Improvement of androgenic alopecia by extracellular vesicles secreted from hyaluronic acid-stimulated induced mesenchymal stem cells
Authors: Oh Hyun Geun , Jung Minyoung , Jeong Seon-Yeong , Kim Jimin , Han Sang‑Deok , Kim Hongduk , Lee Seulki , Lee Yejin , You Haedeun , Park Somi , Kim Eun A. , Kim Tae Min , Kim Soo 
Published: September 2024

Abstract
Background
Androgenetic alopecia (AGA) is a common form of hair loss. Androgens, such as testosterone and dihydrotestosterone, are the main causes of AGA. Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) can reduce AGA. However, preparing therapeutic doses of MSCs for clinical use is challenging. Induced pluripotent stem cell-derived MSCs (iMSCs) are homogenous and easily expandable, enabling scalable production of EVs. Hyaluronic acid (HA) can exert various functions including free radical scavenging, immune regulation, and cell migration. Herein, we examined whether hyaluronic acid (HA) stimulation of iMSCs could produce EVs with enhanced therapeutic outcomes for AGA.

Methods
EVs were collected from iMSCs primed with HA (HA–iMSC–EVs) or without HA (iMSC–EVs). The characteristics of EVs were examined using dynamic light scattering, cryo-transmission electron microscopy, immunoblotting, flow cytometry, and proteomic analysis. In vitro, we compared the potential of EVs in stimulating the survival of hair follicle dermal papilla cells undergoing testosterone-mediated AGA. Additionally, the expression of androgen receptor (AR) and relevant growth factors as well as key proteins of Wnt/β-catenin signaling pathway (β-catenin and phosphorylated GSK3β) was analyzed. Subsequently, AGA was induced in male C57/BL6 mice by testosterone administration, followed by repeated injections of iMSC–EVs, HA–iMSC–EVs, finasteride, or vehicle. Several parameters including hair growth, anagen phase ratio, reactivation of Wnt/β-catenin pathway, and AR expression was examined using qPCR, immunoblotting, and immunofluorescence analysis.

Results
Both types of EVs showed typical characteristics for EVs, such as size distribution, markers, and surface protein expression. In hair follicle dermal papilla cells, the mRNA levels of AR, TGF-β, and IL-6 increased by testosterone was blocked by HA–iMSC–EVs, which also contributed to the augmented expression of trophic genes related to hair regrowth. However, no notable changes were observed in the iMSC–EVs. Re-activation of Wnt/β-catenin was observed in HA–iMSC–EVs but not in iMSC–EVs, as shown by β-catenin stabilization and an increase in phosphorylated GSK3β. Restoration of hair growth was more significant in HA–iMSC–EVs than in iMSC–EVs, and was comparable to that in mice treated with finasteride. Consistently, the decreased anagen ratio induced by testosterone was reversed by HA–iMSC–EVs, but not by iMSC–EVs. An increased expression of hair follicular β-catenin protein, as well as the reduction of AR was observed in the skin tissue of AGA mice receiving HA–iMSC–EVs, but not in those treated with iMSC–EVs.

Conclusions
Our results suggest that HA–iMSC–EVs have potential to improve AGA by regulating growth factors/cytokines and stimulating AR-related Wnt/β-catenin signaling.