Mesenchymal Stem Cell Therapy in Alopecia Areata: Visual and Molecular Evidence from a Mouse Model

Abstract

Recent studies have highlighted the potential of Mesenchymal Stem Cells (MSCs) as an alternative treatment for Alopecia Areata (AA) due to their immunosuppressive properties. While MSCs have shown promise in cell experiments, their effectiveness in vivo remains uncertain. This study aims to validate local administration of MSC therapy's efficacy in AA treatment through animal experiments. AA was induced through Interferon-gamma (IFN-γ) administration in mice, and MSC treatment (MSCT)'s effects were assessed visually and through tissue analysis. The MSC-treated group showed more hair regrowth compared to the control (CTL) group. MSCT notably reduced local inflammatory cytokines (JAK1, JAK2, STAT1, STAT3, IFN-γR, IL-1β, IL-16, IL-17α, and IL-18) in AA-induced mice's skin, but systemic cytokine levels remained unchanged. Furthermore, MSC treatment normalized the expression of Wnt/β-catenin signaling pathway genes (LEF1 and β-catenin) and growth factors (FGF7 and FGF2), which are crucial for hair cycle regulation. This study lays the groundwork for further exploring MSCs as a potential treatment for AA, but more research is needed to fully understand their therapeutic potential.

Keywords: alopecia areata; immunosuppressive; in vivo; local; mesenchymal stem cells; mouse model; systemic; treatment.

Figure 1

Gross images of the dorsal skin of 18 representative Alopecia Areata (AA)-induced mice at week 4, 6, 8, and 10. Mice C5, C6, C13, and C14 belong to the control (CTL) group with partial AA (areas with more than 25% hair coverage), and mice C8-C12 belong to the CTL group with diffuse AA (areas with less than 25% hair coverage). Mice M13–M16 belong to the Mesenchymal Stem Cell (MSC) group with partial AA, and mice M17-M21 belong to the MSC group with diffuse AA. At week 4, extensive AA patches were observed in both groups. In the MSC group, diluted cultured Human Bone Marrow-Derived Mesenchymal Stem Cells (hMSCs) were intradermally injected into eight dorsal sites at week 4 and 5, while saline was injected in the CTL group during the same period. By week 6, hair regrowth was evident in most mice from both groups. However, hair loss recurred during the subsequent hair cycle, with prominent hair loss observed in most mice from both groups at week 8 and continuing through week 10. In fact, overall, the MSC group showed less hair loss compared to the CTL group.

Figure 2

Image showing the part with hair (black) and the part without hair (white). For each mouse at week 4 and 10, the proportion of areas with hair was calculated using pixels and the resulting values were noted. C: CTL group, M: MSC group.

Figure 3

Comparative analysis of changes in the hair-bearing area ratio between the CTL and MSC groups. All mice from the CTL and MSC group were included, with a total of 14 mice in the CTL group and 21 mice in the MSC group. For the diffuse subtype, 8 mice were in the CTL group, and 10 were in the MSC group, while for the partial subtype, 6 mice were in the CTL group, and 11 were in the MSC group. The degree of improvement in the proportion of hair-covered areas between week 4 and week 10 was calculated for each mouse. The MSC group exhibited significantly greater hair regrowth compared to the CTL group. Additionally, when comparing the diffuse AA subtypes between the two groups, the MSC group showed superior results, as did the MSC group with partial AA subtypes. The data represent the means ± SEM; n = 3; results were statistically significant at * p < 0.05 and ** p < 0.01.

Figure 4

Compared relative fold changes in mRNA expression of pro-inflammatory/anti-inflammatory cytokines in mice dorsal skin. The mRNA expression levels of (A) JAK1, (B) JAK2, (C) STAT1, (D) STAT3, (E) IL-18, (F) IL-17α, (G) IL-22, (H) IL-10, (I) IFN-γR, (J) IL-1β, (K) IL-15, and (L) IL-6 in the Healthy Control (HC), CTL, and MSC groups were measured and analyzed. JAK1, JAK2, STAT1, STAT3, IFN-γR, IL-1β, IL-10, and IL-17α were significantly increased in the CTL AA group compared to the HC group. JAK1, JAK2, STAT1, STAT3, IFN-γR, IL-1β, IL-10, IL-18, IL-17α, IL-15, and IL-6 were significantly decreased in the MSC AA group compared to the CTL AA group. The data represent the means ± SEM; n = 3. Results are statistically significant at # p < 0.05 and ## p < 0.01 compared to the HC group and * p < 0.05 and ** p < 0.01 compared to the CTL group.

Figure 5

Compared relative fold changes in mRNA expression of genes within the Wnt/β-catenin pathway (a major signaling pathway in the hair cycle) in mice dorsal skin. The mRNA expression levels of (A) LEF1, (B) β-catenin, (C) DKK1, (D) FGF7, and (E) FGF2 in HC, CTL, and MSC groups were measured and analyzed. β-catenin, FGF7, and FGF2 were significantly increased in the saline-treated CTL AA group compared to the HC group. LEF1, β-catenin, FGF7, and FGF2 were significantly decreased in the MSC-treated AA group compared to the saline-treated CTL AA group. (A–E). The data represent the means ± SEM; n = 3; results are statistically significant at # p < 0.05 and ## p < 0.01 compared to the HC group, and * p < 0.05 and ** p < 0.01 compared to the CTL group.

Figure 6

Histological analysis of mice dorsal skin tissue. Immunofluorescence staining was performed on tissues on Day 70 to compare immune cell infiltration between groups. The images presented include: (A) CD8, (B) CD4, (C) nucleic, and (D) merged of the HC group; (E) CD8, (F) CD4, (G) nucleic, and (H) merged of the CTL group; and (I) CD8, (J) CD4, (K) nucleic, and (L) merged of the MSC group. CD8 expression is shown in green, CD4 in red, and nucleic in blue. Compared to the HC group, the infiltration of CD4+CD8+ T cells in the hair follicles and surrounding subcutaneous tissue is increased in the CTL group. This increased infiltration is reverted in the MSC group. However, MSC treatment (MSCT) confirmed a decrease in CD4+CD8+ T cells in the dermis and subcutaneous fat layer compared to CTL group. Scale bar = 100 μm.

Figure 7

Comparison of expression of inflammatory cytokines in serum. We analyzed serum using the BEADS array kit to measure inflammatory cytokines in blood drawn on Day 70. The expression levels of (A) IL-12p70, (B) TNF, (C) IFN-γ, (D) MCP-1, (E) IL-10, and (F) IL-6 in serum. There were no significant differences between groups. The data represent the means ± SEM; n = 3.

Figure 8

Induction of AA in C3/HeJ mouse via Interferon-gamma (IFN-γ) injections. Daily intraperitoneal administration of IFN-γ on Day 1–5 and intradermal administration of IFN-γ on Day 8, 10, 12, 15, 17, and 19 (3 times a week for 2 weeks) were conducted to develop AA. AA-induced mice were divided into two groups: CTL group, which received saline administration, and MSC group, which received MSC administration.