Exosomes from acellular Wharton's jelly of the human umbilical cord promotes skin wound healing

Abstract

Background: Compromised wound healing has become a global public health challenge which presents a significant psychological, financial, and emotional burden on patients and physicians. We recently reported that acellular gelatinous Wharton's jelly of the human umbilical cord enhances skin wound healing in vitro and in vivo in a murine model; however, the key player in the jelly which enhances wound healing is still unknown.

Methods: We performed mass spectrometry on acellular gelatinous Wharton's jelly to elucidate the chemical structures of the molecules. Using an ultracentrifugation protocol, we isolated exosomes and treated fibroblasts with these exosomes to assess their proliferation and migration. Mice were subjected to a full-thickness skin biopsy experiment and treated with either control vehicle or vehicle containing exosomes. Isolated exosomes were subjected to further mass spectrometry analysis to determine their cargo.

Results: Subjecting the acellular gelatinous Wharton's jelly to proteomics approaches, we detected a large amount of proteins that are characteristic of exosomes. Here, we show that the exosomes isolated from the acellular gelatinous Wharton's jelly enhance cell viability and cell migration in vitro and enhance skin wound healing in the punch biopsy wound model in mice. Mass spectrometry analysis revealed that exosomes of Wharton's jelly umbilical cord contain a large amount of alpha-2-macroglobulin, a protein which mimics the effect of acellular gelatinous Wharton's jelly exosomes on wound healing.

Conclusions: Exosomes are being enriched in the native niche of the umbilical cord and can enhance wound healing in vivo through their cargo. Exosomes from the acellular gelatinous Wharton's jelly and the cargo protein alpha-2-macroglobulin have tremendous potential as a noncellular, off-the-shelf therapeutic modality for wound healing.

Keywords: Exosomes; Skin; Stem cells; Umbilical cord; Wharton’s jelly; Wound healing.

Fig. 1

Analysis of whole exosome treatment on fibroblast viability and migration. a The absorbance level detected from the emitted luminescence from viable cells. Whole exosome (Exo)-treated fibroblasts had the highest viability, followed by acellular gelatinous Wharton’s jelly (AGWJ)-treated cells. Lysed exosomes and no treatment or control (Ctrl) medium-treated cells had the lowest viability. N = 3, *p < 0.05, **p < 0.01. b Crystal violet stained scratch assay showing fibroblast migration without any treatment, whole exosome-treated, and lysed exosome-treated cells at 0 h and 24 h. Cell migration was increased when the cells were treated with whole exosomes; however, lysed exosome-treated cells diminished cell migration after 24 h. c Quantification of the number of cells which have migrated into the scratch zone. Whole exosome-treated cells had a significantly increased migration compared with control medium-treated cells. N = 3; *p < 0.05

Fig. 2

Analysis of exosome interaction with fibroblast cells Exosomes were labeled with CD81 antibody and treated with gold nanoparticles for visualization. a Transmission electron microscopy (TEM) at 7.8k× magnification showing an entire cell. b, c 100k× magnification of the cell focusing on exosomes surrounding the cell. Black arrows point to exosomes. d Red fluorescently labeled exosomes concentrate to the green EYFP fibroblast cells and over a period of 2.5 h, the red fluorescence starts to diminish. e Control green EYFP cells tracked over time without any exosomes. f Magnified images at 30 min, 90 min (1.5 h), and 180 min (3 h) displaying a gradual decrease in red fluorescence and a complete lack of red fluorescence by 3 h. N = 3

Fig. 3

Effect of exosome treatment on wound healing in vivo after 7 days. a A representative image of a Masson’s trichrome stained wound displaying the length of a control wound. Complete wound images were scanned under 2.5× magnification. b A representative exosome-treated wound displaying the healing progress at the end of 7 days. c Representative 40× image from the center of the granulation tissue of a control wound displaying cells and granulation within the wound center. d Representative 40× image from the center of the granulation tissue of exosome-treated wound, displaying cells and granulation within the wound center. e Quantification of the measurement of the granulation length from blue to blue across the wound. f Quantification of the cell number within the wound bed from 40× images. g Quantification of blue granulation intensity from within the wound center, determined by a score out of 10 by three blinded individuals, 1 being lowest and 10 being highest. N = 4 control mice, N = 4 exosome-treated mice. Arrow heads point to the start and end of granulation tissue within the wound bed. * p < 0.05

Fig. 4

Proteomic analysis of exosomes. a Mass spectrometry analysis of exosomal proteins. Central star points to the two most abundant proteins, albumin (ALB) and alpha-2-macroglobulin (α2M). Blue boxes identify proteins involved in wound healing. b Western blot analysis confirming the expression of α2M in exosomes compared with fibroblast (Fibro) cells. c Western blot analysis confirming the presence of exosomes by detecting CD81 protein, a marker of exosomes. N = 3

Fig. 5

Effect of α2M on cell proliferation, migration, and viability. a Immunofluorescence imaging of bromodeoxyuridine (BrdU) incorporation into proliferating cells. DAPI labels the nucleus blue. The row labeled DAPI/BrdU displays the merged images after green FITC-labeled BrdU labels the nucleus of proliferating cells green. The treatments (labeled at the top) were either control (CTRL) DMEM-treated fibroblast cells, exosome-treated cells, or recombinant α2M-treated cells at 100 ng/ml. Images captured under 10× magnification. b Fibroblast scratch assay. Top row images were taken at 0 h and bottom row images were taken at 24 h time points. Treatments were either control (CTRL) DMEM-treated cells, α2M treatment at 100 ng/ml, and α2M treatment at 1 μg/ml. Images captured at 4× magnification. c Quantification of fibroblast cell viability. d Quantification of the percentage of BrdU-positive cells. e Quantification of the number of cells within the scratch zone. N = 4; *p < 0.05, **p < 0.01, ***p < 0.001