Mitochondrial transfer of mesenchymal stem cells in the rescue of acquired and inherited ocular degeneration

Abstract

Mitochondrial diseases are a group of energy metabolic disorders caused by acquired or inherited mitochondrial dysfunction, leading to tissue degeneration and cell death eventually. Mesenchymal stem cell (MSC) -based therapy represents a newly emerging therapeutic approach for tissue regeneration. Therapeutic mechanisms of MSC such as cell differentiation, cell fusion and paracrine effects have been postulated with supporting evidence. Recent studies have demonstrated that MSC could donate mitochondria to injured cells and improve mitochondrial function in heart and lung injuries. The aim of this study is to evaluate the role and mechanisms of mitochondrial transfer from induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) to ocular cells with acquired or hereditary mitochondrial complex I defects. I sought to determine whether MSCs could donate mitochondria to corneal epithelial cells (CECs) and protect them against acquired mitochondrial dysfunction. An in vitro cellular model was created via permanent deprivation of partial mitochondrial function by rotenone (Rot), a mitochondrial complex I inhibitor. It shows mitochondria of MSCs were efficiently transferred to CECs responsive to Rot treatment. The efficient mitochondrial transfer was associated with increased formation of tunneling nanotube (TNT), a tubular structure which allows direct intercellular communication. CECs with or without transferred mitochondria from MSCs displayed a distinct survival capacity and mitochondrial oxygen consumption rate. Separation of MSCs and CECs by a Transwell culture system revealed that intercellular mitochondrial transfer is abolished. Mechanistically, increased filopodia outgrowth in CECs for TNT formation was associated with inflammation-activated NFκB/TNFαip2 signaling pathways. To study iPSC--based regeneration for acquired mitochondrial disease in vivo, a rabbit model of corneal alkaline burn was used. MSCs grown on a decellularized porcine corneal scaffold were transplanted onto the alkali-injured cornea. Enhanced corneal wound healing was evident following healthy MSC scaffold transplantation. Transferred mitochondria from MSCs scaffold were detected in corneal epithelium. To further study iPSC-MSC-mediated mitochondrial transfer for hereditary mitochondrial disorder, a Ndufs4 deficient mice model was used for neurodegeneration and regeneration study. Ndufs4 gene encodes a nuclear-encoded accessory subunit of complex I of the mitochondrial respiratory-chain. The study demonstrated that absence of Ndufs4 results in functional defects and death of retinal ganglion cells (RGCs). iPSC-MSCs delivered through intravitreal injection successfully donate mitochondria to RGCs which could prevent the RGC loss and preserve RGCs functions. In addition, TNT formation and mitochondria transfer were associated with increased inflammatory reaction in mitochondrial Ndufs4 deficiency mice. Glial cells were activated in Ndufs4 gene knockout mice, secreting pro-inflammatory cytokines to stimulate TNT formation and promoting mitochondria transfer. MSCs donated mitochondria into retinal cells, which reduced level of above inflammatory cytokines. Therefore, intravitreal transplantation of MSCs allows them to donate functional mitochondria that effectively protect RGC from death. Taken together, intercellular mitochondrial transfer of MSCs provides the possibility to protect cells from mitochondrial complex I defects. This therapeutic strategy might also be applicable for a broad range of mitochondrial diseases.