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Multiple approaches have been used to characterize and classify colony‐forming cell types. Specific antibodies to the intermediate filament components of the mammalian cell cytoskeleton⁵⁶⁴ provide the means for establishing tentative correlations between cell types in culture and their presumptive in vivo counterparts.⁵⁶⁵

A synopsis of the current classification of human AFCs in culture is provided in Table 3.7, which also summarizes criteria used for classification and the various nomenclatures to which they have led (for a more extensive compilation of the properties of AFCs, see the review by Gosden⁵⁶³). Morphologic criteria were applied first. They quickly led to the realization that a high degree of cytoplasmic and nuclear pleomorphism is the hallmark of cultured AFCs. In contrast to what is known from postnatally derived human skin fibroblast cultures, multinucleation is a frequent and distinctive feature of cultivated AFCs. One report describes 7 percent of AFCs having two nuclei and 1 percent showing three or more nuclei.⁵⁶⁶ Within the clonal progeny of a single AF specimen, considerably more cells appear to be of epithelial than fibroblast origin. A cell type that looks very much like a prototype fibroblast‐like cell at the individual cell level (Figure 3.4) was distinguished by Hoehn et al.⁵⁵⁶ from classic fibroblasts on the basis of its “bull's‐eye” colony pattern. Such a pattern is never observed with classic skin or embryonic lung fibroblasts. Figure 3.5 shows that the typical bull's‐eye colony pattern is displayed by epithelioid (E) and by AF‐type cells. The clonal pattern of F‐type cells exhibits a whorl‐like center and parallel arrays of spindle‐shaped cells. Since shapes of individual cells and clonal units are influenced by culture conditions, these features change during long‐term culture.⁵⁶⁷

Table 3.7 The classification of human second‐trimester amniotic fluid cells in culture (excluding RA cells)

Reference	Melancon et al. 593 Gerbie et al. 594	Sutherland et al.725	Hoehn et al. 556, 588	Priest et al. 566, 569, 571	Virtanen et al. 583	Cremer et al.584 Ochs et al. 587
Criteria	Morphology, enzyme production	Morphology, growth behavior	Morphology, clone patterns, longevity, cytogenetics	Collagen and gonadotropin production, ultrastructure	IIF, intermediate filaments	Intermediate filaments, prokeratin peptides

RA, rapidly adhering; AF, amniotic fluid‐specific; E, epithelioid; ED, epithelial and densely packed; ⁵⁶⁶ F, fibroblastoid; IIF, indirect immunofluorescence microscopy. Dotted lines indicate correspondence between the various nomenclatures (e.g. E3 corresponds to AF and E1).

See also review by Gosden. ⁵⁶³

Figure 3.4 Examples of living F‐, AF‐, and E‐type cells observed by phase‐contrast microscopy. Note the relative homogeneity of F‐type, in comparison to the pleomorphism of AF‐ and E‐type cells.

Figure 3.5 Examples of fixed colonies of F, AF and E clonal types at 2 weeks after plating. The AF‐ and E‐type colonies display typical “bull's‐eye” patterns. Compared with AF clones, the E‐type clones display wider growth margins around the darkly stained central core. The examples of AF and E clones are from primary platings of uncentrifuged amniotic fluid at 17 weeks gestational age. The F‐clone examples are subclones derived from a single F‐type primary clone isolated by a steel cloning cylinder and subsequent dilute plating on 2 × 3 inch glass slides. Crystal violet stain, 4/5 of actual size. Reproduced at 90 percent.