Identification of subsets of cells with stem-like properties in cells derived from normal and malignant human epithelia. Holoclone (h), meroclone (m) and paraclone (p) colonies formed by CaLH3 (A) and Ca1 (D) cell lines viewed by phase contrast microscopy and after staining for CD44 (B and E) illustrate the small, refractile, tightly-packed holoclone cells and their strong cell surface staining for CD44. Representative FACS plots for CaLH3 and CA1 cell lines (C and F), two specimens of HNSCC (G, H), and normal oral keratinocytes (NOK) (I). For HNSCC and Ca1 the subpopulation of CD44high formed quite distinct populations, but CaLH3 and NOK showed a more continuous distribution and in this case the 3-5% of cells with the highest CD44 expression was arbitrarily selected as being the subpopulation of CD44high cells. Representative images of cells plated after FACS sorting for CD44 expression in CaLH3 and Ca1 cell lines (J and K) and early (p2) NOK cultures (L). The subpopulation of CD44high cells (left flask in each panel) showed greater clonogenicity than the subpopulation of CD44low cells in both normal and neoplastic-derived cells. Cell lines formed tumour spheres when growing in soft agar (M - representative image of MCF7 cell line), but the CD44high cell fractions formed a significantly larger proportion of spheres than the CD44low fraction when seeded at clonal density in soft agar (N) or when plated as single cells in culture medium in non-adherent plates (O).