Ebselen Preserves Tissue-Engineered Cell Sheets and their Stem Cells in Hypothermic Conditions

Abstract

Clinical trials have been performed using autologous tissue-engineered epithelial cell sheets for corneal regenerative medicine. To improve stem cell-based therapy for convenient clinical practice, new techniques are required for preserving reconstructed tissues and their stem/progenitor cells until they are ready for use. In the present study, we screened potential preservative agents and developed a novel medium for preserving the cell sheets and their stem/progenitor cells; the effects were evaluated with a luciferase-based viability assay. Nrf2 activators, specifically ebselen, could maintain high ATP levels during preservation. Ebselen also showed a strong influence on maintenance of the viability, morphology, and stem cell function of the cell sheets preserved under hypothermia by protecting them from reactive oxygen species-induced damage. Furthermore, ebselen drastically improved the preservation performance of human cornea tissues and their stem cells. Therefore, ebselen shows good potential as a useful preservation agent in regenerative medicine as well as in cornea transplantation.

Nrf2, a Potential Therapeutic Target against Oxidative Stress in Corneal Diseases

“…The physiological regenerative function of stem cells is lost in corneal limbal stem cell deficiency (LSCD), leading to corneal opacity and vision impairment. Transplantation of stem cells is applied to treat LSCD, using tissue-engineered epithelial cell sheets, such as human oral mucosal epithelial cell (hOEC) sheets [ 25 , 26 ] and human induced pluripotent (iPS) stem cells [ 27 ]. These stem cell-based regenerative cell sheet techniques facilitate the regeneration of the patient's own damaged stem cells and showed positive effects in treating LSCD in clinical trials [ 28 ].…”

“…Ebselen, an organic selenium-containing redox compound and a well-known Nrf2 activator, has exhibited great potentials as a promising medium for the preservation of tissue-engineered cell sheets and the stem/progenitor cells under hypothermia during preservation. The expression of two tight junction-relevant proteins (ZO-16 and MUC165), which maintain the barrier function of the corneal epithelium, were enhanced, and the reduction of p63 (an epithelial stem cell marker) was prevented by Ebselen in the hOEC sheet under hypothermic stress [ 25 ]. Ebselen also maintained the high ATP levels, normal morphology, viability, and function of the hOEC sheet during hypothermic preservation by reducing ROS generation, inducing the Nrf2 activation, decreasing the lactate dehydrogenase (LDH) releasement, and increasing the GSH/oxidized glutathione (GSSG) ratio.…”

“…Though not evidenced during preservation, Nrf2 translocation induced by ebselen in the hOEC sheets was evidenced after 3–6 hours of reculturing at 37°C. These results indicate that ebselen possesses two different biological roles: one is the direct effect of removing hypothermic-induced ROS generation during the preservation period and the other is the indirect effect via Nrf2 translocation during the reculturing period [ 25 ]. Moreover, ebselen maintained the normal morphology of limbal epithelial layer and showed meaningfully higher colony-forming efficiency, when the human corneal limbal tissue was preserved in ebselen, when compared to that of control in which a great number of corneal epithelium was lost [ 25 ].…”

“…These results indicate that ebselen possesses two different biological roles: one is the direct effect of removing hypothermic-induced ROS generation during the preservation period and the other is the indirect effect via Nrf2 translocation during the reculturing period [ 25 ]. Moreover, ebselen maintained the normal morphology of limbal epithelial layer and showed meaningfully higher colony-forming efficiency, when the human corneal limbal tissue was preserved in ebselen, when compared to that of control in which a great number of corneal epithelium was lost [ 25 ]. Therefore, ebselen was demonstrated to be an effective hypothermic preservation medium for tissue-engineered cell sheets, and it is believed to promote the ocular regenerative medicine, such as LSCD management and cornea transplantation.…”

“…It was regulated by decreasing ROS production, reducing oxidative enzymes, such as NADPH oxidase 4 (NOX4), and increasing dual specificity phosphatase 6 (DUSP6). TKE2 also activated Nrf2 signaling and upregulated the expression of antioxidative enzymes (SOD and GSTP), indicating that Nrf2 is involved in maintaining the different hemostasis of corneal stem cells and exhibits strong antioxidant capacity against oxidative stress by regulating ROS generation and elimination [ 25 ].…”