Clonal variation and aging of diploid fibroblasts

Experimental Cell Research 103 (1976) 247-255

CLONAL

VARIATION

AND AGING

OF DIPLQID

FIBROBLASTS Cinematographic P. MARLENE

University

of Vermont,

Studies of Cell Pedigrees ABSHER

College

and R. G. ABSHER

of Medicine,

Burlington,

VT 05401 USA

SUMMARY Time-lapse cinematographic (TLC) analysis of clones of human diploid libl-obla\t\ indicatlc heterogeneity in clonal division bchaviour. Variations arc noted in intcrdivision time. clone kc and generations per clone. Correlation coefficients for interdivision time\ of’ sister pairs are high in young clones and generally low in aged clones. A consistent division pattern at all population doubling levels ih one of low average intcrdivision time for earl); and late generation\ of a clone and high avcragc intcrdivision time for the middle range of gencratron\ of a clone. The clonal di\ ision patterns observed experimentall! have been duplicated in computer simulated pcdigrccb. The computer model is based on an oscillating system which alto\\\ i‘or t111x ol’ regulator wbstance\. The critical concentrations of regulator substances determine the clonal diviblon pattern for a g!\ cn progenitor cell

Throughout

the lifespan

of cultured

diploid

fibroblasts. variations in several cell characteristics occur. and thcsc variations tend to become more pronounced as the cultures age. Variations have been reported in cell size [l-5], cycling time [4-71, migration [4. 51. morphology [2-S. 8, 91, and clone size distribution [7. 81. As fibroblasts in culture age there is a dcclinc in cloning efficiency [ 10. 111, in the proportion of dividing cells in a population [2? 6. IO, 121 and in the ability of the cells to incorporate [“H]TdR into nuclear DNA [ 13j. To assess variations in beha\,iour of individual fibroblast cells. the technique 01 time-lapse cinematography (TLC) has been used. Cell lineage studies reported by Absher et al. [4. 51 illustrated directly maria(ions in intcrdivision time (IDI‘). mor17-7hlXll

phology. and motility of WI38 human cmbryonic lung fihroblasts at population doubling Icvcls of 20-M. We are nw reporting further TLC studies of WI38 fibroblasts at early and late population doubling levels. New findings for di\)ision paiterns deriving from cell lineage data that are chili-acteristic of the proliferative bchaviour oi these cells will be discussed. An integral part of the diploid fibroblas? lineage studies i> ;I mathematical model that has been developed and used to generate computer simulations of the clonai pcdimodel i\ based upon grccs. The computer an oscillating regulatory mechanism which allows for flux of a substance or suhst:incc\. the critical concentration of uhich modulates nnt only interdivision times Of clon,il progeny. but also wntrols Ihe ad\;eu: 01‘

248

Absher and Absher non-dividing cells arising during the growth of a clone. The computer simulations based upon this model exhibit division behaviour similar to clones in the experimentally derived lineages. MATERIALS

Stock cultures of human embryonic lung fibroblasts, strain W138, were propagated in Eagle’s Basal Medium (EBME) supplemented with 10% fetal calf serum and 50 pg aureomycin per ml. Cells were subcultivated at a 1 : 2 split ratio using trypsinization to disperse the monolayer. Hemocytometer counts were made at each subcultivation to calculate actual population doubling levels. Stock cultures were grown in an atmosphere of 5% CO*, 95% air at 37°C in a Wedco incubator. To prepare cultures for filming, approx. 2~ 10’ cells in 8 ml of cloning medium were seeded into a 60 mm Falcon tissue culture dish fitted with a Cooper-type lid. Cloning medium consisted of equal parts of fresh medium and medium taken from the donor culture at confluency. This medium was selected because it enhanced cloning efficiency at the low seeding densities used and resulted in proliferation of approximately twice the number of cells grown in the presence of fresh medium alone. To prevent loss of cells due to migration out of the photographic field, areas of approx. 0.25430 mm? were bordered with sterile Dow-Coming high vacuum grease. The culture chamber was sealed with sterile Vaseline and filming was begun after the cells had attached to the surface of the vessel; usually l-2 h after seeding. The cinematography equipment consists of a Sage model 501 time-lapse unit, 16 mm Bolex camera, Zeiss GFL microscope with long working distance condensor and 10x phase contrast objective. The complete system is housed in a room kept at 37°C. Cell cultures were filmed at a rate of 1 frame/min using Kodak Plus X reversal film. Analysis of films utilizes a manually operated film editor and a Kodak Analyst projector. At the termination of each experiment (usually 9 days) the cells in the chamber were rinsed in PBS, fixed in methanol and stained with Giemsa. Field counts of cells growing within and outside of the silicone-bordered areas were made to determine whether exposure to silicone had any inhibitory effects upon the growth of WI38 fibroblasts. To compare growth of

a

Table 1. Clone size (N,) and maximum generation (GF) offour WI38 clones

b I 0

AND METHODS

1 20

1 40

1 LO

1 80

1 1 IO0 no

1 I I 1 140 160 180 200

Fig. 1. Abscissa: time in culture (hours). Pedigrees of two WI38 clones, PDL 26, cultured together. Numbers on lines indicate interdivision time in hours. L, cells that leave photographic field; 0, large cells. (a) Clone 1; (b) clone 2. Exp CellRes 103 (1576)

Clone

NF

GF

PDL PDL PDL PDL

II3 17 57 30

10

26 no. I 26 no. 2 29 51

ii 7

Clonai 1:arktion ad ngifzg

249

number of generations), mother : daughter and sister : sister relationships. and cell size and morphology. The final clone size (N,) and maximum generation numbers (Gi) for these clones are shown in table I and illustrate two aspects of the heterogeneity in prolifemtive capacity of WI38 fibroblasts. From these, as well as other clones that have been studied, the final cell number and maximum generations achieved is highly variable and does not appear to be directly reiated io whether growth was from an isolated cell or from a small population. As an example, the two clones shown in fig. la and b were grown together. The observation t two clones had differing interdivision times and proliferative capacities suggests that cell cycle regulation is an intrinsic I;rOpeiTJ$ of an individual cell, though it may be / I I 1 I I I I 1 / modulated by environmentai conditions, in0 20 lo 60 80 100 120 140 160 180 200 cluding other cells. Fig. 2. Abscissa: time in culture (hours). Pedigree of WI38 clone. PDL 29. Numbers on lines Of perhaps the greatest interest and sigindicate interdivision time in hours; 0. large cells. nificance in terms of celP cycls regula.tion and clonal proliferation is the fktuation in interdivision times from generation to genthe WI38 clones in open and closed systems, identical cultures were prepared in the Cooper dishes and incubated either sealed or unsealed. in the walk-in 37°C eration. The pedigrees in figs 1G and 2 show two large size clones of early PDL. The room and the CO, incubator. for a period of 6 days. The cells were fixed and stained with Giemsa, and 25 randomly selected fields were counted at 430x magnification. Total cell counts indicated no more than 25 variation between any of the experimental conditions.

RESULTS The clonal pedigrees in figs l-3 are presented to illustrate several aspects of cell division behaviour of WI38 fibroblasts. Four pedigrees are shown of representative clones from cultures at population doubling levels (PDL) of 36, 29 and 51 (figs l-3). Although the clones were grown under the same culture conditions, variations are noted in interdivision time, proliferative capacity (final clone size and maximum

J 0

11 20

40

’ LO

1 80

1 100

120

” 140

160

1 130

2;4

Fig. 3. Abscissa: time in culture (hours). Pedigree of WI38 clone, PDL 3 i. Numbers on iine:, indicate interdivision time in hours: , large
Absher and Absher

Fig. 4. Abscissa:

generation no.; ordinate: interdivision time (hours). A-A, Average IDT; O-O, median IDT. Variation in average and median interdivision time with successive generations. Average and median IDT calculated from PDL 29 clone shown in fig. 2.

Fig.

IDTs increase dramatically beginning in the fourth generation; 25-30% of IDTs are in excess of 25 h and range up to 65 h. The number of cells with long IDTs increases in the fifth generation to approx. 50 % for both clones. These long IDTs are frequently followed by l-3 generations of progeny cells with short IDTs. In the PDL 29 clone (fig. 21, 4 out of 10 long IDTs are followed by l-3 generations of short IDTs and in the PDL 26 clone (fig. la), 14 of 32 long IDTs are followed by daughter progeny with short IDTs. Similar patterns are seen in the lineages shown in figs 1b and 3. When the average IDT of each generation is plotted against the generation num-

ber, basic patterns emerge. The patterns seen in figs 4 and 5 are the most common for clones of all PDL ranges, although considerable variation exists in the magnitude of the peaks. Characteristic of this pattern is a 2-3 generation interval between peaks of mean IDT. This periodicity has been observed in approx. 75% of clones studied. Accounting for another 15% of clones is a pattern consisting of 4 generation intervals between peaks, and the remaining clones have 5-6 generation intervals between peaks. A few clones do not exhibit a decreased average IDT but have a steadily increasing mean IDT [4]. Since the clones are not fed, the marked decrease in mean IDT

E.rp CeiIRes 103 (1976,

5. Abscissa: generation no.; ordinate: interdivision time (hours). A-A, Average IDT; CO, median IDT. Variations in average and median interdivision time with successive generations. Average and median IDT calculated from PDL 51 clone shown in fig. 3.

178 15.6

Clonal

variatiorz

and aging

25 1

ter pairs were between 0.02 and 0.2, of either positive or negative sign, 21.82’.8 I(14 ,158( fps 24.4 In addition to the heterogeneity in inter954 division time and clonal proliferation there 222 238 22.2 are also variations in cell size and mor49.8 23.8 II 8 phology. As a rule. cells that do not divide * 266 during the filming periods of 9-12 days are large, slowly migrating cells with extended 168 amounts of cytoplasm. In size and morphology these WI38 cells have features of 412 17.4 the ‘terminally differentiating’ cells in human skin fibroblast cultures described by Martin et al. [8]. Occasionally a very karge cell will divide after long lag times of up to ’ 40 I. 60 80 1 100 a I?0 m 110 ’ 160 n 180 ” 200 220 3 240 c 120 h, but usually no division of daughier 0I 20 cells follows. An exception is the clone Fin. 5. Abscissa: time (hours). Portionof a cornput&-simblated pedigree. Numbers shown in fig. 1b which was developed from on lines indicate interdivision time in hours. a large cell and, as noted by dark circles on the pedigree diagram, most of the progeny is not due to a replenishment of fresh nu- were also large cells. Large cells are also noted in the same manner on the other trients. Although marked fluctuations in IDT oc- pedigrees. Correlated with most of the large cur from generation to generation, the rela- cells of clone PDL 26 in fig. lb are long tionship of sister : sister pairs is fairly highly IDTs. However, progeny cells are capabie correlated in clones of young and middle of reverting to a smaller size (perhaps due PDLs. The correlation coefficient (r) of all to an asymmetric division such as observed sister pairs was 0.72 for the large clone and in human skin fibroblasts by Martin et ai. 0.58 for the small clone in PDL 26 and 0.77 [8]) with short IDT as can be seen in this for the PDL 29 clone. In all, 65% of the pedigree. A mathematical model has been deearly and middle aged clones exhibited r values in excess of 0.70. Late passage cells veloped as an aid to understanding the reguwere more variable in the correlation coef- lation of cell proliferation, heterogeneity of ficients for sister pairs. The clone of PDL clonal division patterns, and how altera51 shown in fig. 3 did not show a high cor- tions in regulation may lead to senescence. relation between sister pairs, the r value Computer stimulations of the model generbeing 0.15, although other clones in the ate clonal pedigrees that can be compared with those determined from the experisame culture had sister pair correlation coefficients as high as 0.69 for clones with mental films. A portion of a pedigree from a computer more than three sister pairs, Correlation coefficients for mother : daughter pairs at simulation is shown in fig. 6. Comparison all PDL ranges indicated little correlation of this pedigree with the experimental prdiin IDT between mother and daughter cells. grees shown in figs 1-3. illustrates many In two-thirds of the clones measured, the similarities. Similarities are seen in (1) ID1 correlation coefficients for mother : daugh- distribution and variation by generation; (2i 941

llc%

Ex2Ce/!ReJ IO.7 i!57fil

Absher and Abshef

DISCUSSION

161 1

2

3

4

5

6

7

8

9

7. Abscissa: generation no.; ordinate: interdivision time (hours). A-A, Average IDT; O-O, median IDT. Variations in average and median interdivision time with successive generations. Average and median IDT calculated from entire clone of computer-simulated pedigree, of which a portion is shown in fig. 6.

Fig.

cells with very long IDT appearing in the fourth generation and which are followed by daughter cells having short IDT; and (3) the proportions of cells entering a ‘noncycling’ state at successive generations. The mean and median IDT values for each generation of the computer simulated pedigree are shown in fig. 7. IDT fluctuations can be seen to result in a pattern that is similar to the experimental genealogies of figs 4 and 5, and have peaks in the mean IDT occurring at 3 generation periods. Exp Cell Res 103 (1976)

Heterogeneity in proliferative behaviour of human diploid fibroblasts may be categorized as interclonal, intraclonal and ageassociated. The proliferative behaviour includes interdivision time, numbers of generations and clone size. Factors controlling proliferation of fibroblasts that lead to expression of variation may include environmental conditions, cell : cell interactions and synthesis and activity of intracellular regulatory substances. While availability of essential nutrients, space, and numbers of cells present will be limiting factors for overall growth of populations of fibroblasts, there is evidence to suggest that clonal variability is due in part to intrinsic cell regulatory mechanisms. Obviously, there are many levels at which cell cycle regulation could result in variability of clonal proliferation, For example, differing numbers of functioning membrane transport sites, rates of synthetic activity, and production of mitotic stimulators or inhibitors could lead to variability in cellular proliferation. The work of Martin et al. [8] suggests that clone size variation is due to epigenetic factors rather than being a result of genetic changes. As evidence, they showed a bimodal distribution of clone sizes in human skin fibroblasts that was reproducible in secondary, tertiary and quaternary subclones isolated either from rapidly or slowly proliferating primary clones. Merz & Ross [7] found heterogeneity in clone size distribution for WI38 cells and they suggest that the variations are a reflection of highly variable interdivision times for individual cells. Our data indicate that, in addition to the variations in IDT, the variations in proportion of cells that cease to divide with successive generations in the growing clone actually accounts for most of the variation

Clonal variation

in clone size. For example, the clone in fig. 1a had progeny cells into the tenth generation. If all cells divided, the final clone size would be 1024 cells. Because of cells ‘dropping out’ of cycle after generation 3, the clone size was 113 cells. As Martin et al. [8] found, cells derived from rapidly proliferating clones segregate daughters of both high and low proliferative capacity, our data indicate an analogous situation at the intraclonal level. Cells having long or short IDT give rise to cells with both long and short IDT. Most clones have a large proportion of cells in the fourth to sixth generation with long IDT, which usually give rise to progeny having short IDT. While interdivision time may be highly variable for mother : daughter pairs, sister pairs tend to be less variable. Froese [14] argues that cells within sister and cousin pairs are located in closer proximity to each other than to other groups of cells, and, therefore, high correlations of sister pairs are due to interactions between neighboring cells or to a uniformity in microenvironments. Since WI38 cells migrate so freely, sister and cousin pairs of one clone are no more closely situated to each other at any point in time than they are to progeny of other clones. It would seem, then, that high correlation of sister pairs and interclonal variations in IDT cannot be explained by such microenvironmental conditions or cell interactions. The fact that WI38 cells of many clones intermingle freely and yet clonal proliferation can be quite unique for a given progenitor cell suggests that cell: cell contact per se is not of major consequence in determining IDT or clone size. As an example, the two clones shown in fig. la, b were grown together and had different proliferative activity. Plots of migratory pathways and division sites of cells indicated no

and agirzg

253

preferential allocation of space to either progeny of these clones. Martz & Steinberg [ 151 studied cell : cell contact in relation to contact inhibition in films of embryonic Swiss mouse 3T3 cells. They found that maximal contact was not able to inhibit division of a large number of cells and many cells having long IDT did not have maximal contact. If cells do have the capacity to regulate each ot not necessarily involve cell : cell contact. Cellular metabolic products released into the medium could lead to inhibition or perhaps stimulation of division [le. 171.How a given cell responds to the product might be determined by the inembrane structure of that cell, i.e.. number and availability of receptor sites or to the ability of the cells 10 ‘process’ the regulatory product. Thus, a cell of low proliferative capacity growing with cells of high proliferative capacity would not be ‘turned on’ by stimulators produced by other cells if the receptor site was bound or non-functional. This type of system would explain the variability in TDT, proliferative capacity and high correlations of sister pairs. Kubitschek [18] has suggested that growth rate and generation time may be primarily determined by the rmmber of transport or binding sites actively functioning in the uptake of growth factors. We suggests also that slowly growing mother cells may give rise to daughters with very rapid generation rates; large cells would have a large number of transport sites thus leading to rapid generation rates. Since we find that large mother cells segregate both large and small daughter cells having either long or short IDT. the absolute number of transport sites may not be a key dererminant; however, the number offunctioning sites may be. Thus, a small cell with x number of functioning receptor or transport sites would be as likely to segregate daughE.rp Cm!1Rrs l,?.i Ii W,

254

Absher cd Abslzer

ter cells of rapid IDT as a large cell with the same number of functional sites. Thus, it appears that much of the clonal variation observed in proliferation of diploid fibroblasts reflects intrinsic regulatory mechanisms of a cell functioning, in a sense, independent of the cell’s environment. However, while fibroblast clones may vary from each other considerably in their proliferative behaviour, there are division patterns at the intraclonal level that are common to most clones at all population doubling levels. In figs 4 and 5, it was shown that mean IDT values increase in the fourth and fifth generation with lower mean IDT values for earlier and later generations. The patterns noted suggest a consistent flow of cells from cycling to either transient or permanent non-cycling states. The existence of slowly or non-cycling cells may represent a continual shift toward a differentiated state. Whether dedifferentiation also occurs is another point to be considered. Our data shows that cells of normal size, morphology and division behaviour can be derived from cells appearing to have undergone a ‘differentiation’ as described by Martin et al. [8]. On the other hand, one might consider these cells which revert from large, slowly cycling cells to smaller, rapidly cycling cells as having been in a resting or GO state as described by several investigators [ 19, 201. GO may be defined as a true resting state according to Epifanova & Terskikh [20] or resting states may represent blocks in Gl and G2 in various cell types [3, 19, 211. Certainly, alternating cycling and resting states for cells would result in the periodic fluctuation in IDT observed in clones of W138. Many investigators appear to favour the concept of reversible GO state; however, a given cell may remain indefinitely in this state, and for all intents be regarded as a Exp CellRes 103 (19761

non-dividing cell. If such cells accumulate in cell populations during in vitro propagation, as a result of differentiation or some irreversible defect, this will eventually result in a lack of ability of the population to double in cell number. Our data indicate a regularity of periodicity for entry of cells into a resting state. With increasing age the resting state lengthens to an indefinite time period and the cell is functionally considered to be a non-divider. This results in more of the skewed-asynchronous type of division pattern described by Kay [22] as a logarithmic-tangential intermediate characterized by continuation of some branches of a pedigree and termination of other branches. As WI38 cultures age, fewer branches continue and this results in skewing toward a more tangential division pattern. One approach to understanding the parameters of cell proliferation that account for the experimentally observed clonal division patterns, entry of cells into transient or permanent non-cycling states, interclonal variation, and age-associated variations in division behaviour, is to construct mathematical models in which one can incorporate these parameters in a functionally simplistic framework. In the development of models, attempts are often made to identify the formal parameters and variables with concentrations of specific ions or substances. For the purpose of understanding the function of a system, it is not necessary to identify all the variables. In fact, some behaviour such as excitation or inhibition may be due to several factors acting together rather than any one factor acting separately [23]. A mathematical model has been developed which is based upon a simplified feedback regulatory system involving the flux of one or more substances. Oscillations of levels and activities of these sub-

Clonai t~arialion and ttgiilg

stances in cells and the environment control the mitotic cycle. The model is similar to the generalized two-factor reactiondiffusion system discussed by Rosen [23]. In such a model each cell is described by intracellular kinetics using a two-component state vector, by diffusive coupling to the extracellular space, and by diffusive coupling between cells in contact. Behaviour of the cell population is then a direct reflection of these properties ascribed to the individual cells. Our model is based on the same kind of rationale as the twofactor reaction-diffusion scheme but with differences that relate to the complexity of intracellular kinetics, to the nature of coupling with the medium and to the coupling between cells in contact. From the mathematical model, computer simulations of lineages have been obtained which were compared with the experimental data in terms of the distribution of IDT, mother: daughter and sister : sister relationships. frequency of cells with long IDT-producing progeny with short IDT. and proportion of cells ceasing to divide in a generation of a clone. A more comprehensive treatment of the mathematical model and the computer simulation studies of clonal proliferation of diploid fibroblasts will be presented in a separate publication. The model has been introduced at this time because it is important to note that relatively simple model systems can duplicate the cell division patterns observed in experimentally-derived clonal pedigrees. The functional simplicity of the model allows the incorporation of not

255

only clonal proliferation at a given popclation doubling level but also prediction of clonal division behaviour in rela.tion tc aging processes. The authors wish to thank Rosemarv Dovvns and Anne Giroux-Hayward for technical as&iance. This research- was supported by NIH Contract NOl-HD-22755.

REFERENCES 1. Simons. J W I M, Exp cell res 45 (1967) 336. 2. Cristofalo. V J & Kritchevskv. 0. Med es. perimentalis I9 (1969) 313. 3. hlacieira-Coelho, A B: Ponten, J. J cell bio! 43 (1969) 374. 4. Absher, P M, Absher, R G & Barne-. W 19. Exp cell res 88 (1974) 95. 5. Absher. P M. Absher, R G & Barnes. W D. 2e11 impairment in aging and development ted V J Cristofalo & E HoleCkova) p. 91. Plenum Press. New York (1975). 6. Macieira-Coelho, A, PonrCn. J & Philipson, i. Exp cell res 42 (1966) 673. 7. Merz. G S & Ross, J D, J cell physioi 82 (i1973)75~ 8. Martin, G M. Sprague. C .4. Norwood, T H Sr Pendergrass, W R, Am j pathol 74 (1974) 137. 9. Robbins, E. Levine, E M & Eagle. H. J exp med 131(1970) 1311. 10. Merz, G S & Ross. J D. J cell physiol 74 (1969) 219. 11. Smith, J R & Havflick, L, J cell biol62 i 1974)48. 12. Macieira-Coelho, A. PontCn. J & Philipson. L, Exp cell res 43 (1966) 20. 13. Cristofalo, V J & Sharf, B B. Exp cell res76 (1973: 419. 14. Froese, G, Exp cell t-es35 (1964) 415. 15. Martz. E & Steinberg. M S, J cell physioi 79 (1973) 189. 16. Houck, J L. Weil. R L & Sharma. V K, Nature new biol240 (1971) 210. 17. Milo, G E, jr. Exp’crll res 79 (1973) 143. 18. Kubitschek, H E, Cell tissue kinet4(:971) 113. 19. Gelfant, S B Smith. J G. Science 178(1972) 357. 20. Epifanova, 0 I & Tersktkh. V V. Cell tissue binet 2 (1969) 75. 21. Castor, L N, J cell physiol72 (1968) 161. 22. Kay. H E M, Lancet ii (1965) 418. 23. Rosen. R, Bull math biophys 30 (1968) 493. Received June 8, 1976 Accepted July 6. 1976