Biological Characteristics Of Human Limbal Stem Cells Which Cultured On The Intact And Denuded Amniotic Membrane

ObjectiveThe integrity corneal epithelial function is important for maintaining the transparency of cornea. The corneal epithelium has the ability of self-renewing which is based on the regeneration of limbal stem cells. When limbal stem cells partial or total injured, this self-renewing function will be affected, even leads to blindness. Many studies show that we can culture the limbal stem cells in vitro and transplant them to treat LSCD.Many studies which use amniotic membranes as carrier have achieved a measure of success. According to these studies, we observed and compared the biological characteristics of human limbal stem cells which were cultured on the intact and denuded amniotic membranes.Material and method1. The source of eye ball and amniotic membraneThe amniotic membranes and eye balls are from the eye bank of the First Clinical College of China Medical University.2. Preparation of amniotic membrane According to Tseng's method, under sterile conditions, the membranes were obtained and stored at -80℃. Immediately before use, the amniotic membrane was thawed. Half of the amniotic membranes were deprived of their amniotic epithelial cells by incubating with trypsase.3. Culture of LSC and BrdU labelingSmall biopsy specimens of limbus were cultured on intact (I group) and denuded(D group) amniotic membrane. On the 7th day of culture, some limbal cells were incubated with a fresh medium containing 10μmol/L BrdU for another 7 days, and then they were chased for other days by switching to the BrdU free medium.4. ImmunostainingSections were fixed by 4% formaldehyde, then incubated with primary antibodies and FITC conjugated IgG as secondary antibodies at 37℃for 2h. The intact and denuded amniotic membrane was used as control group. The sections were examined and photographed under a fluorescent microscope. Under 100×magnification, positive results were counted in 3 fields at random.5. Scanning electron microscopeSections were fixed in 25g/L glutaraldehyde to prepare the sample.6. Statistics analysisMeans of positive rate were calculated. The differences between two groups were analyzed with non-parametric rank sum test by spss 11.5. A p value of less than 0.05 was considered significant.Result1. Culture of LSCsHuman limbal stem cells can be cultured on these two kinds of amniotic membrane. Scanning electron microscopy showed that there were cell-cell junctions between the cultured cells. 2. ImmunostainingPositive immunostaining of AE5 and Cx43 in I group were distinct lower than that in D group, but positive staining of BrdU and PCNA in I group were more than that in D group. Negative immunostaining of 4 antibodies in intact and denuded amniotic membrane control group.Conclusion1. The intact and denuded amniotic membranes are both helpful for growing of human limbal stem cells.2. The cells which cultured on the denuded amniotic membrane had already differentiated, while the intact amniotic membrane was considered to be better to retain the characteristics of the stem cell.