Christopher S Potten

The intestinal epithelial stem cell: the mucosal governor

All epithelial cells in the small and large intestine are thought to originate from stem cells located towards the base of the crypts of Lieberkühn. To‐date, there are no specific intestinal stem cell markers, hence stem cell properties can only be inferred. A range of experimental techniques have been employed including cell position mapping, radiation regeneration (clonogenic) assays, chimeric and transgenic mice. This review discusses the implications of experiments performed using these techniques in order to deduce the number, location and functional properties of stem cells. Stem cell homeostasis is maintained by cell proliferation and death ‘through apoptosis’. The various growth and matrix factors and genes which may control these processes, and be important for stem cell function, are discussed along with their carcinogenic and clinical implications.

Extreme sensitivity of some intestinal crypt cells to X and γ irradiation

THE destructive effects of radiation have been studied for 80 yr. Most techniques involve looking at the surviving cells, which tend to be the more resistant cells of the tissue. On the assumption that the results are representative of all cells in the tissue, many conclusions have been drawn. On the other hand, Cheng and Leblond have used tritiated thymidine (3HTdR) to kill cells synthesising DNA in the crypts of the small intestine1. Two surprising features of their experiments have provoked little comment. First, very low doses (40–50 µCi per mouse) of 3HTdR caused measurable cell killing and second, the killing (evident from the presence of labelled apoptotic-like2 phagosomes1) was not random throughout the crypt but occurred selectively at the crypt base where relatively few cells are in S (refs 3 and 4) and where the stem cells are presumably located1,3–5. I report here that the presence of hypersensitive cells at the base of the crypts can be demonstrated after whole-body X or γ irradiation, and to describe the time sequence for the production and loss of these killed cells together with their dose-response relationship.

Gut instincts: thoughts on intestinal epithelial stem cells

The surface of the gastrointestinal tract is lined by a simple columnar epithelium that is folded to form a number of invaginations, or crypts, that are embedded in the connective tissue. Each crypt contains approximately 250 cells, depending on its species and anatomical location. Crypt size and organization are generally uniform within a given region of the gastrointestinal tract (1). Of the major gut epithelial cell types, all but one (the Paneth’s cells of the small intestine) move upward toward the lumen of the gut as they mature. Hence, the differentiated, functional cells are found mainly on the villi (small intestine) or toward the top of the colonic crypt — the intercrypt table — in the large intestine. During the latter stages of the process, these mature epithelial cells become senescent and are shed intact into the lumen. Cells shed from the gut must be replaced by a steady supply of cells generated in the low- to mid-crypt region, where, at least in the mouse, up to 60% of the crypt cells divide twice daily. Because of this continuous upward migration, the location of a cell within the migratory stream indicates its stage in the process of maturation. Intestinal stem cells reside at the origin of the migration, which is found just above the crypt base in the small intestine and at the crypt base in the colon (2). As with normal homeostatic proliferation, crypt regeneration after cytotoxic damage also appears to originate at this site. Unfortunately, the stem cells responsible for tissue homeostasis and regeneration cannot be identified morphologically or distinguished from other epithelial cells by any recognized set of markers. Hence, most interpretations of stem-cell behavior are based upon monitoring cohorts of cells before and after perturbation of the tissue. This approach offers direct insights into the dynamics of the crypt-cell population, but only limited steady-state behavioral information on the cells themselves. This limitation may be inevitable in the absence of good molecular markers, but it must be kept in mind when interpreting many current experiments. Estimates of stem-cell number vary widely, from 0.4% to 60% of the crypt cells, with the smallest value implying that a single stem cell occurs in each crypt. This discrepancy arises largely because of differences in the operational definition of the stem cell. It is now generally accepted that “stemness” is not a single property, but a number of properties that can be manifested under different conditions. Thus, a stem cell must be undifferentiated (relative to the other epithelial cell types, but not necessarily relative to embryonic cells) and capable of proliferation and self-maintenance, producing many differentiated progeny, and regenerating the tissue after injury (3). It must also retain the ability to switch between these options when appropriate. Hence, the properties, and probably the number, of stem cells in a crypt may change in response to circumstances, including the choice of experimental manipulations (which often fail to mimic the conditions that these cells normally encounter in vivo). Nevertheless, these manipulations have brought to light some interesting properties of this system, including the possible ability of partially differentiated cells to dedifferentiate and replenish the supply of true stem cells. This degree of plasticity is unexpected based on the traditional understanding of stem-cell function. Various experiments suggest that the number of stem cells per crypt is tightly regulated, implying that stem cells can somehow detect each other’s presence and respond appropriately. The number of functioning stem cells per crypt greatly influences numerous aspects of crypt organization and control. Thus, as discussed below, the changes in stem-cell number can alter stem-cell cycle time, the number of divisions before differentiation, the number of lineages each stem cell normally generates or is capable of generating, and the number of cells capable of tissue regeneration after damage. Finally, because these cells are maintained throughout the lifetime of an animal, the number of stem cells may correspond to the number of cells that are capable of generating a carcinoma. In a crypt with multiple stem cells, a central question is whether each stem cell produces just one cell type among the various differentiated phenotypes, or whether each stem cell is fully pluripotent, capable of producing all the intestinal epithelial cell types under steady-state conditions. A single stem cell is certainly capable of producing more than one lineage, as seen in a regenerative situation where only a single clonogenic cell remains and yet the whole crypt cell repertoire is reestablished. However, this does not prove conclusively that this also occurs in a normal steady-state crypt. However, it is unlikely that each stem cell is unipotent in the steady state, because one would then expect to observe dramatic fluxes in the number of each differentiated cell type as stem cells are deleted or undergo symmetrical division. This has not been reported. Further evidence for steady-state pluripotency comes from following the expression of G6PD polymorphisms or populations of mutant cells with varying lectin-binding properties, as described below. In such studies, crypts contain cells of various phenotypes, and the mutant areas change over time in size and extent. The mechanism by which a cell commits and proceeds to full differentiation is currently unknown. However, certain homeobox transcription factors are likely to be involved. Homeobox genes determine cell fate and general pattern formation in many tissues, particularly in regard to cephalocaudal patterning, and the homeobox-containing proteins cdx-1 and cdx-2 appear to regulate epithelial differentiation, possibly by transducing signals from the underlying mesenchyme.

The effect of age and menstrual cycle upon proliferative activity of the normal human breast.

The aim of this study was to determine the proliferative activity within the epithelial cells of the normal human breast in 122 patients (6 reduction mammoplasties and 116 fibroadenoma excisions) in relation to age and the phase of the menstrual cycle. Thirty three of the patients were on oral contraceptives and 33 were parous. Thin tissue slices were incubated with tritiated thymidine and processed for autoradiography. Other samples were fixed directly and prepared for histology. The labelling, mitotic and apoptotic indices (LI, MI and AI) were determined and all illustrated considerable variability. The labelling indices are significantly (P less than 0.05) influenced by both patient age and stage during the menstrual cycle and ranged from 0-11.5%. Maximum LI values were obtained on the 20.8th day of the cycle. A square root transformation of the data was used to reduce the skewness of the data to a more normal distribution. The square root of the LI declined by 0.22 per decade. The mitotic data showed similar significant (P less than 0.05) correlations against age and day of cycle with a peak on the 21.5th day of the cycle, a decline by 0.072 per decade and a range from 0-0.6%. The data for apoptotic cells were less clearly influenced by the stage of the menstrual cycle but showed a significant (P less than 0.5) decline with age. The AI in parous patients was significantly higher than that in non-parous patients. There was no significant effect of oral contraceptives on any of the parameters measured when age and stage of cycle were taken into account. The considerable variability in the data could not be fully accounted for by either technical factors, the age of the patients, or the day of the cycle. We conclude that proliferation is negatively related to age and is influenced by the menstrual cycle but that additional as yet unknown factors must account for a large part of the variability seen in the data.

Regulation and Significance of Apoptosis in the Stem Cells of the Gastrointestinal Epithelium

In rapidly proliferating tissues the stringent control of cell proliferation and cell death by apoptosis is central to the maintenance of tissue homeostasis. In the gastrointestinal tract most work studying the control of tissue cell number has traditionally focused on the growth factor control of proliferation, and the changes that occur during carcinogenesis. However, in recent years it has become increasingly apparent that the control of apoptosis is also crucial. Apoptosis is an important mechanism for eliminating both excess normal cells and those cells which have sustained damage; therefore maintaining a tissue, i.e., stem cells with preserved DNA integrity.

Slowly cycling (label‐retaining) epidermal cells behave like clonogenic stem cells in vitro

Abstract. Slowly cycling label‐retaining epidermal cells were identified by light microscopic autoradiography in the dorsal epidermis and hair follicles of adult mice 8–10 weeks after twice daily injection of [3H]dT on days three through five after birth. Pulse‐labelled epidermal cells were identified in the epidermis and hair follicles of 7–8 week old mice 1 h after a single injection of [3H]dT at 8.00 a.m. For mice of both groups, epidermal cells including those from the hair follicles were harvested by trypsinization and were cultured from low density on feeder layers of irradiated Swiss mouse 3T3. On days 2, 4, 5, 7, 10 and 12, the cultures were fixed and processed for light microscopic autoradiography, and the distribution of labelled nuclei was quantified. On day 2 of culture, both label‐retaining cells (LRC) and pulse labelled cells (PLC) were found primarily as single cells. After five days, LRC were found as pairs and clusters having silver grain counts consistent with their division. In contrast, PLC remained primarily as single cells. These results suggest that LRC may divide to form colonies (are clonogenic) whereas PLC are rarely clonogenic. The significance of this experiment is that it suggests that the LRC may not only be persistent in the epidermis, but that they may also be cells with relatively greater proliferative potential than the PLC and are thus likely to be stem cells.

Cell migration and organization in the intestinal crypt using a lattice-free model

We present a novel class of spatial models of cell movement and arrangement applied to the two‐dimensional cellular organization of the intestinal crypt. The model differs from earlier approaches in using a dynamic movement on a lattice‐free cylindrical surface. Cell movement is a consequence of mitotic activity. Cells interact by viscoelastic forces. Voronoi tessellation permits simulations of individual cell boundaries. Simulations can be compared with experimental data obtained from cell scoring in sections. Simulation studies show that the model is consistent with the experimental results for the spatial distribution of labelling indices, mitotic indices and other observed phenomena using a fixed number of stem cells and a fixed number of transit cell divisions.

The relationship between ionizing radiation-induced apoptosis and stem cells in the small and large intestine

Apoptosis is observed in the crypts of the small intestine of healthy animals and man (spontaneous apoptosis). The levels can be dramatically elevated 3-6 h following ionizing radiation exposure. Both the spontaneous and radiation-induced apoptosis in the small intestine crypts are most frequently observed at the positions in the crypt associated with stem cells (about four cell positions from the base of the crypt). The number of apoptotic deaths can be counted in routine histological preparations, but interpretation of the counts is complicated by numerous factors. However, recording the number of cells containing one or more apoptotic fragments in crypt sections provides a good estimate for the absolute number of cell deaths in crypts. Similarities are noted in the frequency and cell positional relationship of radiation-induced apoptosis in the small intestine of various strains of mice and one strain of rat. Apoptosis in the large intestine is generally lower in frequency than in the small intestine and, for the mid-colonic and rectal regions, has a different cell positional frequency distribution, with the highest apoptotic yield at the crypt base. The caecal colon has a pattern of apoptotic distribution more similar to that in the small intestine. After exposure to 1 Gy ionizing radiation, the maximum apoptotic yield occurs over a period of 3-6 h in the small intestine. There is some unexplained variability in the values between groups of mice and between different mouse strains. After 8 Gy, the yield remains elevated for several days, however a similar maximum yield is still observed at the early times. In mouse large intestine and rat small intestine, the yield continues to rise until about 6 Gy in mouse large intestine and until at least 10 Gy in rat small intestine. Spontaneous apoptosis is interpreted as part of the homeostatic mechanism regulating stem cell numbers. About 1.6 cells per crypt are dying at any one time. Following irradiation, there is an apparent relationship between mitotic and apoptotic levels, suggesting that these processes are linked. The dose-response relationship suggests that there are about six apoptosis-susceptible cells in crypts of the small intestine, with about 2-4 of these occurring at cell positions in which there are other more resistant clonogenic cells. In the large intestine, the position of these apoptosis-susceptible cells varies with region, but the numbers are similar.

Effects of soy-protein supplementation on epithelial proliferation in the histologically normal human breast.

A high dietary intake of soy products (eg, as in Japan and Singapore) has been associated with a reduction in the incidence of breast cancer in premenopausal women. Phytoestrogens present in soybeans inhibit human breast cancer cell proliferation in vitro and breast cancer development in animal models, but no data exist on the effects of phytoestrogens on histologically normal human breasts. This study examines the effects of dietary soy supplementation on the proliferation rate of premenopausal, histologically normal breast epithelium and the expression of progesterone receptor. Women (n = 48) with benign or malignant breast disease were randomly assigned to receive their normal diet either alone or with a 60-g soy supplement (containing 45 mg isoflavones) taken daily for 14 d. Biopsy samples of normal breasts were labeled with [3H]thymidine to detect the number of cells in S phase and were immunocytochemically stained for the proliferation antigen Ki67. The phytoestrogens genistein, daidzein, equol, enterolactone, and enterodiol were measured in serum samples obtained before and after supplementation. Serum concentrations of the isoflavones genistein and daidzein increased in the soy group at 14 d. Results showed a strong correlation between Ki67 and the thymidine labeling index (r = 0.868, P < or = 0.001). The proliferation rate of breast lobular epithelium significantly increased after 14 d of soy supplementation when both the day of menstrual cycle and the age of patient were accounted for. Progesterone receptor expression increased significantly in the soy group. Short-term dietary soy stimulates breast proliferation; further studies are required to determine whether this is due to estrogen agonist activity and to examine the long-term effects of soy supplementation on the pituitary gland and breast.

Characterization of Radiation-induced Apoptosis in the Small Intestine and Its Biological Implications

The small intestine with its high cell proliferation, well-accepted hierarchy, high radiation susceptibility and low cancer incidence is a useful model for studying the controls of cell replacement. Apoptosis, which represents part of the overall homeostatic process, occurs spontaneously at the stem cell position in the crypts, and very small doses of radiation elevate the levels of apoptosis rapidly in this region. Other cytotoxic agents also target cells in this region including several mutagenic chemicals. Yet other drugs target cells at higher positions in the crypt indicating that all crypt cells possess the programme for apoptosis, but this is normally suppressed in many of the cells. In contrast, high doses of radiation are required to reproductively sterilize the crypts and, using clonal regeneration techniques, the number of clonogenic cells is dependent on the levels of damage induced (dose), i.e. the more injury that is induced the greater number of cells that are recruited into the clonogenic compartment. All doses of radiation trigger rapid changes in proliferation in the stem cell region which suggests that the detection of the induced cell death (even small levels, such as one apoptotic cell per crypt) is efficient and has rapid consequences. p53 may be involved in this damage recognition and apoptosis initiation. The studies to date suggest that apoptosis plays an important role in this tissue in terms of its homeostasis and its protection against carcinogenesis by removal of potentially carcinogenic damaged cells.