Abstract
The aim of this study was to investigate the feasibility of inducing rat neural crest cells (NCC) to differentiate to functional corneal endothelial cell (CEC)-like cells in vitro. Rat NCC were induced with adult CEC-derived conditioned medium. Immunofluorescence, flow cytometry and real time RT-PCR assay were used to detect expression of the corneal endothelium differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2. CFDA SE-labeled CEC-like cells were transplanted to the corneal endothelium of a rat corneal endothelium deficiency model, and an eye-down position was maintained for 24 hours to allow cell attachment. The animals were observed for as long as 2 months after surgery and underwent clinical and histological examination. Spindle-like NCC turned to polygonal CEC-like after induction and expressed N-cadherin, FoxC1, Pitx2, zonula occludens-1 and sodium-potassium pump Na+/K+ ATPase. The corneas of the experimental group were much clearer than those of the control group and the mean corneal thickness in the experimental group was significantly less than in the control group7, 14, 21 and 28 days after surgery. Confocal microscopy through focusing and histological analysis confirmed that green fluorescence-positive CEC-like cells formed a monolayer covering the Descemet’s membrane in the experimental group. In conclusion, CEC-like cells derived from NCCs displayed characters of native CEC, and the induction protocol provides guidance for future human CEC induction from NCC.
Highlights
Tissue engineering of the cornea has been presented as a promising opportunity for overcoming the limitations of conventional corneal replacement, such as shortage of healthy donor corneas and possible allograft rejection [1,2,3,4]
We showed in this study that conditioned medium (CM) derived from corneal endothelial cells (CEC) combining with serum could induce rat neural crest cells (NCC) to differentiate into CEC-like cells, and we tested the effect of CEC-like cell transplantation in a rat model of corneal endothelium deficiency
Low-affinity neurotrophin receptor P75 and the HNK-1 epitope have been used as general markers for neural crest cells, we co-immunostained the cells for both P75 and HNK-1 [18,19,20,21]
Summary
Tissue engineering of the cornea has been presented as a promising opportunity for overcoming the limitations of conventional corneal replacement, such as shortage of healthy donor corneas and possible allograft rejection [1,2,3,4]. It is known that both human and rodent CEC originate from neural crest cells (NCC) [6,7,8,9,10,11]. The neural crest is a transient cell population in the early stage of embryonic development, which gives rise to many kinds of tissues and cell types. At present NCC can be acquired from fetus, and from adult skin, hair follicle and even embryonic stem cell-induction in both human and rodent [12,13,14,15,16,17]. Direct precursor of CEC, multiple differentiation ability in vitro and extended sources make NCC a candidate cell source for CEC induction
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