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Intestinal stem cells protect their genome by selective segregation of template DNA strands

¹ Epithelial Biology Department, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M9 4BX,UK
² EpiStem Limited, Incubator Building, Grafton Street, Manchester M13 9XX, UK

View larger version (11K): [in a new window]   Fig. 1. A diagram showing the segregation of template and newly synthesised DNA strands in one chromosome, according to the Cairns' hypothesis (1975), which proposes that all the chromosomes would behave in this way. The template strands are selectively retained by the stem cell daughter of a cell division, whereas the newly synthesised strands are segregated to the daughter cell destined to enter the dividing transit compartment and be shed from the tissue after a few days, thus removing any replication-induced errors. Label introduced into the newly synthesised strands takes two divisions to be removed from the stem cells. Label in the template strand would persist in the stem cell line.

View larger version (42K): [in a new window]   Fig. 2. A diagram showing the histological appearance of a longitudinal section through a crypt of the small intestine. Paneth cells (with apical granules) are located at the base of the flask-shaped structure, and there are between 15 and 20 proliferating cells arranged along the long axis. Circumferentially, at each position above about the fourth position from the bottom, there are about 16 cells per annulus. Also shown is the current model (on the right) for the proliferative organisation in the crypt. This consists of between four and six cell lineages, each with a self-maintaining lineage ancestor stem cell (closed circle) located within the annulus of 16 cells at about cell position four from the base. It is upon these actual stem cells that the entire cell production process is ultimately dependent in steady state conditions, and in the absence of damage or injury to the tissue these cells maintain cell production for the lifetime of the animal. If, however, some or all of these actual stem cells are killed, the next two to three generations within the cell lineage (open circles) retain the ability to repopulate the stem cell compartment and the entire cell lineage, that is, they retain the ability to function as stem cells (indicated as circles) and are hence potential stem cells, which are normally within the transit population in steady state conditions but are as yet uncommitted to differentiation. Once the commitment to differentiation has been made, the cells retain no stem cell capabilities, but do retain the ability to divide and differentiate further, that is, they are part of the dividing transit population (open squares). A further four to six divisions occur within the dividing transit population, during which differentiation into the enteroendocrine, Paneth, goblet and enterocyte lineages may occur. The other cells shown are: a goblet cell at position 16 and a mitotic cell at cell position 10. An apoptotic fragment in the crypt is shown in cross section.

View larger version (24K): [in a new window]   Fig. 3. A schematic outline of the experimental procedures.

View larger version (15K): [in a new window]   Fig. 4. The results of an experiment where twice daily (9 am and 9 pm) injections of tritiated thymidine were given for three consecutive days (Fig. 3). All mice were then sacrificed in groups of four to six when they were 11 weeks old. The number of non-Paneth LRCs per small intestinal transverse section (circumference) was determined per mouse, and the mean values from the four to six mice per group ± standard errors are shown. For mice less than 20 days old, the number of non-Paneth LRC per circumference in the 11 week old animals was about 1.5 per circumference. If 3HTdR doses are given between about 37 and 57 days, a fairly constant number (about 5.5) of non-Paneth LRCs per circumference was obtained. There were about 84 crypts per circumference in these adult animals and, hence, there are about 6.5% of the crypts in a circumference that contain one or more non-Paneth LRCs. The threshold for detecting a labelled cell was set at five or more grains.

View larger version (19K): [in a new window]   Fig. 5. The distribution of non-Paneth LRCs along the crypt axis [a cell positional analysis; see (Potten et al., 1997; Potten et al., 1992; Potten, 1998; Potten and Grant, 1998) for details] is shown for animals labelled with 3HTdR at 42 days (solid line) and for animals labelled during the crypt regenerative phase following a dose of 8 Gy of gamma rays (red line). Both cell position distributions show peak values at around cell position four-five, with a fairly broad distribution and a long tail to the right. Standard error limits are shown.

View larger version (95K): [in a new window]   Fig. 6. Photomicrographs of two longitudinally sectioned crypts showing a single heavily labelled LRC situated just above the Paneth cells at about cell position four (arrow). These pictures were taken from animals subjected to the post-irradiation regeneration labelling protocol (8 Gy followed by 2 days of labelling with analysis and photography 8 days after labelling). Similar pictures were obtained in the juvenile labelling experiment. There are occasional labelled cells in the pericryptal stroma.

View larger version (78K): [in a new window]   Fig. 7. The upper two panels show a mitotic anaphase figure in two different planes of focus with an asymmetric distribution of grains (same labelling and analysis as in Fig. 6). The middle two panels (arrows) show LRCs undergoing apoptosis 4.5 hours after a dose of 1 Gy rays delivered 16 days after the post-irradiation regeneration labelling protocol described for Fig. 6. The true morphlogical features of apoptosis in these autoradiographs are difficult to photograph and can only be determined by careful optical focusing. The next lower two panels and their different focal plane inserts (bottom panels) show a label-retaining nucleus undergoing DNA synthesis, which is labelled with BrdUrd (left hand panel), and a LRC undergoing mitosis (lower panel right), indicating that the LRCs do indeed progress through the cell cycle and divide (see also data in Table 1). (The same protocol was followed as in Fig. 6.)

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