Corneal Wound Healing Effects of Mesenchymal Stem Cell Secretome Delivered Within a Viscoelastic Gel Carrier

Gabriella Maria Fernandes-Cunha, Kyung-Sun Na, Ilham Putra, Hyun Jong Lee, Sarah Hull, Yu-Chia Cheng, Ignacio Jesus Blanco, Medi Eslani, Ali R Djalilian, David Myung, Gabriella Maria Fernandes-Cunha, Kyung-Sun Na, Ilham Putra, Hyun Jong Lee, Sarah Hull, Yu-Chia Cheng, Ignacio Jesus Blanco, Medi Eslani, Ali R Djalilian, David Myung

Abstract

Severe corneal injuries often result in permanent vision loss and remain a clinical challenge. Human bone marrow-derived mesenchymal stem cells (MSCs) and their secreted factors (secretome) have been studied for their antiscarring, anti-inflammatory, and antiangiogeneic properties. We aimed to deliver lyophilized MSC secretome (MSC-S) within a viscoelastic gel composed of hyaluronic acid (HA) and chondroitin sulfate (CS) as a way to enhance corneal re-epithelialization and reduce complications after mechanical and chemical injuries of the cornea. We hypothesized that delivering MSC-S within HA/CS would have improved wound healing effects compared the with either MSC-S or HA/CS alone. The results showed that a once-daily application of MSC-S in HA/CS enhances epithelial cell proliferation and wound healing after injury to the cornea. It also reduced scar formation, neovascularization, and hemorrhage after alkaline corneal burns. We found that combining MSC-S and HA/CS increased the expression of CD44 receptors colocalized with HA, suggesting that the observed therapeutic effects between the MSC-S and HA/CS are in part mediated by CD44 receptor upregulation and activation by HA. The results from this study demonstrate a reproducible and efficient approach for delivering the MSC-S to the ocular surface for treatment of severe corneal injuries. Stem Cells Translational Medicine 2019;8:478-489.

Keywords: Cellular proliferation; Chondroitin sulfate; Cornea; Mesenchymal stem cells.

Conflict of interest statement

G.F.‐C., H.J.L., A.D., and D.M. are co‐inventors on a patent application related to the technology described in this article. The other authors indicated no potential conflicts of interest.

© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Figures

Figure 1
Figure 1
(A): P63, ABCG2, and CK3 staining were observed in the harvested primary human corneal epithelial cells (HCECs) used in this study, indicating a mixed population of limbal and central stem cells. CK3 was expressed in these cells 24 hours after treating with complete keratinocyte serum‐free medium (KSFM) with growth factors (no treatment, control), MSC‐S, HA/CS, MSC‐S in HA/CS. (B): Live/dead cytotoxicity assay after treatment with complete KSFM with growth factors, MSC‐S, HA/CS, and MSC‐S in HA/CS over 72 hours in primary corneal epithelial cells. (C): Effect of MSC‐S, HA/CS, and MSC‐S in HA/CS on primary HCECs proliferation. Cell proliferation was determined using a cell metabolic activity assay, and (D): DNA concentration, at 24, 48, and 72 hours. The data were normalized to no treatment at 24 hours. In both assays, proliferation was statistically significantly greater when MSC‐S was delivered in HA/CS compared to no treatment (n = 4; *, p < .05; **, p < .01; ****, p < .0001). Abbreviations: CS, chondroitin sulfate; HA, hyaluronic acid; MSC‐S, mesenchymal stem cells secretome.
Figure 2
Figure 2
Results of MSC‐S treatments in a mechanical corneal wound model in rats in vivo. The epithelial layer from rat corneas was debrided and the MSC‐S was applied with and without HA/CS, compared to HA/CS alone and saline alone, using 1 drop of each daily. (A): Fluorescein staining of the treated corneas was used to quantify the size of the epithelial defect on a daily basis for each of the treatment groups. Shown are representative photos under blue light illumination for each of the treatment groups (Saline, MSC‐S, HA/CS, and MSC‐S in HA/CS). (B): After 24 hours, the group that received MSC‐S in HA/CS had smaller wound sizes compared to saline group (**, p < .01), whereas HA/CS and MSC‐S treatments alone did not. (C): Immunostaining of rat corneas 7 days after MSC‐S in HA/CS treatment showing that the epithelial layer was able to form ZO‐1—scale bar 50 μm. (D): Hematoxylin and eosin staining of the cornea from saline, MSC‐S, HA/CS, MSC‐S in HA/CS and no injury. A stratified epithelium formed in all groups by day 7—scale bar 25 μm. Abbreviations: CS, chondroitin sulfate; HA, hyaluronic acid; MSC‐S, mesenchymal stem cells secretome.
Figure 3
Figure 3
Results of different carrier gel treatments in a mechanical corneal wound model in rats in vivo. (A): Fluorescein staining of corneas examined under blue light revealed that MSC‐S delivered in HA/CS yielded smaller wound areas 1 day after treatment compared to saline, MSC‐S in Systane gel (0.4% polyethylene glycol 400% and 0.3% propylene glycol) and BSA in HA/CS. Each treatment was applied right after the injury on day 0. (B): Quantification of fluorescently stained wound areas showed that the MSC‐S in HA/CS treatment led to a statistically significantly smaller wound area at 24 hours compared to all other treatments (*, p < .05). Abbreviations: BSA, bovine serum albumin; CS, chondroitin sulfate; HA, hyaluronic acid; MSC‐S, mesenchymal stem cells secretome.
Figure 4
Figure 4
Results from corneal alkaline burn model experiments in rats in vivo. Sodium hydroxide (NaOH) was applied to the epithelium followed by copious irrigation with balanced salt solution, followed by treatments (saline, MSC‐S, HA/CS, MSC‐S in HA/CS) 1 drop daily for 7 days and the corneas were photographed with and without fluorescein staining out to day 14. (A): Representative photographs for each of the study arms. Yellow arrows show areas of scar formation in the saline group and intrastromal hemorrhage in the HA/CS group (B): Percent (%) wound closure 24 hours after treatment. Eyes treated with MSC‐S in HA/CS yielded significantly smaller wound areas compared to the saline group (*, p < .05), while the HA/CS and MSC‐S treatments alone did not. (C): High‐magnification photographs of representative photos of alkaline burned corneas 14 days after saline, HA/CS, MSC‐S, MSC‐S in HA/CS. (D): Area of corneal neovascularization (NV) 14 days after treatments. The corneas treated with MSC‐S in HA/CS resulted in a lower NV area compared to the saline group (*, p < .05), whereas the HA/CS and MSC‐S groups did not (representative photographs taken at 15× magnification). Abbreviations: CS, chondroitin sulfate; HA, hyaluronic acid; MSC‐S, mesenchymal stem cells secretome.
Figure 5
Figure 5
(A): Immunofluorescence images of rat cornea sections 14 days after treatment with saline, MSC‐S, HA/CS, and MSC‐S in HA/CS—scale bar 50 μm. Alpha smooth muscle actin and CD31 staining were greater in the HA/CS and saline groups compared to both MSC‐S‐containing groups (with and without the HA/CS carrier). Expression of Aldehyde Dehydrogenase 3 Family Member A1 (ALDH3A1) was maintained for both group containing MSC‐S. Decreased ALDH3A1 expression was observed in the corneas that received saline and HA/CS. (B): Quantification of the fluorescence intensities using ImageJ revealed that the alpha smooth muscle actin expression was significantly lower in all treatment groups compared the saline group, and lower in the MSC‐S‐containing groups compared to the HA/CS group, while (C): CD31 expression was significantly lower in the MSC‐S‐containing groups compared to the saline group, and (D): ALDH3A1 expression was similar to normal corneas in MSC‐S containing groups but was significantly lower in the HA/CS and saline groups (*, p < .05; **, p < .01). Abbreviations: CS, chondroitin sulfate; HA, hyaluronic acid; MSC‐S, mesenchymal stem cells secretome.
Figure 6
Figure 6
(A): CD44 expression (green fluorescence) in human corneal epithelial cells after treatment with complete keratinocyte serum free medium with growth factors (no treatment, control), MSC‐S, HA/CS, MSC‐S in HA/CS—scale bar 20 μm. (B): Quantification of the fluorescence intensities using ImageJ revealed that CD44 receptors are upregulated in the cells that received MSC‐S (**, p < .01) and MSC‐S in HA/CS (***, p < .001). (C): CD44 expression in rat corneas in vivo 7 days after treatment with saline, MSC‐S, HA/CS, MSC‐S in HA/CS, and uninjured/untreated corneas—scale bar 50 μm. (D): Quantification of the fluorescence intensities using ImageJ showed that CD44 receptors are upregulated in the cornea treated with MSC‐S in HA/CS (*, p < .05). Abbreviations: CS, chondroitin sulfate; HA, hyaluronic acid; MSC‐S, mesenchymal stem cells secretome.
Figure 7
Figure 7
Localization of HA and CD44 receptors on confluent monolayers of primary human corneal epithelial cells after treatment with complete keratinocyte serum free medium with growth factors (no treatment, control), MSC‐S, HA/CS, MSC‐S in HA/CS after (A): 8 hours and (B): 24 hours. The localization of HA at the cell membranes suggests binding to CD44 (scale bar 20 μm). Abbreviations: CS, chondroitin sulfate; HA, hyaluronic acid; MSC‐S, mesenchymal stem cells secretome.

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