Study On Transdifferentiation Of Rat's Mesenchymal Stem Cells Into Corneal Epithelial Cells In Vitro

Background and AimCorneal stem cells are principally located at the sclerocorneal limbus, and are indispensable for the maintenance of a healthy corneal surface. Loss of limbal stem cells (LSC) can be partial or total. LSC deficiency is associated with persistent epithelial defects, vascularisation of the cornea, conjunctivalisation of the cornea, corneal ulceration and perforation, and band keratopathy. Autologous or allo-limbal transplantation are limited because of the insufficient source of LSC and immune rejection which resulted from allograft. The effect of amniotic membrane transplantation for ocular surface reconstruction is also limited in some serious limbal injuries due to lacking of stem cells. The development of tissue engineering brings the hope to solve these problems.Studies show that there are multi-potential mesenchymal stem cells (MSC) in the bone marrow of human, which can be induced into cardiac muscle cells, fat cells, endothelium cells, neural cells, bone and bone marrow cells, etc. MSC originates accurately, sufficiently, and has great differentiate potential. Moreover, it can avoid some ethics questions which the embryo stem cells (ES) probably faced, thus, it has a better application prospect. The corneal stroma is a microenvironment of corneal epithelial cells and LSCs, which maintains the corneal epithelial cell's characteristic.This research aimed to culture and expand MSC in vitro by using tissue engineering technology, and explore the differentiation potential of transdifferentiation of mesenchymal stem cells (MSC) into corneal epithelial cells, and to discuss the plasticity that make MSC be the seed cell in corneal tissue engineering.Method(1) MSC of adult rats that were isolated and purified by density gradient centrifugation combined with an attachment culture method.(2) Observe the morphology of MSCs which were cultured in vitro, then the cells were identified by immunohistochemistry method.(3) The transdifferentiation of MSC into corneal epithelial cells was induced in vitro by co-cultured with corneal stromal fibroblasts. The expression of CK12 on MSC, which is a marker for corneal epithelial cells, was identified by immunofluorescent staining.ResultsMSC can be cultured and expanded in vitro, which showed great potential of proliferation. Immunocytochemistry showed MSC were positive for CD29, CD44, while negative for CD34 and CD45. The positive rate of CD29 was 81.56 %, CD34 was 0.10 %, CD44 was 88.77 %, CD45 was 0.17 %,CD71 was 10.02%, CD90 was98.43, CD133 was1.56% by flow cytometry. These results indicated that the cultured cells were MSC. One week after co-cultured with corneal stromal fibroblasts, most of MSC differentiated into stroma cells, part of MSC were smaller comparatively and expressed CK12, which indicated they had been transdifferentiated into corneal epithelial cells.ConclusionThe data suggest that MSCs have the potential to transdifferentiate into corneal epithelial cells in vitro, and it can be the option of seed cells in corneal epithelial tissue engineering.