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. 2023 Nov 24;12(23):2698.
doi: 10.3390/cells12232698.

An Exosome-Rich Conditioned Medium from Human Amniotic Membrane Stem Cells Facilitates Wound Healing via Increased Reepithelization, Collagen Synthesis, and Angiogenesis

Chan Ho Noh  1   2 , Sangryong Park  2 , Hye-Rim Seong  2 , Ah-Young Lee  2 , Khan-Erdene Tsolmon  1 , Dongho Geum  3 , Soon-Cheol Hong  4 , Tae Myoung Kim  2 , Ehn-Kyoung Choi  2 , Yun-Bae Kim  1   2
Affiliations

An Exosome-Rich Conditioned Medium from Human Amniotic Membrane Stem Cells Facilitates Wound Healing via Increased Reepithelization, Collagen Synthesis, and Angiogenesis

Chan Ho Noh et al. Cells. .

Abstract

Tissue regeneration is an essential requirement for wound healing and recovery of organs' function. It has been demonstrated that wound healing can be facilitated by activating paracrine signaling mediated by exosomes secreted from stem cells, since exosomes deliver many functional molecules including growth factors (GFs) and neurotrophic factors (NFs) effective for tissue regeneration. In this study, an exosome-rich conditioned medium (ERCM) was collected from human amniotic membrane stem cells (AMSCs) by cultivating the cells under a low oxygen tension (2% O2 and 5% CO2). The contents of GFs and NFs including keratinocyte growth factor, epidermal growth factor, fibroblast growth factor 1, transforming growth factor-β, and vascular endothelial growth factor responsible for skin regeneration were much higher (10-30 folds) in the ERCM than in normal conditioned medium (NCM). In was found that CM-DiI-labeled exosomes readily entered keratinocytes and fibroblasts, and that ERCM not only facilitated the proliferation of keratinocytes in normal condition, but also protected against H2O2 cytotoxicity. In cell-migration assay, the scratch wound in keratinocyte culture dish was rapidly closed by treatment with ERCM. Such wound-healing effects of ERCM were confirmed in a rat whole skin-excision model: i.e., the wound closure was significantly accelerated, remaining minimal crusts, by topical application of ERCM solution (4 × 109 exosome particles/100 μL) at 4-day intervals. In the wounded skin, the deposition of collagens was enhanced by treatment with ERCM, which was supported by the increased production of collagen-1 and collagen-3. In addition, enhanced angiogenesis in ERCM-treated wounds was confirmed by increased von Willebrand factor (vWF)-positive endothelial cells. The results indicate that ERCM from AMSCs with high concentrations of GFs and NFs improves wound healing through tissue regeneration not only by facilitating keratinocyte proliferation for skin repair, but also activating fibroblasts for extracellular matrix production, in addition to the regulation of angiogenesis and scar tissue formation.

Keywords: amniotic membrane stem cell; angiogenesis; collagen synthesis; exosome-rich conditioned medium (ERCM); growth factor; keratinocyte proliferation; neurotrophic factor; wound healing.

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Conflict of interest statement

Authors Chan Ho Noh, Sangryong Park, Hye-Rim Seong, Ah-young Lee, Tae Myoung Kim, Ehn-Kyoung Choi and Yun-Bae Kim were employed by the company Designed Cells Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Isolation and characterization of exosomes from amniotic membrane stem cells (AMSCs). (A) Representative transmission electron microscopic findings of exosomes. (B) Particle size distribution of AMSC exosomes analyzed by a Nanoparticle-Tracking Analysis system. Red line: calibration curve for quantification, blue number: size of each peak. (C) Western blot analysis of CD9-, CD63-, and CD81-positive exosomes in normal conditioned medium (NCM) and exosome-rich conditioned medium (ERCM). (D) Concentrations of growth factors (GFs) and neurotrophic factors (NFs) in NCM (white bars) and ERCM (black bars). KGF: keratinocyte growth factor, EGF: epidermal growth factor, FGF1: fibroblast growth factor 1, TGF–β: transforming growth factor–β, VEGF: vascular endothelial growth factor. * Significantly different from NCM (p < 0.05).
Figure 2
Figure 2
Penetration of CM-DiI-labeled AMSC exosomes into HaCaT keratinocytes (A) and 3T3–L1 fibroblasts (B). The cells (1 × 105/mL, n = 5/group) were treated with 200 µM H2O2 or its vehicle (Normal), and incubated with labeled exosomes (50 μL/mL) for 4 h. Inset: part for each enlarged image (right side).
Figure 3
Figure 3
HaCaT keratinocyte-proliferative and -protective activities of exosome-rich conditioned medium (ERCM). (A) HaCaT cell proliferation by ERCM. (B) HaCaT cell protection by ERCM against oxidative stress (200 μM H2O2). The cells (1 × 104/mL, n = 5/group) were treated with 200 μM H2O2 and ERCM (1–30 μL/mL), and incubated for 24 h. (C,D) Facilitation of HaCaT cell migration (scratch-healing) by ERCM. The cell culture plates (1 × 105/mL, n = 5/group) were scratched, and incubated with ERCM (1–30 μL/mL) for 24 h. # Significantly different from Normal control (p < 0.05). * Significantly different from Scratch alone (p < 0.05).
Figure 4
Figure 4
Collagen synthesis-promoting activity of exosome-rich conditioned medium (ERCM). Western blot (A) and quantitative (B) analysis of collagen–1 (white bars) and collagen–3 (black bars) in 3T3–L1 fibroblasts. The cells (1 × 104/mL, n = 5/group) were treated with ERCM (1–30 μL/mL), and incubated for 24 h. * Significantly different from Normal control (p < 0.05).
Figure 5
Figure 5
Gross findings of wound healing in rats dermally applied with fusidate sodium or exosome-rich conditioned medium (ERCM). (A) Representative findings. (B) Quantitative analysis of wound closure. Full-thickness 20-mm skin wounds (n = 10/group) were applied with ERCM (filled square, 4 × 109 exosome particles/100 μL), Saline solution (open circle) or 3 mg Fucidin® ointment (filled circle, 100 μg as fusidate sodium) on days 0, 4, 8, and 12. * Significantly different from Saline control (p < 0.05).
Figure 6
Figure 6
Microscopic findings of wound healing and myofibroblast differentiation in rats dermally applied with fusidate sodium or exosome-rich conditioned medium (ERCM). (A) Representative findings stained with hematoxylin-eosin, Masson’s trichrome or immunostained with an antibody specific for α-smooth muscle actin (α-SMA). (B) Quantitative analysis of α-SMA-positive cells (n = 5/group). # Significantly different from Normal control (p < 0.05). * Significantly different from Saline (Wound alone) (p < 0.05).
Figure 7
Figure 7
Collagen synthesis and neovascularization in the skin of rats dermally applied with fusidate sodium or exosome-rich conditioned medium (ERCM). (A) Representative findings immunostained with antibodies specific for collagen–1, collagen–3 or von Willebrand Factor (vWF). Arrow: vWF-positive vessel. (B,C) Western blot analysis of collagen–1 (white bars) and collagen–3 (black bars) (n = 5/group). (D) Quantitative analysis of vWF-positive cells (n = 5/group). # Significantly different from Normal control (p < 0.05). * Significantly different from Saline (Wound alone) (p < 0.05).
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