The ethical approval for derivation and use of derived human Wharton's Jelly stem cells (hWJSCs), and the commercial human ovarian cancer cell lines (OVCAR3 and SKOV3) was obtained from the Bioethics Committee of the King Abdulaziz University vide approval number [33-15/KAU], with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki.

Human umbilical cords (n = 10) were obtained following informed consent from patients undergoing full-term derlivery at the Department of Obstetrics and Gynecology, King Abdulaziz University Hospital (KAUH). The umbilical cord was transferred in a sterile container containing Hanks balanced salt solution (HBSS) and antibiotics and processed within 6 h. Derivation of hWJSCs were done according to the protocol published earlier (14, 15). Briefly, the umbilical cord was cut into pieces of ~2 cm and opened length wise. The blood vessels were removed and the opened side exposed to an ezyme cocktail containing collagenase type-I (2 mg/mL), collagenase type-IV (2 mg/mL) and hyaluronidase (100 IU) for 30 min. The enzyme ativity was blocked by addition of medium containing 10% fetal bovine serum (FBS), and the matrix contents were gently scraped and the medium containing cells and matrix substance was centrifuged at 500 g × 5 min. The cell pellet was washed twice with phosphate bufered saline (PBS⁻) devoid of calcium chloride and magensium and centrifuged again. The resultant pellet was resuspended in culture media comprised of DMEM high glucose (DMEM-HG), supplemented with 10% FBS, 2 mM Glutamax, 1% non-essential aminoacids (NEAA), basic fibroblast growth factor (bFGF) 16 ng/mL and 1% antibiotics [pencillin (50 IU/ml), streptomycin (50 μg/ml)] and incuabted at standard culture conditions of 37°C in a 5% CO₂ incubator. The cultures were left undisturbed until cell growth was evident, except for gentle changes of growth media every 72 h. The deirved cells were tested for their biological and stemness properties before being utilized in the study.

The derived hWJSCs were initially analyzed for the presence of MSCs related surface CD markers using fluorescent activated cell sorting (FACS) as reported earlier (14). Briefly, hWJSCs were trypsinized and centrifuged (1000 rpm × 5 min) and the cell pellet was gently resuspended in 5 ml of PBS⁻ to obtain single cell suspension. The cells were counted, aliquoted (1 × 10⁵ cells/15 ml tube per treatment condition) and blocked with 100 μl of 3% FBS to prevent non-specific binding. CD marker cocktails (containing 5 μl per individual CD marker) were freshly prepared and used to assess the MSCs related CD markers (Miltenyi Biotec) as follows: MSC isotype cocktail (negative control); MSC positive cocktail 1 (containing CD45-APC, CD105-FITC, CD73-PERCP) and MSC positive cocktail 2 (containing CD29-PERCP, CD34-PE, CD44-PE-CY, CD90-FITC). Respective CD markers cocktail were added to the different samples and incubated in the dark at 4°C for 20–30min. The cells were washed twice with 3% FBS buffer and centrifuged (1000 rpm × 5 min). The resultant pellet was resuspended in 500 μl of 3% FBS and analyzed using FACS (FACS Aria II, BD Biosciences).

Early passages of hWJSCs were grown under standard culture conditions and the media changed every 48 h. When the cells were 70% confluent, the spent culture media was replaced with fresh media and the cells cultured for upto 72 h. The hWJSC-conditioned media (hWJSC-CM) was then harvested, filter sterilized using 0.2 μm syringe filters and aliquots stored at 4°C until use in experiments (16). Neat samples of hWJSC-CM represented 100%, while the other percentages of hWJSC-CM was prepared by diluting the neat hWJSC-CM with basal culture medium.

Derived hWJSCs were grown under standard culture conditions and the media changed every 48 h. At confluence the cells were trypsinized and centrifuged (1000 rpm × 5 min) and the pellet washed twice in PBS⁻ and centrifuged again. The resultant cell pellet was lysed in cell lysis buffer (Sigma, St Louis, MO). Protease inhibitor cocktail (Sigma, ST Louis, MO) was added to the cell lysis buffer prior to use. The cells were pipetted up and down to lyse the membranes and release the cellular contents. The cells in suspension were incuabted in a rocker platform for 15 min followed by centrifugation (15,000 rpm × 15 min). The clear supernatant was collected, aliquoted and stored at 4°C until use in experiments. The total protein content was quantified using Nanodrop (Nanodrop technologies, Wilmington, CA) (16).

Human commercial ovarian cancer cell lines (OVCAR3 and SKOV3) were purchased from the European Collection of Authenticated Cell Cultures (ECACC). OVCAR3 cells were cultured in Low Glucose Dulbecco's Modified Eagles Medium (DMEM-LG) and SKOV3 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640. Both media were supplemented with 10% FBS, 2 mM Glutamax and 1% penicillin-streptomycin. Both OVCAR3 and SKOV3 cells were rapidly thawed in 37°C waterbath using standard cell thawing procedure and cultured at optimal conditions of 37°C in a 5% CO₂ incubator. Both OVCAR3 and SKOV3 cells represent serous adenocarcinoma having low and high invasion potential, respectively. Both cell types exhibit drug resistance, presence of hormone receptors and tumorigenic potential, and are therefore commonly used in in vitro/in vivo research studies.

The hWJSCs and commercial ovarian cancer cell lines (OVCAR3, SKOV3) were plated at a seeding density of 2 × 10⁴ cells/well of a 24 well plate and allowed to attach overnight. The culture media was then replaced with media containing different concentrations of either hWJSC-CM (12.5, 25, 50, 75, 100%); hWJSC-CL (5, 10, 15, 30, 50 μg/ml); paclitaxel (2.5, 5, 10, 20, 30 nM) or doxorubicin (3, 10, 30, 100, and 300 nM) for 24, 48 and 72 h. Control cells were cultured in normal culture medium without any pharmacological agents. The cell metabolic activity/inhibition following different experimental conditions were analyzed using MTT assay according to manufacturer's instructions. Briefly, the culture media was removed and 200 μl of fresh media containing 10 μl of MTT reagent (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide; Sigma, MO) was added and incubated under standard culture conditions for upto 4 h. The media was removed and 200 μl of solubilization reagent was added. Absorbance was obtained at 570 nM with a reference wavelength of 630 nM using SpectraMax i3 Multimode Reader (Molecular Devices, USA).

OVCAR3 and SKOV3 cells were plated at a seeding density of 2 × 10⁴ cells/well of a 24 well plate and tested with hWJSC-CM (50, 75, 100%); hWJSC-CL (10, 15, 30 μg/ml); paclitaxel (2.5, 5, 10, 20, 30 nM) or doxorubicin (3, 10, 30, 100, 300 nM). Both controls and experimental groups were then cultured at 37°C in a 5% CO₂ incubator for up to 24–72 h. Cell morphology including any visible changes following experimental procedures were imaged every 24 h using inverted phase contrast microscopy (Nikon Instruments, Japan).

OVCAR3 and SKOV3 cells were plated at a seeding density of 1 × 10⁵ cells/T25 cm² tissue culture flask and tested with hWJSC-CM (50, 75, 100%); hWJSC-CL (10, 15, 30 μg/ml); paclitaxel (2.5, 5, 10 nM) or doxorubicin (10, 30, 100 nM). Briefly, the control and treated cells were fixed with ice-cold 70% ethanol for 2–3 h by dropwise addition to avoid clumping of cells. The fixed cells were washed with PBS⁻ and stained with 50 μg/ml propidium iodide (PI) in PBS⁻ containing 0.1% TritonX-100 and 50 μg/ml RNAse-A. The cells were analyzed using FACS Aria III flow cytometer (BD Biosciences, San Jose, CA) and results computed using FACSDiva^TM software (BD Biosciences, San Jose, CA).

Detection of cleaved caspase 3 was done using fluorescin isothiocyante (FITC) conjugated active caspase 3 antibody according to the manufacturer's instructions (BD Biosciences, San Jose, CA). Briefly, both OVCAR3 and SKOV3 cells were plated at a seeding density of 1 × 10⁵ cells/T25 cm² tissue culture flask and allowed to attach overnight. Both OVCAR3 and SKOV3 cells were then treated with hWJSC-CM (50%), hWJSC-CL (15 μg/ml), paclitaxel (5 nM) or doxorubicin (30 nM) for 24 h under standard culture conditions. Following the treatment period the cells were trypsinized, centrifuged (1000 rpm × 5 min) and the pellet washed with ice-cold PBS. The cells were again centrifuged and the pellet was resuspended in 0.5 ml of Cytofix/Cytoperm solution and incubated on ice for 20 min. The cells were then washed twice with Perm/Wash buffer (1x) and incubated with 5 μl of FITC conjugated rabbit anti-caspase 3 antibody for 30 min at room temperature. The cells were washed again and resuspended in 300 μl buffer and analyzed using FACS Aria III flow cytometer (BD Biosciences, San Jose, CA) and results computed using FACSDiva^TM software (BD Biosciences, San Jose, CA).

OVCAR3 and SKOV3 were initially cultured in respective serum-free culture medium for 24 h. The serum-starved cells were then seeded at a density of 1 × 10⁵ cells/well and cultured in the upper chamber of 24-well transwell (8 mm pore size) culture plates (Corning Costar Corporation, Cambridge, MA) in serum-free medium. The lower chamber was filled with respective culture medium containing either hWJSC-CM (50%) or hWJSC-CL (15 μg/ml) and the culture plate was incubated under standard culture conditions of 37°C in a 5% CO₂ incubator. Cell migration from upper to lower chambers was evaluated after 24 h. The culture inserts were treated with the cell dissociation buffer in new wells of 24-well plates, and further washed with respective media to ensure that all the cells attached to the lower surface of the transwell membrane were completely dislodged into the new well. The migrated cells were quantified following treatment with MTT reagent and measuring their absorbance at 570 nM with a reference wavelength of 630 nM using SpectraMax i3 Multimode Reader (Molecular Devices, USA).

OVCAR3 and SKOV3 cells were seeded at a seeding density of 1.5 × 10⁵ cells/well in 6-well ultralow attachment plates (Corning Inc) in serum-free DMEM/F12 medium with 1% of methylcellulose supplemented with 1% penicillin-streptomycin, 20 nM progesterone, 100 μM putrescine, 1% insulin-transferrin-selenium, 10 ng/ml b-FGF and 10 ng/ml human rEGF. After 7 to 12 days of culture, the generated TS were imaged using inverted phase contrast microscope (Nikon Instruments, Japan). Tumor spheres were then filtered to separate uniform sized TS of more than 100 μm size and equal numbers plated in a new 6-well ultralow attachment plates. These TS were then exposed to experimental agents hWJSC-CM (50, 75, 100%); hWJSC-CL (10, 15, 30 μg/ml); paclitaxel (2.5, 5, 10 nM) or doxorubicin (10, 30, 100 nM) and any changes in morphology were imaged using inverted phase contrast microscopy (Nikon Instruments, Japan).

The TS generated from OVCAR3 and SKOV3 as above were plated in 24 well tissue culture plates to have approximately equal sizes and numbers of spheroids per well. The TS were then treated with hWJSC-CM (50%), hWJSC-CL (15 μg/ml) or paclitaxel (10 nM) and cultured for 48 h under standard culture conditions of 37°C in a 5% CO₂ incubator. The untreated TS of both OVCAR3 and SKOV3 served as controls. Following the study period both experimental and control TS were treated with trypsin to enable cell dissociation and was neutralized with media containing 10% FBS. The dissociated cells from each experimental condition were gently mixed to obtain single cell suspension and centrifuged (1000 rpm × 5 min), washed twice with PBS⁻ and centrifuged again. The resultant cell pellet was then resuspended in PBS⁻, cells counted and 1 × 10⁵ cells per tube were used for staining with CSCs related CD marker cocktails (pooled based on fluorescent markers with different wavelengths). Unstained cells were used for isotype controls. Following respective staining for 30 min on ice, the cells were washed with PBS⁻ containing 3% FBS to remove unbound antibodies/labels. The final cell pellet from both control and experimental samples were reconstituted in 100 μl of buffer and analyzed using FACS Aria III flow cytometer (BD Biosciences, San Jose, CA). The results were then computed using FACSDiva^TM software (BD Biosciences, San Jose, CA).

Both OVCAR3 and SKOV3 cells treated with hWJSC-CM (50%), hWJSC-CL (15 μg/ml), paclitaxel (10 nM) and doxorubicin (30 nM) were analyzed for cell cycle, pro-inflammatory and prostaglandin receptor related gene expression using quantitative real-time polymerase chain reaction (qRT-PCR). Briefly, the total ribonucleic acid (RNA) was extracted from the control and treated OVCAR3 and SKOV3 cells using Pure Link® RNA Mini Kit (Ambion^TM, Thermo Fischer Scientific) according to the manufacturer's protocol. First-strand cDNA synthesis was carried out using random hexamers (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems) and on column deoxyribonuclease (DNase-I) treatment was included in the protocol. Genes related to cell cycle (Cyclin A2, Cyclin E1), prostaglandin receptor signaling (EP2, EP4) and inflammation (TNF-α, IL-6) that are involved in cell cycle check, angiogenesis, cancer growth and progression were studied. Primers were taken from earlier published reports (17, 18, 19) and the primer sequences are given in Table 1. The qRT-PCR analysis was performed using the ABI StepOnePlus^TM Real-Time PCR System (Applied Biosystems) using SYBR green dye master mix and relative quantification was performed using the comparative 2 –ΔΔCt method which analyzes the relative change in gene expression based on Ct values (fluorescence denoting cDNA copy numbers) of housekeeping gene and gene of interest.

The differences observed between treated and control cell numbers, cell migration and gene expression assays were analyzed using the Students t-test with the statistical package for Social Sciences (SPSS 13). The results were expressed as mean ± standard error of the mean (SEM) from three different replicates for individual assays and a value of p < 0.05 was considered to be statistically significant.

The hWJSCs were derived from 10 human umbilical cords and the primary cultures of hWJSCs were characterized for their stemness and biological properties. The hWJSCs appeared as short fibroblasts in initial passages which transformed to long fbroblastic shape with subsesequent passages (Figures 1A,B). The hWJSCs expressed MSCs related CD markers namely CD29, CD44, CD73, CD90, and CD105 and their expression levels were more than 90% in almost all the derived hWJSCs. These cells were also negative for the haematopoietic stem cell markers namely CD34 and CD45 (Figure 1C).

The human ovarain cancer cell lines OVCAR3 and SKOV3 were immediately thawed using standard protocol and cultured using respective media. Both cell lines demonstated their characteristic epithelial morphology in culture (Figures 1D,E).

OVCAR3 cells demonstrated concentration dependent decrease in cell metabolic activity following treatment with hWJSC-CM and hWJSC-CL at 24–72 h. The mean decreases in OVCAR3 cell metabolic activity following treatment with hWJSC-CM at different concentrations (12.5, 25, 50, 75, 100%) were 3.66, 3.66, 8.44, 10.99, and 22.43% at 24 h; 7.78, 13.20, 41.43, 46.65, and 50.59% at 48 h, and 14.96, 16.54, 20.47, 28.35, and 55.12% at 72 h (Figure 2A). The mean decreases obtained in OVCAR3 cells following treatment with hWJSC-CL at different concentrations (5, 10, 15, 30, and 50 μg/ml) were 14.63, 12.20, 17.07, 20.73, and 34.15% at 24 h; 4.35, 8.70, 43.48, 53.26, and 65.22% at 48 h, and 15.04, 16.62, 27.86, 65.18, and 70.75% at 72 h (Figure 2B). The mean decreases observed in OVCAR3 cells with hWJSC-CM (100%) at 24 h; hWJSC-CM (50, 75, 100%) at 48 and 72 h; and hWJSC-CL (15, 30, 50 μg/ml) at 24, 48, and 72 h were statistically significant (P < 0.05) compared to the control.

SKOV3 cells demonstrated concentration dependent decrease in cell metabolic activity following treatment with hWJSC-CM and hWJSC-CL at 24–72 h. The mean decreases obtained in SKOV3 cells following treatment with hWJSC-CM at different concentrations (12.5, 25, 50, 75, 100%) were 20.52, 20.52, 22.73, 25.86, 31.61% at 24 h; 10.34, 15.62, 17.07, 21.27, 31.67% at 48 h and 14.20, 32.81, 42.06, 49.03, 65.00% at 72 h (Figure 2C). The mean decreases in SKOV3 cells following treatment with hWJSC-CL at different concentrations (5, 10, 15, 30, 50 μg/ml) were 12.64, 16.82, 22.69, 29.03, 43.36% at 24 h; 6.45, 12.21, 25.48, 35.92, 46.35% at 48 h and 2.28, 8.25, 29.54, 52.02, 55.02% at 72 h (Figure 2D). The mean decreases in SKOV3 cells with higher concentrations of hWJSC-CM (25, 50, 75,100%) at 72 h and hWJSC-CL (15, 30, 50 μg/ml) at 24, 48, and 72 h were statistically significant (p < 0.05) compared to the control.

Both OVCAR3 and SKOV3 cells demonstrated concentration dependent decrease in cell metabolic activity following treatment with paclitaxel at 24–72 h. The mean decreases obtained in OVCAR3 cells following treatment withpaclitaxel at different concentrations (2.5, 5, 10, 20, 30 nM) were 35.86, 46.22, 52.46, 89.24, 90.12% at 24 h; 57.51, 60.53, 73.29, 90.40, 90.64% at 48 h and 89.10, 85.98, 85.76, 91.21, 90.56% at 72 h (Figure 3A). The mean decreases obtained in SKOV3 cells following treatment with paclitaxel were 7.48, 8.51, 17.15, 41.33, 58.93% at 24 h; 27.34, 28.01, 34.14, 45.45, 59.33% at 48 h and 41.95, 62.26, 74.82, 88.85, 93.29% at 72 h (Figure 3B). The mean decreases in OVCAR3 cells for all concentrations of paclitaxel (2.5, 5, 10, 20, 30 nM) at 24, 48, and 72h and the mean decreases in SKOV3 cells with paclitaxel (30 nM at 24 h; 2.5, 5, 10, 20, 30 nM at 48 and 72 h) were statistically significant (p < 0.05) compared to the control.

Both OVCAR3 and SKOV3 cells demonstrated concentration dependent decrease in cell metabolic activity following treatment with doxorubicin at 24–72 h. The mean decreases obtained in OVCAR3 with doxorubicin at different concentrations (3, 10, 30, 100, 300 nM) were 35.64, 3.41, 41.64, 32.27, 26.92% at 24 h; 16.53, 20.90, 44.75, 41.84, 53.98% at 48 h, and 4.11, 13.18, 61.30, 69.09, 69.92% at 72 h (Figure 3C). The mean decreases obtained in SKOV3 cells were 0.64, 0.85, 1.52, 3.71, 6.34% (for 3, 10, 30, 100, 300 nM, respectively) at 24 h; 13.82, 14.84, 34.74% (for 30, 100, 300 nM, respectively) at 48 h, and 0.63, 6.99, 17.22, 52.67% (for 3, 30, 100, 300 nM, respectively) at 72 h (Figure 3D). The mean decreases in OVCAR3 cells with doxorubicin concentrations 3, 10, 30, 100, 300 nM at 48 h as well as the mean decreases with 10, 30, 100, 300 nM at 72 h were statistically significant (P < 0.05) compared to the control. The mean decreases in SKOV3 with doxorubicin concentrations 30, 100, 300 nM at 48 and 72 h were statistically significant (P < 0.05) compared to the control.

OVCAR3 cells showed various morphological changes leading to cell death following treatment with hWJSC-CM (50, 75, 100 %) and hWJSC-CL (15, 30, 50 μg/ml) for 24, 48, and 72 h (Figures 4A,B). The morphological changes included cell shrinkage, membrane damage and cell death. Unlike OVCAR3 the SKOV3 cells showed few cellular changes leading to cell death following treatment with hWJSC-CM (50, 75, 100 %) and hWJSC-CL (15, 30, 50 μg/ml) (Figure 5A,B). In general, the cellular changes were both time and concentration dependent, and was more evident with higher concentrations of hWJSC-CM and hWJSC-CL.

Both OVCAR3 (Supplementary Figures 1A,B) and SKOV3 (Supplementary Figures 2A,B) cells showed various morphological changes including cell shrinkage and membrane damage leading to cell death following treatment with pacliaxel (2.5, 5, 10, 20, 30 nM) and doxorubicin (3, 10, 30, 100, 300 nM) for 24, 48, and 72 h. The cellular changes were both time and concentration dependent (Supplementary Figures 1B, 2B).

Cell cycle assay of OVCAR3 and SKOV3 cells showed changes in the cell cycle profile following treatment with hWJSC-CM (50, 75, 100%) and hWJSC-CL (10, 15, 30 μg/ml) for 48 h, compared to the control. With OVCAR3 cells, there was an increase sub-G1 phase (by 7.9, 10.4, 26.3%) and G2M phase (by 15.3, 14.2, 6.5%) following treatment with hWJSC-CM (Figure 6A, Left panel). Treatment with hWJSC-CL demonstrated an increase in the sub-G1 population (by 16.3, 8.0, 13.7%) and the G2M phase (by 11.7, 12.9, 17.9%) compared to the control (Figure 6A, Right panel). With SKOV3 cells, there was an increase sub-G1 phase (by 21.3, 15.3, 15.0%) and G2M phase (by 11.5, 9.5, 12.9%) following treatment with hWJSC-CM (Figure 6B, Left panel. Treatment with hWJSC-CL demonstrated an increase in the sub-G1 population (by 12.8, 22.3, 13.2%) and the G2M phase (by 22.2, 11.3, 19.0%) compared to the control (Figure 6B, Right panel).

Cell cycle assay of both OVCAR3 and SKOV3 cells at 48 h, showed changes in the cell cycle profile following treatment with paclitaxel (2.5, 5, 10 nM) and doxorubicin (10, 30, 100 nM) compared to the control. The OVCAR3 cells treated with paclitaxel showed an increase sub-G1 phase (by 65.8, 52.0, 60.6%) and G2M phase (by 6.9, 7.7, 4.3%) compared to the control (Figure 6C, Left panel). SKOV3 cells treated with paclitaxel also demonstrated an increase in the sub-G1 population (by 37.6, 22.8, 4.7%) and the G2M phase (by 27.5, 21.3, 11.8%) compared to the control (Figure 6C, Right panel). The OVCAR3 cells treated with doxorubicin showed an increase sub-G1 phase (by 10.2, 13.7, 13.0%) and G2M phase (by 23.6, 33.0, 41.4%) compared to the control (Figure 6D, Left panel). SKOV3 cells also demonstrated an increase in the sub-G1 population (by 9.8, 10.1, 10.3%) and the G2M phase (by 14.1, 25.6, 11.1%) compared to the control (Figure 6D, Right panel). Treatment of both OVCAR3 and SKOV3 cells with hWJSC extracts (hWJSC-CM, hWJSC-CL) as well as the standard anticancer agents (paclitaxel and doxorubicin) demonstrated increases in sub-G1 and the G2M phases of cell cycle compared to the control, indicating that the cancer cells underwent cell death via apoptosis and metaphase arrest.

Both OVCAR3 and SKOV3 cells treated with hWJSC-CM (50%), hWJSC-CL (15 μg/ml), paclitaxel (5 nM) and doxorubicin (30 nM) showed mild positive expression of activated caspase 3 compared to the control (Figures 7A,B). The percentage of cells positve for activated caspase 3 with OVCAR3 cells were 6.0, 2.5, 2.1, and 2.4% following treatment with paclitaxel (5 nM), doxorubicin (30 nM), hWJSC-CM (50%), hWJSC-CL (15 μg/ml), respectively (Figure 7A). The percentage of cells positve for activated caspase 3 with SKOV3 cells were 4.2, 4.1, 2.2, and 3.3% compared to the untreated control (Figure 7B).

The cell migration assay done on both OVCAR3 and SKOV3 cells following treatment with hWJSC-CM (50%) or hWJSC-CL (15 μg/ml) for 48 h showed that the hWJSC extracts (hWJSC-CM and hWJSC-CL) inhibited cancer cell migration compared to the control. The percentage decreases in migration observed with OVCAR3 and SKOV3 cells were 2.67, 8.97, and 17.32% and 34.20% for hWJSC-CM and hWJSC-CL, respectively (Figures 7C,D). However, only those decreases observed in OVCAR3 and SKOV3 cells with hWJSC-CL were statistically significant (P < 0.05).

The tumor spheres of both OVCAR3 and SKOV3 treated with hWJSC-CM (50, 75, 100 %) or hWJSC-CL (10, 15, 30 μg/ml) for 48 h showed changes in morphology, decrease in the number and size of TS compared to the control (Figures 8A–D). The morphological changes were more pronounced following treatment with hWJSC-CL, which showed more cell debris and cell death than hWJSC-CM in both OVCAR3 and SKOV3.

The TS of both OVCAR3 and SKOV3 treated with paclitaxel (2.5, 5, 10 nM) and doxorubicin (10, 30, 100 nM) for 48 h also showed changes in morphology, decrease in the number and size of TS compared to the control (Figures 9A–D). More cystic degeneration was evident in OVCAR3 following treatment with both paclitaxel and doxorubicin. In contrast, there were more cell death and decrease in the sizes of TS in SKOV3 following treatment with both paclitaxel and doxorubicin.

The TS of OVCAR3 treated with paclitaxel (10 nM) for 48 h were positive for CD117 (0.2–0.9%), CD133 (0.2%), CD33 (0.2%), CD309 (0.2%), CD44 (0.2%), CD47 (7.8%), CD90 (0.3%), CD24 (66.0–75.3%), CD105 (0.1%), and CD326 (95.1–96.0%). OVCAR3 treated with paclitaxel were negative for E-Cadherin and CD31 (Supplementary Figure 3A). SKOV3 treated with paclitaxel (10 nM) were positive for CD117 (1.4%), CD133 (2.4–5.7%), CD 33 (0.1%), CD31 (0.3%), CD309 (1.8–5.0%%), CD326 (8.9%), CD34 (0.2%), CD44 (28.5%), CD24 (0.2%), CD105 (1.6–5.3%), and CD47 (0.4%). SKOV3 treated with paclitaxel was negative for E-Cadherin and CD90 (Supplementary Figure 3B).

Screening of TS of OVCAR3 and SKOV3 for CSCs following treatment with hWJSC-CM (50%) and hWJSC-CL (15 μg/ml) and paclitaxel (10 nM) demonstrated minimal presence of some of the CD markers reported to be CSC markers associated with ovarian cancers. There were also differences in the expression pattern of CD markers between OVCAR3 and SKOV3.

The gene expression analysis done on both OVCAR3 and SKOV3 following treatment with hWJSC-CM (50%) or hWJSC-CL (15 μg/ml) for 48 h showed differential expression of the cell cycle regulatory genes (cyclin A2, Cyclin E1), prostaglandin receptors (EP2, EP4) and pro-inflammatory genes (IL-6, TNF-a) (Figures 12A–F).

OVCAR3 cells demonstrated decreases in cyclin A2 and cyclin E1 expression with doxorubicin (30 nM) (by 83.45, 93.61%); hWJC-CM (by 81.06, 80.91%); and hWJSC-CL (by 89.38, 89.16%), respectively. In contrast, treatment with paclitaxel (10 nM) demonstrated increase in cyclin A2 and cyclin E1 expression by 77.33 and 44.31% (Figure 12A). Treatment of SKOV3 cells with hWJC-CM demonstrated an increase in cyclin A2 and cyclin E1 expression (by 93.53 and 82.37%), while there were decreases with paclitaxel (by 28.27, 4.64%), doxorubicin (by 87.17, 90.45%) and hWJSC-CL (by 49.44, 44.24%), respectively (Figure 12B).

Treatment of OVCAR3 cells with paclitaxel demonstrated increases in EP2 and EP4 genes expression by 58.00 and 242.72%. In contrast, decreases in EP2 and EP4 expression were observed with doxorubicin (by 78.98%,85.94%); hWJC-CM (by 65.89, 73.41%); and hWJSC-CL (by 75.31, 51.24%), respectively (Figure 12C). Differential expression of EP2 and EP4 were observed following treatment of SKOV3 cells with paclitaxel (decrease in EP2 by 42.70% and an increase in EP4 by 7.63%) and hWJSC-CM (increase in EP2 by 6.16% and a decrease in EP4 by 36.03%). However, treatment with doxorubicin and hWJSC-CL for 48 h deomonstrated decreases in EP2 and EP4. The percentage decreases were 87.00%, 62.97% with doxorubicin and 50.49%, 45.42% with hWJSC-CL, respectively (Figure 12D).

Treatment of OVCAR3 cells with paclitaxel (10 nM) for 48 h demonstrated increases in IL-6 and TNF-α genes expression by 243.07 and 77.78%. In contrast, decreases of both IL-6 and TNF-α expression were observed with doxorubicin (30 nM) by 75.47, 49.35%; hWJC-CM (50%) by 95.35, 82.86%; and hWJSC-CL (15 μg/ml) by 72.53, 68.10%, respectively (Figure 12E). Treatment of SKOV3 with paclitaxel and hWJC-CM for 48 h demonstrated increases in IL-6 and TNF-α genes expression by 68.57, 39.62 and 141.22, 78.34%, respectively. Treatment of SKOV3 cells with doxorubicin demonstrated decreases in IL-6 and TNF-α genes expression by 6.94 and 89.77%, respectively. However, treatment of SKOV3 cells with hWJSC-CL demonstrated differential expression (a decrease in IL-6 by 21.64% and an increase in TNF-α by 51.03%) (Figure 12F).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.