enhanced chemiluminescence (ECL) using Clarity™ Western ECL Substrate (Bio-Rad, USA). The signals were captured with Amersham Imager 600 (GE Healthcare Life Sciences). For quantitation, the membrane was scanned, and the digital image was saved in black-and-white JPEG format. The digital image was analyzed using ImageJ software available in the public domain at https://imagej.nih.gov/ij/ download.html. Briefly, the digital image was opened in ImageJ software, and the bands were outlined using a squared region tool and added to the ROI (Region of Interest) manager. The image was inverted, and the analysis tool was used to measure the integral optical density (IOD) of the individual bands. The intensity of the protein band was expressed as a ratio to β-actin band intensity.

Quantitative real-time RT-PCR

For qRT-PCR, cells were washed with 1X PBS and total RNA extraction was performed using TRIzol® reagent (Invitrogen, USA). Total RNA was reverse transcribed into cDNA using iScript™ reverse transcription Supermix for RT-qPCR (Bio-Rad, USA) according to the manufacturer's instructions. Gene expression was quantified using the SYBR Green PCR Master Mix (Applied Biosystems, USA). The cycling program was set as follows: thermal activation at 95 °C for 30 s and 40 cycles of PCR (melting at 95 °C for 15 s, annealing at 60 °C for 60 s). The primer sequences are shown in Table 1. Relative expression was evaluated using the 2ΔΔCt method and normalized by the Ct of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The qRT-PCR analyzes were performed using an Applied Biosystems StepOne™ Plus real-time PCR system and StepOne™ software version 2.3 (Applied Biosystems; ABI, USA).

Inhibition of AMPK activity using compound C

PL-MSCs were seeded in 6-well plates (Corning costar®, China) at a density of 4.5 × 103 cells/cm2 and cultured with complete DMEM medium at 37 °C in a humidified incubator with 5% CO2 until 80% confluence. The cells were then washed with 1X PBS and cultured with osteogenic differentiation medium containing 10, 40, 80, 160, and 320 µM metformin with or without 10 µM of Compound C (Sigma-Aldrich, USA). The medium was changed every 3 days. The osteogenic differentiation potential of PL-MSCs after compound C treatment was determined by a gene expression study, an ALP activity assay, and Alizarin Red S staining, as mentioned above.

Statistical analysis

Data were analyzed and presented as mean ± standard error of the mean (SEM). Statistical significance was assessed using one-way analysis of variance (ANOVA) using SPSS version 26, developed by SPSS, Inc. A significance level of p < 0.05 was considered statistically significant.