Analysis of the changes in the proportion of clustered labelled cells in epidermis

A new cell kinetic approach is presented from which the duration of the S and G2 + M phases can be estimated. The technique involves an analysis of the spatial distribution of labelled cells in sections or sheets of epithelium (i.e. an analysis of clustered labelled cells). The technique is largely independent of the absolute number of labelled cells and hence is not influenced by factors which affect the absolute number of labelled cells. The technique is described and experimental data from dorsal murine skin are presented. The technique has also been simulated mathematically so that the phase durations and their variances could be estimated. The advantages of the technique are: it is technically simple; it provides at least two independent estimates of the phase durations; unlabelled cells need not be counted (compare with LI or PLM analysis); it is independent of variations in the absolute yield of labelled cells, and it is applicable if the LI is low and the S phase is short (where the PLM technique tends to fail).     

EVIDENCE OF RAPID AND SLOW PROGRESSION OF CELLS THROUGH G2 PHASE IN MOUSE EPIDERMIS: A COMPARISON BETWEEN PHASE DURATIONS MEASURED BY DIFFERENT METHODS

Percentage labelled mitosis (PLM) measurements were initiated at four different times during a 24-hr period and continued for 24 hr in hairless mouse epidermis. Estimates of G₂ and S phase durations (mean T_G2 and mean T_S) were calculated. A significant number of labelled mitoses (10–20%) was seen after 30 min in all four PLM measurements and the estimated mean T_G2 varied from 1.4 to 2.5 hr and was in agreement with values from PLM measurements in other epithelial tissues. These mean T_G2 values were much shorter than expected from [³H]TdR double labelling experiments and from a multiparameter cell kinetic study in hairless mouse epidermis and did not reflect the circadian variations seen in these studies. the differences in estimates of phase durations can be explained by postulating two G₂ cell populations; one with a rapid and another with a slow rate of cell cycle progression. the cells with the higher rate are mainly registered by the PLM method, whereas those with the lower rate largely escape detection by this method. T_G2 estimates from PLM measurements in mouse epidermis therefore do not reflect the phase duration of the entire G₂ population. It is also concluded that circadian variations in T_S can not be accurately registered by the PLM method.     

Cell Kinetic Studies In the Epidermis of Mouse. Iii. the Percent Labelled Mitosis (Plm) Technique

Full PLM curves have been obtained for four sites in the mouse. the first peaks have been analysed by computer and the duration of the G₂+ M and S phases determined together with their standard deviations. the full curves showed a general similarity for all four sites with no clear second peak. the data are compared with the published data for mouse and human epidermis using the in vivo PLM technique. the timing and shape of the first peak can vary considerably even for one site in mice. Hence, both G₂+ M and S can vary in their durations. Cells labelled at one time of day exhibit different kinetic properties to those labelled at another time of day. the duration of G₂+ M is shortest in dorsum labelled at 03.00 hours (3.2 hr) and longest in tail (up to 7.5 hr). the S-phase is shortest in dorsum (6.3–7.2 hr) and longest in tail or ear (13.3–14.1 hr). There is also a very large standard deviation in tail and foot. There is little general variability when the psoriatic human data are considered, which is surprising. the general variability amongst the data from experimental mice might also be expected amongst humans which might make comparisons between the cell kinetics of normal and diseased skin difficult.     

Analysis of cytokinetics by means of Feulgen cytofluorometry combine with 3H-thymidine autoradiography

DNA cytofluorometry combined with autoradiography after pulse-labelling with ³H-TdR visualizes movement of the labelled cells along the cell cycle. If the specimen is fixed after an adequate waiting time, this method enables us to measure the absolute length of the S phase in a cell population with a single sampling. The method was applied for Yoshida sarcoma cells proliferating in the rat ascites. Using a single specimen fixed after 4 h waiting time, the shortest, the average, and the longest durations of S phase in the population were estimated at 5.8, 7.5 and 13 h, respectively. Measurement on a flash-labelled specimen gives the relative durations of G 1, S, G 2 and M. It is shown that, using these 2 samples, a complete cell cycle time analysis can be performed.     

Cell population kinetics of thyroid follicular cells in infant rats

Abstract. T cell population kinetics of thyroid follicular cells in rats were studied by means of autoradiography and a statmokinetic technique. During the first fortnight after birth no significant changes in the mitotic index (MI) and labelling index (LI) were found. In the next 2 weeks a constant decrease in the number of proliferating cells occurs. In 10-day old animals 40% of the follicular cells were in the cell cycle (GF); 3.25 ± 0.77 (SEM) % in the S phase and 0.18 ± 0.04% in mitoses (MI). Day–night changes in the LI and mitotic rate (MR) indicated a peak value at 13.30 hours with a lowest value at 22.30 hours. The mean LI and MR averaged over the whole 24 hr were 3.1 ± 0.1% and 122.2 ± 18.1%, respectively. In 10-day old animals, using the fraction of labelled mitoses (FLM) method the median cell cycle time ( T _C) was 79 hr and the phase durations were T _G1—64.6 hr, T _s—8.2 hr and T _G2—5.1 hr. The decrease in the number of proliferating cells with the age of the animals is considered to be a result of both cell cycle prolongation and in growth fraction reduction.     

Effects of Pulse Labelling With Tritiated Thymidine On the Circadian Rhythmicity In Epidermal Cell-Cycle Distribution In Mice

The influence of pulse labelling with 50 °Ci tritiated thymidine ([³H]TdR) (2 μCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G₂ phases of the cell cycle were estimated from the obtained DNA frequency distributions. the proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [³H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [³H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G₂ and M phase during the first 32 hr after the injection. the number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 °Ci [³H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

Epidermal cell proliferation. II. A comprehensive mathematical model of cell proliferation and migration in the basal layer predicts some unusual properties of epidermal stem cells

The clustering of 3HTdR labelled cells in the epidermal basal layer and their changes with time have been modelled mathematically and cannot be adequately fitted by an earlier model of the cell kinetic organisation of the skin. A more refined model analysis was performed based on Monte Carlo computer simulations of cell layers which take cell division, cell aging and lateral as well as vertical cell migration into account. A large variety of hypothetical scenarios was tested to see if each could provide a fit to the clustering data. The analysis provides further support for the concept of a cell kinetic heterogeneity with a stem-transit-postmitotic differentiation scheme. In the best overall model scheme three transit divisions are predicted but unlike in the earlier model it is now postulated that postmitotic cells can be produced at all stages in the lineage rather than only at the end of the amplification scheme. Most important, the model predicts that stem cells and most of the transit cells differ in the way they process 3HTdR label. Grain dilution is an important mechanism to explain the fate of some labelled cells in the tissue, but on its own it can only consistently explain the data if the stem cells have a very low labelling index (LI less than or equal to 1%) which implies a very short biologically unreasonable S-phase. If a higher LI (longer S-phase) is assumed for the stem-cells other mechanisms must be predicted to explain the lack of large clusters and the increase in time of the singles. The selective segregation of chromosomes at mitosis is one such mechanism. However, on its own a large number of cells would have to behave in this way (i.e. both stem and T1 cells). If combined with other assumptions such as some grain dilution this selective segregation may be restricted only to stem cells. In addition the model allows cell production and migration rates to be estimated and the analysis can be related to the EPU-concept. Indeed the model itself would tend to automatically generate an EPU like structure. The model quantitatively reproduces LI, PLM, CL and clustering data.     

Ultraviolet B irradiation induces epidermal regeneration with rapidly cycling cells

The left flank of hairless mouse skin was irradiated with a minimal erythema dose of ultraviolet B (UVB) light at 297 nm (25 mJcm-2), while the right flank served as untreated control. The alterations in epidermal growth kinetics induced by this UVB dose were studied with the percentage of labelled mitoses (PLM) technique during the period of increased proliferation. Thirty hours after irradiation, when a large cohort of cells appears in S phase, each animal was injected intra-peritoneally with 50 microCi tritiated thymidine [( 3H]-TdR). The number of labelled basal and suprabasal cells, as well as their localization in epidermis were registered in histological sections at short intervals up to 48 h after the [3H]-TdR pulse. Labelled mitoses were also counted in the same specimens. The results showed a four-fold increase of the high initial number of labelled cells in UVB-exposed epidermis within 18 h of the pulse injection, and a six-fold increase after 36 h. In control epidermis, where the starting value of the labelling index was much lower, there was only a three to four-fold increase in the number of labelled cells during the period studied. The PLM and the labelling index data were consistent with an average cell cycle time of approximately 10-12 h for UVB-exposed cells, in contrast to about 30 h for the fastest cycling population in control epidermis. The PLM curve also indicated a prolonged S phase duration in UVB-exposed epidermis compared with controls. In addition, labelled cells were seen in the suprabasal layer as early as 6 h after the [3H]-TdR injection and within 36 h labelled cells had reached the outermost layer of nucleated cells, indicating a reduced transit time through epidermis. The present study shows that a minimal erythema dose of UVB light at 297 nm induced a period of increased transit time through the S phase, combined with rapid cell proliferation, leading to an overall shortening of the epidermal cell cycle time. The cohort of cells labelled with [3H]-TdR 30 h after irradiation seemed to proceed as a wave of partially synchronized cells through the cell cycle for more than two rounds, which is comparable with the cell kinetic perturbations observed in regenerating mouse epidermis.     

Ultraviolet B irradiation induces epidermal regeneration with rapidly cycling cells

Abstract. The left flank of hairless mouse skin was irradiated with a minimal erythema dose of ultraviolet B (UVB) light at 297 nm (25 mJcm^-2), while the right flank served as untreated control. The alterations in epidermal growth kinetics induced by this UVB dose were studied with the percentage of labelled mitoses (PLM) technique during the period of increased proliferation. Thirty hours after irradiation, when a large cohort of cells appears in S phase, each animal was injected intra-peritoneally with 50 /iCi tritiated thymidine ([³H]-TdR). The number of labelled basal and suprabasal cells, as well as their localization in epidermis were registered in histological sections at short intervals up to 48 h after the [³H]-TdR pulse. Labelled mitoses were also counted in the same specimens. The results showed a four-fold increase of the high initial number of labelled cells in UVB-exposed epidermis within 18 h of the pulse injection, and a sixfold increase after 36 h. In control epidermis, where the starting value of the labelling index was much lower, there was only a three to four-fold increase in the number of labelled cells during the period studied. The PLM and the labelling index data were consistent with an average cell cycle time of approximately 10–12 h for UVB-exposed cells, in contrast to about 30 h for the fastest cycling population in control epidermis. The PLM curve also indicated a prolonged S phase duration in UVB-exposed epidermis compared with controls. In addition, labelled cells were seen in the suprabasal layer as early as 6 h after the [³H]-TdR injection and within 36 h labelled cells had reached the outermost layer of nucleated cells, indicating a reduced transit time through epidermis. The present study shows that a minimal erythema dose of UVB light at 297 nm induced a period of increased transit time through the S phase, combined with rapid cell proliferation, leading to an overall shortening of the epidermal cell cycle time. The cohort of cells labelled with [³H]-TdR 30 h after irradiation seemed to proceed as a wave of partially synchronized cells through the cell cycle for more than two rounds, which is comparable with the cell kinetic perturbations observed in regenerating mouse epidermis.     

The effects of partial and complete denervation on adult newt forelimb blastema cell-cycle parameters

Abstract. The adult newt blastema cell-cycle time (cct) was measured by the percentage of labeled mitoses (PLM) method at the early-bud and mid-bud stages and was found to be 42.9 and 42.7 h, respectively. At both stages, the DNA synthetic phase (S) occupied the majority (75%) of the cct. However, the blastema labeling index (LI) after a 2-h pulse of ³H-thymidine was less than 30% i.e., considerably less than predicted from the ratio of the duration of S over the cct. Compared to that of controls, the PLM plot for partially denervated blastemas exhibited a coincident and equal-sized first peak of labeled mitoses and a coincident but smaller second peak of labeled mitoses. After 24 h of continuous labeling, the LI of control blastemas reached 53%, whereas the LIs of partially denervated and completely denervated blastemas reached only 33% and 20%, respectively. These results are consistent with the view that many cells of adult newt blastemas are not actively progressing through the cell cycle and that the number of noncycling cells is increased by partial or complete denervation. The noncycling cells are probably in the G₁ phase of the cell cycle.     

Cell kinetic studies in the epidermis of mouse. III. The percent labelled mitosis (PLM) technique

Full PLM curves have been obtained for four sites in the mouse. The first peaks have been analysed by computer and the duration of the G2 + M and S phases determined together with their standard deviations. The full curves showed a general similarity for all four sites with no clear second peak. The data are compared with the published data for mouse and human epidermis using the in vivo PLM technique. The timing and shape of the first peak can vary considerably even for one site in mice. Hence, both G2 + M and S can vary in their durations. Cells labelled at one time of day exhibit different kinetic properties to those labelled at another time of day. The duration of G2 + M is shortest in dorsum labelled at 03.00 hours (3 X 2 hr) and longest in tail (up to 7 X 5 hr). The S-phase is shortest in dorsum (6 X 3-7 X 2 hr) and longest in tail or ear (13 X 3-14 X 1 hr). There is also a very large standard deviation in tail and foot. There is little general variability when the psoriatic human data are considered, which is surprising. The general variability amongst the data from experimental mice might also be expected amongst humans which might make comparisons between the cell kinetics of normal and diseased skin difficult.     

In vitro response of lymphocytes to phytohemagglutinin (PHA) as studied with antiserum to PHA : II. Cell cycle analyses

The cell cycle and phase times of human lymphocytes responding to PHA have been analysed with the percent labelled metaphases (PLM) technique. The range of generation times (13–18 h) and DNA synthesis times (6.5–10.5 h) reported here compare well with previous measurements in the literature. Cycle analyses of the early responding cells of the initial response, selected with partial anti-PHA serum inhibition, and of restimulated cells yield relatively well-defined PLM curves. The short cycle times measured from these curves may reflect the early cycles after stimulation or a subpopulation of responding cells. Analyses at two times during both the initial and restimulation responses suggest that cycles lengthen with time after stimulation. The poor PLM curves of the initial response and the restimulation response of cells released from anti-PHA inhibition indicate considerable intercellular variation in cycle times. Cells in the initial long G 1 phase contribute to this variation. PHA dose does not appear to affect the cycle time.     

Kinetics of the calcium induced stratification of human keratinocytes in vitro

In a low concentration of calcium (0.1 mM), keratinocytes form a monolayer with about 30% of cells synthesizing involucrin. After addition of calcium to the culture medium to a concentration of 1.2 mM, the monolayer stratifies within 24 h, with a preferential migration of involucrin positive keratinocytes. In the present study, we tried to determine if keratinocytes control the decision to migrate at a distinct cell cycle point. A percentage labelled mitosis (PLM) curve was constructed for keratinocytes grown in low calcium medium and values for the length of the cell cycle (47 h), S phase duration (11 h) and G2+M period (6 h), were obtained. Monolayer cultures at 80% confluence were switched to high calcium concentration at various times (from 0 to 48 h), after pulse labelling with [3H]-thymidine. Based on the PLM data, the behaviour of cells known to be in S, G1 and G2 at the time of the migration stimulus were followed. No significant difference in the percentage of labelled suprabasal cells was found for any point of the cell cycle. For cells submitting to stratification, in S phase involucrin staining showed that about 60% of the [3H]-thymidine labelled cells were also involucrin negative. These results indicate that upward migration of keratinocytes in cultured epithelium can be triggered at all points in the cell cycle with equal probability and is not restricted to those cells that already contained involucrin.     

Intestinal Cell Proliferation. I. A Comprehensive Model of Steady-State Proliferation In the Crypt

Abstract. Cell replacement in the crypt of the murine small intestine has been studied and modelled mathematically under steady-state conditions. A great deal of information is available for this system, e.g. cell cycle times, S phase durations, the rate of daily cell production, the Paneth cell distribution etc. the purpose of the present work was to consider simultaneously as much of these data as possible and to formulate a model based upon the behaviour of individual cells which adequately accounted for them. A simple mathematical representation of the crypt has been developed. This consists of sixteen stem cells per crypt (T_c= 16 hr, T_s= 9 hr), and four subsequent transit cell divisions (T_c= 11 to 12 hr, T_s= 8 hr) before maturation. Experimental data considered to test the modelling were LI and data on the number of vertical runs of similarly labelled cells. All data were obtained from the ileum after 25 μCi [³H]TdR given at 09.00 hours. A number of alternative assumptions have been considered and either accepted or rejected. Two alternative model concepts of cell displacement explain the data equally well. One is dependent upon strong local cell generation age determinance while the other could accommodate any weak local cell displacement process in conjunction with an environmental cut-off determinant at the middle of the crypt. Both models provide new interpretations of the data, e.g. certain rates of lateral cell exchange between neighbouring columns (250 to 350 per crypt per day out of a total of 420 cell divisions per day) can be concluded from run data, while LI data provide information about the mechanisms involved in maintaining a position-related age order in the crypt.     

Circadian variations in the DNA synthesis of the rat corneal epithelium. A study using double labelling with tritiated thymidine

A prominent circadian rhythm was found in the labelling indices (LI) of the peripheral rat corneal epithelium and of the adjacent conjunctival epithelium, while almost no diurnal variation was found in the central area. Application of a double labelling technique indicated that there are rhythmic pulses of high and low influx of cells into the S phase and similar pulses of efflux of cells from the S phase. Results of the study indicate that there are different cohorts of cycling cells all over the rat corneal epithelium. Cells belonging to a rapidly proliferating cohort are observed in the peripheral cornea. There is a gradual reduction in the fraction of labelled DNA-synthesizing cells towards the centre. The considerably lower fraction of cells taking up tritiated thymidine (3H)TdR in the central cornea may be due to a higher fraction of basal cells having reached higher levels of differentiation. This may result in a shift from the salvage to the de novo pathway. The slowly proliferating cohort seems to have a prolonged S phase duration and displays practically no diurnal variation in the LI. The DNA-synthesizing cells belonging to this latter cohort probably use the salvage pathway for DNA synthesis resulting in uptake of (3H)TdR all over the cornea. The LI is thus not a reliable indicator of cell proliferation in the corneal epithelium, due both to the heterogeneity of the cell proliferation, and in particular due to the lack of labelling of the centrally located DNA-synthesizing cells. To what extent these properties may also be present in other proliferating tissues with different levels of differentiations, may be questioned.     

Effects of pulse labelling with tritiated thymidine on the circadian rhythmicity in epidermal cell-cycle distribution in mice

The influence of pulse labelling with 50 microCi tritiated thymidine ( [3H]TdR) (2 microCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G2 phases of the cell cycle were estimated from the obtained DNA frequency distributions. The proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [3H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [3H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G2 and M phase during the first 32 hr after the injection. The number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 microCi [3H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

The decay of autoradiographic grain number over crypt base columnar cells in murine ileum as a measure of their generation time

The decay in the number of grains over [3H]-thymidine labelled crypt base columnar cells (BCC) in autoradiographs of the ileum of BDF1 mice has been studied. The results revealed that using the conventional grain count halving (GCH) method it is possible to obtain an estimation of the generation time (Tc) of the proliferative BCC cells in the Paneth cell zone (PC-zone) of 18.8 +/- 0.74 h. This lies within the range obtained by the percent labelled mitoses (PLM) method, but is shorter than most values obtained by stathmokinetic methods. The present data show no evidence for a shortening of the cell cycle 3 days after irradiation (8 Gy) which is contrary to some earlier observations. Some reasons for this discrepancy are discussed. The comparatively high labelling index of the BCC allows a larger amount of data to be easily collected, compared with the PLM technique, and correction factors which take into account the complicated shape of the bottom of the crypt are not required.     

Age-related changes in the cell kinetics of rat foot epidermis

The durations of the cell cycle and its component phases have been determined for the basal layer of the epidermis of the skin from the upper surface of the hind foot of the rat using single pulse [3H]-thymidine labelling and the percent labelled mitosis (PLM) technique. Rats of three age groups were used, namely 7, 14 and 52 weeks. The duration of DNA synthesis (Ts) and the G2 plus M phase (TG2 + M) were comparable in 7-week and 52-week-old rats (P greater than 0.1). The major difference between 7-week and 52-week-old rats was in the duration of the G1 phase (TG1). In 7-week-old rats TG1 was 15.0 +/- 0.8 h and in 52-week-old rats TG1 was 31.2 +/- 3.5 h. A consequence of this variation was that the overall duration of the cell cycle was longer in 52-week-old rats (53.9 +/- 5.3 h) than in 7-week-old rats (30.1 +/- 1.3 h). Difficulties were found in fitting a simple curve to the PLM data for 14-week-old rats. This suggests that the proliferative cell population of the epidermis of rats of this age group may be heterogeneous. A satisfactory fit to the data was obtained using a computer model which assumed that the proliferative population of the epidermis of 14-week-old rats was a mixture of cells with cell cycle parameters the same as those of the 7-week and the 52-week-old rats. These two sub-populations of relatively slowly and rapidly proliferating cells were present in the ratio of 2:1.     

Age-related changes in the cell kinetics of rat foot epidermis

Abstract. The durations of the cell cycle and its component phases have been determined for the basal layer of the epidermis of the skin from the upper surface of the hind foot of the rat using single pulse [³H]-thymidine labelling and the percent labelled mitosis (PLM) technique. Rats of three age groups were used, namely 7, 14 and 52 weeks. The duration of DNA synthesis (T_s) and the G₂ plus M phase (Tg₂± m) were comparable in 7-week and 52-week-old rats ( P > 0–1). The major difference between 7-week and 52-week-old rats was in the duration of the G₁ phase (Tg₁). In 7-week-old rats Tg₁ was 15.0 ± 0.8 h and in 52-week-old rats Tg₁ was 31.2 ± 3.5 h. A consequence of this variation was that the overall duration of the cell cycle was longer in 52-week-old rats (53.9 ± 5.3 h) than in 7-week-old rats (30.1 ± 1.3 h).
Difficulties were found in fitting a simple curve to the PLM data for 14-week-old rats. This suggests that the proliferative cell population of the epidermis of rats of this age group may be heterogeneous. A satisfactory fit to the data was obtained using a computer model which assumed that the proliferative population of the epidermis of 14-week-old rats was a mixture of cells with cell cycle parameters the same as those of the 7-week and the 52-week-old rats. These two sub-populations of relatively slowly and rapidly proliferating cells were present in the ratio of 2:1.     

Subpopulations of Slowly Cycling Cells In S and G2 Phase In Mouse Epidermis

Evidence has been presented supporting the existence of heterogeneity in cell-cycle progression in mouse epidermis, the present study was undertaken to characterize this heterogeneity in more detail. Hairless mice were continuously labelled with tritiated thymidine every 4 hr for 4 days. Basal cell suspensions were prepared from slices of mouse skin at intervals during the experiment and subjected to DNA flow cytometry. Cell-cycle analysis was combined with sorting of cells from windows in G¹, S and G², phase, and the proportion of labelled cells within each window was determined in autoradiographs. Reanalysis and resorting to control the purity of sorted fractions were performed. Computer simulations of the data were made using a mathematical model assuming different S and G² phase characteristics. A good fit to the data was only obtained when heterogeneity in mouse epidermal cell-cycle progression was assumed, indicating the existence of slowly traversing, distinct subpopulations of cells in G² and S phase. These cells are assumed to contribute to about 40% of all cells in S phase and to about 70% of all in G² phase. the estimated residence times in the resting states were 38 and 32 hr in S and G² phase, respectively. Two-parameter sorting based on DNA and light scatter indicated that slowly cycling cells were larger than the average. There is no evidence of significant subpopulations of permanently non-proliferating keratinocytes in any of the cell-cycle phases.