Effect of amniotic membrane-derived mesenchymal stem cells on innate immune system and a their usefulness in ocular cell therapy

Abstract

Purpose: Several conditions like infections and mechanical or chemical stimulus can produce corneal injury. The major source of epithelial regeneration in cornea is the sclerocorneal limbal stem cells; the lack of these cells results in chronic inflammation, epithelial defects, stromal damage and corneal neovascularization. The regeneration of the corneal epithelium using bone marrow-derived mesenchymal stem cells has been documented in a rat model. On the other hand, regenerative and immunosuppressive capacities of amniotic membrane have been demonstrated on ocular surface inflammation. In this work, we evaluated the effect of amniotic membrane-derived mesenchymal stem cells (A-MSC) over components of innate immune system and their possible use in the regenerative ocular therapy.

Methods: The A-MSC were enzimatically isolated from amniotic membrane and were cultivated in DMEM/F12 supplemented with 10% fetal bovine serum (FBS). The A-MSC were immunophenotyped by flow cytometry using anti-CD105, CD73, CD44, CD29 and CD45 antibodies. The in vitro tri-linage differentiation in hepatocytes, chondrocytes and neurons was performed with condicionated medium. The xenotrasplant of A-MSC was made in a murine corneal burn model with an ethanol solution (R-OH 99.6%). To determinate the effect of the A-MSC on the innate immune system we used peripherial polymorphonuclear cells isolated from peripheral blood (PBPMN) and we evaluated the formation of neutrophil extracellular traps (NETs) with PMA in the presence or absence of A-MSC supernatant (A-MSC SN).

Results: Cells with fibroblastic morphology and the ability to generate fibroblast-colony forming units were obtained. The phenotype of the cells was CD105+CD73+CD44+, CD29+ and CD45-, which suggest that these cells were mesenchymal cells. Their ability to differentiate into hepatocytes, chondrocytes and neurons was confirmed by the acquisition of albumin, collagen-II and nestin proteins, respectively. The xenotransplant of A-MSC significantly improved the transparency of the murine damaged corneas and induced the reduction of the inflammatory infiltrate. On the other hand the interaction of PBPMN with the supernatant of A-MSC decreased the NETs production.

Conclusions: These data suggest that A-MSC can be applied in the ocular regenerative therapy as an immunosuppressor treatment in ocular and inflammatory diseases.

Keywords: 482 cornea: epithelium • 555 immunomodulation/immunoregulation • 557 inflammation