Generally, the utility of primary cells is limited by their restricted lifetime (13). karyotyping was performed and expression of stem cell markers and cell cycleassociated genes was analysed. Results:MCCs, as primary cells, could be cultured for more than 60 passages. We observed Batyl alcohol increased cell proliferation and high number of colony forming units (CFUs) after extensive culture. Interestingly, there were no changes in expression of MCC markers. Furthermore, cell differentiation potentials remained comparable with MCCs at early passages. However, karyotyping revealed numeric chromosomal aberrations at Batyl alcohol higher passages. Moreover, tumour suppressor genes such asp16andp21were found to be downregulated after largescale cell culture. Conclusions:MCCs immortalize spontaneously after extensive cell culture, but still demonstrate stem celllike qualities. == Introduction == As the outermost surface of the eye, the cornea not only functions as a strong protective shield against external insults, but also provides optical function by transmitting and focusing light on the retina. It is comprised of three structurally unique and highly specialized cell layers the epithelium, stroma and endothelium whose homeostasis, deturgescence and integrity are essential for visual acuity. These layers are constituted of several different cell types including epithelia, endothelial cells and keratocytes, the last shaping the corneal stroma (1,2,3,4). Among these heterogeneous corneal cell populations, which all provide Rabbit polyclonal to CDC25C the cornea with its essential character, different types of stem or progenitor cells have been identified and the cornea has become of interest as an area of stem cell research (3,4,5,6,7,8,9). Stem cells have proven to be an intriguing source of tissues Batyl alcohol for use in cellbased therapy and corneal tissue engineering. Keratoplasty, currently the only effective method of providing recovery of vision after corneal blindness, raises many problems and there is significant interest in alternatives. Stem cells are promising tools for new therapeutic strategies, but to properly exploit the recent advances of corneal stem cell research, many questions remain to be answered (4,10). In a previous study, we have identified a neural crestderived corneal stemlike cell population (named murine corneal cells, MCCs). MCCs originate from the corneal limbus of juvenile mice before the stage of eyelid opening. They express a unique marker profile including typical neural crestoriginated stem cell transcripts such as Sca1 and other stem/progenitor cell markers such as Notch1, Nes, Abcg2 and Cd34. MCCs have further prove stem celllike attributes, such as cell migration, proliferation and, most interestingly, multipotency underin vitroconditions after differentiation into cells with features resembling adipocytes, osteoblasts and neuronal cells (11). To use MCCs in cellbased therapies of corneal tissue, high cell numbers are recommended. MCCs are somatic stemlike cells and have a limited lifespan that generally restricts their value for cellbased therapies. In our previous study, we observed a significant decrease in cell proliferation during the first cell passages although our investigations were conducted up to passage 10 (11). In the present study, we have focused on MCCs at later passages and investigated to what extent their subculture would be possible and whether they maintain their stem celllike qualities and genetic stability after extensive proliferation. == Materials and methods == == Isolation and cell culture == The MCCs were established from wildtype C57Bl6/J mice purchased from Charles River Laboratories (Wilmington, MA, USA). All animals were housed and handled in full accordance with institutional guidelines and killed at maximum 8 days of age. Cells were isolated as described previously (11). In brief, discs of whole corneas including the limbal area of at least five eyes were chopped into fine pieces, rinsed and collected in PBS (PAA, Pasching, Austria). After being centrifuged at 800gfor 2 min, tissue pellets were resuspended in growth medium, highglucose DMEM (4.5 g/l of glucose) withlglutamine (PAA), supplemented with 10% foetal Batyl alcohol bovine serum (PAA) and 1x penicillin/ streptomycin (PAA). Tissue pieces were subsequently seeded in 25 cm2culture flasks (Nunc, Denmark); they attached to culture dish surface and after 35 days, an exodus of single cells could be observed. Cultures were maintained in humidified air (5% CO2) at 37 C and medium was changed twice a week. Upon reaching confluence, cells were detached using trypsinEDTA (PAA) and again subcultured in growth medium, being plated at initial densities of 58 103cell/cm2. == Colonyforming unit assay == For measurement of colonyforming units (CFUs), MCCs were plated in.