Efficient Treatment of Psoriasis Using Conditioned Media from Mesenchymal Stem Cell Spheroids Cultured to Produce Transforming Growth Factor- β 1-Enriched Small-Sized Extracellular Vesicles

Abstract

Psoriasis is a common chronic inflammatory disease in which keratinocytes proliferate abnormally due to excessive immune action. Psoriasis can be associated with various comorbidities and has a significant impact on health-related quality of life. Although many systemic treatments, including biologic agents, have been developed, topical treatment remains the main option for psoriasis management. Consequently, there is an urgent need to develop topical treatments with minimal side effects and high efficacy. Mesenchymal stem cells (MSCs) exhibit excellent immune regulation, anti-inflammatory activities, and therapeutic effects, and MSC-derived extracellular vesicles (EVs) can serve as crucial mediators of functional transfer from MSCs. Therefore, this study aimed to develop a safe and easy-to-use emulsion cream for treating psoriasis using MSC conditioned media (CM) containing EVs. We developed an enhanced Wharton's jelly MSC (WJ-MSC) culture method through a three-dimensional (3D) culture containing exogenous transforming growth factor-β3. Using the 3D culture system, we obtained CM from WJ-MSCs, which yielded a higher EV production compared to that of conventional WJ-MSC culture methods, and investigated the effect of EV-enriched 3D-WJ-MSC-CM cream on psoriasis-related inflammation. Administration of the EV-enriched 3D-WJ-MSC-CM cream significantly reduced erythema, thickness, and scaling of skin lesions, alleviated imiquimod-induced psoriasiform lesions in mice, and ameliorated histopathological changes in mouse skin. The upregulated mRNA expression of inflammatory cytokines, including IL-17a, IL-22, IL-23, and IL-36, decreased in the lesions. In conclusion, we present here a new topical treatment for psoriasis using an MSC EV-enriched cream.

Keywords: Emulsions; Extracellular vesicles; Immunomodulation; Mesenchymal stem cells; Psoriasis.

Fig. 1

Schematic diagram of the topical treatment. (A) Differences in the 2D and 3D culture methods, as well as the advantages of MSCs and 3D MSCs. The arrow represents the shaking motion applied to the culture medium within the culture flask. (B) Schedule of the cream processing and experimental procedures. IMQ was topically applied from days 0∼6, and MSC conditioned media cream applied from days 8∼10. The experiment was terminated on day 12. 2D: two-dimensional, WJ: Wharton’s jelly, CM: conditioned media, 3D: three-dimensional, MSCs: mesenchymal stem cells, IMQ: imiquimod.

Fig. 2

Characterization of WJ-MSCs and their CM. (A) Comparative analysis of nanoparticles in 2D-WJ-CM and 3D-WJ-CM. The distribution of nanoparticles by size, total concentration per milliliter, peak size (nm), and median size (nm) were measured using ZetaView (TWIN PMX-220; Particle Metrix). (B) Comparison of protein volume and their EV purity (particle number/protein concentration) in 2D-WJ-CM and 3D-WJ-CM. (C) Total expression of TGF-β1 per 1×10⁹ particles in EVs. TGF-β1 levels were significantly higher in 3D-WJ-CM compared to those in 2D-WJ-CM. (D) Flow cytometry analysis of MSC-positive surface marker CD90/105 and negative surface marker CD45 in 2D-MSCs and 3D-MSCs. 2D: two-dimensional, WJ: Wharton’s jelly, CM: conditioned media, 3D: three-dimensional, MSC: mesenchymal stem cell, EV: extracellular vesicle, TGF-β1: transforming growth factor-β1. Results with a p-value<0.05 were considered statistically significant. **p<0.01, ***p<0.001, ****p<0.0001.

Fig. 3

Emulsion cream selection. (A) Formulation shape according to oil and emulsifier ratios in 2D-WJ-CM and 3D-WJ-CM. (B) The degree of phase separation according to oil and emulsifier ratios in 2D-WJ-CM and 3D-WJ-CM. The left-angled red line indicates the phase-separated position. (C) Proportion of maintaining the emulsion form. 2D: two-dimensional, WJ: Wharton’s jelly, CM: conditioned media, 3D: three-dimensional.

Fig. 4

Phenotype of 3D-WJ-CM cream-treated psoriasis-like mice. Mice were randomly divided into five groups: normal, IMQ＋vehicle, IMQ＋MTX, IMQ＋2D-WJ-CM, and IMQ＋3D-WJ-CM groups. (A) Two representative photographs of mice phenotypes before sacrifice. (B) In the back skin of BALB/c mice, the thickness, erythema, and scaling were scored from 0∼4 points from the start of treatment to the date of sacrifice. In addition, the cumulative score combining thickness, erythema, and scaling was presented. Symbols represent the mean standard deviation of six mice. IMQ: imiquimod, MTX: methotrexate, 2D: two-dimensional, WJ: Wharton’s jelly, CM: conditioned media, 3D: three-dimensional. Two-way ANOVA showed significant differences between groups, ^###p<0.001, ^####p<0.0001 compared to Vehicle and 3D-WJ-CM. *p<0.05, **p<0.01, ***p<0.001 compared to 2D-WJ-CM and 3D-WJ-CM.

Fig. 5

3D-WJ-CM cream recovery in psoriasis-like mice. (A) H&E staining of mice skin by group. Scale bar=50 μm. (B) Levels of inflammatory cytokines (TLR7, IL-17α, IL-23α, IL-22, and IL-36) in the back skin of psoriasis-like mice treated with 3D-WJ-CM. IMQ: imiquimod, MTX: methotrexate, 2D: two-dimensional, WJ: Wharton’s jelly, CM: conditioned media, 3D: three-dimensional. Results with a p-value<0.05 were considered statistically significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and ns=not significant compared to the IMQ＋vehicle group.

Fig. 6

3D-WJ-CM attenuates inflammation in vitro. (A) The concentration of nitric oxide in the supernatant of LPS-stimulated RAW 264.7 cells. (B) Protein expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α). Protein expression was detected in the cell supernatant by ELISA. LPS: lipopolysaccharide, MTX: methotrexate, 2D: two-dimensional, WJ: Wharton’s jelly, CM: conditioned media, 3D: three-dimensional. ***p<0.001, ****p<0.0001 compared to the LPS group.