https://orcid.org/0000-0002-2968-2856
Figures
Abstract
Background
Mesenchymal stem cells derived from Wharton’s Jelly (WJ-MSCs) are an attractive cell source for regenerative medicine due to high proliferative capacity, non-invasive accessibility, and minimal ethical constraints. However, their therapeutic efficacy may vary with isolation technique and culture conditions.
Methods
We compared three WJ-MSC isolation methods; two explant approaches (non-scraped and scraped) and one enzymatic method – each cultured with or without basic fibroblast growth factor (bFGF). WJ-MSCs were obtained from three full-term umbilical cords, and subsequently evaluated for cell viability, proliferation kinetics, immunophenotypic surface marker expression, multilineage differentiation potential, and proteomic profiles through mass spectrometry coupled with bioinformatics analyses.
Results
All methods produced viable WJ-MSCs, although enzymatic isolation without bFGF resulted in early culture failure in 2/3 donors and was excluded from downstream analyses. Highest viability was observed with the non-scraped explant method supplemented with bFGF, and bFGF significantly enhanced proliferation by reducing cell doubling time. All groups consistently expressed canonical MSC markers, along with WJ-MSC-specific surface proteins. Osteogenic differentiation was robust across all groups, whereas adipogenic differentiation was limited. Proteomic profiling revealed 2,372 proteins commonly expressed across all groups, indicating a largely stable core proteome, with isolation- and bFGF-dependent modulation observed primarily at the pathway level. Gene set enrichment analysis showed that bFGF-treated cultures were enriched for metabolic pathways, including oxidative phosphorylation and fatty acid metabolism, whereas bFGF-free and enzymatic isolation methods showed increased inflammatory and stress-related signatures. Differential expression analysis further identified 36 proteins uniquely regulated by isolation method and bFGF treatment, associated with cell adhesion, tissue morphogenesis, and immunomodulatory functions.
Conclusion
This study clarifies how isolation- and growth factor–driven effects shape the functional properties and paracrine identity of WJ-MSCs. The non-scraped explant method with bFGF emerges as a robust, reproducible approach, yielding high-viability, phenotypically stable, and metabolically resilient MSCs. These findings provide a framework for standardized WJ-MSC production optimized for regenerative and immunomodulatory applications.
Citation: Nguyen JL, Mohamed-Ahmed S, Saleem R, Mustafa K, Al-Sharabi N, Bentsen MHL (2026) Isolation methods influence the biological properties of Wharton’s Jelly-derived mesenchymal stem cells: A comparative study of yield, viability, proliferation, differentiation potential, and proteomic profiles. PLoS One 21(3): e0345081. https://doi.org/10.1371/journal.pone.0345081
Editor: Vikash Chandra, ICAR-Indian Veterinary Research Institute: Indian Veterinary Research Institute, INDIA
Received: September 2, 2025; Accepted: March 2, 2026; Published: March 20, 2026
Copyright: © 2026 Nguyen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (https://www.proteomexchange.org/) Data are available via ProteomeXchange with identifier PXD063958.
Funding: This work was supported by the The Western Norway Regional Health Authority, Trond Mohn Foundation (grant ID: TMS2021TMT08), Research Council of Norway (M-ERA.NET project number 353453) and the University of Bergen. The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Mass spectrometry-based proteomic analyses were performed by the Proteomics Unit at the University of Bergen (PROBE). This facility is a member of the National Network of Advanced Proteomics Infrastructure (NAPI), which is funded by the Research Council of Norway (INFRASTRUKTUR-program project number: 295910). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into osteogenic, chondrogenic, and adipogenic lineages. Their versatility and immunomodulatory properties make them a promising tool for regenerative medicine [1]. While MSCs can be derived from various tissues, Wharton’s Jelly-derived MSCs (WJ-MSCs) have emerged as a particularly valuable source due to their accessibility, high proliferation rate, and minimal ethical concerns – factors that have driven ongoing research into optimizing their isolation and therapeutic potential [2–4].
The isolation method can be a key factor influencing the yield, quality, and therapeutic potential of WJ-MSCs [5]. Two primary approaches are commonly used: explant-based techniques and enzymatic digestion [6]. The explant-based methods rely on the natural migration of MSCs from Wharton’s Jelly onto the culture surface, preserving cell integrity but often yielding lower initial cell numbers. In contrast, enzymatic digestion uses enzymes like collagenase to break down the extracellular matrix, enabling faster cell release but potentially compromising cell surface markers and viability [7,8]. Within explant-based methods, the degree of physical manipulation of the Wharton’s Jelly tissue can vary. Comparing scraped and non-scraped explant methods allows assessment of how differing degrees of mechanical disruption to Wharton’s Jelly and its extracellular matrix influence early cell stress, outgrowth behavior, and phenotypic stability of WJ-MSCs. In the scraping technique, the gelatinous matrix is mechanically scraped from the inner surface of the umbilical cord to release embedded cells, whereas in the non-scraping method, intact cord fragments are simply placed in culture to allow cells to migrate out naturally. These two approaches differ in the degree of mechanical stress applied to the tissue, which can influence both cell yield and phenotype. Therefore, refining different isolation protocols is essential to fully harness the therapeutic potential of WJ-MSCs, and systematic comparisons are needed to identify the most effective approach. Beyond isolation techniques, optimizing culture conditions is essential for enhancing the therapeutic potential of WJ-MSCs. Supplementing the culture conditions with growth factors, such as basic fibroblast growth factor (bFGF), has shown promise in promoting MSC proliferation and preserving their undifferentiated state [9]. Although bFGF has been studied in MSCs derived from bone marrow and adipose tissue, its specific effects on WJ-MSCs remain underexplored [10]. Investigating its role could provide valuable insights for improving WJ-MSC expansion and stemness maintenance across therapeutic applications.
Standard evaluation criteria for MSCs, including WJ-MSCs, typically encompass proliferation capacity, surface marker expression, and multi-lineage differentiation potential [11]. Identifying unique surface markers is vital for distinguishing WJ-MSCs from other cells within the Wharton’s Jelly microenvironment, such as neonatal fibroblasts [12,13]. In addition, proteomic profiling offers valuable insights into the functional properties of WJ-MSCs, potentially revealing distinct proteomic signatures that differentiate them from MSCs of other tissue origins [14]. Such advanced molecular characterization is crucial for deepening our understanding of WJ-MSC biology and refining their therapeutic applications.
The study addresses critical gaps in WJ-MSC research by systematically comparing the commonly used explant-based and enzymatic isolation techniques. Importantly, this study employs a donor-matched comparison of three WJ-MSC isolation strategies (non-scraped explant, scraped explant, and enzymatic digestion) with and without bFGF supplementation. Key parameters assessed include proliferation rate, immunophenotypic profile, and differentiation capacity. Furthermore, the study integrates extracellular proteomic profiling to define how isolation method and growth-factor signaling jointly shape the WJ-MSC paracrine secretome.
Methods
Isolation and expansion of WJ-MSCs
Human umbilical cords were collected from three independent donors (n = 3), all born at term (gestational age 39–41 weeks) from uncomplicated pregnancies, at the Women’s Ward at Haukeland University Hospital, Bergen, Norway. Each umbilical cord was processed using all three isolation methods. Within one hour of birth, the umbilical cords (UC) were rinsed of blood and clots using sterile 0.9% NaCl and cut into 2–3 cm pieces. These pieces were transported on ice in sterile 50 mL tubes containing 0.9% sterile NaCl supplemented with 2% penicillin/streptomycin (Cytevia) and 0.01% amphotericin B (Cytevia) and stored at 4 °C for approximately 2 hours. Following incubation, the cord pieces were rinsed three times with sterile phosphate-buffered saline (PBS) (Thermo Fisher Scientific). Each donor sample was divided into six experimental groups based on the isolation method and growth-factor supplementation: non-scraped explant, scraped explant, and enzymatic digestion, each cultured with or without 5 ng/mL bFGF. For each experimental group, 3–5 Biological replicates were used in each experiment.
Explant Method without Scraping (Non-Scraped): The UC segments were longitudinally split using tweezers and a scalpel, and the blood vessels were carefully dissected and removed. The segments were then cut into 3–5 mm pieces and placed in 150 mm tissue culture dishes. To promote adherence, the pieces were air-dried for approximately 5 minutes. Growth medium – composed of Minimum Essential Medium (αMEM) (Thermo Fisher Scientific) supplemented with 15% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Cytevia) – was added to one dish. The second dish received the same medium, further supplemented with 5 ng/mL bFGF. The UC explants were removed after 8–11 days, once sufficient cell outgrowth was observed.
Explant Method with Scraping (Scraped): After dissection and removal of the blood vessels, the gelatinous Wharton’s Jelly tissue was carefully scraped off using a scalpel, ensuring that the external UC membrane was excluded. The tissue was then cut into 3–5 mm pieces and processed identically to the non-scraping samples. Growth medium, with or without 5 ng/mL bFGF, was added to separate dishes. The UC explants were removed after 8–11 days, once sufficient cell outgrowth was observed.
Enzymatic method: After dissection and removal of the blood vessels, the UC pieces (2–3 cm) were further cut into smaller pieces and placed into two sterile 50 mL centrifuge tubes. The tissue was resuspended in collagenase type 1 (195 U/mg; Worthington Biochemical) prepared in PBS (4 mg/ml), and incubated at 37°C for 30 minutes, with gentle agitation every 10 minutes. Enzyme activity was neutralized by adding an equal volume of complete growth medium. The cell suspension was then filtered through sterile 70 µm Falcon cell strainers (Thermo Fisher Scientific) to remove undigested material. The filtrate was centrifuged at 1500 rpm (~380 × g) for 5 minutes, and the resulting cell pellet was resuspended in complete medium, supplemented with or without 5 ng/mL bFGF, and plated in T75 culture flasks (Thermo Fisher Scientific). All enzymatic digestions were performed using identical conditions for each donor.
Medium was refreshed every 3 days. Cell morphology and growth were monitored using an inverted microscope (Eclipse TS100, Nikon). After two weeks in culture, cells were detached using Trypsin/EDTA solution (Lonza) and sub-cultured. Upon reaching 70–80% confluence, the sub-cultured cells were detached again and further expanded.
Ethical considerations
This study was conducted in accordance with the Declaration of Helsinki. The use of human umbilical cord tissue was approved by the Regional Committee on Medical and Health Research Ethics of Western Norway (REK Vest) (approval number 175565). Written informed consent was obtained from all mothers prior to collection of umbilical cords. The umbilical cords were obtained between March 2021 and August 2021, and samples were anonymized prior to processing and analysis. No additional interventions were performed on donors, and no identifiable information was collected.
Population doubling time (PDT)
The growth rate was determined by calculating the population doubling time. Cells at passage 3 were seeded at a density of 2000 cells/cm² and expanded in growth medium. After 7 days, cells were harvested and counted. The growth rate (r) was calculated using the formula:
Where Cf is the final cell count, Ci is the initial cell count, ti is the initial time and tf is the final time (in hours). The doubling time (td) was then calculated using the formula:
Cell viability assay
Cells at passage 3 were seeded at a density of 3000 cells/cm² and incubated at 37°C with 5% CO2. Cell viability was assessed on days 1, 4 and 7 using the Alamar Blue assay (Thermo Fisher Scientific), following the manufacturer’s recommendation. Fluorescence was measured at 560 nm excitation and 590 nm emission using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific).
Cell surface marker analysis by flow cytometry
Cells at passages 2–3 were immunophenotypically characterized as previously described [15]. Adherent cells were harvested and incubated with anti-human antibodies against MSC surface markers. Negative markers included CD34-FITC, CD45-APC and HLA-DR-PE, while positive markers included CD73-FITC, CD90-PerCP-Cy5.5, and CD105-PE (all from BD Biosciences), following the manufacturer’s instructions. In addition, cells were incubated with anti-human antibodies targeting the following markers: CD56-PE (BD Biosciences); EphA2-Alexa Fluor® 647 (Novus Biological); and N-cadherin-Alexa Fluor® 488 (CDH2-Alexa Fluor® 488;Novus Biologicals), also according to the manufacturer’s protocols. Stained samples were analyzed using a BD Accuri C6 Cell Analyzer (BD Biosciences), and data were processed with FlowJo software (FlowJo V10).
Differentiation potential
Osteogenic differentiation potential.
WJ-MSCs at passage 3 were seeded at a density of 3000 cells/cm2 and cultured in osteogenic medium (OM), consisting of growth medium supplemented with 0.05 mM L-ascorbic acid 2-phosphate, 10 nM dexamethasone, and 10 mM β-glycerophosphate (all from Sigma-Aldrich). Cells maintained in standard growth medium served as controls. After 21 days, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich), and extracellular matrix mineralization was assessed using Alizarin Red S staining (Sigma-Aldrich). The stain was dissolved in cetylpyridinium chloride (Sigma-Aldrich) and absorbance at 540 nm was measured using the microplate reader.
Adipogenic differentiation potential.
WJ-MSCs at passage 3 were seeded at a density of 5000 cells/cm2 and cultured in adipogenic growth medium (AM) using the StemProTM Adipogenesis Differentiation Kit (Thermo Fisher Scientific). Cells maintained in standard growth medium served as controls. After 21 days, cells were fixed with 4% paraformaldehyde, and intracellular lipid droplet formation was assessed using Oil Red O staining.
Production of condition medium for proteomics
WJ-MSCs at passage 3 were seeded at a density of 5000 cells/cm2 and cultured in the previously described growth medium until reaching 70–80% confluence. The cells were then washed three times with PBS and incubated in serum-free αMEM for 48 hours. Following incubation, the culture medium was collected and centrifuged at 300 x g for 5 minutes. The supernatant was carefully transferred to new tubes and centrifuged again at 2000 x g for 20 minutes to eliminate residual cell debris and apoptotic bodies. The resulting supernatant was filtered through a 0.2 μm pore-sized filter to ensure sterility and clarity, yielding the WJ-MSC conditioned medium (WJ-MSC-CM). The WJ-MSC-CM was aliquoted and stored at -80oC until further analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Bioinformatic analysis
The LC-MS/MS raw data were processed using Proteome Discoverer software (version 2.5.0.400; Thermo Scientific) and analyzed with Perseus (version 2.3.0.1). Common contaminants and proteins identified by fewer than two peptide ions were excluded from all datasets. To ensure accurate identification of proteins within each group and across all groups, proteins not detected in at least two donors per group were filtered out. To evaluate functional enrichment of proteins shared across all groups, a ranked gene list was generated and analyzed using Gene Set Enrichment Analysis (GSEA, version 4.3.2). The human hallmark gene sets (h.all.v2024.1.Hs.symbols.gmt) were used without collapsing, and “gene set” was selected as the permutation type. Gene sets were considered significantly enriched if they met the criteria of a false discovery rate (FDR) q-value < 0.25 and a normalized enrichment score (NES) > 1. Differentially expressed proteins (DEPs) among the common proteins were identified using pairwise comparisons: scraped vs. non-scraped, scraped vs. scraped plus, non-scraped vs. non-scraped plus, and enzymatic plus vs. bFGF groups (scraped plus and non-scraped plus). Statistical analysis was performed using a Multiple T-Test followed by post hoc Tukey’s HSD test for one-way ANOVA in Perseus software with a significance threshold of p < 0.05. DEPs were visualized as a venn diagram and heatmap in RStudio (v2024.12.0). To explore protein-protein interaction networks and functional associations, the upregulated proteins were further analyzed using the STRING database (http://string-db.org). The network analysis focused on Gene Ontology (GO) categories - Biological Process (BP) and Molecular Function (MF) – and incorporated text mining, experimental results, and curated databases, using a medium confidence interaction score cutoff of 0.4. Finally, to infer the biological relevance of the common and differentially expressed proteins, relevant GO terms were retrieved from QuickGO (https://www.ebi.ac.uk/QuickGO/, EMBL-EMI) and cross-referenced with the identified proteins.
Statistical analysis
All data are presented as mean ± standard deviation (SD) for each donor. Statistical analyses were conducted using GraphPad Prism 9 (GraphPad). Depending on the experimental design, comparisons between groups were performed using unpaired t-tests, one-way ANOVA or two-way ANOVA followed by Tukey’s post hoc. A p-value of less than 0.05 was considered statistically significant.
Results
Isolation of WJ-MSCs
WJ-MSCs isolated using all tested methods, exhibited plastic adherence and a spindle-shaped, fibroblast-like morphology characteristic of MSCs (Fig 1A). In the enzymatic isolation group cultured without bFGF, cells from two of three donors showed markedly reduced viability and proliferation, resulting in early culture failure. Due to insufficient biological replicates, this group was excluded from further analyses.
(A) Cell morphology at day 5 post-isolation. (B) Cell viability measured using Alamar Blue assay up to day 7. (C) Population doubling time calculated after 7 days in culture. * indicates significant differences between the tested groups at the same time-point. # indicates significant differences within the same group from the previous time-point. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Cell viability and population doubling time (PDT)
WJ-MSCs isolated using all tested methods exhibited a progressive and statistically significant increase in cell viability over the 7-day culture period (Fig 1B). By day 7, cells cultured with bFGF consistently showed higher viability compared to their counterparts cultured without bFGF within each respective isolation method.
Among all conditions, the non-scraped isolation method supplemented with bFGF resulted in the highest cell viability, followed by the enzymatic and scraped methods, both also supplemented with bFGF.
PDT analysis demonstrated that bFGF supplementation significantly enhanced cell proliferation, as indicated by shorter doubling times across all isolation methods (Fig 1C). In contrast, WJ-MSCs cultured without bFGF exhibited markedly longer PDTs, with the scraped and non-scraped methods yielding the slowest proliferation rates. Importantly, no statistically significant differences in PDT were found among the bFGF-supplemented groups, suggesting a comparable proliferative benefit across all methods when bFGF was present.
Surface marker characterization of WJ-MSCs
The classical mesenchymal stem cell markers CD73, CD90, and CD105 were robustly expressed (>95%) across all isolation methods, confirming the mesenchymal identity of the WJ-MSCs. In contrast, the hematopoietic and immunogenic markers CD34, CD45, and HLA-DR were consistently low (<2%) in all groups, indicating minimal contamination with hematopoietic or immune cells (Fig 2).
Flow cytometry analysis of MSC-associated surface markers across different isolation techniques. Blue histograms show unstained controls, while red histograms indicate stained cells. Positive cell surface marker expression is indicated in percentages.
To further characterize WJ-MSCs based on surface marker expression and to distinguish them from other cell populations present in Wharton’s Jelly, such as fibroblasts, we evaluated the expression of additional markers: EphA2, CDH2, and CD56 (Fig 3). WJ-MSCs isolated using all methods exhibited very high expression of EphA2 (>95%) and high expression of CD56 (>75%), while CDH2 expression remained low (<10%) across all groups.
Flow cytometry analysis of WJ-MSCs specific surface markers – EphA2, CDH2, and CD56 – across different isolation techniques. Blue histograms show unstained controls, while red histograms indicate stained cells. Positive cell surface marker expression is indicated in percentages.
Osteogenic and adipogenic potentials
WJ-MSCs isolated using all tested methods demonstrated the capacity for osteogenic differentiation following 21 days of culture in osteogenic induction medium. This was confirmed by the formation of mineralized extracellular matrix, as evidenced by positive staining with Alizarin Red S (Fig 4A and B). The mineralized matrix formation was statistically significantly higher in the scraped group than the non-scraped groups, with or without bFGF supplementation. Similarly, after 21 days in adipogenic induction medium, WJ-MSCs from all isolation techniques exhibited signs of adipogenic differentiation, indicated by the presence of intracellular lipid vacuoles that stained positively with Oil Red O (Fig 4C). However, generally in the cells from all isolation techniques, this formation of intracellular lipid vacuoles was not detected in the whole population, with only a small fraction of cells showing intracellular lipid vacuoles. Some of these vacuoles tended to fuse forming a few larger vacuoles.
(A and B) Osteogenic differentiation based on Alizarin red S staining after 21 days, microscopic images, scale bar 100 μm (A) and quantification of the stain (B). (C) Adipogenic differentiation based on Oil red O staining after 21 days, scale bar 50 μm. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test. *Indicates significant differences (p < 0.01) between the tested groups under similar culture condition. #indicates significant differences from undifferentiated cells of the same group.
Protein profiles
Proteomic analysis identified a comparable number of proteins across all groups: 2582 proteins in the scraped group, 2604 in scraped plus, 2582 in non-scraped, 2619 in non-scraped plus, and 2611 in the enzymatic plus group. Protein quantification was based on LFQ intensity detected in at least two donors per group, resulting in 2493 proteins in the scraped group, 2511 in scraped plus, 2515 in non-scraped, 2514 in non-scraped plus, and 2476 in the enzymatic plus group. Venn diagram analysis revealed that 2372 proteins were shared across all experimental groups. Additionally, two proteins were uniquely identified in the scraped group, nine in the non-scraped group, seven in the scraped plus group, and six in the non-scraped plus group. Furthermore, three proteins were uniquely identified in the enzymatic plus group (Fig 5A). Heatmap analysis of the commonly identified proteins highlighted systematic differences in protein expression profiles between the groups (Fig 5B).
(A) Venn diagram showing the overlap of proteins identified in WJ-MSC conditioned media across the five experimental groups. (B) Hierarchical clustering heatmap of proteins commonly detected across groups; rows represent individual proteins and columns represent donor-matched samples across isolation methods. Color scale indicates Z-scored LFQ intensity (green = low, red = high). (C) Gene Set Enrichment Analysis (GSEA) of shared proteins highlighting significantly enriched Hallmark pathways in pairwise comparisons. Hallmark gene sets were considered significant (highlighted in red) at an FDR < 25%, according to standard GSEA recommendations.
Gene set enrichment analysis (GSEA) among common proteins
GSEA was performed on the 2372 proteins shared across all groups to evaluate differential gene set expression based on isolation method (scraping vs. non-scraping) and the presence or absence of bFGF. In the comparison between the scraped and non-scraped groups, two gene sets were upregulated in the scraped group, although none reached statistical significance at a false discovery rate (FDR) < 25%. In contrast, 41 gene sets were upregulated in the non-scraped group, with the “myc_targets_v2” gene set showing significant enrichment (NES = −1.65, FDR < 25%) (Fig 5Ci). When comparing scraped and scraped plus groups, 17 gene sets were upregulated in the scraped group, but again none were significant at the chosen FDR threshold. In the scraped plus group, 26 gene sets were upregulated, with “oxidative phosphorylation” (NES = −1.99, FDR < 25%), “fatty acid metabolism” (NES = −1.77, FDR < 25%), and “adipogenesis” (NES = −1.68, FDR < 25%) showing significant enrichment (Fig 5Cii). In the non-scraped vs. non-scraped plus comparison, 35 gene sets were upregulated in the non-scraped group. Among them, “epithelial-mesenchymal transition” (NES = 1.88, FDR < 25%), “UV response D” (NES = 1.51, FDR < 25%), and “inflammatory response” (NES = 1.52, FDR < 25%) were significantly enriched. In contrast, eight gene sets were upregulated in the non-scraped plus group, with significant enrichment observed for “oxidative phosphorylation” (NES = −2.14, FDR < 25%), “fatty acid metabolism” (NES = −1.48, FDR < 25%), and “interferon alpha response” (NES = −1.38, FDR < 25%) (Fig 5Ciii). In the comparison between scraped plus and non-scraped plus groups, 23 gene sets were upregulated in the scraped plus group, with “inflammatory response” significantly enriched (NES = 1.72, FDR < 25%). Although 20 gene sets were upregulated in the non-scraped plus groups, none reached statistical significance (Fig 5Civ). When comparing enzymatic plus with scraped plus, 30 gene sets were upregulated in the enzymatic plus group and 13 in the scraped plus group; however, none of these enrichments were statistically significant at FDR < 25% (Fig 5Cv). Similarly, the comparison between enzymatic plus and non-scraped plus revealed 30 gene sets upregulated in enzymatic plus, including “inflammatory response” (NES = 1.55, FDR < 25%) as significantly enriched. Thirteen gene sets were upregulated in the non-scraped plus group, but none were statistically significant (Fig 5Cvi).
Identification of differentially expressed proteins (DEPs) among common proteins
Quantitative analysis of the 2372 proteins shared across all groups identified a total of 59 DEPs. Pairwise comparisons based on isolation method (scraping vs. non-scraping, and bFGF treatment (presence vs. absence) revealed that 36 of these DEPs were unique to specific comparisons. In the comparison between scraped and non-scraped groups, only one protein was upregulated in the non-scraped group, and no DEPs in the scraped group. When comparing scraped and scraped plus groups, six proteins were upregulated in the scraped groups, and three in the scraped plus group. In the non-scraped vs. non-scraped plus comparison, 30 proteins were upregulated in the non-scraped group and one in the non-scraped plus group. Analysis of the enzymatic plus group against bFGF-treated groups (scraped plus and non-scraped plus), revealed one protein upregulated in enzymatic plus. Additionally, two proteins were upregulated in non-scraped plus compared to enzymatic plus, and one protein was differentially expressed in the comparison between scraped plus and enzymatic plus (Fig 6Ai). A heatmap of all DEPs across pairwise comparisons highlighted distinct differences in protein expression profiles between the groups (Fig 6Aii).
(Ai) Venn diagram illustrating the distribution of unique DEPs across pairwise comparisons between isolation methods and culture conditions. (Aii) Heatmap of the 36 unique DEPs identified across all comparisons; rows represent individual proteins and columns represent donor-matched experimental groups. The color scale indicates Z-scored LFQ intensity (green = low, red = high). (Bi) Protein–protein interaction network of the unique DEPs generated using STRING (medium confidence). (Bii) Gene Ontology (GO) Biological Process enrichment analysis of DEPs, displaying the top 10 most significantly enriched terms ranked by false discovery rate (FDR). (Ci) GO Molecular Function enrichment analysis showing the top significantly enriched terms ranked by FDR.
WJ-MSCs contain proteins involved in developmental and inflammatory processes
To explore the biological relevance of the 2372 common and 36 unique differentially expressed proteins (DEPs), gene names were analyzed using QuickGO terms related to development and healing. Several key processes were analyzed (Table 1).
Within the category “growth factor activity” (37 common proteins), the unique TGFB1 was upregulated in the non-scraped vs. non-scraped plus comparison. The term lung development” (53 common proteins) included five unique DEPs. Among these, MAP2K1 was upregulated in non-scraped plus vs. non-scraped, while FBN1, TGFB1, and LTBP3 were upregulated in non-scraped compared to non-scraped plus. LTBP3 was also identified in the scraped vs. scraped plus comparison. For “endothelial cell differentiation” (23 common proteins), ICAM1 was upregulated in non-scraped vs. non-scraped plus. In “neuron differentiation” (74 common proteins), two unique DEPs were observed: MAP2K1 was upregulated in non-scraped plus vs. non-scraped, and THY1 was upregulated in non-scraped vs. non-scraped plus. The term “muscle cell differentiation” (56 common proteins) involved three unique DEPs - SPAG9, TPM1, and LMOD1 – upregulated in non-scraped vs. non-scraped plus, with TPM1 also upregulated in scraped vs. scraped plus. No unique DEPs were associated with “osteoblast differentiation” (40 common proteins). For “inflammatory response” (60 common proteins), SAA1, THBS1, and TGFB1 were upregulated in non-scraped vs. non-scraped plus, with THBS1 also identified in scraped vs. scraped plus. In “regulation of inflammatory response” (63 common proteins) SAA1, ADAMTS12, and TGFB1 were differentially expressed in non-scraped vs. non-scraped plus. Although four proteins were annotated under “immune response”, no unique DEPs were detected. However, in “regulation of immune response” (39 common proteins), THY1 and TGFB1 were differentially expressed in non-scraped vs. non-scraped plus. Due to the limited number of unique DEPs per pairwise comparison, functional enrichment analysis was performed on all 36 unique DEPs using the STRING database. This revealed association with 92 Gene Ontology Biological Process (GOBP) terms. Notable enriched biological processes included “cell adhesion” (13 proteins, FDR = 0.00013), “regulation of cellular response to growth factor stimulus” (8 proteins, FDR = 0.00037), “regulation of multicellular organismal processes” (18 proteins, FDR = 0.00094), “positive regulation of leukocyte migration” (6 proteins, FDR = 0.00096), and “anatomical structure formation involved in morphogenesis” (11 proteins, FDR = 0.0013) (Fig 6Bi and ii). In addition, six Gene Ontology Molecular Function (GOMF) terms were enriched among the unique DEPS, including “integrin binding” (6 proteins, FDR = 0.0024), “calcium ion binding” (9 proteins, FDR = 0.0087), “protein binding” (27 proteins, FDR = 0.0087), “cell adhesion molecule binding” (8 proteins, FDR = 0.0087), “protein-containing complex binding” (11 proteins, FDR = 0.0101), and “signaling receptor binding (11 proteins, FDR = 0.0418) (Fig 6Ci and ii).
Discussion
Our results demonstrate that both the isolation method and bFGF supplementation influence the biological and molecular features of WJ-MSCs. By combining a donor-matched comparison of three isolation strategies with extracellular proteomic profiling, this study extends previous explant–enzymatic comparisons by adding molecular insight into bFGF-mediated stabilization of WJ-MSCs. Among all methods tested, the non-scraped explant approach with bFGF supplementation produced the most consistent proliferation and stable phenotype, whereas enzymatic digestion without bFGF yielded poor viability in most donors. Despite procedural differences, the core WJ-MSC proteome remained stable across all conditions, underscoring the cells’ intrinsic molecular robustness and regenerative potential.
Comparing scraped and non-scraped explants highlights how mechanical handling influences early cell behavior. Preserving the native ECM supported higher viability and proliferation, likely because intact matrix cues sustain adhesion and cytoskeletal organization. Partial removal of the jelly matrix reduced proliferation, suggesting that mechanical stress transiently disturbs cell–matrix signaling. These findings agree with reports that minimally invasive explant culture maintains ECM architecture, stemness, and reduces contamination [8,16]. Although ECM signaling was not directly analyzed in the current study, future studies examining integrin expression, actin dynamics, and matrix remodeling could clarify how physical disruption modulates downstream pathways governing WJ-MSC stability. Enzymatic digestion without bFGF reduced cell viability, indicating that Wharton’s Jelly is sensitive to proteolytic damage. The exclusion of the enzymatic isolation group without bFGF from downstream analyses underscores this sensitivity and highlights that enzymatic digestion of Wharton’s Jelly requires growth-factor support to ensure reproducible cell recovery across donors. Enzyme exposure likely disrupts anchorage and membrane receptors crucial for survival [17–19]. Prior studies using collagenase or hyaluronidase [6–8,20] achieved variable success, suggesting that enzyme type, digestion time, and donor ECM composition influence outcome. The high hyaluronic-acid content of Wharton’s Jelly probably contributes to this sensitivity. The current methodologies in tissue processing involve the integration of partial digestion with explant outgrowth techniques or the immediate addition of growth factors, such as bFGF. These approaches are designed to reduce enzymatic stress on tissues, thereby enhancing cell viability and culture success rates. Future research should focus on optimizing enzyme formulations, adjusting exposure durations, and implementing post-digestion rescue strategies with trophic factors to further improve reproducibility and consistency in experimental outcomes. Such refinements are essential for advancing tissue engineering protocols and ensuring reliable results across different laboratories and applications.
Despite early differences in proliferation, all isolation methods ultimately produced viable and proliferative WJ-MSC populations, highlighting the cells’ intrinsic regenerative capacity. The non-scraped explant technique consistently achieved the most stable growth, aligning with studies demonstrating that preserving ECM contact supports phenotypic stability and sustained self-renewal [8,16]. Although enzymatic digestion accelerates initial outgrowth, it can transiently alter receptor expression and signaling balance. Chu et al. [20] reported that Collagenase NB6-based enzymatic isolation produced cells comparable to explant-derived MSCs, though proliferation was faster initially. Overall, explant-based isolation remains the most reliable approach for generating structurally and functionally stable [16]. Cryopreservation is another factor that may contribute to variability in WJ-MSC recovery. While WJ-MSCs generally tolerate freezing well [21], post-thaw viability is strongly influenced by cryoprotectant composition. Serum-free cryoprotectants improve cell recovery compared with DMSO-based solutions [22], suggesting that standardized freezing media and rates are needed to ensure consistency in cell banking.
A key finding of this study is the pronounced effect of bFGF supplementation in enhancing WJ-MSC proliferation across all isolation methods. The addition of bFGF consistently shortened population doubling time and reduced inter-donor variability, consistent with activation of PI3K/Akt and ERK signaling pathways that promote cell-cycle progression and survival [23]. Although this has not been investigated in our study, these cascades are likely to increase cyclin-CDK expression and enhance survival. bFGF also appeared to buffer isolation-induced stress, stabilizing both cellular and proteomic behavior. Endogenous bFGF secreted from residual Wharton’s Jelly may partly explain the superior early proliferation observed in these groups [24].
Flow-cytometric analysis confirmed that bFGF did not alter canonical MSC markers (CD73, CD90, CD105 high; CD34, CD45, HLA-DR low) [25]. Our findings were also aligned with reports by Ramasamy et al. [26] and Hagmann et al. [27] who showed that bFGF enhances proliferation via ERK-dependent cyclin activation without impairing differentiation, though prolonged exposure slightly reduced CD90 and CD146 levels. Both studies also noted alterations in functional markers, including CD340 and CD49a, and shifts in cytokine secretion such as decreased VEGF and MMP3, that could influence immunomodulation in vivo. In our work, extended phenotypic analysis revealed consistent expression of EphA2 and CD56 across all groups, distinguishing WJ-MSCs from fibroblasts, while CDH2, a neonatal fibroblast marker [12,13,28] was slightly elevated in non-scraped explant cultures with bFGF, suggesting minimal fibroblast carryover. Although these changes were modest, they emphasize the need for longitudinal immunophenotypic monitoring to ensure prolonged bFGF exposure does not induce lineage drift or phenotypic heterogeneity before clinical translation. Therefore, future work should use phospho-proteomics and long-term immunophenotyping to verify that extended bFGF exposure preserves functional identity.
Previous studies reported that bFGF safely accelerates WJ-MSCs expansion without compromising phenotype or multilineage differentiation potential [26,29]. Our results demonstrate strong osteogenic differentiation, across all experimental groups of WJ-MSCs, while their adipogenic differentiation remained limited, with formation of mainly small lipid vacuoles only in a small subset of cells, rather than across the entire population. This partial formation of small lipid vacuoles and the delay in their subsequent fusion into larger vacuoles suggest incomplete or delayed adipocyte maturation, consistent with the slower lipid accumulation dynamics previously described for WJ-MSCs [30]. This limited adipogenic response is in agreement with previous studies reporting that WJ-MSCs possess inherently lower adipogenic potential compared to MSCs derived from adipose or bone marrow tissues [30,31], likely reflecting their intrinsic metabolic orientation toward matrix production and tissue regeneration rather than lipid storage. Further modulation of the adipogenic culture conditions with additional supplements may improve adipogenic differentiation in WJ-MSCs [30].
The extracellular proteomic landscape reinforces the cellular observations, revealing a stable and functionally rich secretome [32]. Across all isolation strategies, 2,372 extracellular proteins were consistently detected, with only 59 differentially expressed and 36 uniquely regulated across specific comparisons. It should be noted that the relatively small number of unique differentially expressed proteins identified in this study reflects the overall stability of the WJ-MSC proteome across isolation methods and culture conditions. This finding suggests that isolation strategy and bFGF supplementation act primarily as modulators rather than drivers of large-scale proteomic reprogramming [33]. Importantly, functional differences may therefore be governed not by extensive changes in protein abundance, but by subtle regulation of key signaling nodes, pathway-level coordination, or post-translational modifications that are not captured by standard label-free quantitative proteomics. This interpretation is supported by the GSEA results, which revealed consistent pathway-level shifts in metabolic and inflammatory signaling despite limited changes in individual protein expression. Future studies integrating phospho-proteomics, redox-sensitive proteomics, or targeted analyses of regulatory proteins will be essential to resolve how such subtle molecular alterations translate into functional differences in WJ-MSC behavior. Notably, bFGF supplementation reduced the number of DEPs compared with bFGF-free cultures, indicating that bFGF stabilizes the secretome by buffering stress-induced variability [34,35]. The GSEA revealed that bFGF treatment enriched pathways linked to oxidative phosphorylation, fatty-acid metabolism, and adipogenesis, indicating a coordinated shift toward oxidative energy metabolism that supports biosynthetic and secretory functions. In contrast, non-bFGF supplemented groups showed enrichment of inflammatory-response and epithelial–mesenchymal-transition gene sets, consistent with transient stress signaling following isolation. Taken together, our data support a model in which isolation-induced stress transiently activates inflammatory and stress-response pathways in WJ-MSCs, as reflected by enrichment of inflammatory-response gene sets in bFGF-free conditions, consistent with previous reports linking metabolic state to MSC functional stability [36]. Such signatures likely represent a primed, stress-responsive state rather than overt immune activation, consistent with the known immunomodulatory behavior of MSCs. bFGF supplementation appears to buffer this stress response by stabilizing cellular metabolism and reducing proteomic variability, shifting pathway enrichment toward oxidative phosphorylation and biosynthetic support. In this model, isolation method determines the magnitude of initial stress signaling, while bFGF acts as a molecular stabilizer that promotes a metabolically resilient and functionally consistent WJ-MSC phenotype with preserved immunomodulatory potential. Although signaling intermediates were not directly studied, future phospho-proteomic and metabolic-flux analyses should clarify how bFGF couples mitochondrial metabolism with secretory stability. The identified extracellular proteins in our current study, including VEGFA, HGF, PDGFC, BMP6, IL6, TGFB1, and ICAM1, compose a multifunctional paracrine network integrating angiogenic, immunomodulatory, and morphogenic functions. This composition aligns with previous reports showing that WJ-MSC-conditioned media and exosomes contain trophic molecules such as VEGF, IL-8, GM-CSF, MCP-1, and α2-macroglobulin that promote angiogenesis, dampen inflammation, and support tissue repair [37–40]. Similarly, WJ-MSC-conditioned medium has been shown to contain abundant developmental regulators involved in neurogenesis, myogenesis, and tissue regeneration [41]. These data, together with our findings, reinforce the concept that WJ-MSCs secrete a multifunctional and context-responsive secretome capable of coordinating vascular, immune, and regenerative processes. Secretome studies highlight the inherent heterogeneity of MSCs from different tissues [42–44]. Paliwal et al. [44] and Kupcowa et al. [45] showed that donor age, tissue origin, and microenvironment shape secretome composition. High-resolution proteomics of WJ-MSCs identified abundant α2-macroglobulin, IL-6, IL-8, GM-CSF, and chemokines such as MCP-1 and RANTES [38,39,46], key regulators of angiogenesis, immune recruitment, and tissue remodeling. Barrett et al [40] also noted that WJ-MSCs express hundreds of proteins linked to immunomodulation, wound healing, and chemotaxis, emphasizing their broader therapeutic potential compared with adult MSC sources. The enrichment of oxidative phosphorylation pathways in bFGF-treated cultures suggests enhanced redox resilience. Although not directly identified in our current study, prior studies show that perinatal stem cells upregulate antioxidant enzymes such as SOD, CAT, and GPx under stress [43], implying adaptive control of oxidative metabolism during expansion. Future work should integrate targeted redox proteomics and functional secretome assays to assess whether bFGF-induced metabolic stability improves paracrine potency and consistency. Collectively, these proteomic and secretome data indicate that bFGF functions not only as a mitogenic stimulus but also as a molecular stabilizer that preserves metabolic balance and paracrine coherence during WJ-MSC expansion.
From a translational perspective, the findings of this study have several important implications for the clinical development and manufacturing of WJ-MSC–based therapies. First, the donor-matched design demonstrates that preserving native extracellular matrix interactions through non-scraped explant isolation, combined with bFGF supplementation, yields reproducible expansion while maintaining phenotypic and molecular stability. Such consistency is critical for GMP-compliant MSC production, where batch-to-batch variability remains a major challenge.
Second, the observation that bFGF reduces secretome variability and limits stress-associated proteomic alterations suggests a stabilizing effect that may improve product predictability and safety. A more consistent paracrine profile is particularly relevant for MSC therapies that rely on immunomodulatory and trophic mechanisms rather than long-term engraftment. The enrichment of angiogenic, immunoregulatory, and developmental factors in the WJ-MSC secretome further supports the therapeutic relevance of these cells for applications such as tissue repair, inflammation modulation, and regenerative signaling.
Finally, the identification of isolation- and culture-dependent molecular signatures provides a framework for rational protocol selection tailored to specific clinical indications. Collectively, these findings bridge in vitro characterization with translational requirements by linking isolation strategy and growth-factor supplementation to molecular consistency, functional relevance, and manufacturing robustness.
Despite these advances, several limitations remain. Donor variability, including gestational age, maternal health, and cord quality, may have contributed to heterogeneity; increasing the sample size would strengthen statistical confidence. Proteomic results also require validation by Western blot, ELISA, or targeted mass spectrometry. In addition, osteogenic and adipogenic differentiation capacities were only assessed using lineage-specific staining, and performing deeper analyses of differentiation efficiency at the molecular level may provide better insights of the capacities of these cells. Although the proteomic and secretome analyses suggest immunomodulatory and regenerative potential, functional assays directly assessing immunomodulation or tissue-regenerative efficacy represent an important focus for future studies. Addressing these points will help translate WJ-MSC research into reproducible therapeutic applications.
Conclusion
The minimally manipulating non-scraped explant isolation method of WJ-MSCs supplemented with bFGF emerges as the preferred strategy for generating reproducible, phenotypically stable, and metabolically resilient WJ-MSCs. The choice of isolation method and growth-factor supplementation critically determines WJ-MSC quality and function. By applying a donor-matched comparison of three isolation strategies combined with extracellular proteomic and pathway-level analyses, this study provides molecular insight beyond previous explant–enzymatic comparisons. Enzymatic digestion, while faster, requires careful optimization to preserve ECM cues and cell viability. At the molecular level, WJ-MSCs maintain a resilient proteome and a secretome enriched in angiogenic, immunomodulatory, and redox-regulatory proteins. These results clarify how methodological and molecular factors converge to shape WJ-MSC biology and provide a framework for standardized production optimized for regenerative and immunomodulatory applications.
Acknowledgments
We gratefully acknowledge the infants and their mothers who generously donated umbilical cords for this research.
Declaration of Generative AI and AI-assisted technologies in the writing process
During the preparation of this work the authors used AI technologies in order to improve language and readability. After using these tools, the authors reviewed and edited the content as needed and took full responsibility for the content of the publication.
References
- 1. Pittenger MF, Discher DE, Péault BM, Phinney DG, Hare JM, Caplan AI. Mesenchymal stem cell perspective: cell biology to clinical progress. NPJ Regen Med. 2019;4:22. pmid:31815001
- 2. El Omar R, Beroud J, Stoltz J-F, Menu P, Velot E, Decot V. Umbilical cord mesenchymal stem cells: the new gold standard for mesenchymal stem cell-based therapies?. Tissue Eng Part B Rev. 2014;20(5):523–44. pmid:24552279
- 3. Yea J-H, Kim Y, Jo CH. Comparison of mesenchymal stem cells from bone marrow, umbilical cord blood, and umbilical cord tissue in regeneration of a full-thickness tendon defect in vitro and in vivo. Biochem Biophys Rep. 2023;34:101486. pmid:37234487
- 4. Batsali AK, Kastrinaki M-C, Papadaki HA, Pontikoglou C. Mesenchymal stem cells derived from Wharton’s Jelly of the umbilical cord: biological properties and emerging clinical applications. Curr Stem Cell Res Ther. 2013;8(2):144–55. pmid:23279098
- 5. Zhang X, Hirai M, Cantero S, Ciubotariu R, Dobrila L, Hirsh A, et al. Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue. J Cell Biochem. 2011;112(4):1206–18. pmid:21312238
- 6. Varaa N, Azandeh S, Khodabandeh Z, Gharravi AM. Wharton’s Jelly Mesenchymal Stem Cell: Various Protocols for Isolation and Differentiation of Hepatocyte-Like Cells; Narrative Review. Iran J Med Sci. 2019;44(6):437–48. pmid:31875078
- 7. Salehinejad P, Alitheen NB, Ali AM, Omar AR, Mohit M, Janzamin E, et al. Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton’s jelly. In Vitro Cell Dev Biol Anim. 2012;48(2):75–83. pmid:22274909
- 8. Hendijani F. Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues. Cell Prolif. 2017;50(2):e12334. pmid:28144997
- 9. Fujimoto Y, Yokozeki T, Yokoyama A, Tabata Y. Basic fibroblast growth factor enhances proliferation and hepatocyte growth factor expression of feline mesenchymal stem cells. Regen Ther. 2020;15:10–7. pmid:32490062
- 10. Neubauer M, Hacker M, Bauer-Kreisel P, Weiser B, Fischbach C, Schulz MB, et al. Adipose Tissue Engineering Based on Mesenchymal Stem Cells and Basic Fibroblast Growth Factor in Vitro. Tissue Eng 2005;11:1840–51.
- 11. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315–7. pmid:16923606
- 12. Al Madhoun A, Marafie SK, Haddad D, Melhem M, Abu-Farha M, Ali H, et al. Comparative Proteomic Analysis Identifies EphA2 as a Specific Cell Surface Marker for Wharton’s Jelly-Derived Mesenchymal Stem Cells. Int J Mol Sci. 2020;21(17):6437. pmid:32899389
- 13. Bosch J, Houben AP, Radke TF, Stapelkamp D, Bünemann E, Balan P. Distinct differentiation potential of “MSC” derived from cord blood and umbilical cord: are cord-derived cells true mesenchymal stromal cells?. Stem Cells Dev. 2012;21:1977–88.
- 14. Fong C-Y, Chak L-L, Biswas A, Tan J-H, Gauthaman K, Chan W-K, et al. Human Wharton’s jelly stem cells have unique transcriptome profiles compared to human embryonic stem cells and other mesenchymal stem cells. Stem Cell Rev Rep. 2011;7(1):1–16. pmid:20602182
- 15. Mohamed-Ahmed S, Fristad I, Lie SA, Suliman S, Mustafa K, Vindenes H, et al. Adipose-derived and bone marrow mesenchymal stem cells: a donor-matched comparison. Stem Cell Res Ther. 2018;9(1):168. pmid:29921311
- 16. Otte A, Bucan V, Reimers K, Hass R. Mesenchymal stem cells maintain long-term in vitro stemness during explant culture. Tissue Eng Part C Methods. 2013;19(12):937–48. pmid:23560527
- 17. Daviran M, Caram HS, Schultz KM. Role of Cell-Mediated Enzymatic Degradation and Cytoskeletal Tension on Dynamic Changes in the Rheology of the Pericellular Region Prior to Human Mesenchymal Stem Cell Motility. ACS Biomater Sci Eng. 2018;4(2):468–72. pmid:29862316
- 18. Jean C, Gravelle P, Fournie J-J, Laurent G. Influence of stress on extracellular matrix and integrin biology. Oncogene. 2011;30(24):2697–706. pmid:21339741
- 19. Reing JE, Zhang L, Myers-Irvin J, Cordero KE, Freytes DO, Heber-Katz E. Degradation products of extracellular matrix affect cell migration and proliferation. Tissue Eng Part A. 2009;15:605–14.
- 20. Chu W, Zhang F, Zeng X, He F, Shang G, Guo T, et al. A GMP-compliant manufacturing method for Wharton’s jelly-derived mesenchymal stromal cells. Stem Cell Res Ther. 2024;15(1):131. pmid:38702793
- 21. Laroye C, Gauthier M, Morello J, Charif N, Cannard VL, Bonnet C, et al. Scale-Up of Academic Mesenchymal Stromal Cell Production. J Clin Med. 2023;12(13):4414. pmid:37445448
- 22. Shivakumar SB, Bharti D, Subbarao RB, Jang S-J, Park J-S, Ullah I, et al. DMSO- and Serum-Free Cryopreservation of Wharton’s Jelly Tissue Isolated From Human Umbilical Cord. J Cell Biochem. 2016;117(10):2397–412. pmid:27038129
- 23. Huang F, Gao T, Wang W, Wang L, Xie Y, Tai C, et al. Engineered basic fibroblast growth factor-overexpressing human umbilical cord-derived mesenchymal stem cells improve the proliferation and neuronal differentiation of endogenous neural stem cells and functional recovery of spinal cord injury by activating the PI3K-Akt-GSK-3β signaling pathway. Stem Cell Res Ther. 2021;12(1):468. pmid:34419172
- 24. Yoon JH, Roh EY, Shin S, Jung NH, Song EY, Chang JY, et al. Comparison of explant-derived and enzymatic digestion-derived MSCs and the growth factors from Wharton’s jelly. Biomed Res Int. 2013;2013:428726. pmid:23653895
- 25. Priya N, Sarcar S, Majumdar AS, SundarRaj S. Explant culture: a simple, reproducible, efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate. J Tissue Eng Regen Med. 2014;8(9):706–16. pmid:22837175
- 26. Ramasamy R, Tong CK, Yip WK, Vellasamy S, Tan BC, Seow HF. Basic fibroblast growth factor modulates cell cycle of human umbilical cord-derived mesenchymal stem cells. Cell Prolif. 2012;45(2):132–9. pmid:22309282
- 27. Hagmann S, Moradi B, Frank S, Dreher T, Kämmerer PW, Richter W, et al. FGF-2 addition during expansion of human bone marrow-derived stromal cells alters MSC surface marker distribution and chondrogenic differentiation potential. Cell Prolif. 2013;46(4):396–407. pmid:23869761
- 28. Widowati W, Gunanegara RF, Rizal R, Widodo WS, Amalia A, Wibowo SHB. Comparative analysis of Wharton’s jelly mesenchymal stem cell (WJ-MSCs) isolated using explant and enzymatic methods. J Phys Conf Ser. 2019;1374:012024.
- 29. Solchaga LA, Penick K, Porter JD, Goldberg VM, Caplan AI, Welter JF. FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells. J Cell Physiol. 2005;203(2):398–409. pmid:15521064
- 30. Rogulska O, Vavrinova E, Vackova I, Smolkova K, Petrenko Y. Enhancing adipogenesis in Wharton’s jelly multipotent mesenchymal stromal cells through lipidomic insights and fatty acid supplementation. Sci Rep. 2025;15(1):30962. pmid:40847078
- 31. Kim GY, Choi GT, Park J, Lee J, Do JT. Comparative Analysis of Porcine Adipose- and Wharton’s Jelly-Derived Mesenchymal Stem Cells. Animals (Basel). 2023;13(18):2947. pmid:37760347
- 32. Billing AM, Ben Hamidane H, Dib SS, Cotton RJ, Bhagwat AM, Kumar P, et al. Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers. Sci Rep. 2016;6:21507. pmid:26857143
- 33. Mootha VK, Lindgren CM, Eriksson K-F, Subramanian A, Sihag S, Lehar J, et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003;34(3):267–73. pmid:12808457
- 34. Zhang H-Y, Zhang X, Wang Z-G, Shi H-X, Wu F-Z, Lin B-B, et al. Exogenous basic fibroblast growth factor inhibits ER stress-induced apoptosis and improves recovery from spinal cord injury. CNS Neurosci Ther. 2013;19(1):20–9. pmid:23082997
- 35. Bendall SC, Stewart MH, Menendez P, George D, Vijayaragavan K, Werbowetski-Ogilvie T, et al. IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature. 2007;448(7157):1015–21. pmid:17625568
- 36. Jarquín-Yáñez K, Aguilar-Sandoval DM, Piñón-Zárate G, Herrera-Enríquez MA, Avila-Campos ID, Hernandez-Tellez B, et al. Growth Factor-Primed WJ-MSC Secretome Enhances Fibroblast Expansion In Vitro. Biomedicines. 2025;13(12):2863. pmid:41462878
- 37. Yoo KH, Jang IK, Lee MW, Kim HE, Yang MS, Eom Y, et al. Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues. Cell Immunol. 2009;259(2):150–6. pmid:19608159
- 38. Shin S, Lee J, Kwon Y, Park K-S, Jeong J-H, Choi S-J, et al. Comparative Proteomic Analysis of the Mesenchymal Stem Cells Secretome from Adipose, Bone Marrow, Placenta and Wharton’s Jelly. Int J Mol Sci. 2021;22(2):845. pmid:33467726
- 39. Bakhtyar N, Jeschke MG, Herer E, Sheikholeslam M, Amini-Nik S. Exosomes from acellular Wharton’s jelly of the human umbilical cord promotes skin wound healing. Stem Cell Res Ther. 2018;9(1):193. pmid:30005703
- 40. Barrett AN, Fong C-Y, Subramanian A, Liu W, Feng Y, Choolani M, et al. Human Wharton’s Jelly Mesenchymal Stem Cells Show Unique Gene Expression Compared with Bone Marrow Mesenchymal Stem Cells Using Single-Cell RNA-Sequencing. Stem Cells Dev. 2019;28(3):196–211. pmid:30484393
- 41. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545–50. pmid:16199517
- 42. Mateos J, Pernas PF, Labora JF, Blanco F, Arufe MDC. Proteomic Applications in the Study of Human Mesenchymal Stem Cells. Proteomes. 2014;2(1):53–71. pmid:28250369
- 43. Ge Z, Qiu C, Zhou J, Yang Z, Jiang T, Yuan W, et al. Proteomic analysis of human Wharton’s jelly mesenchymal stem/stromal cells and human amniotic epithelial stem cells: a comparison of therapeutic potential. Sci Rep. 2024;14(1):28061. pmid:39543366
- 44. Paliwal S, Chaudhuri R, Agrawal A, Mohanty S. Human tissue-specific MSCs demonstrate differential mitochondria transfer abilities that may determine their regenerative abilities. Stem Cell Res Ther. 2018;9(1):298. pmid:30409230
- 45. Kupcova Skalnikova H. Proteomic techniques for characterisation of mesenchymal stem cell secretome. Biochimie. 2013;95(12):2196–211. pmid:23880644
- 46. Mirabdollahi M, Haghjooy Javanmard S, Sadeghi-Aliabadi H. In Vitro Assessment of Cytokine Expression Profile of MCF-7 Cells in Response to hWJ-MSCs Secretome. Adv Pharm Bull. 2019;9(4):649–54. pmid:31857970