Growth dynamics of individual clones of normal human keratinocytes: observations and theoretical considerations

Abstract

The life histories of 429 individual epidermal keratinocyte clones picked at random were studied. Individual basal keratinocytes were derived from asynchronous rapidly proliferating subconfluent cultures propagated in either a low calcium (0.1mM) or a high calcium (2mM) serum-free medium. Single-celled clones were isolated by seeding trypsin-EDTA dissociated cells into a Petri dish containing cloning chips. Chips with only one cell per chip were transferred into dishes containing either low calcium or high calcium growth factor replete serum-free medium. Clone formation was monitored microscopically and the number of cells in each colony tallied at least twice daily for further analysis. A total of 369 clones were established from seven different neonatal foreskin cell strains (A-F), and 60 clones were derived from one adult human skin cell strain (G). During a five-day culture interval, among 32 clones of strain A, 83% divided at least once, 50% divided once in 24 hours, 86% divided at least three times within three days, and more than 50% divided at least four to five times in five days. Of 231 clones amongst the other five cell strains (B-F), an average of 63% (±12 S,E) divided more than three times in an eight day period, the remainder divided either once, twice or not at all. Of the 106 clones of strain G, reared in high calcium serum-free medium, 67% divided more than three times in a six-day period, and 55% divided five or more times in 6 days. Clones derived from adult skin strain H had a lower clone forming potential with 70% dividing at least once in seven days, and only 30% dividing three or more times. By contrast, the average generation time (AvGT) for second and third passage keratinocytes derived from neonatal foreskin cultures was 24 hrs. Detailed dendrograms were constructed for many of the proliferating clones. The majority of clones expressed a synblastic division pattern with every cell dividing at least once per day. A fraction of clones either exceeded this circadian division rate or displayed a biphasic division pattern with all cells initially dividing once a day and then abruptly slowing to once every other day or to an intermediate rate. A minority of clones was committed to a few terminal divisions. The division patterns of the non-synblastic clones fit an alternating bifurcated branching mode of clonal expansion expressed by the Fibonacci sequence for numbers of accumulated cells per clone per day. These results were analyzed in terms of deterministic, probabilistic and a limit cycle oscillator models of cell division timing

Occurrence of Fibonacci numbers in development and structure of animal forms: Phylogenetic observations and epigenetic significance

“…The existence of such a clock whose temporal periodicity can be translated spatially into periodic boundaries of somites was originally proposed in theoretical models [31]. We have proposed a similar biochemical anharmonic oscillator in cells [32] and as an explanation for the phenomenon of Fibonacci numbers in the development of staggered cell cycles during clonal growth of normal human epidermal keratrinocytes undergoing commitment to terminal differentiation [33]. The existence of cyclic genes in the segmentation clock has now been identified in fish, frog, chick and mouse embryos [34].…”

“…A particular example of this correspondence was reported earlier [33]. A proliferating normal human clone of epidermal keratinocytes committed to terminal differentiation adopts a pattern of division in which two different branches of the binary division tree diverge with one of the two daughter cells at each nodal division time dividing on time, while its sister cells fails to divide, but resumes division exactly one cycle later (Modulo 1).…”

“…Whatever, the exact role of cycle genes are that set clonal division boundaries will have to await further dissection of developmental events that are associated with clonal division mechanics as proposed here, and schematically diagrammed by the hypothetical dendrogram of committed epidermal keratinocyte clones [33]. In summary, our survey of zoological literature has affirmed the wide occurrence of Fibonacci numbers in the organization of acellular and prokaryotic life forms as well as in some eukaryotic protistans and in the embryonic development and adult forms of many living and fossil remains of metazoan animals.…”