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

John J Wille

.

DOI: 10.4236/ns.2011.38094                

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.

Keywords

Biochemical Oscillators; Clonal Growth Dynamics; Human Keratinocytes; INTERDIVISION Times; Temporal Control of Mitosis Models

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Riley, P.A. and Hola M. (1980) Clonal differences in generation times of GPK epithelial cells in monolayer culture, Experimental Cell Biology, 48, 310-320.
[2]	Kitano, Y., Nagase, N., Okada, N. and Okano, M. (1982) Cinemicrograhic study of cell proliferation patterns and interdivision times of human keratinocytes in primary culture, Normal and Abnormal Epidermal Differentiation, University of Tokyo Press, Tokyo, 97-108.
[3]	Barrandon, Y. and Green, H. (1985) Cell size as a determinant of the clone-forming ability of human keratinocytes, Proceedings of the National Academy of Sciences of the United States of America, 82, 5390-5394.doi.org/10.1073/pnas.82.16.5390
[4]	Barrandon, Y. and Green H. (1987) Three clonal types of keratinocytes with different capacities for multiplication, Proceedings of the National Academy of Sciences of the United States of Amrica, 84, 2302-2306. doi.org/10.1073/pnas.84.8.2302
[5]	Wille, J.J. (1999) Heterogeneity in clonal potential and life histories in individual keratinocytes propagated in serum-free medium, Journal of Inventigative Dermatology, 112, 573.
[6]	Wille, J.J., Pittelkow, M.R., Shipley, G.R. and Scott, R.E. (1984) Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: clonal analyses, growth kinetics, and cell cycle studies, Journal of Celluar Physiology, 121, 31-44. doi.org/10.1002/jcp.1041210106
[7]	Sheffey, C. and Wille, J.J. (1978) Cycloheximide-induced mitotic delay in Physarum polycephalum, Experimental Cell Research, 113, 259-262. doi.org/10.1016/0014-4827(78)90365-8
[8]	Bass, J. and Takahashi, J.S. (2010) Circadian integration of metabolism and energetic, Science, 330, 1349-1354. doi.org/10.1126/science.1195027
[9]	Thompson, D.W. (1943) On growth and form. Cambridge University press, New York.
[10]	Hofstadter, D.R. (1980) Godel, esher, bach: An eternal golden braid. Chapter V: Recursive Structures and Processes, Vintage Books, A Division of Random House, New York, 131-137.
[11]	Kauffman, S.A. and Wille, J.J. (1975) The mitotic oscillator in Physarum Polycephalum, Journal Theory Biology, 55, 47-93. doi.org/10.1016/S0022-5193(75)80108-1.
[12]	Barnett, A. Ehret, C. and Wille, J.J. (1969) Testing the chronic theory of circadian timekeeping, Biochronometry, Proceeding of the Symposium on Biochronometry, Friday Harbor, Washington, D.C.
[13]	C.F. Ehret and E.Trucco E, (1967) “Molecular models for the circadian clock. I. the chronic concept,” Journal Theory Biology, 15, 240-262. doi.org/10.1016/0022-5193(67)90206-8
[14]	Glass, L. and Mackey, M. (1988) From Clocks to Chaos. The Rhythms of Life, Chapter 2, Princeton University Press, Princeton.
[15]	Tyson, J.J. and Novak, B. (2008) Temporal organization of the cell cycle, Current Biology, 18, R759-R768.doi.org/10.1016/j.cub.2008.07.001
[16]	Novak, B. and Tyson, J.J. (2008) Design principles of biochemical oscillators, Nature Reviews, 9, 981-991. doi.org/10.1038/nrm2530
[17]	Brooks, R.F. Bennett, D.C. and Smith, J.A. (1980) Mammalian cell cycles need two random transitions, Cell, 19, 493-504. doi.org/10.1016/0092-8674(80)90524-3
[18]	Wille, J.J. and Scott, R.E. (1984) Cell cycle-dependent integrated control of cell proliferation and differentiation in normal and neoplastic mammalian cells, Cell Cycle Clocks, New York, 433-453.
[19]	Tyson, J.J. and Hannsgen, K.B. (1985) The distributions of cell size and generation time in a model of the cell cycle incorporating size control and random transitions, Journal Theory Biology, 113, 29-62. doi.org/10.1016/S0022-5193(85)80074-6
[20]	Kar, S. Baumann, W.T. Paul, M.R. and Tyson, J.J. (2009) Exploring the roles of noise in the eukaryotic cell cycle, http://www.pnas.org/cgi/doi/10.1073/pnas.0819934106
[21]	Mackey, M.C. Santavy, M. and Selepova, P. (1986) A mitotic oscillator model for cell cycle with a strange attractor, Non-linear Oscillations in Biology and Chemistry, Springer-Verlag, Berlin, 354-454.
[22]	Schafer, E. and Cleffmann, G. (1982) Division and growth kinetics of the division mutant “conical” of Tetrahymena, Experimental Cell Research, 137, 277-284.
[23]	Zhang, R. Aguila, D. Schneider, C. and Schneider, B.L. (2005) The importance of being big, Journal Invest Dermatol Symp Products, 10, 131-140. doi.org/10.1111/j.1087-0024.2005.200414.x
[24]	Mitchison, M. (1971) The Biology of the Cell Cycle, Cambridge University press, London.
[25]	Kubitschek, H.W.E. (1970) Evidence for the generality of linear cell growth, Journal Theoretic Biology, 28, 15-29. doi.org/10.1016/0022-5193(70)90061-5.
[26]	Baserga R., (1976) Multiplication and Division in Mammalian cells, Marcel Dekker, New York