Therapeutic Potential of Mesenchymal Stem Cell-Secreted Factors on Delay in Corneal Wound Healing by Nitrogen Mustard

Abstract

Ocular surface exposure to nitrogen mustard (NM) leads to severe ocular toxicity which includes the separation of epithelial and stromal layers, loss of endothelial cells, cell death, and severe loss of tissue function. No definitive treatment for mustard gas-induced ocular surface disorders is currently available. The research was conducted to investigate the therapeutic potential of mesenchymal stem cell-conditioned media (MSC-CM) in NM-induced corneal wounds. NM was added to different types of corneal cells, the ocular surface of porcine, and the ocular surface of mice, followed by MSC-CM treatment. NM significantly induced apoptotic cell death, cellular ROS (Reactive oxygen species), and reduced cell viability, metabolic gene expression, and mitochondrial function, and, in turn, delayed wound healing. The application of MSC-CM post NM exposure partially restored mitochondrial function and decreased intracellular ROS generation which promoted cell survival. MSC-CM therapy enhanced wound healing process. MSC-CM inhibited NM-induced apoptotic cell death in murine and porcine corneal tissue. The application of MSC-CM following a chemical insult led to significant improvements in the preservation of corneal structure and wound healing. In vitro, ex vivo, and in vivo results suggest that MSC-CM can potentially provide targeted therapy for the treatment of chemical eye injuries, including mustard gas keratopathy (MGK) which presents with significant loss of vision alongside numerous corneal pathologies.

Wound-Healing Effects of Mesenchymal Stromal Cell Secretome in the Cornea and the Role of Exosomes

“…A human corneal epithelial cell (HCEC) was cultured in 10% fetal bovine serum (FBS; #F2442, Sigma-Aldrich, St. Lois, MO, USA), 1X L-glutamine (#MT25005CI, Corning, Corning, NY, USA), 1X NEAA (#11140050, Gibco, Bilings, MT, USA), and 1% penicillin–streptomycin (P/S; #MT30002CI, Corning, Corning, NY, USA) in 5% CO 2 at 37 °C [ 3 ]. A human corneal limbal epithelial (HCLE) cell line was cultured in 1% P/S and keratinocyte serum-free medium (KSFM; #17005-042, Thermo, Waltham, MA, USA) with 0.05 mg/mL of Bovine Pituitary Extract and 5 ng/mL of Epidermal Growth Factor [ 3 ]. Human bone marrow (BM)-derived MSCs were purchased from RoosterBio (# MSC-CC040, Ballenger Creek, MD, USA) and cultured in MEM alpha (MEMa; #4106102, ThermoFisher Scientific, Waltham, MA, USA) media supplemented with 10% FBS, 1X L-glutamine, and 1X NEAA [ 3 , 60 ].…”

“…A human corneal limbal epithelial (HCLE) cell line was cultured in 1% P/S and keratinocyte serum-free medium (KSFM; #17005-042, Thermo, Waltham, MA, USA) with 0.05 mg/mL of Bovine Pituitary Extract and 5 ng/mL of Epidermal Growth Factor [ 3 ]. Human bone marrow (BM)-derived MSCs were purchased from RoosterBio (# MSC-CC040, Ballenger Creek, MD, USA) and cultured in MEM alpha (MEMa; #4106102, ThermoFisher Scientific, Waltham, MA, USA) media supplemented with 10% FBS, 1X L-glutamine, and 1X NEAA [ 3 , 60 ].…”

“…The number of blue-stained cells and the number of total cells were counted to determine the number of live and dead cells, and to the calculate cell viability: % of Viable cells = [1.00 − (Number of blue cells ÷ Number of total cells)] × 100. These analyses were performed in triplicate ( n = 3) [ 3 ].…”

“…Mesenchymal stem cells (MSCs) are adult stem cells characterized by specific cell surface markers and their ability to differentiate into bone, cartilage, and fat cells [ 1 , 2 , 3 , 4 , 5 ]. MSCs are found in many tissues throughout the body, including bone marrow, adipose tissue, and umbilical cord tissue [ 1 , 2 ].…”

“…Often referred to as MSC-conditioned media (MSC-CM), this approach can recapitulate many of the therapeutic effects of cell therapy with fewer side effects. Studies have shown that MSC-CM can promote the repair of chemical and mechanical injuries [ 3 , 31 , 32 , 33 ]. Furthermore, cell-free therapies can be manufactured in higher volumes with lower production costs as well [ 34 ].…”