Figure 4. Analysis of spheres and their progeny. A: Immunocytochemical analysis of sphere colonies on day 7. Bright-field images and immunostaining of spheres for p63 (an epidermal stem/progenitor cell marker), p75^NTR (an epidermal basal progenitor cell marker), cytokeratins 3 and 12 (differentiated epithelial cell markers, nestin (a neural stem cell marker), microtubule-associated protein 2 (MAP2: a differentiated neuronal cell marker), and neuron-specific enolase (NSE: a differentiated neuronal cell marker). Each colony has been labeled by BrdU. As a control, IgG was used instead of the primary antibody. Scale bar=100 µm. B: Double immunocytochemical staining of a sphere colony. The spheres are double immunostained by nestin and cytokeratin 12 or by p63 and alpha smooth muscle actin (αSMA). Scale bar=100 µm. C: RT–PCR of corneal epithelial tissues, spheres, and their progeny. Genes for P63, keratin 3, keratin 12, and nestin are present in corneal epithelial tissues, spheres, and their progeny derived from the limbal or central regions, but not in total RNA processed without reverse-transcription. D: Immunocytochemical analysis of differentiated cells obtained from spheres. Cells migrating out of the spheres express both cytokeratin 3 and cytokeratin 12 (differentiated epithelial cell markers). These cells are also positive for MAP2, and NSE. Scale bar=100 µm.