The small intestine as a model for evaluating adult tissue stem cell drug targets

Adult tissue stem cells are defined and some current controversies are discussed. These crucial cells are responsible for all cell production in renewing tissues, and play a vital role in tissue regeneration. Although reliable stem cell markers are generally unavailable for adult epithelial tissues, the small intestinal crypts are an excellent in vivo model system to study stem cells. Within this tissue, the stem cells have a very well-defined cell position, allowing accurate definition of stem cell specific events. Clonal regeneration assays for the small intestine allow stem cell survival and functional competence to be studied. The ultimate lineage ancestor stem cells are extremely efficiently protected from genetic damage, which accounts for the low cancer incidence in this tissue. Some of the regulatory networks governing stem and transit cell behaviour are beginning to be understood and it is postulated that p53 plays a crucial role in these processes.     

Cell death (apoptosis) in hair follicles and consequent changes in the width of hairs after irradiation of growing follicles

Irradiation of anagen (growing) hair follicles results in a dose-dependent increase in the number of histologically identifiable fragments of dead cells (apoptotic fragments). The incidence of apoptotic fragments is linearly related to dose, increasing at a rate of 2.92 fragments per follicle section per Gy. The effects of doses of 0.2 Gy can be easily detected. Subjective attempts to associate clusters of fragments with dead or dying cells suggests that the number of fragments per cell increases with dose (about 1.7 fragments per cell after 1 Gy to about 2.7 fragments per cell after 5 Gy). There is a natural incidence of cell death in controls (0.13 +/- 0.06 fragments per follicle section with about 1.4 fragments per dead or dying cell). Damage to the follicle cells is expressed in the differentiated product of the follicle, the hair, by a reduction in width. This is probably the cellular basis for the production of dysplastic hairs. The hair width has been measured and is reduced by about 7 per cent for every gray of radiation. The value of the hair and hair follicles as potential biological dosimeters is discussed.     

Estimates of the number of clonogenic cells in crypts of murine small intestine

There is a proliferative cell hierarchy in the mouse intestinal crypt with ancestral stem cells which can regenerate all components of the lineage after injury (clonogenic cells). The number of these clonogenic or regenerative cells per crypt can be estimated from radiobiological experiments where doses of radiation are used to kill cells and ablate crypts. Various approaches can be adopted which provide different estimates of this number of cells. One of the conventional approaches used in the past provided estimates of about 70-80 clonogenic cells per crypt (i.e. about 50% of the proliferative or 30% of all crypt cells). A technically simpler approach has recently been suggested. This has been used here to provide many independent estimates of the number of crypt clonogenic cells. These suggest about 32 clonogenic cells exist per crypt i.e. about half the previous estimate and about twice the number of putative "functional" stem cells (those which operate as stem cells in the normal steady-state crypt). The reasons for the differences are discussed. The new estimates are compatible with the hypothesis that the crypt contains a ring of about 16 functional stem cells which are expected to be clonogenic, besides which there is a second ring of 16 clonogenic cells which represent early transit cells (the immediate daughters of the stem cells) which can act as clonogenic cells if required after radiation injury.     

Cell cycles in cell hierarchies

In the replacing tissues of the body, namely the bone marrow, testis, and the surface epithelia with their appendages, cell replacement would appear to be achieved using an hierarchically organized proliferative compartment with relatively few ultimate stem cells producing dividing transit cells which eventually differentiate and mature into the functional cells of the tissue. The cell cycle times of the various constituents of the hierarchy differ, and the stem cells apparently have a longer cell cycle than the transit cells. There may be variations in the cell cycle as cells pass through the transit population in some cases, e.g. in the bone marrow, while in others the cycle time remains fairly constant, e.g. in the testis. The difference in the cell cycle time between stem cells and transit cells is not completely unequivocal, and there is little or no difference in cycle time in the epithelium on the dorsal surface of the tongue while in other cases the experimental evidence for long stem-cell cycles is somewhat imprecise. However, the epithelium in the small intestine and the spermatogonia in the testis have been fairly extensively studied and here the evidence clearly shows a lengthening of the cell cycle as more primitive cells are considered.     

Double Labelling With Bromodeoxyuridine and (3H)-Thymidine of Proliferative Cells In Small Intestinal Epithelium In Steady State and After Irradiation

Abstract. The simultaneous immunohistochemical detection of bromodeoxyuridine (BrdU) and [³H]-thymidine ([³H]TdR), by conventional autoradiography, was performed on the mouse small intestine (ileum). Proliferation was studied under normal conditions as well as after 3 Gy of γ-rays. the BrdU method in conjunction with [³H]TdR autoradiography appears to be reliable and useful for the study of cell kinetics especially in disturbed states, on condition that [³H]TdR is delivered to the animals before BrdU. It has been found that cells in the crypt are delayed by irradiation in their progression through the cell cycle predominantly in late S phase. the cells at the bottom of the crypt are more affected than the more differentiated but proliferating cells in the upper part of the crypt.     

Molecular cloning and sequencing of a murine pgk-1 pseudogene family

Seven genomic mouse DNA fragments carrying pgk-1-homologous regions have been cloned and sequenced. They have to be classified as processed genes because intervening sequences, present in their productive counterpart, are absent. Four pseudogenes (I-IV) represent nearly the complete sequence of pgk-1 cDNA. Two of these genes (I and II), although rather different from the published mouse pgk-1 cDNA in the 3'-untranslated region, represent the actual mouse pgk-1 cDNA sequence in the coding part except for substitutions in the third position of three codons. These genes can code for a functional PGK protein but, lacking as they do classical promoter structures, are probably not expressed. They show the typical characteristics of retroposons, being flanked by A-rich regions and direct repeats which are localized at the positions where the homology with the mouse pgk-1 cDNA is interrupted. Pseudogenes III and IV have numerous mutations. Gene III is also flanked by direct repeats, whereas gene IV is flanked by inverted repeats. The other three genes are flanked by direct repeats localized further inside the target sites. They are truncated and mutated extensively as usually observed with pseudogenes.     

Radiosensitivity of normal human epidermal cells in culture

Using an in vitro culture system we have derived radiation survival curves for the clonogenic cells of normal human epidermis. The culture system used allows the epidermal cells to stratify and form a multi-layered sheet of keratinizing cells. The cultures appear to be a very good model for epidermis in vivo. The survival curves show a population which is apparently more sensitive than murine epidermis in vivo. It remains unclear whether this is an intrinsic difference between the species or is a consequence of the in vitro cultivation of the human cells.     

A long-lived thymidine pool in epithelial stem cells

Abstract. The labelling index (LI) of the individual basal cell positions of the anterior column of mouse tongue filiform papillae was assessed with time after an injection of [³H]TdR at 12.00 hours (the minimum point in the circadian LI rhythm). An initial doubling of the LI in the stem cell zone due to cell division was followed by a second rise of 14–16% 16 hr after injection and this occurred even in the presence of vincristine. Although the uptake of [³H]TdR and the initial LI doubling were largely prevented by a preceding injection of hydroxyurea, the 14–16% LI rise was still observed. The possible explanations are discussed, the favoured one being that an average of one of the six or seven cells (the stem cell) in each stem cell zone can store [³H]TdR in a long-lived precursor pool for at least 16 hr before being utilized for DNA synthesis. This complements previously published work which suggested that one cell in each stem cell zone may selectively segregate DNA at mitosis.     

FURTHER OBSERVATIONS ON THE LATE LABELLING ASSOCIATED WITH STIMULUS-RESPONSIVE CELLS IN SKIN

The presence of a long-lived DNA precursor pool which may show some specificity for stimulus-responsive cells has been demonstrated. Autoradiography, biochemical analysis, hydroxyurea sensitivity, and the temporal response all suggest that the late incorporation is into DNA in cells in the basal layer of the skin that respond to stimulation. The effect is observed with various doses of tritiated thymidine and both methyl and 6 labelled material. ¹²⁵Iododeoxyuridine also shows late labelling in skin. It is believed that the late labelling is readily distinguishable from reutilization and from possible slow utilization of catabolites into molecules other than DNA. Skin of the ear and dorsum shows similar degrees of late incorporation, while tail and foot skin apparently have smaller long-lived pools. This may indicate smaller stimulus-responsive cell populations. If tritiated thymidine is given to cells after stimulation there is still some slow or delayed uptake. The distribution of labelled cells in the autoradiographs suggests that the late labelling cells may be associated somehow with pre-existing labelled cells (cells in S at the time of thymidine administration).     

CELL KINETICS IN THE SEBACEOUS GLANDS OF THE MOUSE. II. THE GLANDS DURING HAIR GROWTH

The sebaceous glands of the mouse have been studied during hair growth initiated either spontaneously or artificially. The labelling index of the glands increases early in the spontaneous hair growth period. That of the epidermis is much lower and hardly changes during the growth period. After the initiation of hair growth by plucking, changes in cell proliferation in the sebaceous glands appear to follow those in the epidermis. The size of the gland and the number of cells in it also change after plucking. These variations can be related to the stages of hair growth.