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).     

Growth factor regulation of proliferation in primary cultures of small intestinal epithelium

Summary Although the intestinal epithelium is one of the most rapidly renewing tissues, little is known about the major growth factors that control the rate of cell replacement and migration. Recently, a primary culture model has been described for the developing rat small intestinal epithelium, which permits epithelial growth while maintaining interactions with associated stromal cells, thereby possessing several contextual advantages over established cell lines (Evans et al., 1992). We have used this model to begin to determine the factors that may be involved in controlling intestinal epithelial cell proliferation. Under the conditions examined, no single growth factor promoted exclusive proliferation of epithelial cells; stromal cell proliferation was also apparent. The most potent stimulators of epithelial proliferation were insulin and insulin-like growth factor 1 (IGF-1). These factors also appeared to inhibit migration of the epithelial cells. 5–10 ng/ml EGF, 5–20 ng/ml TGFα, and 10–20 ng/ml PDGF also slightly increased epithelial cell numbers. Cell proliferation was inhibited by 0.1 ng/ml TGFβ-1. In Dulbecco’s modified Eagle’s medium (DMEM) containing 0.25 IU/ml insulin, glucose levels of 2–3 g/liter permitted epithelial growth with limited expansion of the stromal cell population. Higher levels of glucose further stimulated the nonepithelial cell types. Transferrin was also a potent stimulator of both cell types.     

The recruitability and cell-cycle state of intestinal stem cells

Evidence is presented which suggests that the crypts of the small intestine contain at least two discrete but interdependent classes of stem cells, some with discrete cell kinetic properties and some with discrete radiation responses or radiosensitivities. Very low doses of X rays or gamma rays, or neutrons, kill a few cells in the stem cell regions of the crypt in a sensitive dose-dependent manner. Similar doses generate several different cell kinetic responses within either the clonogenic fraction or the cells at the stem cell position within the crypt. The cell kinetic responses range from apparent recruitment of G0 clonogenic cells into cycle, to a marked shortening of the average cell cycle of the cells at the stem cell position. It is suggested that the cell kinetic changes may be the consequence of the cell destruction.     

Evidence for discrete cell kinetic subpopulations in mouse epidermis based on mathematical analysis

Abstract. Continuous (repeated) labelling studies in mouse epidermis indicate that nearly all cells are labelled after about 100 hr. Percentage labelled mitoses studies ([³H]TdR at 15.00 and 03.00 hours) have a first peak that does not reach 100% and has a half-width of about 10 hr. Small second and third peaks can be detected at about 90 and 180 hr, respectively. The changes with time in the number of labelled cells show a difference dependent on the time of day of [³H]TdR administration. Both curves show an early doubling in labelled cells which then decline, forming a peak of labelled cells. A second peak occurs at about 120 hr. This is followed by a progressive decline with no further peaks until values of about 1% labelling are obtained at 340 hr.
These experiments have been investigated mathematically. A computer programme has been devized that permits all three types of experiments to be analysed simultaneously. More importantly, it can analyse situations with a heterogeneity in cell cycle parameters in all proliferative subpopulations.
Various models for epidermal cell replacement have been considered. The data as a whole can best be explained if the basal layer contains at least two distinct subpopulations of cells and an exponentially decaying post-mitotic population with a half-life of about 30 hr. The proliferative sub-populations must be characterized by near integer differences in the length of cycle, the precursor (stem) compartment having the longer cycle. An inverse relationship is required for the length of S, i.e. the shortest time for the stem cells.
A full range of cell kinetic parameters can be calculated and are tabulated for the most appropriate model system which is one involving three transit proliferating subpopulations.     

Regeneration and dose-response characteristics of irradiated mouse dorsal epidermal cells.

A microcolony technique is described for measuring epidermal cell survival 3 days after whole-body X-irradiation. This assay provides a cell D0 value of 233 +/- 11 rad and a zero-dose extrapolate of 1-23 x 10(4) cells/cm2 for mice irradiated in oxygen 20 hours after hair plucking. The microcolony cellularity had an apparent doubling-time of 25 hours which may be an upper limit at least for some clones. The clones appeared to fragment continually and form new daughter clones, suggesting that few would form macroscopic nodules. Many of the clones were also apparently associated with hair follicles.     

Recruitment of cells in the small intestine into rapid cell cycle by small doses of external gamma or internal beta-radiation

Epithelial cell recruitment was examined in mouse ileum after external gamma-irradiation (50 cGy) or internal beta-irradiation (0.148 MBq/g of [3H]thymidine), using the per cent-labelled-mitoses method and by analysing the distribution of mitotic cells in the crypts. In the presumptive stem cell zone at the lower cell positions of the crypt, the slowly cycling cells decreased their cell cycle 6 or 12 hours after a dose of 50 cGy. In the higher cell positions, a slight shortening of the cell cycle was also observed. After administration of a high dose of [3H]thymidine, dormant (G0) cells also entered the cell cycle in the lower cell positions. The results suggest that stem cells in the crypt may react to irradiation in two ways: first, by shortening the cell cycle in cycling cells; secondly, by an entry into the cell cycle by other dormant cells. There was destruction of some cycling stem cells before any recruitment. The data support the idea that the stem cell population in the crypt is heterogeneous.