In the present study, BMSCs were previously isolated from 2 male and 2 female non-smoking patients (25-45 years old), who were referred to the Orthopedic Department of Jordan University Hospital (JUH)/University of Jordan (Amman, Jordan) after sustaining orthopedic fractures from road traffic accidents (51). Institutional Review Board (IRB) approval (approval no. IRB/7/2019) for obtaining BMSCs was issued by the IRB Committee of the Cell Therapy Center (CTC)/University of Jordan. Informed consent was also obtained from all patients to donate their tissues, as specified in The Declaration of Helsinki, prior to participation.

Bone marrow samples were collected by iliac crest aspiration. The inclusion criteria for patient selection were as follows: i) Males or females, 25-45 years old; ii) those with orthopedic fractures or trauma resulting from traffic accidents requiring orthopedic intervention, in which iliac crest bone marrow aspiration did not pose any additional risk; and iii) hemodynamically stable (normal vital signs or stabilized post-resuscitation). While the exclusion criteria were as follows: i) History of, or evidence of, hematologic disorders, including leukemia, lymphoma, or aplastic anemia; ii) major mental health disorders that preclude participation in the present study; iii) type I or type II diabetes mellitus, chronic hypertension, or receiving antidiabetic medications; iv) severe anemia (hemoglobin <8 g/dl); v) active systemic infection (such as sepsis and osteomyelitis) that may contaminate the bone marrow; vi) a clinically active autoimmune condition; vii) history of chemotherapy or radiation therapy, which may alter bone marrow cell composition; and viii) positive serological evidence of HIV I and II, HBV, HCV, and VDRL.

All participants met the inclusion criteria and had none of the exclusion conditions, confirming their suitability for the present study.

Bone marrow sample collection began on December 1, 2019, and continued for 1 year until January 1, 2021. Bone marrow samples were aseptically collected in 12-16 EDTA tubes. Subsequently, the buffy coat was isolated by centrifugation [300 x g, 6 min, at room temperature (RT)], suspended in 1.5 ml phosphate-buffered saline (PBS), and used for culture. Then, 5 ml of the separated buffy coat was layered onto an equal volume of Ficoll (Cytiva) and centrifuged (500 x g, 30 min, at RT with the centrifuge brake turned to off).

For the present study, all the experimental procedures were approved (approval no. IRB-7-2019-8) by the IRB Committee at the CTC, University of Jordan. BMSCs were cultured at passage 3 in minimum essential medium Eagle-alpha modification (αMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 15% fetal bovine serum (FBS; Cytiva), 1% of 100X Glutamax (Gibco; Thermo Fisher Scientific, Inc.), and 1% of 100X antibiotic-antimycotic mixture (Gibco; Thermo Fisher Scientific, Inc.). Breast cancer (MCF7, ATCC^® HTB-22^™) and alveolar basal epithelial adenocarcinoma (A549, ATCC^® CCL-185^™) were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 1% of 100X Glutamax, and 1% of 100X antibiotic-antimycotic. Moreover, human dermal fibroblast cells used were previously prepared (49,50) and cultured in Advanced DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 1% of 100X Glutamax, and 1% of 100X antibiotic-antimycotic mixture. All cells were maintained and incubated at 37˚C, 21% O₂, and 5% CO₂, with media changes every 2 days.

The four iPSC lines used in the present study are as follows: JUCTCi010-A, derived from skin dermal fibroblasts of a healthy 27-year-old Jordanian female (49); JUCTCi010-B, derived from MSCs obtained from skin dermal fibroblasts of a 27-year-old Jordanian female (49); JUCTCi011-A and JUCTCi011-B, derived from skin dermal fibroblasts of a 34-year-old Jordanian male (50). The generation of these lines was approved (approval no. IRB/07/2017) by the Cell therapy Center (CTC), University of Jordan, Amman, Jordan, which covered JUCTCi010-A and B as well as JUCTCi011-A and B. This IRB approval has been documented in previous publications by the authors on female (52) and male (53) iPSC lines.

For coating plates used to culture iPSCs in mTeSR media (STEMCELL Technologies), Matrigel was diluted 1:100 in DMEM/F12 to achieve a final concentration of ~500 µg/ml. For a 6-well plate, 1 ml of the diluted Matrigel solution was added per well and incubated for at least 1 h at 37˚C before use (51). For differentiation, the iPSC monolayer cultures were detached using 0.5 M EDTA (Gibco; Thermo Fisher Scientific, Inc.), and the resulting cell suspensions were plated in MSC differentiation media on ultra-low attachment plates to form EBs (54). The MSC differentiation media consisted of αMEM, supplemented with 15% FBS, 1% 100X Glutamax, and 1% 100X antibiotic-antimycotic mixture (54).

On days 2 and 4 of differentiation, the media were replaced with fresh media containing 10 µM retinoic acid (RA; Merck KGaA) and 0.1 µM RA, respectively. On day 6, the media were switched to RA-free differentiation media and on day 7, the EBs were plated on Matrigel-coated plates and maintained in MSC differentiation media. The differentiation media were replaced every two days. On day 12, 2.5 ng/ml basic fibroblast growth factor (R&D Systems, Inc.) was added, with media changes occurring every 2 days thereafter. iMSCs were passaged upon reaching 80-90% confluency and were then cryopreserved in 1X freezing media composed of 90% FBS and 10% DMSO and stored in liquid nitrogen.

Cells were seeded in triplicate at a density of 200,000 cells per well in 6-well plates and cultured in complete culture medium (CCM) until reaching 50% confluency. The medium was then replaced with osteogenic differentiation medium consisting of αMEM supplemented with 15% FBS, 1% 100X Glutamax, 1% 100X antibiotic-antimycotic mixture, 10 mM dexamethasone (Merck KGaA), 50 µg/ml ascorbic acid 2-phosphate (Merck KGaA), and 10 mM β-glycerophosphate (Carbosynth Ltd.) for 21-28 days, or until calcium deposits appeared. Control cells remained in CCM with medium changes every 2-3 days. Upon observation of mineral deposits, one well from each sample was stained with Alizarin Red (Merck KGaA) for 5 min at RT to visualize calcium deposits, which were then imaged using the EVOS XL Core Imaging System (Thermo Fisher Scientific, Inc.).

EB-iMSCs and BMSCs were seeded in triplicate at a density of 200,000 cells per well and cultured in CCM until reaching 50% confluency. They were then switched to adipogenic differentiation medium consisting of αMEM supplemented with 15% FBS, 1% 100X Glutamax, 1% 100X antibiotic-antimycotic mixture, 10 mM dexamethasone, 500 µM 3-isobutyl-1-methylxanthine (IBMX), 0.2 mM indomethacin, and 10 µg/ml insulin (all from Merck KGaA) and maintained for 14-21 days, with medium changes every 2-3 days, until fat vacuoles were visible. Control cells remained in CCM. Upon differentiation, the cells were stained with Oil Red-O (Merck KGaA) for 5 min at RT to visualize fat vacuoles, which were then imaged using the EVOS XL Core Imaging System.

Cells were assessed by flow cytometry using BD Stem Flow hMSC Analysis kit (cat. no. 562245, BD Biosciences) and according to the manufacturer's instructions. iMSCs and BMSCs were trypsinized at early passages (passage <8) and washed with 1X PBS. The cells were then resuspended in 800 µl of 1% bovine serum albumin (BSA) staining buffer. Next, the cells were incubated for 30 min with fluorescently conjugated antibodies targeting human MSC surface markers (FITC anti-CD90, PerCP anti-CD105, APC anti-CD73, and PE anti-CD44; all included in th aforementioned kit) or with appropriate isotype controls (all from BD Biosciences) at a concentration of 50 µg/ml for FITC anti-CD90, PerCP anti-CD105, and APC anti-CD73, and 20 µg/ml for anti-CD44 and the isotype controls. The absence of negative cocktail surface markers was also. Following incubation, the cells were washed twice with 1X PBS to remove any unbound antibodies and then resuspended in 200 µl of PBS. The expression of surface markers was analyzed using a BD FACSCanto II flow cytometer (BD Biosciences), and the data were processed using BD FACSDiva software (BD Biosciences) (55).

After reaching 70-80% confluency, cells of iMSCs and BMSCs at passage 3 were washed with PBS and then cultured in serum-free α-MEM for 48 h. The conditioned media were then collected and centrifuged at 300 x g for 10 min at 4˚C, followed by centrifugation in 2,000 x g for 20 min at 4˚C. Following centrifugation, the supernatants were filtered through 0.22-µm filter units to remove any remaining cell debris, which could contribute to exosome aggregation during the filtration process. To ensure consistent exosome pelleting, the filtered supernatant was then ultracentrifuged at 110,000 x g for 2 h at 4˚C using a fixed-angle rotor. After purification, the exosome pellets (iMSC-Exos and BMSC-Exos) were gently resuspended in 500 µl filtered PBS to avoid disrupting the lipid bilayer, which could promote aggregation. The concentration of the exosomes was measured using the Micro BCA^™ Protein Assay Kit (cat. no. 23235; Thermo Fisher Scientific, Inc.), strictly following the manufacturer's instructions. The measured concentration was standardized to 1 mg/ml. The exosomes were then aliquoted, resuspended in filtered PBS, and stored at -80˚C to preserve their structural integrity and reduce aggregation over time for future use (56).

A total of ~100 µg of isolated exosomes were incubated with 3 µl of aldehyde/sulfate latex beads (4% w/v, 4 µm, Invitrogen; Thermo Fisher Scientific, Inc.) (57) for 15 min and then incubated overnight at RT with gentle shaking (270˚ shaking) after adding 10 µl of filtered 1X PBS. Subsequently, the exosome-bead binding was blocked by adding 1M glycine for 15 min at RT, which was prepared by dissolving 0.7507 g of 0.01 M glycine (Millipore, Sigma), 200 mg of 2% BSA (Abcam), and 10 ml of 1X PBS, followed by filtration using 0.22 µm filter units. The exosome-coated beads were then incubated for 40 min at 37˚C with various antibodies (A5488 anti-CD9 (cat. no. FAB1880G), AF647 anti-CD81 (cat. no. FAB4615R), and APC anti-CD63 (cat no. FAB5417A) or their respective isotype controls (all from BioTechne; R&D Systems, Inc.) according to the manufacturer's instructions. Finally, 150 µl of filtered 1X PBS was added to the samples, which were then processed on a BD FACSCanto II and analyzed using BD FACSDiva software.

A total of 100 µg of purified exosomes were mixed 1:1 with 2% paraformaldehyde (PFA; MilliporeSigma), and applied to Formvar-carbon-coated electron microscopy grids [Electron Microscopy Sciences (EMS)] to promote membrane absorption for 20 min in a dry environment at RT. The grids were then rinsed with 1X PBS and subsequently immersed in 1% glutaraldehyde for 5 min at RT to eliminate any negative background. Following this, the grids were washed seven times with distilled water, with each wash lasting 2 min. To enhance contrast, the grids were treated with uranyl oxalate for 5 min at RT and then placed on methyl cellulose-UA for 10 min on ice. Finally, the grids were allowed to air dry for 10 min at RT before being examined under TEM with a magnification of 500 nm at an acceleration voltage of 30 kV, using VERSA 3D (FEI; Thermo Fisher Scientific, Inc.).

Size distribution was measured using a dynamic light scattering (DLS) nanosizer, the ZetaView (serial no. MAL1137709) (Malvern Nano ZS; Malvern Panalytical, Ltd.), which analyzes particle sizes ranging from 0.3 nm to 10 µm. Data were processed using Zetasizer software version 7.11 (Malvern Panalytical, Ltd.). The temperature was maintained at 24˚C throughout the process. The data acquisition settings were configured as follows: A measurement angle of 173˚ backscatter, 10 runs per measurement, 60 sec per run, 3 total measurements, and a 10-sec delay between measurements. All settings followed the manufacturer's recommendations for EV analysis. Prior to measurement, samples were diluted with filtered PBS.

iMSC-Exos and BMSC-Exos were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-O; Thermo Fisher Scientific, Inc.) fluorescent dye according to the manufacturer's instructions. Briefly, 200 µl of the isolated exosomes were incubated with 5-8 µl/1 ml of the dye for 1 h at RT, then added to 4 ml of filtered 1X PBS, ultracentrifuged at 110,000 x g for 1.5 h at 4˚C, and resuspended in 700 µl of filtered 1X PBS. The medium of MCF7 or A549 or fibroblasts cells (all at 1.5x10⁵ cells per well) was then replaced with serum-free medium (SFM) containing 50 µg/ml of iMSC-Exos or BMSC-Exos. After 16 h of treatment, the cells were washed with 1X PBS and incubated for an additional 16 h. Subsequently, the cells were incubated for 30 min at RT with 5-chloromethylfluorescein diacetate dye (CMFDA; Invitrogen; Thermo Fisher Scientific, Inc.), fixed with 4% PFA, incubated for 15 min at RT, washed with 1X PBS, and stained for 5 min at RT with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen; Thermo Fisher Scientific, Inc.). Finally, the cells were mounted with mounting medium (Abcam). The cellular internalization of iMSC-Exos or BMSC-Exos was observed under a time-lapse microscope (Carl Zeiss).

MCF7, A549 and fibroblast cells (8x10³ cells per well) were cultured in 96-well plates with 100 µl of RPMI-1640 medium for 24 h at 37˚C and 5% CO₂. Following this initial incubation, the medium was replaced with serum-free RPMI-1640 containing 350 µl of either iMSC-Exos or BMSC-Exos (50 µg/ml). Cell proliferation was measured after 24, 48, and 72 h by adding 10 µl of thiazolyl blue tetrazolium bromide reagent (MTT; Promega) to each well, followed by a 3-h incubation at 37˚C. Subsequently, 100 µl of solubilization stop solution (Promega Corporation) was added, and the plates were incubated for an additional 30 min at 37˚C. The absorbance of the cells was then measured at 570 nm using a BioTek microplate reader (5), and the data were analyzed with BioTek Gen 5 data analysis software (BioTek; Agilent Technologies, Inc.).

Cell apoptosis was assessed using the eBioscience^™ Annexin V-FITC Apoptosis Detection Kit (cat. no. 88-8005-74; Invitrogen; Thermo Fisher Scientific, Inc.) and analyzed by flow cytometry. Briefly, A549 and MCF7 cell lines were seeded at a density of 3x10⁵ cells per well and treated with or without 50 µg/ml of iMSC-Exos or BMSC-Exos for 48 h after replacing the medium with serum-free RPMI-1640. Following treatment, cells were collected and washed with 1X PBS. The cell pellet was then resuspended in 100 µl of 1X Binding Buffer, mixed with 5 µl of FITC-conjugated Annexin V and incubated in the dark at RT for 15 min. Subsequently, 5 µl of propidium iodide (PI) was added to each sample, followed by 100 µl of 1X Binding Buffer to dilute the cell suspension. The samples were then analyzed using a BD FACSCanto II flow cytometer and BD FACSDiva software.

MCF7 or A549 cells were seeded at a density of 3x10⁵ cells per well. Upon reaching 90% confluency, an artificial wound was created by scratching the cell monolayer with a 200-µl pipette tip. The medium was then changed to serum-free RPMI-1640, and the cells were washed twice with 1X PBS before being treated with 50 µg/ml of iMSC-Exos or BMSC-Exos. Images were recorded using EVOS XL Core Imaging System (Thermo Fisher Scientific, Inc.), at 0, 9, 20, and 47 h after treatment and the distance of cell migration was measured using ImageJ software (version 1.53; National Institutes of Health) (5).

Senescence-associated β-galactosidase (SA-βGal) staining was performed on treated cells using a senescence detection kit (cat. no. ab65351; Abcam). Briefly, MCF7 or A549 cells were seeded at a density of 3x10⁵ cells per well in RPMI-1640 medium. The following day, the medium was replaced with serum-free RPMI-1640 containing 50 µg/ml of either iMSC-Exos or BMSC-Exos. After 48 h, the medium was aspirated, and the cells were washed once with 1 ml of 1X PBS and fixed with 0.5 ml of fixative solution provided in the kit for 15 min at RT. Subsequently, 0.5 ml of staining solution, prepared by mixing 5 µl of 100X staining supplement, and 25 µl of 20 mg/ml X-Gal in dimethylsulfoxide with 470 µl of staining solution (all included in the aforementioned kit), was added, and the cells were incubated at 37˚C for 12 h. The cells were then observed under a microscope (EVOS XL Core Imaging System; Thermo Fisher Scientific, Inc.) to investigate the development of blue color, which indicates senescence, and were manually counted and compared to untreated cells.

All data were analyzed using GraphPad Prism version 9.1.0 (Dotmatics). The statistical tests included an unpaired Student's t-test or a two-way analysis of variance (ANOVA), followed by a Bonferroni post-hoc test when indicated. Data are presented as the mean ± standard error (SE), and a P≤0.05 was considered to indicate a statistically significant difference. All the experiments were conducted in three independent replicates (n=3).

A flow cytometric analysis was initially performed to detect the expression of stem cell surface markers on iMSCs and BMSCs. The analysis clearly revealed the expression of CD105, CD90, CD73, and CD44 on both iMSCs and BMSCs (Fig. 1A), with negative expression of CD34, CD11b, CD19, CD45, and HLA-DR surface markers. The expression percentages of the aforementioned markers in the BMSCs were as follows: CD105 was expressed in 97% of the cells, CD90 was expressed in 98.6%, CD73 was expressed in 99%, and CD44 was expressed in 98.7% (Fig. 1B). In comparison, the average expression percentages for iMSCs were 91% for CD105, 98.3% for CD90, 99% for CD73, and 99% for CD44 (Fig. 1B). Additionally, both iMSCs and BMSCs were tested for their ability to differentiate into osteogenic (Fig. 1C) and adipogenic (Fig. 1D) lineages. The results obtained from the present study revealed the positive differentiation potential of both cell types, with BMSCs showing higher levels of adipogenic differentiation than iMSCs.

The exosomes were bound to sulfate-latex beads to detect exosome surface markers using flow cytometry (CD81, CD9, and CD63), as aforementioned. The analysis confirmed the successful expression of these markers in both the iMSC-Exos and the BMSC-Exos. Notably, both types of exosomes presented higher expression levels of the CD9 tetraspanin protein than CD81 and CD63. However, no significant differences in the expression of exosome surface markers were observed between the two groups (Fig. 2A and B). The average percentages of these markers in iMSC-Exos were as follows: CD9 (99%), CD81 (79%), and CD63 (64%). In comparison, BMSC-Exos exhibited average percentages of CD9 of 95%, CD81 of 77%, and CD63 of 40% (Fig. 2A and B). The size distribution analysis revealed that both iMSC- and BMSC-derived exosomes exhibited heterogeneous size ranges of 88-220 nm and 32-117 nm, respectively, with iMSC-Exos having a larger average size than BMSC-Exos (Fig. 2D). TEM revealed a typical cup-like shape of both iMSC-Exos and BMSC-Exos (Fig. 2C). Additionally, the aggregates observed for the iMSC-Exos were consistent with the DLS findings, which indicated a larger average size for the iMSC-Exos than for the BMSC-Exos. Overall, the characteristics of the isolated EVs were consistent with those of the exosomes.

Subsequently, the uptake efficiency of exosomes by two cancer cell lines was assessed (namely, MCF7 and A549, as well as nonmalignant fibroblasts) by incubating them with either iMSC-derived or BMSC-derived exosomes for 12 h. The exosomes were prelabeled with DiI-O, a lipophilic red-orange, fluorescent dye. The cells were also stained with CMFDA (green) to visualize the cytoplasm, and DAPI (blue) was used for nuclear staining. After 12 h of incubation with DiI-O-labeled exosomes, the cells were fixed and examined under a fluorescence microscope. Imaging data confirmed the successful internalization of iMSC-Exos and BMSC-Exos into MCF7 (Fig. 3A) and A549 cells (Fig. 3B), as well as nonmalignant fibroblasts (Fig. S1).

The effects of both exosome types on cancer cell proliferation and survival were examined. Cancer cell proliferation was assessed using an MTT assay to evaluate the effects of iMSC-Exos and BMSC-Exos at 24 and 48 h of treatment. Compared to untreated SFM cells, both MCF7 and A549 cells showed a significant suppression of proliferation after 24 h of treatment with either type of exosome (P≤0.0001 for both) (Fig. 4A and B). Moreover, BMSC-Exos had a greater inhibitory effect than iMSC-Exos on MCF7 cells at 24 h after treatment (P≤0.01).

Notably, the significant antitumor effect observed at 24 h compared with that of SFM did not persist at 48 h in MCF7 cells treated with both types of exosomes or in A549 cells treated with BMSC-Exos, indicating a transient effect of BMSC-Exos on MCF7 and A549 cells and of iMSC-Exos on MCF7 cells. However, within 48 h of treating the MCF7 cells with the BMSC-Exos, cell proliferation decreased compared with that of the MCF7 cells treated with the iMSC-Exos (P≤0.01). In A549 cells, the suppressive effect of iMSC-Exos persisted over time, as evidenced by a continued reduction in proliferation at 48 h compared with that of the SFM control (P≤0.001) and BMSC-Exos (P≤0.01). Additionally, the treatment of fibroblasts that were used as control cells with either type of exosome had no significant effect on proliferation (Fig. 4C). The findings of the present study suggested that the antiproliferative effect may be cancer-specific.

Next, the effect of the BMSC-Exos and iMSC-Exos on the induction of apoptosis in the MCF7 and A549 cells was assessed using flow cytometry of the Annexin V/PI-stained cells. Treatment of MCF7 cells with either iMSC-Exos or BMSC-Exos for 48 h resulted in no significant increase in the number of early apoptotic cells (Annexin V⁺/PI⁻) or late apoptotic cells (Annexin V⁺/PI⁺) (Fig. 4E). Similarly, no significant differences in apoptosis were observed in A549 cells treated with either BMSC-Exos or iMSC-Exos (Fig. 4G).

The percentage of cells positive for SA-βGal activity, which reflects cellular senescence, was determined by counting the number of blue cells in the total population, as described by Debacq-Chainiaux et al (58). In MCF7 cells treated with iMSC-Exos or BMSC-Exos, the percentage of SA-βGal-positive cells was not significantly different (Fig. 5A and B). The findings of the present study indicated that no significant induction of cellular senescence occurred in MCF7 cells treated with either type of exosome. By contrast, treatment of A549 cells with either BMSC-Exos or iMSC-Exos resulted in a significant increase in senescence compared with that of the control (both P<0.0001) (Fig. 5C and D). These results indicate that the effect of exosome treatment on cellular senescence in tumor cells may be minimal.

A scratch wound healing assay was performed to assess the effects of iMSC- and BMSC-derived exosomes on MCF7 and A549 cancer cells. A scratch was made through the monolayers of cancer cells, and microscopy images of the scratch wounds were captured at ~9, 20, and 47 h after treatment with iMSC- or BMSC-derived exosomes. In MCF7 cells treated with BMSC-derived exosomes, a significant increase in migration was observed at ~20 h (P≤0.01) (Fig. 6A and B). However, this effect was reversed at ~47 h (P≤0.05), indicating a time-dependent effect of the BMSC-Exos on these cells. By contrast, no significant difference in migration was observed in A549 cells treated with either type of exosomes compared with the SFM control (Fig. 6C and D).