Constructive And Transplantated Studies Of Goat Limbal Stem Cell (Venus) Strain

The integrity and transparency of cornea plays a key role in vision. Limbal Stem Cells (LSCs) are precurors of cornea, which are responsible for self-renewal and replenishing corneal epithelium. Though it is successful to cell replacement theapy for impair ocular surface by Limbal Stem Cell Transplantation (LSCT), the mechanism of renew is unclear after LSCT. To real time follow-up the migration and differentiation of corneal transplanted epithelial cells afer transplanting, we screened a goat limbal stem cell strain (GLSC-V) which labeled Venus gene with fluorescent protein by G418, and cultivated the cells on denude amnion membrane to reconstruct fluorescent corneal epithelial grafts, then transplanted the fluorescent corneal epithelial grafts onto limbal stem cell deficiency (LSCD) models. We investigated the trace of GLSC-V on the reconstructed ocular surfaces by a series of methods. This provide a foundation for the studies on the mechanisms of corneal epithelial cell regeneration after LSCT. The details of the studies following as:1.The islotion, culture and identification of GLSCsWe detached limbal biopsy tissue from goat ocular surface in vitro, and separated the epithelium into single cells by using Dispase II and trypsin in order. The cells were adhered on Collagen IV-coated dishes for 20 min to enrich partially GLSCs. The specific growth property of GLSCs cultured in vitro were similar to other mammalian LSCs. Furthermore, GLSCs were undifferentiated and had high proliferative capacity. These results showed that GLSCs could be used for transgenic study.2.Establishment of GLSC-V strainWe transfected venus (a fluorescent protein gene) into goat LSCs, selected with G418 and established a stable transfected cell line, named GLSC-V. These cells showed green fluorescence, and which could maintain for at least 3 months. Immunofluorescent staining and semiquantitative RT-PCR results showed that the physical property of GLSC-V were similar to untransfected GLSCs. This demonstrated that GLSC-V could be as seeding cells for constructing corneal epithelial grafts.3.Transplatation and detection of constructed fluorescent epithelial graft with GLSC-VWe seeded GLSC-V onto denude amniotic membrane, and incubated the cells up to 15~21 days until formed multiple cellular strata. By transmission electron microscope observation, the fluorescent corneal epithelial cells had more desmosomal junctions and the cells were riched in tonofilament surrounding cellular nucleus. The fluorescent corneal epithelial sheets showed multiple cellular strata by H&E staining and the lowest basal cells expressed P63 that were similar to normal corneal epithelium by using immunofluorescent staining.We transplanted the fluorescent corneal epithelial grafts onto limbal stem cell deficiency (LSCD) models and observated the models for 4 months after operation. Afer 4 months, 67% LSCD models (2/3) were transplanted with fluorescent corneal epithelial graft got transparent basically, and 33% corneal epithelium (1/3) reconstructed partially. The negative control was entirely covered with blood vessels and conjunctiva. The all reconstructed ocular surfaces got transparent were originated from the nasal and temporal limbus. Although reconstructed corneal epithelium showed multiple cellular strata was similar to normal cornea, the reconstructed cellular strata were distributed on the stroma unevenly. The cells shape were irregular in the unrepaired areas of reconstructed central corneal epithelium. Immunofluorescent staining analysis that reconstructed superficial corneal epithelial cells were differentiated. Stem cells were distributed among different areas of reconstructed limbal epithelium. A few of stem cells were existed in basal and middle cells of normal central epithelium, however, there were a lot of stem cells in reconstructed central epithelium.We founded venus not only was expressed in reconstructed corneal epithelium, but conjunctival epithelium, and venus expression of reconstructed central epithelium more than limbal epithelium.We supposed the mechanisms of corneal epithelial cells therapy that corneal epithelial cells not only exsit in the reconstructed corneal epithelium after LSCT, but migrate into conjunctival epithelium. Donor cells reconstruct the superficial corneal epithelium mainly, and don't be replaced by recipient cells for long-term. The high proliferative cells of donor and recipient cells participate in repairing the damaged goat cornea in chorus. In the wake of repairing corneal epithelium, donor cells are replaced by recipient cells gradually, so it is happened that repairing and renewing coreal epithelium are concurrent after LSCT.