Hair-growth-promoting effect of conditioned medium of high integrin α6 and low CD 71 (α6bri/CD71dim) positive keratinocyte cells

Abstract

Keratinocyte stem/progenitor cells (KSCs) reside in the bulge region of the hair follicles and may be involved in hair growth. Hair follicle dermal papilla cells (HFDPCs) and outer root sheath (ORS) cells were treated with conditioned medium (CM) of KSCs. Moreover, the effects of KSC-CM on hair growth were examined ex vivo and in vivo. A human growth factor chip array and RT-PCR were employed to identify enriched proteins in KSC-CM as compared with CM from keratinocytes. KSC-CM significantly increased the proliferation of HFDPCs and ORS cells, and increased the S-phase of the cell cycle in HFDPCs. KSC-CM led to the phosphorylation of ATK and ERK1/2 in both cell types. After subcutaneous injection of KSC-CM in C3H/HeN mice, a significant increase in hair growth and increased proliferation of hair matrix keratinocytes ex vivo was observed. We identified six proteins enriched in KSC-CM (amphiregulin, insulin-like growth factor binding protein-2, insulin-like growth factor binding protein-5, granulocyte macrophage-colony stimulating factor, Platelet-derived growth factor-AA, and vascular endothelial growth factor). A growth-factor cocktail that contains these six recombinant growth factors significantly increased the proliferation of HFDPCs and ORS cells and enhanced the hair growth of mouse models. These results collectively indicate that KSC-CM has the potential to increase hair growth via the proliferative capacity of HFDPCs and ORS cells.

Figure 1

Effects of keratinocyte stem/progenitor cells conditioned medium (KSC-CM) on the proliferation of hair follicle dermal papilla cells (HFDPCs). (A) Cell proliferation was measured 48 h after concentrated KSC-CM treatment; (B) Western blot analysis against phospho-AKT and phospho-ERK1/2 showed that these pathways are activated by KSC-CM; (C,D) Cell cycle analysis of HFDPCs was performed by flow cytometry, and we found that the fraction of S phase cells was increased following 24 h (C) and 48 h (D) of concentrated KSC-CM treatment. ** p < 0.01.

Figure 1

Effects of keratinocyte stem/progenitor cells conditioned medium (KSC-CM) on the proliferation of hair follicle dermal papilla cells (HFDPCs). (A) Cell proliferation was measured 48 h after concentrated KSC-CM treatment; (B) Western blot analysis against phospho-AKT and phospho-ERK1/2 showed that these pathways are activated by KSC-CM; (C,D) Cell cycle analysis of HFDPCs was performed by flow cytometry, and we found that the fraction of S phase cells was increased following 24 h (C) and 48 h (D) of concentrated KSC-CM treatment. ** p < 0.01.

Figure 2

Effects of keratinocyte stem/progenitor cells conditioned medium (KSC-CM) on outer root sheath (ORS) cell proliferation. (A) Cell proliferation was measured 48 h after concentrated KSC-CM treatment; (B) Western blot analysis against phospho-AKT and phospho-ERK1/2 showed that these pathways are activated by KSC-CM. * p < 0.05; ** p < 0.01.

Figure 3

The promoting effects of keratinocyte stem/progenitor cells conditioned medium (KSC-CM) on hair growth in mice. Telogen-matched, seven-week-old C3H/NeH mice were shaved and subcutaneously injected three times with concentrated KSC-CM. (A) Photographs were taken two weeks after the injections; (B) The new hair was shaved, and the hair weight was measured. ** p < 0.01.

Figure 4

Effects of conditioned medium of keratinocyte stem/progenitor cells (KSC-CM) on ex vivo organ culture of human hair follicles. Isolated human anagen hairs were treated with KSC-CM (0%, 2.5% and 10%) in William’s E medium for two days. The Ki67-positive cells (green) were significantly increased by KSC-CM treatment; cells were counter-stained with 4',6-diamidino-2-phenylindole (DAPI).

Figure 5

Altered protein secretion and mRNA expression in keratinocyte stem/progenitor cells (KSCs) and keratinocytes. (A) The growth-factor chip array showed that secretion of AREG, IGFBP2, IGFBP5, GM-CSF, PDGF-AA, and VEGF were significantly increased; (B) In addition, the mRNA expression of these growth factors is highly expressed in KSCs.

Figure 6

Effect of the growth-factor cocktail (GFC) on hair growth. (A) Seven-week-old C3H/NeH mice were shaved and subcutaneously injected three times with GFC. The hair weight was significantly increased by GFC treatment. In addition; GFC treatment significantly increased the proliferation of hair follicle dermal papilla cells (HFDPCs) (B) and outer root sheath (ORS) (C) cells. ** p < 0.01.

Figure 6

Effect of the growth-factor cocktail (GFC) on hair growth. (A) Seven-week-old C3H/NeH mice were shaved and subcutaneously injected three times with GFC. The hair weight was significantly increased by GFC treatment. In addition; GFC treatment significantly increased the proliferation of hair follicle dermal papilla cells (HFDPCs) (B) and outer root sheath (ORS) (C) cells. ** p < 0.01.