FIG. 6.

Loss of clonal growth and outgrowth by disrupting close contact with niche cells by T/E. In defined keratinocyte serum-free medium, single cells derived from dispase-isolated sheets (37°C/2 h) by T/E did not generate any clonal growth as judged by crystal violet staining on day 10 in six-well plates, unless 3T3 fibroblast feeder layers were added (A). Similarly, single cells derived from the remaining stroma released by collagenase and treated with T/E (Fig. 1) also did not generate any clonal growth unless 3T3 fibroblast feeder layers were added (B, T/E+). In contrast, cells released from the remaining stroma by collagenase without T/E, which showed epithelial clusters associated with nonepithelial cells (Fig. 1), could generate vivid clonal growth without 3T3 fibroblast feeder layers (B, T/E−). Clones generated on 3T3 fibroblast feeder layers consisted of a monolayer of epithelial cells, whereas those without 3T3 feeder layers exhibited compacted small cells with scattered mounts (B, Phase). In SHEM, an outgrowth on dAM inserts in six-well plates, judged by crystal violet staining on day 7, was consistently generated by collagenase-isolated clusters without T/E (C-T/E) but abolished with T/E (C+T/E), and to a lesser extent by dispase-isolated sheets without T/E (D-T/E) (C, two representative segments from the same donor). Using more limbal segments from different donors, C-T/E gave an overall success rate of 84% and achieved a round shape outgrowth within the diameter of 19.9 ± 2.7 mm in 7 days. In contrast, C+T/E gave a success rate of 11% and no measurable diameter, whereas D-T/E yielded a 46% success rate and generated an uneven round shape with a smaller diameter of 12.3 ± 3.6 mm (D, p < 0.05). Scale bar = 100 μm in (B). dAM, denuded amniotic membrane. Color images available online at www.liebertonline.com/tec