Murine embryonic stem cells derived from the inner cell mass of mouse blastocysts can be maintained in culture for extended periods by using feeder layers and leukemia inhibitory factor (LIF). Maintenance of undifferentiated status occurs via LIF-mediated signalling pathways. In this study we cultured embryonic stem (ES) cells in Knockout-DMEM with serum replacement on a three-dimensional matrix, non-woven polyester fabric (NWPF), which is formed from non-arrayed polyethylene teraphthalate fibers. The surface of the fibers was modified by immobilizing LIF. While stimulating the matrix-bound form of LIF in vitro, we also tried to induce LIF-mediated signalling pathways continually. Our goal was to constitute a synthetic microenvironment that would support the undifferentiated growth of murine ES cells. Experimental groups were examined according to colony morphology, alkaline phosphatase activity, SSEA-1 antibody immunoreactivity, and SEM analyses. It was shown that three dimensional macroporous fibrous matrix, NWPF could support growth of undifferentiated ES cells. However, the ratio of undifferentiated colonies was higher on feeder layers than an polymeric surfaces (93% on mouse embryonic fibroblasts; 63,7% on hydrolized polymeric surface, P < 0,05). Results showed that LIF-immobilized surfaces supported undifferentiated growth of ES cells better than hydrolyzed surfaces. Colonies cultured on LIF-immobilized surfaces, had higher alkaline phosphatase activity and undifferentiated phenotype ratio than those on hydrolyzed surfaces. When the soluble or the matrix-bound form of LIF was used, the number of undifferentiated colonies increased in the polymeric groups (77.8% soluble LIF; 81.6% matrix bound LIF P < 0,05). On NWPF discs, ES cells formed big cell aggregates which had high alkaline phosphatase activity but low SSEA-1 immunoreactivity . When they were passaged to feeder layers, SSEA-1 activity increased. We managed to obtain undifferentiated colonies on NWPF discs by using LIF but the skeletal structure of polymeric matrix would be more convenient for differentiation studies.
This study was performed in TUBITAK-RIGEB and supported by a part of grant from Hacettepe University (0102601001).
169 CULTURE OF MURINE EMBRYONIC STEM CELLS ON NWPF DISCS
