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1 Haemopoietic tissue ERIC G. WRIGHT BRIAN I. LORD
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Haemopoietic tissue can broadly be divided into three types of cell population: multipotent progenitor cells, committed progenitor cells and maturing/mature cells. The first two of these are the populations responsible for the maintenance, growth and differentiation of the tissue into functional blood cells. In this short introductory chapter, their roles will be described briefly and their interrelationships defined. Subsequent chapters will deal in more detail with many of the separate parts. HAEMOPOIETIC STEM CELLS Mammalian blood cells have finite life spans, considerably shorter than the life span of the organism, and the maintenance of constant numbers of cells in the peripheral blood is achieved by the proliferation and differentiation of precursor cells which are located primarily in the bone marrow. These precursors are all derived from a common self-maintaining population of stem cells established during embryogenesis (Metcalf and Moore, 1971). Stem cells are regarded as cells with extensive self-maintenance (selfrenewal) capacity, extending throughout the whole (or most) of the life span of the organism, and whilst multilineage differentiation potential may be a property of stem cells it is not an essential feature of 'sternness'. In studies of murine haemopoiesis, spleen colony-forming units (CFU-S) have been widely regarded as the cells most closely fitting this definition, due to their considerable proliferative potential, self-renewal characteristics and capacity for multilineage differentiation (Till and McCulloch, 1961, 1980). Recently Potten and Loeffler (1990) defined stem cells as'... capable of, (a) proliferation, (b) self-maintenance, (c) the production of a large number of differentiated, functional progeny, (d) regenerating the tissue after injury and (e) flexibility in the use of these options'. The repopulating potential of stem cells is particularly relevant to haemopoiesis as it is now clear that sple~n colony-~orm~ng cells ar~ heterogeneous with respect to many biophysical and biological properties and most do not have significant longterm repopulating capacity (Muller-Sieberg et al, 1991). ' Whilst many experiments have clearly demonstrated kinetic heterogeneity of colony formation by CFU-S, developments in cell separation Bailliere's Clinical HaematologyVol. 5, No.3. July 1992 ISBN 0-7020-1628-4
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E. G. WRIGHT AND B. I. LORD
techniques have led to the physical isolation of subsets of cells that are capable of generating spleen colonies 8 or 12 days post-transplantation (Visser et al, 1984). An important development in cell sorting was the use of rhodamine-123, a supravital, cationic, fluorescent dye with a relatively high affinity for mitochondrial membranes. Day-12 CFU-S with low affinity were shown to have a greater marrow repopulating ability than day-12 CFU-S with high affinity (Bertoncello et aI, 1985) and only about half of the day-l2 CFU-S (with low affinity for rhodamine-123) had radioprotective ability (Mulder and Visser, 1987). Using a combination of negative and positive selection with immunomagnetic beads and fluorescence-activated cell sorter (FACS) sorting that did not include rhodamine-I23 , co-enrichment of day-12 CFU-S and cells providing 30-day radioprotection has been reported (Spangrude et al, 1988); more recently, these authors showed that whilst their sorted cells are able to generate all haemopoietic lineages in vivo, only a subset undergoes long-term self-renewal (Smith et al, 1991). Using a different protocol, Ploemacher and Brons (1988, 1989), separated cells forming spleen colonies from cells responsible for reconstitution of the bone marrow stem cell compartment following irradiation. These experiments demonstrated for the first time, and without the application of negative selection by the use of cytotoxic agents, that marrow repopulating cells can be separated from day-12 CFU-S and should be considered as pre-CFU-S. That pre-CFU-S are the primary marrow repopulating cells has received support from experiments demonstrating that 5-fluorouracil, a drug that preferentially spares pre-CFU-S (Hodgson and Bradley, 1979), also spares cells responsible for long-term myeloid and lymphoid repopulation in a competitive repopulation assay (Lerner and Harrison, 1990). A competitive strategem was used to establish a quantitative assay for stem cells capable of long-term repopulation (Szilvassy et al, 1990) and the incidence of this cell (approximately 1 per 100000 marrow cells) is comparable to the estimated frequency obtained in earlier transplantation studies (Boggs et al, 1982; Micklem et al, 1987). It is probable that the lymphoid and myeloid lineages diverge from this pre-CFU-S stage. An interpretation of information currently available is that the stem cell compartment is a developmentally structured continuum. In this continuum the most primitive members have the greatest long-term repopulating ability and are the most resistant to proliferation and differentiation stimuli. In subsequent divisions, cells become detectable as CFU-S with decreasing self-renewal capacity and increasing probability of becoming committed to the various haemopoietic lineages. In the steady state, day-12 CFU-S are more primitive than day-7/8 CFU-S but are not the most primitive stem cells. Those cells with long-term repopulating ability, as assessed by transplantation into lethally irradiated recipients, reside in that pre-CFU-S compartment. A variety of in vitro c1onogenic assays that detect cells from within the stem cell continuum have been reported, although there is still some uncertainty as to how and where many of these cells fit in the development of the stem cell population and what contribution they make to normal steadystate haemopoiesis (Muller-Sieburg et aI, 1991). Nevertheless, because they
exhibit stem cell properties they have been useful in studies of potential stem cell regulators. A developmentally structured stem cell compartment may have important functional significance in vivo, since recent transplantation experiments have demonstrated two phases of bone marrow engraftment: an initial but transient engraftment (essential for survival following the conditioning irradiation) followed by a delayed but long-term reconstitution of the haemopoietic system. These two phases can probably be attributed respectively to the later and earlier members of the continuum, with the later members being detected by spleen colony formation and the earliest members only by their long-term repopulating capacity (Jones et al, 1989, 1990). HAEMOPOIETIC PROGENITOR CELLS Progenitor cells are the immediate progeny of multipotent stem cell differentiation and possess limited further differentiation potential; they are also known as lineage-restricted or committed progenitors in recognition of this characteristic (Metcalf and Moore, 1971; Metcalf, 1977, 1988; Testa, 1985). Progenitor cells are detected by their ability to give rise to colonies of morphologically recognizable differentiation progeny in semisolid clonal cultures. The original culture assays that identified murine progenitor cells were characterized by colonies of mast cells, granulocytes and/or macrophages (Pluznik and Sachs, 1965; Bradley and Metcalf, 1966; Ichikawa et ai, 1966) and led to the demonstration that neutrophils and mononuclear phagocytes are closely related populations, in me~y cases sharing a common progenitor cell. The clonogemc cells were operationally defined as colonyforming cells (abbreviated to CFC) and prefixed by letters denoting the cell type to which they gave rise, e.g. GM-CFC for cells generating colonies of granulocytes and macrophages. The development of these colonies required the presence of unidentified activities produced by feeder cells or present in media conditioned by the growth of a variety of connective tissue cells. These activities were operationally defined as colony-stimulating factors (CSFs) (Metcalf, 1977, 1984). Subsequent studies in a number of laboratories led to the identification of human progenitor cells committed to the various haemopoietic lineages (Table 1) and to the characterization of the four major CSFs (Table 2). Table 1. Chronology of clonal cultures for human progenitor cells. Cell lineage
Granulocytes and Macrophages Eosinophils Erythroid Cells T-Iymphocytes Mixed Lineage Megakaryocytes Mast Cells B-Iymphocytes Multilineage Blast Cells
Pike and Robinson (1970) Chervenick and Boggs (1971) Tepperman et al (1974) Rozenszajn et al (1975) Fauser and Messner (1978) Vainchenker et al (1979) McCarthy et al (1980) Izaguirre et al (1980) Nakahata and Ogawa (1982)
E. G. WRIGHT AND B. I. LORD
Thus, haemopoiesis may be regarded as a three-tiered hierarchy in which the most ancestral haemopoietic stem cell, identified as the long-term repopulating cell in marrow transplantation studies, heads a developmentally structured stem cell compartment, eventually giving rise to the multipotential CFC which are able to produce multi- or mixed-lineage differentiation in vitro (Muller-Sieburg et ai, 1991). These, so-called Multi-CFC or CFC-Mix or CFU-GEMM (mixed granulocyte, erythroid, macrophage , megakaryocyte colony-forming units) in human marrow cultures undergo one or more differentiation steps to produce a variety of developmentally more restricted progenitor cells. Table 3 illustrates this structure and indicates the assays available to enumerate the cells. The colonies derived from progenitor cells are clones and consist of up to lOS cells after 7-14 days culture. Progenitor cells have little, if any, capacity for self-replicative divisions and most mature colonies contain no stem or Table 2. Characteristics of human colony-stimulating factors. Major cellular sources
Monocytes Fibroblasts Endothelial cells
T-Iymphocytes Fibroblasts Endothelial cells
The M-CSF proteins are homodimers and the mRNAs are readily detected in the cellular sources . The mRNAs for the other factors are those detected after stimulation with mitogens or cytokines and the protein size ranges are for factors expressed in mammalian cells. Table 3. The three-tiered haemopoietic system: developmental structure of haemopoietic tissue and assays appropriate to the various stages. Cell compartment
Stem cells (a) Primitive (b) Multipotent
Assays In Vivo (murine)
In Vitro (murine /human)
Haemopoietic repopulation Marrow repopulating ability Pre-CFU-S CFU-S
Colony-forming cell (·CFC) responds to Colony-stimulating factor (-CSF)
Maturing and mature cells
HPP-CFC Multi-CFC (CFC·Mix) CFU-GEMM (human)
Cell kinetics (murine and human)
Morphology Quantitation of cell production
progenitor cells. Progenitors are committed to a programme of differentiation and maturation, with any self-replicative capacity serving only to amplify the population prior to the terminal maturation of the cells. Most progenitors are unipotential cells, generating colonies of third-tier maturing/mature cells of a single lineage, and the incidence of any particular progenitor cell in adult haemopoietic tissues is of the order of 1% or less (Metcalf, 1977, 1984, 1988). They are not identifiable by definitive morphological characteristics. The survival, proliferation and differentiation of progenitor cells in vitro is absolutely dependent on the continuous presence of CSFs (Metcalf, 1977, 1984) and these have been the subject of a number of recent reviews (Clark and Kamen, 1987;Morstyn and Burgess, 1988;Dexter et al, 1990; DiPersio, 1990; Moore, 1990). The functions of these factors are complex but recent biochemical characterization and the molecular cloning of their structural genes has facilitated large-scale production of recombinant materials. These developments have made possible more detailed studies of progenitor cells which, in turn, have led to the therapeutic application of the CSFs (Devereux and Linch, 1989; Dexter, 1990; Morstyn, 1990; Moore, 1991; and see Chapter 13). Several cell types in culture are able to produce one or more of the CSFs either constitutively or, more commonly, following a variety of stimuli. Granulocyte (G)-CSF and macrophage (M)-CSF are produced by fibroblasts and endothelial cells following stimulation with interleukin 1 (IL-l), endotoxin or tumour necrosis factor (TNF) (Quesenberry and Gimbrone, 1980; Broudy et al, 1986; Koeffler et al, 1987; Munker et al, 1986; Zucali et al, 1986; Bagby et al, 1987); G-CSF and M-CSF by monocytes following phorbol ester and 'V-interferon treatments (Ramaldi et al, 1987); M-CSF by monocytes after stimulation with GM-CSF or TNF (Horiguchi et al, 1987; Oster et al, 1987; Ramaldi et al, 1987); G-CSF by monocytes exposed to Multi-CSF and M-CSF (Metcalf and Nicola, 1985); and GM-CSF and MultiCSF are produced by activated lymphocytes (Ihle, 1983; Yang et aI, 1986; Clark and Kamen, 1987). In addition to the classicallydefined CSFs, several other cytokines that influence the proliferation and differentiation of haemopoietic cells have been identified (Balkwill and Burke, 1989; Jones and Millar, 1989; Steel and Hutchins, 1989; Moore, 1990, 1991). Many are Iymphokines that directly affect lymphopoiesis and synergize with CSFs to affect myelopoiesis, with some having effects at the stem cellleveJ. Under appropriate conditions, synergistic interactions allow members of the stem cell compartment to proliferate and differentiate in vitro (Heyworth et al, 1988; McNiece et aI, 1989, 1990; Lorimore et aI, 1990; Moore, 1990). At present, however, it is unclear how many of these observations relate to haemopoiesis in vivo. MATURING AND MATURE CELLS
The third stage of ha~mop?iesis en~omI?asses the b.uIk (- 95%) of the cells. It represents the proliferative amplification of the differentiated cells as they
E. G. WRIGHT AND B. I. LORD
mature to become fully functional blood cells (Table 3). These cells are recognizable and classified by their classical morphological characteristics. Their performance, which is responsive and adaptive to a variety of stress situations (see Chapter 3), is assessed by auto radiographic techniques which are designed to measure the kinetic parameters of cell proliferation. When mature, the cells leave the marrow environment via the central venous sinus and enter the peripheral circulation, from where they carry out their appropriate functions. THE MICROENVIRONMENT In vivo, the production of haemopoietic cells occurs in association with stromal cells, and while there is considerable evidence that these cells and their products create what has been termed the 'haemopoietic microenvironment', the mechanisms underlying the physiological regulation of haemopoiesis by stromal cells are poorly understood. Haemopoietic cells removed from the body can be maintained for short periods of time in the presence of growth factors, but in the absence of growth factors they die (Metcalf, 1977, 1984). Cultured in the absence of growth factors, but in association with marrow-derived stromal cells, haemopoietic cells will proliferate and differentiate (Dexter et al, 1977a; Wright and Greenberger, 1984), with stem cell replication and commitment to differentiation being maintained for many weeks in these long-term bone marrow cultures (Dexter et aI, 1977b; Cashman et aI, 1985). A possible explanation for the ability of stromal cells to sustain haemopoietic cells for long periods of time in vitro lies in the finding that extracellular matrix molecules of stromal cells can bind CSFs and present them to haemopoietic cells in a biologically active form (Gordon et aI, 1987; Roberts et aI, 1988). In long-term bone marrow cultures, most progenitors are located in association with extracellular matrix (CoulombeI et ai, 1983) and there is evidence that this is due, at least in part, to their interaction with cell adhesion molecules (Gordon, 1988; Gordon et al, 1990; see Chapter 7). It is possible, therefore, that particular stromal cells secrete and/or sequester growth factors that direct or permit particular differentiation programmes and that the localization of particular progenitor cells to particular stromal cells may be due to specific adhesive interactions. It is also possible that similar interactions may provide microenvironments which are able to control the 'decision' made by a stem cell between replication/self-renewal and commitment to differentiation. Acknowledgements Supported by grants from the Medical Research Council and the Cancer Research Campaign.
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