The corneal epithelium is renewed by limbal stem cells (LSCs). Absence, damage or loss of the LSC population leads to the painful and blinding condition of LSC deficiency (LSCD). In vitro expansion of LSCs is an increasingly well recognized treatment modality for LSCD. The purpose of this study was to analyze the outgrowths from human cadaveric limbal explants cultured on cell culture inserts and denuded human amniotic membrane (dHAM) for properties associated with LSCs and to understand the human limbal stem-cell biology in vitro with and without 3T3 feeder cells. In particular, the expression of stem cell markers and colony-forming efficiency in different culture models were investigated. The research can be divided into three sections a) development of tissue culture models, b) translation of limbal-corneal cells towards clinical trial. c) Culture, and characterisation of limbal fibroblast-like cells (LFLc), The limbal explants were cultured, the outgrowths were measured, and colony-forming efficiencies (CFEs) of cells were calculated. In addition, the expression of LSC and corneal differentiation markers were assessed using immunofluorescence, western blot, and quantitative reverse transcription PCR (RT-PCR). Increased CFE in cells from explants grown on dHAM was observed. The expression of differentiation markers CK12, connexin 43 decreased in dHAM cultures, possibly indicating maintenance of the stem cell population. We demonstrate the outgrowths from human cadaveric limbal explants, in six different culture systems, show a steady increase in a wide range of stem cell properties with dHAM and 3T3 feeder cells compared to no-3T3 condition. These findings support the importance of dHAM and 3T3 feeder cells in maintaining the undifferentiated state of culture limbal epithelial stem cells. These findings suggest potential modifications of existing techniques to ensure maximum number of LSCs following in vitro expansion protocols, which might contribute to even greater success of subsequent engraftment. We developed a non-enzymatic method for isolation of LFLc, and demonstrated for the first time the invasion and motility of LFLc in vitro. These findings suggest an important role for LFLc in wound healing. LFLc have shown differentiation potential to adipo and osteogenic lineage.