Objective: Limbal stem cells (LSC) have a prominent role in regeneration of cornea epithelial cells and have been applied in treatment of a vision disabling pathological condition known as limbal stem cell deficiency (LSCD). One the applied methodology is to culture LSCs followed by transplantation on patient’s corneal surface. But this method has the non-deniable disadvantage of premature differentiation of stem cells. Amniotic membrane (AM) can support a convenient nurturing condition for LSCs, because of its adhesive and nutrimental properties. In the current study we focused on separation of LSCs and culturing them on AM in order to stop their premature differentiation before corneal transplantation. Materials and Methods: Slices of cornea were obtained from eye bank and after being transferred to the lab, following procedure were applied to them: washing with PBS followed by immersing in Collagenase for 30 minutes and rewashing, cutting the digested tissue into very small pieces, incubation of slices for 48-72 hours in DMEM media containing 10% FBS and appropriate antibiotics, separating colony of cells by enzymatic digestion followed by transferring them onto AM surface. Results: Cultured cells were morphologically confirmed to be undifferentiated limbal stem cells. After 3rd passage the cells were transferred on amniotic membrane surface. The cells formed colonies on amniotic membrane surface and also stayed undifferentiated. Conclusion: Our results suggest for the achievement of transferring and culturing of LSCs onto the amniotic surface. This study is planned to be followed by transplanting these cells on corneal surface and differentiating them into epithelial cells as an approach for treatment of LSCD. For further evaluations, we suggest to study the cell surface markers to confirm that the cultured cells are undifferentiated limbal stem cells.