Culture and characterization of hUC-MSCs

The hUC-MSCs were cultured and expanded in MSCCult I medium (Regenmedlab, Ho Chi Minh City, Vietnam) at 37 °C in a humidified atmosphere containing 5% CO. Cells at passage 7 (P7) were used for all experiments.

Prior to in vivo administration, hUC-MSCs were characterized in accordance with the minimal criteria established by the International Society for Cell & Gene Therapy (ISCT).

For injection, cells were prepared as a single-cell suspension. Briefly, adherent cells were detached using Deattachment solution (Regenmedlab, Ho Chi Minh City, Vietnam), followed by centrifugation at 500 × g for 5 min. The cell pellet was resuspended in phosphate-buffered saline (PBS). Cell viability was assessed using the trypan blue exclusion assay and was consistently greater than 90% at the time of injection. Cells were adjusted to a final concentration of 2.5 × 10⁶ cells in 200 µL PBS per dose.

Differentiation assays

hUC-MSCs were detached by incubation at 37C for 2 minutes using Deattachment solution (Regenmedlab, Ho Chi Minh City, Vietnam). The cells were then centrifuged at 500 x g for 5 min, and the resulting pellets were resuspended in MSCCult I medium. Subsequently, the cells were seeded into 96-well plate at a density of 1x10 cells per well in 200 µL of medium. After 24 hours, the culture medium was replaced with lineage-specific differentiation media.

For osteogenic differentiation, hUC-MSCs were cultured in StemPro Osteogenesis Differentiation Kit (Thermo Scientific, Waltham, MA, USA) for 21 days. Chondrogenic differentiation was induced using StemPro Chondrogenesis Differentiation Kit (Thermo Scientific, Waltham, MA, USA) for 28 days. Adipose differentiation was performed by culturing hUC-MSCs in Adipogenesis Diffmed (Regenmedlab, Ho Chi Minh city, Vietnam) for 14 days. The differentiation media were refreshed every seven days.

At the end of the differentiation periods, cells were fixed in 4% paraformaldehyde (Sigma Aldrich, USA) and stained with Alizarin Red S (Sigma-Aldrich, St. Louis, MO, USA) to detect calcium accumulation, Alcian Blue (Sigma-Aldrich, St. Louis, MO, USA) to assess proteoglycan accumulation, and Oil Red O dye (Sigma-Aldrich, St. Louis, MO, USA) to detect the presence of lipid droplets.

Immunophenotyping of hUC-MSCs

For immunophenotypic analysis, human umbilical cord–derived mesenchymal stem cells (hUC-MSCs) at passage 7 (P7) were harvested and resuspended in phosphate-buffered saline (PBS). Cells were incubated with fluorochrome-conjugated monoclonal antibodies against CD44 (clone DB105, #130-113-342), CD73 (clone AD2, #130-120-066), CD90 (clone REA897, #130-114-860), CD105 (clone REA794, #130-112-170), CD14 (clone TÜK4, #130-113-146), CD34 (clone AC136, #130-113-178), and HLA-DR (clone AC122, #130-113-401) (all from Miltenyi Biotec, Germany) at a dilution of 1:100 for 15–20 minutes at room temperature in the dark.

Following incubation, cells were washed with PBS to remove unbound antibodies and analyzed using a flow cytometer (BD FACSCalibur™, BD Biosciences, USA) with BD CellQuest™ Pro software.

During analysis, cell populations were initially gated based on forward scatter (FSC) and side scatter (SSC) to exclude debris. The main cell population was then selected for further analysis. At least 10,000 events were acquired per sample. Unstained controls were used to define background fluorescence.

Cell viability assessment

Cell viability was determined immediately prior to administration using trypan blue exclusion assay. Only cell suspensions with viability greater than 90% were used for in vivo injection to ensure consistency and reliability of therapeutic outcomes.

Psoriasis Mouse Model and Cell Treatment

BALB/c male mice (8–12 weeks old) were housed under standard conditions with a 12 hour light/dark cycle and ad libitum access to food and water. All animal procedures were approved by the Animal Ethics Committee of Stem Cells Institute, University of Science, VNU-HCM (No. 230107/SCI-AEC) and conducted in accordance with relevant institutional guidelines.

Mice were randomly assigned to five groups (n=6 per group): (i) normal group (Normal); (ii) IMQ-induced group (IMQ); (iii) PBS-treated IMQ group (PBS); 28 hUC-MSC-treated IMQ group (hUC-MSCs); and (v) corticosteroid-treated IMQ group 29.

One day prior to treatement (day -1), dorsal hair was shaved. Day 0 was defined as the first day of IMQ application, representing the initiation of psoriasis-like inflammation.

For psoriasis induction, mice in the IMQ groups received a daily topical application of 5% Imiquad cream (, Maharashtra, India; 63 mg of 5% cream per mouse, equivalent to 3.15 mg IMQ) on the dorsal skin for consecutive 6 days (day 0 to day 5). The cream was evenly applied using a sterile spatula and allowed to absorb for approximately 10–15 min before the animals were returned to their cages.

On day 2 (D2), mice in the PBS group received a subcutaneous injection of 200 µL PBS, while mice in the hUC-MSCs treatment group received a subcutaneous injection of hUC-MSCs (2.5 × 10⁶ cells suspended in 200 µL PBS). The injection was administered locally into the dorsal skin at the site of IMQ application. Mice in the Cort group were treated with topical corticosteroid (Dermavate 0.05%, Glaxo Operations UK Limited, London, UK; 20 mg) from day 2 to day 5.

Euthanasia and tissue collection

Mice were euthanized on days 6 and 12. Euthanasia was performed by carbon dioxide (CO₂) inhalation using a gradual fill method. Briefly, animals were placed in a euthanasia chamber and exposed to CO₂ with a controlled displacement rate (approximately 20–30% of chamber volume per minute) until loss of consciousness, followed by continued exposure for at least 1–2 minutes after respiratory arrest to ensure death. Death was confirmed by cessation of respiration and lack of response to physical stimuli prior to tissue harvesting. Dorsal skin samples were then collected for subsequent analyses.

Experimental design and sample allocation

A total of 6 mice per group were included in this study and followed longitudinally. Non-terminal parameters, including clinical scoring and skin thickness measurements, were assessed in all animals (n = 6 per group) from day 0 to day 6.

At day 6, a subset of mice (n = 3 per group) was randomly selected and euthanized for tissue collection, including histological, gene expression, and immunofluorescence analyses. The remaining mice (n = 3 per group) were further monitored from day 7 to day 12 and subsequently euthanized on day 12 for endpoint analyses.

Thus, terminal assays at each time point (day 6 and day 12) were performed using independent biological samples (n = 3), whereas longitudinal measurements prior to day 6 were conducted with n = 6 per group. No animals were reused across terminal endpoints.

Psoriasis Area and Severity Index (PASI) scoring

Disease severity was evaluated daily using a modified PASI scoring system assessing erythema, scaling, and skin thickness. Erythema and scaling were each scored on a scale from 0 to 4 as follows: 0, none; 1, slight; 2, moderate; 3, marked, and 4, very marked.

Skin thickness was scored on the percentage increase relative to baseline (day 0) as follows: 0, no increase; 1, 15 - 20%; 2, 20 – 40%; 3, 40 – 60%; 4, >60%.

The cumulative PASI score was calculated as the sum of erythema, scaling, and thickness score, with a maximum score of 12.

Immunohistochemistry

Skin samples were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), embedded in paraffin, and sectioned at a thickness 4 μm. Tissue sections were deparaffinized in xylene (5 minutes, twice) and rehydrated through a graded ethanol series (100%, 95%, and 80%) before rinsing in distilled water

The sample sections were incubated in 10% goat serum for 30 minutes at room temperature to block nonspecific binding sites. After removing excess serum, the sections were incubated with diluted primary antibodies, including IL-17 (1:200, Cat#PA5-106856, Thermo Scientific, Waltham, MA, USA) and IL-23p19 (1:200, Cat#PA5-20239, Thermo Scientific, Waltham, MA, USA) for 30 minutes at room temperature. Subsequently, the slides were gently washed with phosphate-buffered saline (PBS) and treated with Goat Anti-Rabbit (IgG) secondary antibody FITC (1:200, Cat#ab6717, Abcam Limited, Cambridge, UK) for 30 minutes at room temperature, followed by another PBS wash. Finally, the sections were mounted with Invitrogen™ Fluoromount-G™ Mounting Medium, with DAPI (1:20, Cat#00-4959-52, Thermo Scientific, Waltham, MA, USA) and observed using an Observer A1 microscope (Zeiss, Germany). Image analysis was performed using ImageJ software.

Immunofluorescence image quantification

Immunofluorescence images were acquired under identical exposure settings using a fluorescence microscope (Observer A1, Zeiss, Germany) to ensure comparability across experimental groups. For each sample, 3–5 non-overlapping fields were randomly selected from comparable regions of the tissue section.

Quantitative analysis was performed using ImageJ software (NIH, USA). Images were converted to grayscale, and a consistent threshold was applied across all groups to define positive staining. Mean fluorescence intensity (MFI) and/or the percentage of positively stained area were quantified within regions of interest (ROIs) of equal size. Background fluorescence was subtracted using adjacent unstained regions.

RT-qPCR assay

Total RNA was isolated from mouse skin samples using the easy-BLUE™ Total RNA Extraction Kit (iNtRON Biotechnology, MA, USA) according to the manufacturer’s instructions. RNA quantity and purity were assessed spectrophotometrically.

Gene expression analysis was quantified via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the Luna® Universal SYBR Green One-Step RT-qPCR Kit (New England Biolabs, USA). Thermocycling conditions were followed as per the manufacturer’s protocol. Cycle threshold (Ct) values were calculated and normalized to the housekeeping gene GAPDH. The primers used for gene expression analysis are listed in Table 1. Primer specificity was confirmed by melt curve analysis.

Cycle threshold (Ct) values were obtained and normalized to the housekeeping gene GAPDH to generate ΔCt values. Relative gene expression levels were calculated using the 2^−ΔΔCt method, with the normal (untreated) group serving as the calibrator.

For statistical analysis, ΔCt values were used to ensure appropriate handling of approximately normally distributed data, whereas relative expression values (2^−ΔΔCt) are presented for visualization.

Blinding procedure

Outcome assessments were conducted by an investigator independent of the experimental procedures to minimize potential bias. However, formal blinding through coded sample allocation was not implemented, which is acknowledged as a limitation of the study.

Statistical analysis

Data are presented as biological replicates (n = 3–6 mice per group, depending on the experimental endpoint). The entire experiment was independently repeated at least three times using separate cohorts of animals under identical conditions to ensure reproducibility.

Statistical analysis was conducted by GraphPad Prism software version 9.5.1. Data are presented as means ± standard error of the mean (SEM).

Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test. A p-value < 0.05 was considered statistically significant.

Culture and characterization of hUC-MSCs.

hUC-MSCs adhered to the flask surface, exhibiting an elongated spindle shape (Figure 1A). In the chondrogenic medium, hUC-MSCs formed clusters and displayed blue staining with Alcian Blue, demonstrating chondrogenic potential after 28 days (Figure 1B). After 21 days of culture in the osteogenic differentiation medium, Alizarin Red staining revealed calcium accumulation, indicating osteogenesis (Figure 1C). Additionally, adipogenic differentiation was confirmed by the presence of lipid droplets stained red with Oil Red O dye after 14 days (Figure 1D).

Flow cytometry analysis showed that hUC-MSCs were positive for CD44 (99.28%), CD73 (99.50%), CD90 (99.20%), and CD105 (99.91%), while negative for CD14 (0.11%), CD34 (0.16%), and HLA-DR (0.83%) (Figure 1E).

hUC-MSCs alleviated symptoms in the IMQ-induced psoriasis mice model

In the normal group (Figure 2A1–A6) and on day 0 in all treatment groups (Figure 2B1–E6), no signs of erythema, irritation, or inflammation were observed, suggesting that the procedures did not induce adverse skin reactions.

In contrast, mice in the IMQ group exhibited a progressive development of psoriasis-like symptoms. Erythema appeared as early as day 1, accompanied by increased skin thickness and mild inflammation. By day 3, scaling became evident, and a dark red rash covered the dorsal skin, along with silvery scales and peeling. Clinical severity peaked on day 6, with widespread thickening, scaling, and pronounced erythema (Figure 2B4, C4, D4, E4). On days 5 and 6, the back skin was covered with thick silver-white scales, and skin thickness markedly increased. The Psoriasis Area and Severity Index (PASI) reached its maximum score of 11–12 during this period (Figure 3D). The total PASI score in the IMQ group remained significantly elevated compared to the normal group from day 1 to day 12 (p < 0.05).

hUC-MSCs Treatment Alleviates Clinical Severity in the IMQ-Induced Psoriasis Model

In the IMQ and PBS groups, all clinical parameters progressively worsened following IMQ application. Skin thickness increased markedly from day 1 and peaked around day 5-6, followed by a decline after cessation of IMQ treatment (after day 6) (Figure 3A). Similarly, erythema and scaling score increase overtime, with maximal severity observed between days 5 and 6 (Figure 3B–C). The cumulative PASI score reached 11–12 points in both IMQ and PBS groups, indicating severe disease activity (Figure 3D). After IMQ withdrawal, clinical symptoms gradually subsided; however, residual erythema and scaling were still evident in the PBS group at day 12, suggesting incomplete resolution.

In contrast, mice treated with hUC-MSCs exhibited attenuated disease progression. Reductions in skin thickness and erythema were observed from approximately day 5–6 onward, with a more rapid decline compared to PBS group (p<0.05) (Figure 3A-B). The scaling score was also significantly reduced beginning on day 5 (p < 0.05) (Figure 3C). The cumulative PASI score in the hUC-MSCs group remained consistently lower than in the IMQ and PBS groups, with a peak of 7–8, corresponding to an approximate 33% reduction compared to PBS controls (p < 0.05) (Figure 3D). By day 12, clinical scores in the hUC-MSCs group approached baseline levels.

Treatment with Cort produced comparable improvements. Clinical parameters in Cort group followed a similar trajectory to the observed in the hUC-MSCs group, with accelerated resolution during the recovery phase (day 7–12). No statistically significant differences were observed between the hUC-MSCs and Cort groups across all clinical parameters throughout the study period (p > 0.05).

Collectively, these results demonstrate that hUC-MSCs administration attenuates psoriasis-like skin inflammation in the IMQ-induced mouse model, as evidenced by reduced peak disease severity and accelerated resolution following IMQ withdrawal.

Histological Restoration of Psoriatic Skin by hUC-MSCs Treatment

Histological evaluation of dorsal skin tissues on days 6 and 12 revealed marked structural differences among experimental groups (Figure 4A–K).

In the normal control group, skin architecture remained intact at both time points (Figure 4A, G), characterized by a thin epidermis (19.57 ± 3.35 µm) composed of 2–3 layers of keratinocytes layer, a well-organized dermis with densely collagen fibers, and preserved skin appendages, including hair follicles and sebaceous glands (green arrows). No pathological alteration was observed.

In contrast, the IMQ group exhibited characteristic psoriasis features on day 6 (Figure 4B), including pronounced epidermal hyperplasia (116.48 ± 6.57 µm), elongation of rete ridges, loss of the granular layer, and marked inflammatory cell infiltration (Figure 4C). Adition hallmarks, parakeratosis, Munro microabscesses, and vascular dilation were also observed (Figure 4C). Although partial resolution was evident by 12 (Figure 4H), epidermal thickness and dermal inflammation remained elevated compared to the normal group.

The PBS group displayed histologically feature comparable to those of the IMQ group, with severe epidermal thickness on day 6 (120.05 ± 12.45 µm) and only partial improvement by day 12 (Figure 4D, I), indicating the absence of a therapeutic effect.

In the hUC-MSCs group, a marked attenuation of psoriasis feature was observed. On day 6, epidermal thickness was significantly reduced to 39.40 ± 3.31 µm compared to the PBS group (p < 0.001 vs.), accompanied by decreased parakeratosis, reduced inflammation infiltration, and diminished vascular dilation (Figure 4E). By day 12 (Figure 4J), the skin architecture was largely restored, with minimal residual abnormalities and morphology approaching that of the normal group.

Similarly, the Cort group also demonstrated substantial histological improvement, with reduced epidermal thickness (49.58 ± 4.89 µm) on day 6 and near-complete restoration of skin structure by day 12 (Figure 4F, K). The extent of recovery was comparable to that observed in the hUC-MSCs group

Quantitative analysis of epidermal thickness (Figure 4L) confirmed significant differences among groups. Both hUC-MSCs and Cort groups resulted in significantly lower epidermal thickness compared to PBS and IMQ groups (p < 0.01), with no significant difference observed between hUC-MSC and Cord Group.

Collectively, these findings indicate that hUC-MSCs treatment effectively attenuate IMQ-induced histopathological alterations, as reflected by reduced epidermal hyperplasia and inflammation features.

hUC-MSCs Suppress IL-17A and IL-23 Expression in Psoriatic Skin

To evaluate the anti-inflammatory effects of hUC-MSCs, IL-17A and IL-23 expression in skin lesions was assessed by immunofluorescence staining and semi-quantitative image analysis on 6 and day 12 (Figure 5A–C).

IL-17A and IL-23 signals (green) were predominantly localized in the epidermis and upper dermis, while nuclei were counterstained with DAPI (blue). In the normal group, both cytokines were absent or minimally detected at both time points, indicating basal expression levels.

In contrast, the IMQ group exhibited strong IL-17A and IL-23 signals on day 6, particularly within the epidermis and dermal inflammatory infiltrates, consistent with active psoriatic inflammation. Similar expression patterns were observed in the PBS group, indicating a lack of therapeutic effect. By day 12, cytokine expression decreased compared to day 6 but remained elevated relative to the normal group, suggesting incomplete resolution.

In the hUC-MSC-treated group, a marked reduction in IL-17A and IL-23 expression was observed on day 6, with fewer positive cells detected in both epidermal and dermal regions. By day 12, cytokine signals were further reduced and approached those observed in the normal group. A similar trend was observed in the Cort group, with reduced cytokine expression at day 6 and further attenuation by day 12.

Semi-quantitative analysis of fluorescence intensity (Figure 5B, C) supported these observations. On day 6, IL-17A and IL-23 levels were significantly increased in the IMQ and PBS groups compared to the normal group, whereas hUC-MSC treatment significantly reduced cytokine expression (p < 0.05). By day 12, fluorescence intensities declined across all groups, and no significant differences were observed between the hUC-MSC, Cort, and normal groups (p > 0.05).

Together, these results indicate that hUC-MSC treatment is associated with reduced expression of IL-17A and IL-23 in psoriatic skin, consistent with attenuation of inflammatory responses.

hUC-MSCs Attenuate the Expression of Psoriasis-Associated Inflammatory Genes

On day 6, IMQ-treated mice exhibited significant upregulation of proinflammatory genes compared to the normal group (p < 0.05), confirming successful induction of psoriasis-like inflammation. The relative expression levels were markedly increased for Il17a (130.39 ± 21.67), Il23 (8.16 ± 2.75), Ifng (4.26 ± 0.82), Tnf (6.15 ± 1.15), and Ccl20 (3.68 ± 1.17) (Figure 6).

In the PBS-treated group, expression levels remained elevated and were not significantly different from those in the IMQ group (p > 0.05), indicating a lack of therapeutic effect. Notably, Tnf expression was further increased in the PBS group (17.30 ± 2.78, p < 0.01 vs IMQ), accompanied by a trend toward increased Ccl20 expression, which may reflect local responses to injection or tissue manipulation.

In contrast, hUC-MSC treatment resulted in a marked reduction in inflammatory gene expression. The most pronounced effects were observed for Tnf (0.9 ± 0.3, p < 0.0001 vs PBS) and Ccl20 (1.0 ± 0.2, p < 0.05 vs PBS). Reductions in Il17a (39.28 ± 17.56), Il23 (3.53 ± 0.23), and Ifng (2.16 ± 0.20) were also observed; however, these changes were less pronounced and did not consistently reach statistical significance, suggesting a moderate effect on upstream cytokine expression within the IL-23/IL-17 axis.

A similar suppression pattern was observed in the Cort-treated group, with no statistically significant differences between hUC-MSC and Cort treatments (p > 0.05).

By day 12, expression levels of all genes decreased across groups and approached baseline, with no significant differences detected between treated and untreated groups (p > 0.05), consistent with the self-limiting nature of IMQ-induced inflammation.

Overall, these results indicate that hUC-MSC treatment is associated with reduced expression of key inflammatory mediators in psoriatic skin, particularly Tnf and Ccl20.