Alkaline phosphatase “constitutive” clones: Evidence for de-novo heterogeneity of established human skin fibroblast strains

Abstract

Histochemical stains for alkaline phosphatase were performed on colonies derived from mass cultures of six strains of human fibroblasts grown under conditions of relative repression of the enzyme. Under these conditions, alkaline phosphatase “constitutive” clones (> 16 cells) appeared with frequencies varying from 0.38 × 10 −3 to 19.1 × 10 −3, depending upon the strain of fibroblast, its passage number, the nature of the cloning environment and the mode of scoring. Not a single constitutive clone was found among 52,457 sub-colonies from two strains with comparatively high frequencies of constitutive clones. Evidence is discussed which suggests that there is selection against constitutive clones during prolonged in vitro culture. Comparatively large numbers of such cells can be observed histochemically among the early migrating cells around an explant. These results indicate a de-novo hetero-geneity of established “diploid human fibroblast” cultures. In vivo histochemical studies suggest that the constitutive cells are endothelial cells. The constitutive clones of “fibroblasts” may therefore represent morphologically transformed endothelial cells which undergo replication in the presence of fibroblasts. Comparatively high frequencies of such clones can be found for at least 23 cell doublings after a culture has been established. In view of the observed heterogeneity, it is concluded that quantitative somatic cell genetic studies with such material require prior purification of the strains by cloning.