Exosomes from human umbilical cord mesenchymal stem cells promote the growth of human hair dermal papilla cells

Abstract

Human hair dermal papilla cells (HHDPCs) play a significant role in hair growth. This study found that human umbilical cord mesenchymal stem cell-derived exosomes (UC-MSC-Es) effectively enhanced cell growth of HHDPCs. UC-MSC-Es has a size range of 30-180 nm and expression of CD9, CD63, CD81, CD73, and TSG101. UC-MSC-Es significantly increased cell populations of HHDPCs in the S and G2/M phases. UC-MSC-Es also increased the expression of cell cycle-related proteins, β-catenin, and cyclin D1. Further mechanistic studies demonstrated that UC-MSC-Es promoted the phosphorylation of Akt and GSK-3β, and the inhibition of PI3K and Akt reduced the proliferative effects of UC-MSC-Es. Collectively, these findings suggest that UC-MSC-Es have a potential effect in treating hair loss through modulating PI3K and Akt-dependent pathways in HHDPCs.

Fig 1. Identification of exosomes derived from UC-MSCs (UC-MSC-Es).

(A) Exosome morphology and sizes were measured using transmission electron microscopy (up) and nanoparticle tracking analysis assay (down), respectively. (B) Protein expression levels of conditioned medium (CM) and extracellular vesicles (EV) were determined by western blotting. The results were conducted with three independent experiments (n = 4).

Fig 2. UC-MSC-Es enhance the proliferation of human hair dermal papilla cells.

HHDPCs (2 × 10³ cells) were incubated with UC-MSC-Es (1 × 10¹⁰, 3 × 10¹⁰, and 1 × 10¹¹ particles/mL) or minoxidil (3 and 10 μM) for 72 h. Cell proliferation was determined using the WST-1 assay. Data are expressed as the mean ± SEM (n = 3 - 12). *p < 0.05, compared with the control.

Fig 3. UC-MSC-Es promote the cell cycle in HHDPC.

HHDPCs (2 x 10⁴) were incubated with PBS or UC-MSC-Es (1 × 10¹¹ particles/mL) for 48 h. Cell cycle distribution was assessed using propidium iodide staining and measured by flow cytometry. Quantified flow cytometry results showed cell percentages in the G0/G1, S, and G2/M phases. Data are expressed as the mean ± SEM (n = 3). *p < 0.05, compared with the control.

Fig 4. UC-MSC-Es elevate cyclin D1 and β-catenin expression in HHDPCs.

HHDPCs (1 x 10⁶ cells) were incubated with PBS or UC-MSC-Es (1 × 10¹¹ particles/mL) for 48 h. (A) Cyclin D1 and (B) β-catenin were analyzed by western blotting. The upper panel displays representative images, while the lower panel shows the quantified protein analysis results. Data are expressed as the mean ± SEM (n = 4). *p < 0.05, compared with the control.

Fig 5. UC-MSC-Es elevate Akt and GSK3-β phosphorylation in HHDPCs.

HHDPCs (1 x 10⁶ cells) were exposed to PBS or UC-MSC-Es (1 × 10¹¹ particles/mL) for 24 h. The phosphorylation and total levels of (A) Akt and (B) GSK-3β were determined by immunoblotting, using antibodies that recognize phosphorylated and total forms of Akt and GSK-3β. The upper panel shows the representative images of immunoblots, while the lower panel shows the quantified results of protein bands. Data were shown as mean ± S.E.M. (n = 4). *p < 0.05, compared with the control.

Fig 6. PI3K and Akt inhibitors attenuate UC-MSC-Es-induced HHDPC proliferation.

HHDPCs (2 × 10³ cells) were pretreated with idelalisib (10 µM) or MK-2206 (10 µM) for 1 h and then treated with either PBS or UC-MSC-Es (1 × 10¹¹ particles/mL) for 72 h. (A) Cell proliferation was assessed using the WST-1 assay. (B) The phosphorylation and total levels of β-catenin were determined by immunoblotting, using antibodies that recognize phosphorylated and total forms. Cell number was assessed via the WST-1 assay. Data were shown as mean ± S.E.M. (n = 3 - 4). *p < 0.05, compared with the control.

Fig 7. A schematic overview of UC-MSC-Es preparation and their effects on HHDPC proliferation.

Human umbilical cord mesenchymal stem cell-derived exosomes (UC-MSC-Es) were concentrated from the culture medium and then applied to human hair dermal papilla cells (HHDPCs). The UC-MSC-Es facilitated the proliferation of HHDPCs by activating the PI3K and Akt signaling pathways. This activation resulted in the phosphorylation (inactivation) of GSK3β, which subsequently activated β-catenin and led to the upregulation of the cell cycle-related protein cyclin D1. The figure was created using BioRender (https://BioRender.com) under the appropriate license.