Stem cells: the generation and maintenance of cellular diversity

Cell divisions during embryonic development give rise to new differentiated cell types or increase the total number of cells in the embryo. In contrast, the major role of cell division in adult life is to maintain the number of differentiated cells at a constant level: to replace cells that have died or been lost through injury.The rate at which new cells are produced in the adult is a measure of how rapidly the cell population is turning over and, on this basis, tissues can be divided into three broad categories (Leblond, 1963). In tissues with static cell populations, such as nerve and skeletal muscle, there is no cell division and most of the cells formed during development persist throughout adult life. In tissues containing conditional renewal populations, such as liver, there is generally little cell division, but in response to an appropriate stimulus most cells can divide to produce daughters of the same differentiated phenotype. Finally, tissues with perma-nently renewing populations, including blood, testis and stratified squamous epithelia, are characterized by rapid and continuous cell turnover in the adult: the terminally differentiated cells have a short lifespan and are replaced through proliferation of a distinct sub-population of cells, known as stem cells.The simplest definition of a stem cell is that it is any cell with a high capacity for self-renewal, extending throughout adult life. In addition, stem cells are usually considered to have the potential to produce differen-tiated progeny and, as such, a stem cell may have a less 'mature' or less 'differentiated' phenotype than its daughters (Lajtha, 1979). Using these criteria, most of the progenitor cell populations that arise during embry-onic development are not stem cells, since they do not self-renew; however, as we shall describe, they have a number of properties in common with the stem cells of adult organisms, including differentiation potential and capacity for asymmetric cell division.Three adult mammalian tissues in which stem cells have been extensively studied are the haemopoietic system, the epidermis and intestinal epithelium. In our review, we shall begin by describing these stem cell populations: the evidence that they exist; their identifi-cation, location and differentiated progeny. Three com-mon features that emerge are illustrated in Fig. 1A. First, stem cells have the capacity for unlimited self-renewal, in the context of the fifespan of the organism. Second, they have the potential for asymmetric div-isions, such that one daughter is itself a stem cell while the other is committed to undergo terminal differen-tiation. Third, the differentiation process is irreversible, since the daughters of a cell that is committed to terminal differentiation are never stem cells.In haemopoiesis and in intestinal epithelium, the stem cells are pluripotential, giving rise to more than one type of differentiated cell. In the epidermis, in contrast, there appears to be only a single pathway of terminal differentiation (Fig. IB). In all three tissues, (here is evidence that differentiation of stem cell progeny occurs via transit amplifying, or progenitor, populations that have a more limited capacity for self-renewal (Fig. 1B).We shall discuss what is known of the mechanisms that regulate these aspects of stem cell behaviour, drawing on the properties of analogous cells in a range of other tissues and organisms.The terminally differentiated cell types of blood, shown in Fig. 2, are all derived from pluripotential stem cells that are located in bone marrow. The first evidence for the existence of such a stem cell population came from experiments in which bone marrow cells from healthy mice were injected into mice that had received lethal doses of radiation (Ford et al. 1956; Till Joyner et al. 1983; Williams et al. 1984; Dick et al. 1985; Lemischka et al. 1986).The haemopoietic stem cells do not give rise to terminally differentiated cells directly, but via prolifer-ating progenitor populations, as illustrated in Fig. 2. In this way, a relatively small number of stem cells (estimated as 0·4% of the total population of haemo-poietic cells; Lord Bradley Dexter et al. 1977).Although the existence of haemopoietic stem cells is well established, their identification has proved diffi-cult. The strategy has been to separate subpopulations of marrow cells on the basis of buoyant density, sensitivity to antimitotic agents or expression of cell surface antigens, and to look for enrichment of stem cells on the basis of in vitro or marrow reconstitution assays. In mouse, the following profile of the bone marrow reconstituting cells is emerging: they do not express any markers of either granulocyte, macro-phage, B or T cell lineages (Müller-Sieburg et al. 1988), but bind wheat germ agglutinin (Visser et al. 1984; Lord these cells include virtually all the stem cells detected by in vitro assays, and baboon cells bearing CD34 are able to reconstitute the marrow of irradiated animals (Berenson et al. 1988). However, although subpopulations of bone marrow cells that contain haemopoietic stem cells can be defined, no unique markers of stem cell populations have yet been identified.Cells within the bone marrow appear to lack the high degree of spatial organization characteristic of cells in epithelia. Nevertheless, studies of the distribution of different subpopulations of bone marrow cells suggest that it is not random (Western Lord Potten Potten Fig. 3).Further evidence for proliferative heterogeneity has come from studies of cultured human epidermal kéra-tinocytes. Sheets of cultured cells grafted onto suitable recipients form epidermis that persists for years, indi-cating that stem cells are not lost in culture (Gallico et al. 1984). However, only a small proportion of the cells in culture undergo extensive proliferation; the rest either fail to divide or form small abortive colonies in which all the cells terminally differentiate (Barrandon, and see Fig. 1A).Although extensive proliferation in culture or during recovery from radiation is thought to be characteristic of epidermal stem cells, there is evidence that under normal steady-state conditions the stem cells divide more slowly than the transit amplifying population. Thus kinetic analysis of mouse epidermis and in vitro experiments with cultured human kératinocytes suggest that stem cells have a longer cell cycle time and shorter S phase than transit amplifying cells, and that they retain 3Hthymidine for much longer than the transit population (Bickenbach, 1981; Dover Clausen et al. 1984; Jensen et al. 1985b; Morris et al. 1985; Albers et al. 1986). In addition, cells with stem-like characteristics (high capacity for self-renewal) are resistant to treatments that induce premature terminal differentiation in culture, such as exposure to TPA or cultivation in suspension (Parkinson Parkinson et al. 1983; Hall Cotsarelis et al. 1989) and, in tongue papilla, they are found in those regions of the basal layer that project deeply into the underlying connective tissue (Hume 1983).In some regions, such as the dorsal skin of the mouse, the basal layer is flat and kératinocytes in the suprabasal layers are arranged in columns, like a stack of coins (Mackenzie, 1970; Christophers, 1971). It has been proposed that each stack corresponds to an epidermal proliferative unit (EPU), consisting, in the basal layer, of a stem cell surrounded by 5-6 transit amplifying cells and less than four postmitotic cells (i.e. committed to terminal differentiation) (Potten, 1974, 1981). The model is based on cell kinetic analysis and is supported by the findings that the basal cells that retain 3Hthymi-dine (putative stem cells -see earlier) fie at the centre of the stacks of suprabasal cells in mouse dorsal epidermis (Morris et al. 1985) and that gap junctional communication compartments are approximately the same size as EPUs (Pitts et al. 1988). However, a columnar arrangement of kératinocytes is lacking in most body sites, and the pattern of mosaicism revealed in the epidermis of chimaeric mice is not consistent with the EPU model (Schmidt et al. 1987).The lining of the small intestine is a. monolayer of epithelial cells organized into crypts and villi (Fig. 4). Cell proliferation is confined to the crypts; differen-tiated cells migrate to the surface and are shed from the tips of the villi. There are four differentiated cell types: Paneth cells at the base of the crypts and columnar, goblet and entero-endocrine cells in the rest of the crypts and on the villi; none of these mature cells divide, suggesting that they are renewed by prolifer-ation of a stem cell population. Evidence for the existence of a single stem cell compartment generating all four types of progeny came first from the observation that, after radiation-induced cell death, surviving pro-liferative cells phagocytose cell debris and the phago-somes can be used as markers of their progeny; within 30 h phagosomes are found in all the differentiated cell types (Cheng Kirkland, 1988).The stem cells are thought to be located at or near the base of each crypt, since proliferating cells higher in the crypt migrate upwards and are not therefore permanent residents (Potten et al. 1987). It has been proposed that stem cells give rise to committed progenitors for each differentiated cell type (Cheng see Fig. 4). Kinetic studies suggest that there are 4-16 stem cells per crypt (Potten Potten et al. 1987) .However, studies with mouse aggregation chim-eras or mice heterozygous for markers that can be detected histochemically show that the epithelium of individual crypts in the small and large intestine of adult mice is always composed of cells of a single parental type, and hence that each crypt is probably derived from (and maintained by) a single stem cell during development (Ponder et al. 1985; Griffiths et al. 1988; Schmidt et al. 1988; Winton et al. 1988). In adult colonic epithelium there is evidence that each crypt is main-tained by proliferation of one stem cell (Griffiths et al. 1988; Winton et al. 1988; Fig. 5). No molecular markers for intestinal stem cells have so far been reported.In the previous sections, we described evidence for the existence of stem cells in bone marrow, epidermis and intestinal epithelium, and what progress has been made towards their identification. In all three tissues, a relatively small number of stem cells give rise to a large number of terminally differentiated cells via a transit amplifying (progenitor) population and the stem cells differ from their progeny in phenotype and/or location. In each tissue, the processes of stem cell renewal and production of differentiated progeny must be tightly coordinated, in order to meet the varying requirements of the body for differentiated cells. These common features enable us to ask a number of general questions about stem cells:-1. What mechanisms determine asymmetry of fate, such that stem cells produce both stem cells and daughters committed to terminal differentiation?2. What mechanism allows for unlimited self-renewal of stem cells, but a finite number of rounds of division within the transit/progenitor populations?3. Is differentiation from the stem cell compartment irreversible?Observations from a range of different tissues and organisms provide partial answers to these questions.One feature of all three stem cell systems is their capacity to produce both stem cells and cells that are committed to terminal differentiation, represented symbolically as A→ A + B. This can, theoretically, be achieved in two ways. In the first, the outcome of every division is predetermined and invariant: A→ A + B. In the second, individual stem cell divisions may have different outcomes, such that A→A+A or B + B or A + B, but, on a population basis, the end result is the production of equal numbers of A and B. This second situation could arise in three ways: if the outcome of each division is stochastic; if it is environmentally regulated; or if the stem cell population is hetero-geneous.Some of the best examples of invariant asymmetric divisions are found in yeast, Caenorhabditis elegans and plants, in which lineage maps of cell fate can be constructed. In cases where lineage is not the sole determinant of the outcome of divisions, such as in adult mammalian tissues, in Hydra and certain C. elegans tissues, some progress has been made in ident-ifying regulatory factors.Yeast cells can either be haploid or diploid, the diploid state resulting from fusion of two haploid cells that differ in mating type. Although the progeny of haploid cells normally inherit the mating type of the parental cell, switching of mating type can occur. It is the ability to switch that is inherited asymmetrically (Strathern Nasmyth, 1983; Klar, 1987a; Nas-myth Sternberg et al. 1987). The ability of mother, but not daughter, cells to transcribe HO appears to be due to asymmetric distribution of these gene products at div-ision (Klar, 1987b).In the fission yeast, S. pombe, mating type switching occurs such that of the four progeny of a single cell obtained after two generations, only one is switched; in other words, only one of the two daughters in the first generation is competent to produce a switched cell in the subsequent generation. As in 5. cerevisiae, mating type is determined by gene conversion but, in S. pombe, the ability to switch is inherited chromosomally, not through unequal distribution of cytoplasmic or nuclear 1984; Klar, suggest that asymmetry from of parental (Klar, elegans development occurs via an of cell divisions and lineage maps of the of every cell in the adult can be 1988). There is some evidence that lineages normal For example, in the lineages of the each cell to one cell and one cell There is evidence for in individual cells were cultured and the differentiation of their clones after The experiments that within the first three divisions of a haemopoietic stem cell and its at least one cell with can be produced and at least two committed progeny can arise from the two or three divisions et al. et al. This has to the that the stem and transit populations are not shown in Fig. but a from cells of high self-renewal capacity and low of differentiation to those of low self-renewal capacity and high to differentiation et al. 1977; et al. et al. source of heterogeneity could be stem cell This has not been for haemopoietic cells within the of individual since the capacity of or mouse marrow is the same 1988) and the of in the adult germ et al. 1987). The number of the stem cells in different body regions most are found in the with in the or In than lineage the major role in the fate of stem cell spatial in the distribution of different differentiated cell types is a of stem cell A called has been isolated which is by nerve cells and stem cells to differentiate into nerve cells & 1981). is for as to growth and In addition, there is an in the and in the body is when gap junctional communication is et al. further of on stem cell in Hydra is the mechanism that the of stem cells to epithelial cells is the number of stem cells is there is a rapid increase in the size of the stem cell population the normal stem cell is This is achieved by an increase in the of self-renewal of the stem cells et al. 1987). mechanisms presumably maintain stem cell in other organisms, but they have to be