Improvement of androgenic alopecia by extracellular vesicles secreted from hyaluronic acid-stimulated induced mesenchymal stem cells

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.

1. Introduction

Androgenetic alopecia (AGA) is a common form of hair loss which is affected by genetic, hormonal, and environmental factors, as well as aging [1, 2]. Notably, the hair follicles of patients with AGA has a higher sensitivity to androgens such as dihydrotestosterone (DHT). In hair follicles, testosterone is converted to DHT by 5α-reductase and prolonged exposure to DHT makes hair follicles shrink, eventually leading to hair loss [3]. Several strategies for AGA are available, but only finasteride and minoxidil are approved by the US Food and Drug Administration [4]. However, prolonged use of finasteride can cause sexual dysfunction, while minoxidil has side effects such as irritation, swelling, and lightheadedness [5–7].

The Wnt/β-catenin pathway plays an essential role in the hair follicle development from dermal papilla cells (DPCs) [8–10]. In the presence of Wnt ligands, the phosphorylation of β-catenin by glycogen synthase kinase 3β (GSK3β) is inhibited, stabilizing β-catenin. Without the Wnt ligand, GSK3β phosphorylates β-catenin leading to the degradation and ubiquitination of β-catenin [11]. In patients with AGA, DHT inhibits the Wnt/β-catenin pathway by activating GSK3β, contributing to the inhibition of hair follicular stem cell differentiation in DPCs [12]. Consistently, the inhibition of GSK3β via phosphorylation (at Ser9) by human placental extract caused β-catenin stabilization and promoted the hair-inductive capacity of DPCs [13]. Collectively, activation of the Wnt/β-catenin pathway by blocking GSK3β activity is required to block AGA progression.

Extracellular vesicles (EVs), which are nano-sized particles released from almost all cell types, play an essential role in cell-to-cell communication [14]. In addition, the content of EVs in body fluids can provide biological information about the original tissues, making EVs invaluable tools as non-invasive diagnostic markers [15]. Importantly, stem cell-derived EVs have potential for therapeutic purposes i