Sphere-forming cells from peripheral cornea represent a potential source of progenitor cells for treatment of corneal degenerative diseases. Control of cellular repopulation on transplantable substrates is important to prevent uncontrolled growth in unfavourable directions. The coordination of cellular outgrowth may be in response to environmental cues and/or cellular signals from other spheres. To investigate this, cell migration patterns were observed following placement of spheres on an adhesive surface. Human peripheral corneal cells were maintained using a sphere-forming assay and their behaviour on collagen substrate recorded by time-lapse imaging. Immunocytochemistry and proliferation assays were used to detect protein expression and cell division. Proliferation assays showed that spheres formed by a combination of cell division and aggregation. Cell division continued within spheres for up to 4 months and was up-regulated when exposed to differentiation medium and collagen substrate. The spheres expressed both epithelial and stromal cell markers. When exposed to collagen; (1) 25% of the spheres showed spontaneous polarised outgrowth. (2) One sphere initially showed polarised outgrowth followed by collective migration with discrete morphological changes to form leading and trailing compartments. (3) A sphere which did not show polarised outgrowth was also capable of collective migration using cell protrusion and retraction. (4) Active recruitment of cells into spheres was observed. (5) Placement of spheres in close proximity led to production of a cell exclusion area adjacent to spheres. Thus peripheral corneal cell spheres are dynamic entities capable of developing polarity and modifying migration in response to their environment.