Indonesian Journal of Medical Laboratory Science and Technology
Open Access

p-ISSN 2684-6748
e-ISSN 2656-9825

RESEARCH ARTICLE

Vitamin D enhances migration but decreases gene expression
of vascular endothelial growth factor and tumor necrosis
factor-α in Wharton’s jelly mesenchymal stem cells
Vitriyanna Mutiara Yuhendri
Ismawati

1, Nurfi Pratiwi

4, Nicko Pisceski Kusika Saputra

1,2, Ariza Julia Paulina
5, Arfianti Arfianti

1,3, Ismawati

6

1Master of Biomedical Science Program, Faculty of Medicine, Universitas Riau, Pekanbaru, Indonesia
2Department of Histology, Faculty of Medicine, Universitas Riau, Pekanbaru, Indonesia
3Department of Clinical Pathology, Faculty of Medicine, Universitas Riau, Pekanbaru, Indonesia
4Department of Biochemistry, Faculty of Medicine, Universitas Riau, Pekanbaru, Indonesia
5Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Riau, Pekanbaru, Indonesia
6Department of Medical Biology, Faculty of Medicine, Universitas Riau, Pekanbaru, Indonesia

Correspondence:
Arfianti
Department of Medical Biology, Faculty of
Medicine, Universitas Riau, Pekanbaru,
28133, Indonesia

Email: evi_anti@yahoo.com
Article history:
Received: 2024-11-28
Revised: 2025-02-28
Accepted: 2025-03-17
Available online: 2025-04-22

Keywords:
Interleukin-6
Mesenchymal stem cells
Tumor necrosis factor-α
Vascular endothelial growth factor
Vitamin D
https://doi.org/10.33086/ijmlst.v7i1.6698

1.

Abstract

Mesenchymal stem cells (MSCs) have gained significant interest for their
potential
application
in
repairing
damaged
tissues
through
immunomodulatory functions. Vitamin D has been shown to regulate both the
innate and adaptive immune systems. Active forms of Vitamin D, such as 1,25dihydroxyvitamin D3 (1,25(OH)2D3) are known to influence the action of
mesenchymal stem cells (MSCs) in several mechanisms, such as stimulating
their proliferation and osteogenic differentiation, as well as
tissue regeneration. This study aimed to investigate the effects of vitamin D
supplementation on the biological properties and expression of cytokines and
growth factor genes in MSCs isolated from the human umbilical cord (UC).
MSCs were isolated from Wharton’s jelly (WJ-MSC) of UC, cultured, and
supplemented with various concentrations of vitamin D3. The Cell Counting Kit8 (CCK-8) assay was used to measure cell viability, and a scratch wound healing
assay was conducted to evaluate the migration capacity of MSCs. The mRNA
expression levels of vascular endothelial growth factor (VEGF), tumor necrosis
factor (TNF)-α, and interleukin (IL)-6 were quantified using reverse
transcription-polymerase chain reaction (RT-PCR). This study showed that
supplementation with 50 nM of vitamin D3 for 48 h significantly increased the
viability and migratory capacity of MSCs. Furthermore, vitamin D
supplementation significantly decreased the mRNA levels of TNF-α and VEGF
but did not affect IL-6 gene expression compared to the control group. These
findings suggest that vitamin D supplementation can enhance the biological
characteristics and modulate the expression of key immunomodulatory factors
in MSCs, potentially improving the effectiveness of MSC-based therapies.

INTRODUCTION

Regenerative medicine has recently attracted significant interest due to its potential to repair damaged tissues and
organs. Mesenchymal stem cells (MSCs) have emerged as a promising resource for tissue repair due to their self-renewal
ability, potential for multilineage differentiation, and immunoregulatory properties (1). These multipotent progenitor cells
can be obtained from various adult and fetal tissues, including the placenta, umbilical cord, bone marrow, adipose tissue,
and dental pulp (2). Fetal-derived MSCs demonstrate a higher proliferation rate and can undergo more passages in vitro
before senescence than MSCs obtained from adult tissues. However, umbilical cord mesenchymal stem cells (UC MSCs) offer
Citation: Yuhendri VM, Pratiwi N, Paulina AJ, Ismawati I, Saputra NPK, Arfianti A. Vitamin D enhances migration but decreases gene
expression of vascular endothelial growth factor and tumor necrosis factor-α in Wharton’s jelly mesenchymal stem cells. Indones J Med
Lab Sci Technol. 2025;7(1):49–59. https://doi.org/10.33086/ijmlst.v7i1.6698
This is an open access article distributed under the Creative Commons Attribution-ShareAlike 4.0 International License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited. ©2024 The Author(s).
https://journal2.unusa.ac.id/index.php/IJMLST

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several advantages over other MSC sources, including their non-invasive tissue collection, ethical acceptability, and most
notably, their high proliferation rate, which significantly enhances their practicality for clinical application (3–5).
Mesenchymal stem cells produce a secretome of bioactive molecules, such as growth factors, chemokines, cytokines,
proteins, lipids, and miRNA (6). Increasing evidence suggests that the secretome derived from MSCs could significantly
impacts the positive effects of MSCs by influencing the cellular immune response and increasing the regenerative capacity
of resident cells within the injured tissue (7–9). In particular, the ability of MSCs to regulate the immune response has been
linked to the release of Tumour Necrosis Factor alpha (TNF-α), Interleukin (IL)-6, and Vascular Endothelial Growth Factor
(VEGF) (10,11). In addition to having previously described therapeutic properties, MSCs must migrate to damaged tissues
to be involved in regeneration. The migration of MSCs into injured tissues after transplantation is a complex and intriguing
process that depends on various mechanical and chemical factors (12).
Despite the promising evidence from preclinical studies, the therapeutic efficacy of MSCs in clinical studies remains
limited. The translation of MSCs-based therapies into clinical practices is challenging with issues such as low survival,
engraftment, limited migration capacity, and increased cellular senescence of MSCs (13). To address these challenges, it is
crucial to focus on reducing cellular senescence and increasing cellular viability, proliferation, and migration capacity.
Various strategies have been explored to achieve these goals. Furthermore, preconditioning methods like hypoxia, cytokine
priming, three-dimensional (3D) cell culture, genetic modification, and bioactive compounds have been employed to
enhance the immunomodulatory effects of MSCs (14,15).
Vitamin D, a pivotal pro-hormone, regulates a multitude of physiological processes, from bone metabolism to
immune regulation, and cellular differentiation (16). Notably, previous studies have shown that vitamin D can modulate
the immune system (17) and influence the differentiation and proliferation of adipose- and bone marrow-derived MSCs
(18). The most recent evidence suggests a compelling potential, vitamin D may significantly affect the therapeutic efficacy
of MSCs (19), a discovery that piques curiosity and invites further investigation.
Despite the promising preclinical evidence, the therapeutic efficacy of MSC in clinical applications remains limited
due to challenges such as low survival rates, limited migration capacity, and increased cellular senescence. While various
strategies, including preconditioning approaches like hypoxia and genetic modifications, have been explored, the potential
of vitamin D supplementation as a priming agent for MSCs remains under investigation. Previous studies have primarily
focused on the effects of vitamin D on MSCs derived from adipose or bone marrow tissues, leaving a significant knowledge
gap regarding its impact on umbilical cord-derived MSCs (UC-MSCs). These UC-MSCs, with their unique biological
advantages, are a fascinating area of study. This study aims to fill the gap by examining the effects of vitamin D
supplementation on the viability, migratory capacity, and gene expression of key immunomodulatory factors in UC-MSCs.
The novelty of this study lies in its focus on utilizing vitamin D as a supplementation agent to enhance the therapeutic
potential of UC-MSCs, offering new insights into the optimization of MSC-based therapies.

2.

MATERIALS AND METHODS

2.1. Isolation and Expansion of WJ-MSC
Wharton's jelly (WJ) from the umbilical cord (UC) of cesarean mothers aged 20–35 years was used as the source of
MSCs (WJ-MSC). All participants (cesarean mothers) provided informed consent before umbilical cord donation. The WJ
MSCs were isolated and cultured with the utmost care and precision using the explant method (20) as previously published
(21) with a few modifications. Briefly, the UC was transversely incised to a length of 5 cm. Wharton's jelly was separated
from the UC, both umbilical arteries and veins were removed, and the WJ was cut into small pieces. The WJ fragments were
then placed in a 100 mm diameter culture dish (Corning, New York, USA) containing a culture medium composed of
Dulbecco’s Modified Eagle’s Medium (DMEM) (Corning, New York, USA) supplemented with 5% human platelet lysate
(HPL), 3 IU heparin, 1% Glutamax, and 1% penicillin-streptomycin. The MSCs were incubated in a humidified incubator at
37°C with 5% CO2. The culture medium was changed every 2–3 days, and cells were split when they reached 80–90%
confluence using 0.25% trypsin-EDTA (Sigma-Aldrich, Darmstadt, Germany). The WJ-MSC used in these experiments were
in passages 3–5.

2.2. Cell Viability Assay
The WJ-MSC were seeded at a 5.0x104 cells/cm2 density on a 96-well culture plate. The cells were then cultured in
DMEM supplemented with vitamin D3 (cholecalciferol) (Sigma-Aldrich, Taufkirchen, Germany), at 0–100 concentrations
nM for 24 and 48 h. The viability of WJ-MSC was thoroughly examined using the Cell Counting Kit-8 assay (CCK-8)
(ABBKINE, Wuhan, China), according to the manufacturer’s protocols. Wells without cells were used as blanks. The
absorbance was measured at a wavelength of 450 nm. The percentage of cell viability was calculated using the formula 1
where ODs represent the optical density of the sample, ODb represents the optical density of the blank, and ODc represents
the optical density of the control, where the control refers to WJ-MSC cultured in media without the addition of vitamin D3.
Cell Viability (%) =

ODs−ODb
ODc−ODb

× 100% ........................................................................................................................................................................... (1)

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2.3. Migration Assay
The WJ-MSC were seeded in a six-well culture plate (5.0x104 cells/cm2) for migration assay. After overnight
incubation or cells reached 70-80% confluence, the old medium was replaced with DMEM containing vitamin D 3. The
migration capacity of WJ-MSC was assessed using the scratch wound healing assay. The scratched cell-free zone was
manually made across the cell monolayer using a sterile 1000 μL pipette tip. The cells were then washed off twice with
Dulbecco's Phosphate Buffered Saline (DPBS) (Sigma-Aldrich, Taufkirchen, Germany) to remove cellular debris.
Subsequently, a culture medium containing 50 nM of vitamin D3 was added, and cells were cultured for 24 and 48 hours.
The migration of WJ-MSC was observed and photographed using an inverted microscope (Olympus CKX53, Olympus,
Tokyo, Japan). The wound area was calculated with the reliable ImageJ software, ensuring the accuracy of the results. The
wound closure percentage was obtained using the following formula 2, where T0 was the wound area at 0 h and Tt was the
remaining area at the designated time (22).
Wound Closure Rate (%) =

(T0−Tt)
T0

× 100% .............................................................................................................................................................. (2)

2.4. Real-Time Quantitative PCR
The WJ-MSC used for Real-time quantitative PCR (RT-qPCR) experiments were seeded in six-well culture plates
(5.0x104 cells/cm2). The cells were cultured in a medium supplemented with vitamin D3 at a concentration of 50 nM and
incubated for 48 h in a CO2 incubator. Total RNA was extracted using TRNzol Universal Reagent (Tiangen Biotech, Beijing,
China), according to the manufacturer’s instructions. RNA concentration and purity were examined using a Multiskan
SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). A total of 1 microgram
RNA was transcribed into cDNA using the GoTaq® 2-Step RT-qPCR system (Promega Corporation, Madison, WI, USA). The
targeted genes were amplified using primer sequences shown in Table 1 (23–25) on Heal Force Real-Time PCR X 960 under
the following conditions (polymerase activation at 95°C, denaturation at 95°C, and annealing at 60°C). Real-time
quantitative PCR (RT qPCR) was performed using GoTaq® 2-Step RT-qPCR. The gene expression levels were compared
using the robust and reliable relative quantification (2-ΔΔCt) method, with. GAPDH serving as an internal reference to
normalize the mRNA expression levels. In addition, melting curve analysis was performed to determine the specificity of
the PCR products.
Table 1. qPCR Primer Sequences
Gene
Forward
GAPDH
5’-GACCTGCCGTCTAGAAAAAC-3’
TNF- α
5’-TGGCCAATGGCGTGGAGCTG-3’

Reverse
5’-TTGAAGTCAGAGGAGACCAC-3’
5’-GTAGGAGACGGCGATGCGGC-3’

VEGF-A
IL-6

5’-GGGCAGAATCATCAC GAAG-3’
5’-TGTCCTGCAGCCACTGGTTC-3’

5’-ATCTGCATGGTGATGTTGGA-3’
5’-AAGCCAGAGCTGTGCAGATGAGTA-3’

Reference
(23)
Designed using the PrimerBLAST
(24)
(25)

2.5. Statistical Analysis
The experimental data, expressed as the mean ± standard deviation (SD), were analyzed using the reliable GraphPad
Prism v 9. We applied the One-way ANOVA test and Tukey's multiple comparisons test to compare the cell viability between
experimental groups. The effects of vitamin D supplementation on the migration analysis were examined using two-way
ANOVA, and the effects of vitamin D supplementation on mRNA expression of IL-6, TNF-α, and VEGF were analyzed using
an unpaired t-test Statistical significance was determined if p-value<0.05, further reinforcing the accuracy of our
conclusions.

3.

RESULTS AND DISCUSSION

3.1. Effect of Vitamin D on The Viability of WJ-MSCs
The viability of WJ-MSC following supplementation with various vitamin D concentrations for 24 and 48 h was
examined using the CCK-8 Assay. After 24 h of vitamin D3 supplementation at 1 nM and 50 nM, viability increased. However,
cell viability tended to decrease at higher concentrations of 100 nM (Figure 1). Despite these changes, the statistical test
did not identify significant differences between the experimental groups (p > 0.05). After 48 hours of vitamin D 3
supplementation at 1–100 nM concentrations, a significant increase in WJ-MSC viability was observed at 50 nM compared
to the control group (p = 0.0437 and p = 0.0091, respectively) (Figure 2). Notably, prolonged incubation with 50 nM of
vitamin D3 for 48 hours resulted in a significant increase in WJ-MSC viability. These results underscore the significant

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impact of vitamin D3 on WJ-MSC viability and provide a solid foundation for further analysis of cell migration and the
expression of TNF-α, IL-6, and VEGF.
The effects of vitamin D on MSC survival vary depending on the tissue source of MSCs, species of origin, and the
concentration of vitamin D (26). Consistent with our study, adding calcitriol (vitamin D) at concentrations exceeding 50 nM
was cytotoxic to adipose-derived MSCs (27,28). Similarly, their viability decreases when dental pulp-derived MSCs were
treated with vitamin D at concentrations ranging from 1 to 100 nM (27). However, bone marrow-derived MSCs (BM-MSCs)
did not significantly impact cell viability at the same concentrations of vitamin D (28), even though their osteogenic
differentiation capacity increased. In contrast, 20 nM and 40 nM of vitamin D3, even for more extended incubation periods
of 1–5 d, significantly increased the viability of BM-MSCs (27). Therefore, our study and other studies’ results indicate that
lower concentrations of vitamin D (less than 100 nM) do not have a toxic effect on MSCs.

Figure 1. The effect of vitamin D3 supplementation for 24 h on the viability of Wharton’s jelly -derived mesenchymal stem
cells. Cell viability was measured using the cell counting kit-8 (CCK-8) assay. Data are presented as mean ± SD (n=5)

Figure 2. The effect of vitamin D3 supplementation for 48 h on the viability of Wharton’s jelly-derived mesenchymal stem
cells. Cell viability was measured using the CCK-8 assay. Data are presented as mean ± SD (n=5). *p<0.05
compared to controls using one-way ANOVA and post hoc Tukey's multiple comparison analysis.
Vitamin D may have a profound impact on the cellular viability of MSCs through several mechanisms. One proposed
pathways is sirtuin 1 (SIRT1) signaling, which has been shown to enhance MSC proliferation and maintain stemness by
upregulating pluripotency markers (26). Although Vitamin D has been reported to induce osteogenic differentiation in
adipose-derived MSCs via BMP2 signaling (29), the direct link between this pathway and MSC viability remains unclear.
This potential for Vitamin D to affect MSC survival or highlights an exciting area for further research in the field of
regenerative medicine. Further studies are needed to determine whether the observed differentiation enhances MSC
survival or simply alters their cellular function.
In addition to its stimulatory effects on cell viability, vitamin D promotes osteoblastic differentiation of MSC
isolated from dental tissues (30) and alveolar periosteum (31). Several mechanisms are involved in how vitamin D
increases the differentiation capacity of MSCs, including enhancing the expression of RUNX2 (a member of the
PEBP2/CBF transcription factors family controlling the expression of genes whose products are essential for
bone formation) and collagen I, leading to increased production of mineralized matrix nodules (30). Furthermore,

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vitamin D upregulates specific osteogenic and adipogenic genes (32), which are crucial for the differentiation process, and
stimulates MSCs adhesion by increasing Alpha-v beta-3 (αvβ3) integrin expression and focal adhesion formation, which is
crucial for cell commitment and differentiation (33).
The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) also known as calcitriol, has been found to
influence multiple cellular signaling pathways that impact cell viability. This vitamin is crucial role in balancing
autophagy and apoptosis, which is fundamental for cell survival. It enhances the expression of pro-autophagic proteins
like Beclin 1 and induces the phosphorylation of Extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, components
of survival signaling pathways (34). Consequently, this reduces of apoptosis-related gene expression and helps maintain
mitochondrial function. Moreover, the interaction between vitamin D 3-up-regulated protein-1 and thioredoxin, a critical
element in redox signaling, regulates cardiomyocyte viability under stress conditions (35). This interaction is essential for
managing oxidative stress and preventing apoptosis. Additionally, vitamin D interacts with the TGF-beta signaling pathway
via SMAD proteins, which is significant in regulating cell proliferation and differentiation (36). Furthermore, recent studies
have demonstrated that vitamin D3 can significantly attenuate H₂O₂-induced cell injury in MSCs through the Sirt1/FoxO1
signaling pathway. This protective effect is achieved by reducing intracellular oxidative stress caused by H₂O₂ exposure,
thereby enhancing the resilience of MSCs under oxidative conditions. These findings further emphasize the crucial role of
vitamin D in cellular defense mechanisms and its potential therapeutic applications in regenerative medicine (37).

3.2. Effect of Vitamin D on Migration of WJ-MSCs
The migration capacity of WJ-MSC supplemented with vitamin D was assessed using the scratch wound
healing assay. Images were captured at fixed intervals of 0, 24, and 48 h (Figure 3). A noticeable gap in the control
group remained in the scratched region at 24 and 48 h. In contrast, the scratched area began to close in WJ-MSC
administered with 50 nM vitamin D3 for 24 h (Figure 3D), and there was no visible space at 48 h (Figure 3F).
Figure 3G shows the percentage of wound closure in the WJ-MSC group. Exposure to vitamin D3 for 24 h significantly
increased the percentage of wound closure in WJ-MSCs compared with the control group (33.7% vs 55.12%,
p=0.0178). Similarly, after 48 h, a highly significant difference was observed in the percentage of closure area between WJMSC supplemented with 50 nM vitamin D3 and control WJ-MSC (88.67% vs. 49.36%, p=0.0020). These data demonstrate
that vitamin D3 supplementation increased the wound closure rate of WJ-MSC, indicating that vitamin D3 enhances
the migratory ability of WJ-MSC. To the best of our knowledge, this is the first study to investigate the effects of vitamin D
on the migration of MSCs.
Vitamin D has been demonstrated to enhance the migration rates of various cell types. For instance, calcipotriol, a
synthetic analog of vitamin D3, has been shown to increase the rate of wound closure in keratinocyte cells (38). Additionally,
vitamin D has been found to promote migration and inhibit apoptosis of vascular smooth muscle cells (VSMCs) obtained
from rats (39). Human umbilical vein endothelial cells (HUVECs) cultured in a 3-dimensional matrix and supplemented
with vitamin D exhibited increased proliferation and migration capacity, which was associated with increasing the
expression of matrix metalloproteinase-2 (MMP-2) (40). Furthermore, vitamin D3 has been shown to enhance the
promoting effects of nano hydroxyapatite composite scaffolds on the osteogenic differentiation capacity of human adiposeMSCs (41), inspiring new avenues for bone regeneration research.
The capacity to migrate and home to damaged tissues is critical to the ability of MSCs to repair damaged tissue (42).
Various factors, including chemical signals, growth factors, and environmental conditions, influence the migration of
mesenchymal stem cells (MSCs). These factors play crucial roles in guiding MSCs to sites of injury or disease, which is
crucial for their therapeutic efficacy. Chemotactic agents such as stromal cell-derived factor-1 (SDF-1), transforming
growth factor-beta (TGF-β), and substance P (SP) play pivotal roles in regulating MSC mobilization and movement,
often interacting in complex ways. Furthermore, MSCs secrete matrix metalloproteinase 1 (MMP-1), which plays a
role in facilitating MSC movement by activating protease-activated receptor 1 (PAR1). The production of
MMP-1 is amplified by inflammatory mediators such as IL-1β (43,44). In addition to these internal factors,
chemokines, growth factors, and inflammatory mediators in the surrounding environment can profoundly influence MSC
migration by triggering specific signaling pathways. To the best of our knowledge, there is no direct evidence regarding
the effects of vitamin D on the aforementioned factors associated with the migration of MSCs. Therefore, the urgency
and significance of further studies to confirm the effect of vitamin D on the migration capacity of WJ-MSC (45) cannot
be overstated.
Vitamin D has been extensively studied for its inhibitory effect on cancer cell migration. An in vitro
study demonstrated that 1 µM of vitamin D inhibited the migration of thyroid cancer cells (46) and ovarian cancer
cells (47). The latter study also found that the inhibitory effect of vitamin D on cancer cell growth was mediated
through the upregulation of the galectin-3 (GAL-3) gene expression (47). One study examined the effects of
vitamin D supplementation on human umbilical vein endothelial cells (HUVECs) subjected to high glucose levels.
Vitamin D decreased apoptosis, increased migration, and enhanced the viability of HUVECs exposed to high glucose (48).
These findings underscore Vitamin D’s complex and multifaceted role in cellular processes, highlighting its
potential significance in cancer therapeutics and MSC biological properties.

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3.3. Effect of Vitamin D Supplementation on The Gene Expression Levels of TNF-α, IL-6, and
VEGF
The gene expression levels of TNF-α, IL-6, and VEGF in WJ-MSC after vitamin D3 administration were examined
using RT qPCR for mRNA expression analysis (Figure 4). The gene expression was normalized to that of GAPDH, which was
used as the reference control. Figure 4A shows a significant decrease in TNF-α expression in WJ-MSC treated with 50 nM
vitamin D3 compared with the control group (p=0.0435). Similarly, treatment with 50 nM vitamin D 3 for 48 h reduced the
mRNA expression of VEGF in WJ-MSC compared to those without vitamin D3 supplementation (Figure 4B, p=0.0197).
However, administration of 50 nM vitamin D3 for 48 h did not affect the mRNA expression of IL-6 in WJ-MSC compared to
the control group (Figure 4C, p=0.5538). These findings have significant implications for the potential therapeutic
applications of vitamin D3 supplementation.
The immunomodulatory effects of MSCs hold significant promise for their therapeutic potential in managing
pathological conditions associated with immune responses. These conditions include autoimmune diseases, chronic
inflammation, or organ transplantation. Mesenchymal stem cells have been shown to produce a range of cytokines and
growth factors that can regulate immune responses. In the present study, WJ-MSC were found to express TNF-α. However,
preconditioning with vitamin D3 for 48 hours significantly decreased the expression of TNF-α. This finding contrasts with
a previous study using human bone marrow-derived MSCs, which did not to detect TNF-α protein expression using the
enzyme-linked immunosorbent assay (ELISA) method (49).
Tumor necrosis factor-alpha (TNF-α) is a versatile factor in modulating the therapeutic effects of MSCs by
influencing their immunoregulatory and differentiation capacities. TNF-α’s interaction with tumor necrosis factor-alpha
receptor (TNFR)1 and TNFR2, mediates pro-inflammatory and anti-inflammatory responses, showcasing its dual function
(50). This versatility is crucial in MSC-based therapies for autoimmune and inflammatory conditions, with TNFR2 being
particularly vital in enhancing the MSCs’ immunosuppressive functions, such as stimulating regulatory T cells and
suppressing effector T cells (50). Additionally, TNF-α’s impacts on MSC differentiation, such as promoting neural
differentiation and enhancing migration towards gliomas, presents exciting possibilities for neurodegenerative therapies
(51). In inflammatory environments, TNF-α’s involvement in the regulation of MSC fate and functional reprogramming
significantly influences their capacity for tissue regeneration and repair.
The relationship between vitamin D and VEGF expression in MSCs has not been directly demonstrated in previous
studies. However, it is known that the active form of vitamin D, 1,25-dihydroxyvitamin D3, can directly increase VEGF
expression in vascular smooth muscle cells by binding to response elements in the VEGF promoter (52). This suggests that
vitamin D may have complex regulatory effects on VEGF expression, depending on the cell type and specific conditions. The
observed decrease in VEGF expression in WJ-MSC following vitamin D3 preconditioning in the present study underscores
the intricate nature of this relationship and the need for further investigation into the mechanisms underlying the
interaction between vitamin D and growth factor expression in different MSCs.
VEGF promotes blood vessel formation by stimulating the growth of vascular endothelial cells in arteries, veins, and
lymphatic vessels (53). Umbilical cord-derived MSCs have been shown to express VEGF, which can stimulate angiogenesis
and increase the therapeutic effects of MSC therapy (54). According to our study, vitamin D supplementation significantly
decreased the expression of VEGF on WJ-MSC, which contradicts the results of a study that utilized hypoxic conditions in
MSCs, increasing VEGF expression (21). These results suggest that different preconditioning treatments may have distinct
effects on gene expression despite employing the exact source of MSCs. This difference may be attributed to different
sources of the MSCs used in the experiments. Mass spectrometry analysis of conditioned media of MSCs isolated from
various tissues revealed that MSCs obtained from fetal tissues, such as the placenta and umbilical cord, secreted more
diverse proteins than MSCs from adipose tissue and bone marrow (55,56). Interestingly, despite these differences, MSCs
were predicted to exert comparable biological properties, including promotion migration and inhibiting cell apoptosis
regardless of their tissue of origin. This may be due to the similar enrichment of proteins involved in the promotion of
migration and the reduction of apoptosis in MSC from all sources.
Another cytokine MSCs express is IL-6, a pro-inflammatory cytokine involved in various biological processes. IL-6
is the most abundantly produced cytokine by MSCs derived from human bone marrow (49). IL-6 secreted by MSCs can
shift the macrophage phenotypes towards M2 anti-inflammatory (57). Our study showed that vitamin D3 preconditioning
tended to decrease the expression of IL-6, although the difference was not statistically significant. While there is no study
on the effect of vitamin D on IL-6 expression in MSCs, a study using stromal vascular cells (SVCs) demonstrated that vitamin
D supplementation decreased IL-6 expression (58). Interleukin-6 (IL-6) plays a significant role in the therapeutic effects of
mesenchymal stem cells (MSCs) by modulating immune responses and promoting tissue repair. IL-6 secreted by MSCs
promotes the polarization of macrophages towards an anti-inflammatory M2 phenotype, which is crucial for reducing
inflammation and promoting tissue repair in conditions like peritoneal fibrosis and systemic lupus erythematosus (57,59).
In hypoxic-ischemic brain damage, IL-6 from MSCs activates signaling pathways that reduce the proliferation of reactive
astrocytes, aiding in functional recovery (60). Given that IL-6 plays a crucial role in the therapeutic effects of MSCs, targeting
IL-6 could be a promising approach to improve the effectiveness of MSC-based treatments for various inflammatory and

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degenerative disorders. To our knowledge, this is the first study to examine IL-6 expression in MSCs. However, further
research is urgently needed to validate and expand upon these findings, underscoring the importance of your work in
advancing the field of regenerative medicine.

Vitamin D3 50 nM

Control
A

B

0h

D

C

24 h

E

F

48 h

G

Control
50 nM

Figure 3. Scratch Wound Healing Assay on Wharton’s jelly-derived mesenchymal stem cells. (A-B) WJ-MSC
immediately after the scratch was made (T0); (C-D) after 24 h of exposure (T24); (E-F) after 48 h of exposure (T48)
(n=3; Magnification 40X). (G) closure percentage after preconditioning with 50 nM vitamin D3 on WJ-MSCs.
Data are presented as mean ± SD and analyzed using two-way ANOVA. *p < 0.05 and **p < 0.01 compared
to the control group. The control refers to WJ-MSC cultured in media without adding vitamin D3.

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A

Indones J Med Lab Sci Technol. April 2025;7(1):49-59

B

C

Figure 4. Wharton’s jelly-derived mesenchymal stem cells supplementation with vitamin D3 affects gene expression.
RT qPCR analysis was conducted on Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC) after 48 h of
supplementation with the expression of TNF-α (A) IL-6 (B) and VEGF (C) mRNA was normalized to GAPDH.
The data are presented as mean ± SD (n=6). *p<0.05 compared to controls using the t-test.
The control refers to WJ-MSC cultured in media without adding vitamin D3.
Rigorous evaluation of preconditioning effects on MSCs is essential for their successful translation into clinical
applications. This evaluation encompasses various aspects of MSC functionality, including survival, migration, proliferation,
regeneration potential, and immunoregulatory effects in response to preconditioning or priming strategies. These factors
must be thoroughly investigated to ensure the optimal performance of MSCs in therapeutic settings (61,62). The MSCs
secretome, which comprises the bioactive molecules secreted by these cells, also plays a crucial role in their regenerative
potential. The composition of the secretome is significantly influenced by the tissue of origin, the local microenvironment,
and the conditions under which the cells are cultured (6).
Given the importance of the MSCs secretome in determining therapeutic efficacy, it is imperative to explore and
develop potential preconditioning or priming strategies that can enhance the production of bioactive molecules by MSCs.
These strategies may involve manipulating culture conditions, exposing MSCs to specific stimuli, or modifying their genetic
makeup to optimize their secretory profile (63,64). By boosting the production of beneficial factors, such as growth factors,
cytokines, and extracellular vesicles, these approaches may augment the overall therapeutic efficacy of MSCs. However, in
this study, we focused on evaluating gene expression changes following vitamin D3 preconditioning and did not perform
functional assays to validate these findings further. It is important to note that future studies incorporating functional
validation will be necessary to confirm the biological significance of these molecular changes in MSC-based therapies.

4. CONCLUSIONS
In conclusion, the study's findings highlight the potential of vitamin D supplementation to significantly enhance the
functionality and therapeutic efficacy of on WJ-MSC. The observed increase in cell viability and migration capacity at a 50
nM concentration of vitamin D suggests potential enhancements in these cells’ overall functionality and therapeutic efficacy.
Moreover, the modulation of TNF-α and VEGF expression indicates that vitamin D may play a crucial role in regulating the
immunomodulatory properties of WJ-MSC, which are essential for their therapeutic applications in various diseases and
conditions.
These results provide compelling evidence for using vitamin D3 as a supplementation strategy to enhance the
therapeutic capacity of MSCs. However, it is important to note that while these findings are promising, they are primarily
based on in vitro studies. Further research is necessary to fully elucidate the mechanisms underlying vitamin D's effects on
MSCs and to determine whether these benefits translate to improved clinical outcomes when vitamin D-supplemented
MSCs are administered to patients. This research is crucial to assess the long-term safety and efficacy of this approach and
optimize the dosage and timing of vitamin D supplementation for maximum therapeutic benefit.
Author contributions: AA: Conceptualization, methodology, validation, data curation, supervision. AA, VMY: Software,
writing—review and editing. AA, NP, AJP: Formal analysis. II: Investigation. NPKS: Resources. AA, VMY, NP, AJP, II, NPKS:
Writing—original draft preparation. All authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by the Ministry of Education, Culture, Research, and Technology, Directorate of
Research,
Technology,
and
Community
2024
(Contract
No.
20588/UN19.5.1.3/AL.04/2024
and
20713/UN19.5.1.3/AL.04/2024.
Acknowledgements: We would like to thank the medical staff of the Department of Obstetrics and Gynecology, Hermina
Hospital for their valuable contributions in obtaining umbilical cord samples.

56

Vitriyanna Mutiara Yuhendri, et al.

Indones J Med Lab Sci Technol. April 2025;7(1):49-59

Ethics statement: This research was approved by the Research Etichs Committee of Faculty of Medicen Universitas Riau
with No: B/ 047/ UN19.5.1.1.8/UEPKK/2021_Adendum2.
Conflict of interest: The authors have no conflict of interest to disclose.

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