Abstract
BackgroundCorneal endothelial dysfunction causes severe impairment of vision. The only solution is corneal transplantation. However, this treatment is hampered by a worldwide shortage of donor corneas. New therapies may replace the conventional donor corneal transplantation alongside the developments in regenerative medicine and tissue engineering, but sufficient functional corneal endothelial cells (CECs) are essential. The aim of this study was to promote the expansion and function of human corneal endothelial cells (HCECs) in vitro and in vivo.MethodsThe phenotypes of human orbital adipose-derived stem cells (OASCs) were detected by flow cytometry and immunofluorescence. HCECs were isolated and cultured using a conditioned medium obtained from OASCs (OASC-CM) in vitro. Related cell markers of HCECs were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence. The cell counting kit-8 (CCK-8) assay and the wound healing assay were performed to evaluate the proliferation ability of the cells. The cultured HCECs were then transplanted into rabbit and monkey corneal endothelial dysfunction models by cell injection.ResultsCD29, CD105, CD49e, CD166, and vimentin were highly expressed in cultured human OASCs. The CEC-relative markers zonula occludens-1 (ZO-1), Na+/K+ ATPase, N-cadherin, Col8a2, and SLC4A4 were expressed in HCECs cultured by OASC-CM. The HCECs were able to maintain polygonal cell morphology and good proliferative capacity. In animal experiments, corneal transparency was achieved after the injection of HCECs, which demonstrated the good repair capacity of the cells.ConclusionsThe proliferation abilities of the cells were significantly enhanced, and related functional markers were strongly positive, while HCEC morphology was maintained using OASC-CM. HCECs obtained some stem cell-like properties. This preclinical study confirmed the therapeutic ability of the HCECs in vivo. Our findings demonstrated that cultured HCECs with OASC-CM might be a promising source for research and clinical treatment.
Highlights
Corneal endothelial dysfunction causes severe impairment of vision
Immunofluorescence staining showed that Orbital adipose-derived stem cell (OASC) strongly expressed vimentin in the cell plasma which is a kind of intermediate filament protein found in normal Human corneal endothelial cell (HCEC) (Fig. 1c) [22]
Under phase-contrast microscopy, the cultured primary HCECs passage 0 (P0) had a mosaic pattern (Fig. 1f), but after 4–5 passages cultured in basal culture medium (BM) HCECs began a fibroblastic change, became larger with vacuoles, and showed endothelial-to-mesenchymal transition (EMT)
Summary
Corneal endothelial dysfunction causes severe impairment of vision. This treatment is hampered by a worldwide shortage of donor corneas. New therapies may replace the conventional donor corneal transplantation alongside the developments in regenerative medicine and tissue engineering, but sufficient functional corneal endothelial cells (CECs) are essential. The aim of this study was to promote the expansion and function of human corneal endothelial cells (HCECs) in vitro and in vivo. Corneal endothelial cells (CECs) have limited proliferative capacity in vivo [2,3,4]. The treatment relies on a sufficient number of functional human corneal endothelial cells (HCECs). The worldwide shortage of transplantable donor corneal tissues remains a big problem [8]. There is a pressing need to find optimum protocols to expand HCECs in vitro since the procedures involved in the isolation and subsequent cultivation protocols greatly vary between laboratories [13,14,15,16]
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