Mark J. Koury
Nadim Mahmud
Melissa M. Rhodes
Blood Cells
The blood contains several different types of cells. Each of these cell types is quite distinct in appearance, and each has a specific biologic function. Erythrocytes are anucleate, biconcave discoid cells filled with hemoglobin, the major protein that binds oxygen. The erythrocytes transport the respiratory gases oxygen and carbon dioxide. Granulocytes and monocytes are cells that can exit from blood vessels and migrate among the cells of many tissues. These two cell types play key roles in inflammation and phagocytosis. Platelets are very small, anucleate cells that contain molecules required for hemostasis. In addition, platelets provide hemostasis through their abilities to adhere, aggregate, and provide a surface for coagulation reactions. Lymphocytes mediate highly specific immunity against microorganisms and other sources of foreign macromolecules. B lymphocytes confer immunity through the production of specific, soluble antibodies, whereas T lymphocytes direct a large variety of immune functions, including killing cells that bear foreign molecules on their surface membranes. Despite these extreme structural and functional differences among the cells of the blood, strong evidence exists that all of the blood cells are the progeny of a single type of cell: The hematopoietic stem cell (HSC). The processes involved in the production of all of the various cells of the blood from the HSCs are collectively called hematopoiesis. Hematopoiesis includes HSC self-renewal, HSC commitment to specific lineages, and maturation of lineage-committed progenitors into functional blood cells. Self-renewal may occur by symmetric HSC division, such as expansion of the HSC pool during fetal life or post-HSC transplantation. Other possible fates of HSC divisions include apoptosis or mobilization to the peripheral circulation following stress such as growth factor stimulation or depletion of marrow cells by irradiation or chemotherapy. During normal steady state conditions, HSCs reside mainly in the marrow cavity, but under certain stress conditions HSCs can migrate and colonize other organs like liver and spleen in a process termed extramedullary hematopoiesis.
Hematopoiesis begins early during embryogenesis and undergoes many changes during fetal and neonatal development. Unlike some organ systems that form in early life and are not continually replaced, turnover and replenishment of the hematopoietic system continue throughout life. The cells of the blood have finite lifespans, which vary depending on the cell type. In humans, granulocytes and platelets have lifespans of only a few days, whereas some lymphocytes can exist for many months. Cells are replaced as the older cells are removed and the newly formed, mature cells are added. The numbers of the various cell types in the blood are normally kept in relatively constant ranges. In particular, variations in the erythrocyte number are normally minimal, and values 30% above or below the norm for the population have significant health effects. Although the numbers of other blood cell types are not as constant as the number of erythrocytes, the production of other blood cells is also highly regulated. The regulation of hematopoiesis is complex. Some regulatory factors influence overall hematopoiesis by affecting very early progenitor cells: The HSCs and/or their progeny that have not undergone commitment to a single cell lineage. Also, specific regulatory growth factors play key roles in fostering the production of cells in each lineage. Lineage-specific regulation is necessary because of the widely varying lifespans and widely varying functions of the different mature blood cell types.
This chapter presents an overview of human hematopoiesis. However, many experiments on hematopoiesis have been done with mice, and many of the conclusions presented here are based on those experiments. All cell lineages that compose the blood are discussed. Several cell types are derived from the HSCs but are not found in the blood; the final steps of differentiation of these last types of cells occur in the tissues in which they reside. Such cells include dendritic cells of the lymphoid tissues and the skin (Langerhans cells), specialized macrophages of all types, and mast cells. Figure 5.1 is an illustration of the cell types associated with hematopoiesis and their distribution among bone marrow, blood, and lymphatic tissues.
Hematopoietic Organs
Development of Hematopoietic Organs
During prenatal development, the sites of hematopoiesis change several times (1,2,3,4,5,6,7,8). In humans and other vertebrates, the first hematopoietic cells arise during late gastrulation in the extraembryonic yolk sac in structures known as blood islands. This initial hematopoiesis is termed primitive hematopoiesis and serves a supportive role to very quickly produce erythroid cells as the circulatory system is being formed. Primitive hematopoiesis is transient, occurring on embryonic days (E) 7.5 through 13 in mice and day 19 through week 8 in humans. Primitive hematopoiesis provides erythrocytes and macrophages, but not lymphocytes or granulocytes; evidence also exists for primitive platelets (9,10). The primitive erythrocytes are large nucleated cells that have reduced erythropoietin (EPO) requirements during their development compared to definitive erythroid cells (11) that develop later. Primitive erythrocytes lose size and can enucleate while in circulation (12), and their hemoglobin contains embryonic forms of the “α family” and “β family” of globin chains (13).
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Figure 5.1. The cells of the blood and lymphoid organs and their precursors in the bone marrow. EB, erythroblast; MK, megakaryocyte. |
Definitive hematopoietic tissue arises from the mesodermal tissue located in the anterior portion of the aorta-gonad-mesonephros (AGM) region. Definitive hematopoiesis begins approximately 1 to 2 days later than primitive hematopoiesis, with colony-forming unit–spleen (CFU-S) cells arising in the mouse AGM on embryonic day 9. CFU-S cells are the first lineage of multipotent hematopoietic stem cells and arise in much smaller numbers in the yolk sac, also on day 9 in mice (14). On day 10 in mice, long-term repopulating hematopoietic stem cells (LTR-HSCs) first appear in the AGM, but not in the yolk sac. LTR-HSCs are the adult-type definitive stem cells that are able to produce all lineages of hematopoietic cells over the entire lifespan of an animal, mouse or human. Once definitive hematopoiesis begins, lymphocytes, monocytes, granulocytes, and platelets are formed as well as definitive erythrocytes. Definitive erythrocytes differ from primitive erythrocytes in that they are smaller, enucleate prior to entry into the circulation, require erythropoietin to survive, and contain fetal and adult hemoglobins instead of embryonic globin chains. One day after appearing in the AGM, LTR-HSCs are found in very small numbers in the yolk sac, and 2 days later they are also found in the liver. It is thus believed that LTR-HSCs arise in the AGM and seed the liver, the site of fetal hematopoiesis. Yolk sac–derived hematopoiesis becomes unnecessary at this point and disappears. Eventually, LTR-HSCs migrate from the fetal liver to the bone marrow in the circulation, and the bone marrow becomes the primary site of hematopoiesis, with a very small reserve of stem cells remaining in the liver.
When primitive hematopoiesis arises in the yolk sac blood islands, visceral endoderm induces both endothelial and blood cell development in the adjacent mesoderm through a member of the hedgehog family of proteins, Indian hedgehog (IHH). IHH up-regulates the expression of bone morphogenic protein-4 (BMP-4) in the developing mesodermal cells (15). When BMP-4 is up-regulated on the surface of a cell, a serine/threonine protein kinase is activated, which in turn phosphorylates Smad proteins. Members of the Smad family of proteins then translocate to the nucleus, where they target specific nuclear genes that are important for the development of both the endothelial cells that form blood vessels and the primitive blood cells located within these blood vessels (8,15). Slightly later in development, but in a similar manner, BMP-4 is expressed in the endothelial cells in the ventral portion of the developing dorsal aorta and the subjacent mesoderm inducing definitive hematopoiesis. Like primitive hematopoiesis, the first definitive hematopoiesis occurs in newly formed blood vessels, specifically in the ventral part of the dorsal aorta and in the proximal vitelline and umbilical arteries. BMP-4 has been shown to determine cell fate by inducing hematopoietic and endothelial differentiation, but also to increase proliferative and regenerative capacities of LTR-HSCs (16,17,18). Under stress conditions of anemia or hypoxia, BMP-4 is also induced in the spleens of mice, where it leads to proliferation of erythroid cells (19).
Both the definitive LTR-HSCs and the endothelial cells that form the vasculature display contain many of the same molecular cell markers. These include CD34, FLT3 ligand (FL), Flk-1, TAL-1, GATA-2, LMO-2, AML-1, and CD31 (PECAM-1 [platelet-endothelial cell adhesion molecule-1]) (6,7,20,21,22). The close physical and temporal association of endothelial and hematopoietic cells during their differentiation in the embryo, as well as the numerous shared markers, has led to the question as to whether these two cell types share a common progenitor cell, the hemangioblast (20,23). Studies with embryonic stem cells in vitro support the existence of the hemangioblast, but direct evidence of this cell type in vivo has not been demonstrated (20,23,24,25). Alternative hypotheses suggest that the intraembryonic HSCs originate from the mesoderm/mesenchyme adjacent to the aorta (26) or from subaortic patches (27), and migrate through the endothelium into the vascular lumen and subsequently mature to definitive HSCs. Other evidence indicates that vascular tissue can generate signals that promote the survival (28) and repopulating activity (29) of HSCs. Although it is possible that mesodermal cells in the splanchnopleura/AGM directly differentiate into either endothelial or hematopoietic cells without a hemangioblast stage (30), more likely, some endothelial cells are hematogenic and give rise to hematopoietic cells (25,31,32,33). The persistence of hemangioblasts during the postnatal period has been proposed (25,34,35), but further studies will be needed to provide conclusive evidence in support of this proposition. If hemangioblasts do exist during the postnatal life, two important questions to answer will be whether or not they participate in vascular repair and whether they can be identified prospectively with immune phenotypic markers and utilized in cellular therapies.
Although it is an extraembryonic organ, the placenta may be a site of hematopoiesis because HSCs are found there prior to being in the fetal circulation or fetal liver (35). Definitive hematopoietic progeny from HSCs isolated from placenta are detectable around E10.5 to E11.0, which corresponds with HSC activity observed in the AGM region. The HSC pool in the placenta continues to expand until E12.5 to E13.5 and contains much greater numbers of HSCs than are detectable in the AGM (35). Once the definitive hematopoietic cells have been formed, they rapidly seed the fetal liver, which is the major site of hematopoiesis in the midgestational period. The spleen also participates in hematopoiesis during this period. The erythrocytes produced in the fetal liver of mammals are anucleate like the erythrocytes of the adult, and they have the same α-family globin chains as in adults. In humans and some other species, the β-globin chains are a fetal type that is different from the β-globin chains in adults. In the late stages of mammalian fetal development, the bone marrow becomes the main site of hematopoiesis. In the human, the bone marrow is the exclusive site of postnatal hematopoiesis under normal circumstances, whereas in the mouse, the spleen is also a hematopoietic organ throughout life. In humans, during late fetal development, erythrocytes begin to be produced that contain adult types of β-family chains in their hemoglobin molecules, and by the end of the first year of postnatal life, this type of erythrocyte is the only one normally produced.
From naturally occurring and experimentally induced mutations in various murine genes known to be expressed in developing hematopoietic cells, a number of proteins that regulate blood cell development have been identified. Among these are several proteins that are required at various stages of hematopoietic development (i.e., the null state leads to prenatal death) (6,8). Among those proteins that are essential for blood island development in the yolk sac are the vascular endothelial growth factor receptor Flk-1 and the transcription factor TAL-1, which are expressed in both the endothelial and the hematopoietic cells, and GATA-1 and Rbtn-1, which are transcription factors required for primitive erythroid cell development. Definitive hematopoietic cells have additional proteins that are required for their development. These proteins include transcription factors GATA-2, c-myb, AML-1, and GATA-3 that are required by hematopoietic cells when they first are formed in the dorsal aorta. Other transcription factors or growth factors and their respective cell-surface receptors are required during prenatal hematopoiesis for the survival and growth of more differentiated cell types. Examples are the myeloid transcription factor PU.1, the erythroid transcription factor EKLF (erythroid Kruppel-like factor), Kit ligand/stem cell factor (KL/SCF) and its receptor Kit, and EPO and its receptor.
Structure of Hematopoietic Organs
The hematopoietic organs can be considered in terms of several operational components. First, there is the anatomic structure—the three-dimensional organization of different tissue types and their component cells (e.g., the structure of the blood vessels in proximity to the hematopoietic cells). A second operational component of hematopoietic organs is the stroma. Stroma is the term used to refer to the various cells as well as the extracellular macromolecules that occupy the hematopoietic tissue along with the hematopoietic cells. The stromal cells include specialized fibro-blasts, adipocytes, macrophages, and lymphocytes, as well as the endothelial cells of capillaries and sinuses (only the macrophages and lymphocytes are derived from HSCs). The stroma thus constitutes the microenvironment in which the hematopoietic progenitor cells grow and differentiate, and there is strong evidence that various stromal cells as well as extracellular matrix molecules play critical and diverse roles in hematopoiesis. Third, there are the hematopoietic progenitor cells: the HSCs and their progeny that become the blood cells of all lineages. The characterization of the hematopoietic progenitor component and its various differentiation processes are the main focus of this chapter.
Functional Aspects of Bone Marrow Anatomy
Studies of the marrow by electron microscopy have greatly refined our knowledge about the structures present and the topologic associations between the various cell types (36,37,38,39). Largely, the roles of the anatomic features of hematopoietic organs are unknown. However, some functions of the cells of the venous sinuses within the marrow are understood in general terms. The venous structure of the marrow cavity is a complex maze of sinuses that eventually drains into central veins. In the marrow or fetal liver of mammals, hematopoietic progenitors differentiate outside the sinuses, unlike in the marrow of birds, in which hematopoiesis occurs within the sinuses. The sinuses of the marrow in mammals are formed by a continuous layer of endothelial cells that, on the extraluminal side, are partially covered by a discontinuous layer of adventitial reticular cells: fibroblast (myofibroblast) cells that have processes forming a network throughout the extraluminal space of the marrow cavity. The endothelial cells of the sinuses are thought to play a role in the selective exiting of mature blood cells from the marrow into the bloodstream. These mature cells pass through the cytoplasm of the endothelial cells rather than between cells at their borders (38,39). The mechanisms involved in this remarkable process are not well understood. The endothelial cells are also presumed to play a specific role in the movement of circulating hematopoietic progenitor cells from the blood into the marrow stroma (homing).
Hematopoietic Stem Cell Niches in Bone Marrow
The concept of the HSC niche as a functioning unit was proposed by Schofield in 1978 (40), but only recently has the bone marrow stromal niche’s role in supporting HSC activity been identified in experimental animal models (41). The composition and cellular interactions of the HSC niches in humans have yet to be identified. In mammals, hematopoietic cells intermingle with stromal cells and intercellular matrix within the marrow cavity of developing long bones or spongy parts of flat bones. In the marrow cavity, at least two types of HSC niches exist: an osteoblastic niche and a vascular niche. A rare population of bone marrow hematopoietic cells can be identified adjacent to the endosteal layer of bones lined by osteoblast cells. A subfraction of these osteoblasts (spindle-shaped N-cadherin–positive CD45-negative cells termed “SNO” cells) appear to interact with HSCs and likely regulate the activity and fate of HSCs. The osteoblasts also produce hematopoietic growth factors for which HSCs appear to be targets (42). Recent evidence suggests that a quiescent HSC adjacent to an osteoblastic niche undergoes a cell division in which one of the daughter cells differentiates into committed progenitor cells, shifting toward the center of the marrow cavity close to the vascular zone and away from the peripheral endosteal area. The vascular zone at the center of the marrow cavity consisting of a thin meshwork of fenestrated sinusoidal vessels is currently considered to be the site of a possible vascular niche (43). Thus, emerging evidence supports the concept that the marrow stromal niche maintains the quiescence of immature HSCs and releases committed progenitor cells, while the vascular niche harbors the differentiating progenitor cells prior to release into the circulation. In a murine model, the osteoblastic niche has been shown to regulate the number of HSCs (41). Recent studies indicate that HSCs not only can be localized adjacent to endosteal bone surfaces, but also can be found close to endothelial cells in the bone marrow and spleen (44). Furthermore, sinusoidal cells in the bone marrow have been shown to support and promote maturation of hematopoietic cells (44), and endothelial cells can provide signals for survival and possible expansion of marrow repopulating cells (29).
Stroma of Hematopoietic Organs
The stroma contains cells that include myofibroblasts (adventitial reticular cells), adipocytes that are derived from the myofibro-blasts, macrophages, lymphocytes, plasma cells, endothelial cells of the marrow sinuses, and stem cells of various types other than the HSCs. Although bone marrow is considered to harbor mainly HSCs, evidence indicates that nonhematopoietic stem cells also reside in the bone marrow (45,46). These nonhematopoietic stem cells can give rise to mesodermal-derived cells (47), endothelial cells (48), or even diverse cell types associated with multiple embryonic germ layers (49). Among nonhematopoietic stem cells in the bone marrow, mesenchymal stem cells (MSCs) were originally described as undifferentiated cells capable of differentiating in vitro into multiple mesenchymal lineages including bone and cartilage (45). MSCs are plastic-adherent, fibroblastlike multipotent cells that do not express hematopoietic markers (CD45, CD34, CD14, CD11b, CD79a or CD19, and HLA-DR) but do express other specific surface antigens (CD105, CD73, and CD90) (50,51). A relative lack of immunogenicity of MSCs has allowed repair or regeneration of damaged or mutated bone, cartilage, and cardiac tissues (51). The transplantability of MSCs remains controversial since numerous studies have indicated that following allogeneic, unfractionated whole bone marrow cell transplantation, the bone marrow stroma remains entirely of host origin, while the hematopoietic cells are completely of donor origin (52). Injured tissue or a constitutive defect in a tissue may provide additional signals for the induction of differentiation of the MSCs present in infused bone marrow cells (53,54). Horwitz et al. (55) have reported that allogeneic whole bone marrow grafts contain sufficient osteoblast progenitor cells to alter the clinical course of children with osteogenesis imperfecta, a disease caused by mutation of a gene encoding type 1 collagen, the major structural protein of bone. MSCs and HSCs are believed to be derived from two distinct stem cells in the bone marrow, but transplantation of HSCs encoding green fluorescent protein (GFP) have indicated that fibroblasts and myofibroblasts in other organs (lungs, intestine, liver, skin, etc.) may be derived from HSCs (56). Although these results supporting a common origin of hematopoietic and stromal cells were suggested earlier by other investigators (57,58), they remain controversial.
The stroma also contains an extracellular matrix that provides a structural network to which hematopoietic progenitors and stromal cells are anchored. This matrix is composed of various fibrous proteins, glycoproteins, and proteoglycans that are produced by the stromal cells (59). These include collagens (types I, III, IV, V, and VI) (60,61), fibronectin (62,63,64,65,66), laminin (67), hemonectin (68,69,70), tenascin (71), thrombospondin (72,73,74,75,76), and proteoglycans (69,77,78,79,80).
The stroma is functionally important in hematopoiesis through its regulation of hematopoietic progenitor cell proliferation and differentiation, HSC renewal, homing of HSCs to the hematopoietic organs, and egress of mature hematopoietic cells from the bone marrow into the blood. The stroma aids in these functions through the synthesis and secretion of growth factors by stromal cells, direct cell–cell interactions between stromal and hematopoietic cells, and molecular interactions between hematopoietic cells and the extracellular matrix of the hematopoietic organs. One example that illustrates the multiple functions and mechanisms of stromal– hematopoietic interactions was discovered in studies of mice that have mutations in either of two particular genes (81): The white spotting locus (nonfunctional allele W) and the steel locus (nonfunctional allele Sl). Each of these genes is essential for hematopoiesis. Mouse embryos that are homozygous for null alleles of either of these genes die at an early stage of embryogenesis without forming any blood cells. However, mice have been found and bred that bear mutant alleles of each of the two genes that retain partial function (Wv and Sld alleles). Heterozygous mice of the Sl/Sld or the W/Wv genotypes are phenotypically similar to one another, with a lack of cutaneous pigment, sterility, and congenital anemia (81). Reciprocal bone marrow transplantation studies between normal, wild-type mice and heterozygous mice, Sl/Sld and W/Wv, revealed that W/Wv mice have defective HSCs but a functional microenvironment that can support transplants of normal HSCs. Conversely, the Sl/Sld mice have functional HSCs and can thus serve as donors for marrow transplants, but these mice have a defective microenvironment (stroma) for hematopoiesis; thus, their defect cannot be corrected by the receipt of HSCs from normal donor mice. The mechanism of impaired hematopoiesis caused by mutations in these two genes was understood after the cloning of the genes at the W and Sl loci. The W gene encodes the cell-surface receptor protein Kit (gene designated c-kit) (82,83), and the Sl gene encodes the ligand for that receptor, which is variably called steel factor, kit ligand (KL), or stem cell factor (SCF) (84,85,86). The Kit protein is a cell-surface receptor on HSCs and hematopoietic progenitor cells, and KL is expressed by stromal cells. KL is produced in two forms due to alternative splicing of the messenger RNA (mRNA): A soluble form and an integral membrane form (87,88). Both the soluble form and the stromal cell membrane–bound form of KL can stimulate HSCs, the former by free ligand–receptor binding and the latter by cell–cell contact. Activation of Kit is essential for the survival and development of immature hematopoietic progenitors. Kit and KL are not only important in hematopoiesis, but are also produced in certain other developing tissues, where they have roles in pigmentation and gonadal function. KL is just one of a large number of hematopoietic growth factors produced by stromal cells, some of which also exist in both soluble and membrane-bound forms, including Flt-3 ligand (FL) (88a,89,90) and macrophage colony-stimulating factor (colony-stimulating factor-1 [CSF-1]) (91,92,93).
Homing of Hematopoietic Stem Cells
Besides providing a source of growth factors for hematopoietic cells, the stroma of hematopoietic organs directs the movement of these cells. Interactions with stromal elements play a role in the egress of mature blood cells from the hematopoietic organs. Stromal elements are important in the “trafficking” of immature hematopoietic cells or HSCs into and out of hematopoietic organs and the blood. This trafficking occurs during embryonic development as the primary organs of hematopoiesis change from the AGM to the fetal liver to the spleen and bone marrow. Also, some HSCs and other immature progenitors migrate continuously between bone marrow and blood in normal adult animals (94). The processes by which HSCs leave the bloodstream and selectively move into marrow stroma where they interact through highly specific events and initiate hematopoiesis is collectively defined as homing (95). Homing occurs not only for hematopoietic progenitor cells that are circulating normally, but also for HSCs that are introduced into the bloodstream artificially through stem cell transplantation. It is not clear whether the homing mechanisms are the same for transplanted cells and for those circulating normally, although in both cases an adhesive interaction between circulating progenitors and the endothelial cells of the venous sinuses is followed by a transmigration of the progenitors through the endothelium. Homing is the process by which circulating HSCs migrate into the extravascular space within the bone marrow stroma where they selectively interact with specific stromal cells and matrix proteins to initiate and sustain long-term hematopoiesis (95). Homing of intravenously administered HSCs had previously been considered an inefficient process, but recent experiments with very highly purified HSCs demonstrated that they home to the bone marrow with almost absolute efficiency (96,97). In contrast, other investigators using highly purified HSCs found that homing was not an absolutely efficient process (98). Similarly, inhibited or deleted peptidase activity of endogenous CD26 on the surface of highly purified HSC enhanced homing and engraftment (99), indicating that the HSC engraftment efficiency can be increased.
A small fraction of HSCs normally circulate during both fetal and adult life, and these circulating HSCs appear to be essential for the establishment of the HSC pool as well as for the maintenance of HSC numbers. However, during normal adult life, the most immature HSCs, which maintain the stem cell reserve, most likely reside in a quiescent state in the bone marrow stromal niches. Maintenance of quiescence is an important attribute of HSCs (100), and several groups have demonstrated that the number of bone marrow stromal niches determines the size of the HSC reserve (41). On the other hand, following intravenous administration, HSCs rapidly migrate out of the circulation and home to their specific niche (94). It is not known whether homing of intravenously administered HSCs and normally circulating HSCs during steady state conditions utilize the same mechanisms to home to their niches. Experiments with unconditioned parabiotic mice have demonstrated that a small number of HSC niches are constantly being emptied and that these niches can be filled by HSCs that enter the circulation from distant anatomic sites under normal physiologic conditions or after growth factor–stimulated mobilization of HSCs (94,101).
Mobilization of Hematopoietic Stem Cells
Hematopoietic cells at various stages of their differentiation and maturation can be identified within the marrow cavity localized in close contact with specialized stromal niches. Terminally differentiated mature cells leave the marrow and enter the peripheral blood under normal physiologic conditions. As described in the preceding section, a very small number of HSCs will normally enter the blood and circulate transiently before homing to another niche in the bone marrow. The significance of these circulating HSCs is still not clear, but their increased numbers in the circulation following various stresses such as cytoreductive chemotherapy or hematopoietic growth factor administration have been utilized in clinical medicine. These pharmacologically induced increases in circulating HSCs of patients or donors for transplantation is termed stem cell mobilization (102). The most frequently used source of HSCs as hematopoietic grafts are the peripheral blood HSCs. These mobilized, circulating HSCs have several advantages over the HSCs in bone marrow for transplantation, including ease in harvesting, higher HSC yields, and faster hematopoietic engraftment following transplantation. Granulocyte colony-stimulating factor (G-CSF) has become the cytokine most commonly used to mobilize HSCs; granulocyte-macrophage CSF (GM-CSF) is another potent HSC mobilizer (103). KL/SCF, when used in combination with G-CSF, is an excellent HSC mobilizer, but unusual allergic reactions after administration of KL/SCF have restricted its clinical use. Among newly developed HSC mobilizers is the investigational agent AMD3100, a CXCR4 antagonist, which reversibly inhibits binding of the stromal-derived factor-1 (SDF-1) protein to CXCR4, its receptor on HSCs. AMD3100 is capable of mobilizing HSCs within hours and synergizes with the mobilizing effects of G-CSF when it is administered on the last day of a course of G-CSF administration (104).
The mechanism of HSC mobilization is complex (102,104). In brief, mobilization of HSCs appears to be initiated following stress signals (injury, inflammation), which activate neutrophils and osteoclasts. Administration of chemotherapy or G-CSF mimics these stress signals, activating primarily granulocytes that release proteolytic enzymes including elastase and various matrix metalloproteinases (MMPs). SDF-1, the specific stromal ligand for the chemokine receptor CXCR4 on HSCs, is inactivated while adhesion molecules including α4β1 integrin (VLA-4) and the P/E selectins are degraded. These enzymatic disruptions of the HSC interactions with their stromal niches lead to HSC detachment and entry into the circulation.
Adhesion Molecule Interactions of Hematopoietic Stem Cells and Stroma
Direct molecular interactions between the hematopoietic cells and stromal cells involve ligand–receptor relationships between adhesion molecules on the surfaces of the hematopoietic and stromal cells. Similar interactions occur between adhesion molecules on the hematopoietic cell surfaces with specific domains within certain extracellular matrix molecules (see reviews [105,106]). There are many cytoadhesion molecules known, and they generally can be classified into several families: sialomucins, selectins, integrins, and members of the immunoglobulin superfamily (106). CD44 is an additional adhesion molecule, not belonging to one of the above families. Numerous interactions are possible, and there appears in some cases to be redundancy in the systems involved in trafficking, homing, and the other processes within the hematopoietic microenvironment.
Integrins are heterodimeric, transmembrane proteins in which the α and β subunits are joined noncovalently. Both subunits have extracellular and intracellular domains. Eighteen types of α subunits and eight types of β subunits are known, although only a few of the possible heterodimer combinations have been found on hematopoietic or stromal cells and implicated in hematopoiesis. The integrin subunits α4 and β1 clearly have important functions in hematopoiesis. Although they each can pair with certain other subunits of the heterologous type, the α4β1 pair (very late antigen [VLA]-4) appears to be the most important. The integrin α4β1 (VLA-4) is widely expressed on hematopoietic progenitor cells, and it binds to the vascular cell adhesion molecule (VCAM)-1, an adhesion molecule of the immunoglobulin superfamily present on stromal cells, macrophages, and venous sinus endothelium in bone marrow (reviewed in 107). VLA-4 also binds to fibronectin (105,107,108). Chimeric mouse embryos, in which the integrin α4 subunit gene was deleted in a substantial portion of the cells, formed no α4-null erythrocytes, almost no B-lymphoid cells, and few myeloid cells (109,110). In vitro hematopoietic colony formation of α4-null cells appeared normal, so the defect is likely due to impaired interaction of the hematopoietic progenitors with the stroma. Mice with a conditional knockout of α4 integrin accumulate HSCs and progenitors in their peripheral blood and spleen with a concomitant decrease in HSCs and progenitors in the marrow. These mice demonstrate stable hematopoiesis under normal conditions, but have delayed response to erythropoietic stress, homing after HSC transplantation, and short-term engraftment (111). Likewise, chimeric mouse embryos containing β1-null cells demonstrated an essential role for the β1-integrin subunit during hematopoietic development (112). β1 integrin is not essential for the generation of hematopoietic progenitor cells in the aorta-gonad-mesonephros or the yolk sac or for their differentiation in vitro, but it is necessary for their colonization of the fetal liver, spleen, and bone marrow during embryogenesis. HSCs rendered β1 null by conditional gene ablation are unable to colonize hematopoietic organs or to rescue lethally irradiated mice on transplantation (112). Nevertheless, conditional deletion of the β1 gene in adult mice does not lead to defects in stem cell retention in the marrow or in hematopoiesis (113).
Treatment of animals with antibodies to VLA-4 or VCAM-1 induces release (mobilization) of HSCs and more differentiated progenitor cells from the bone marrow (114,115) and inhibits hematopoietic progenitor cell homing after transplantation (116,117). CD34+ marrow progenitors from patients with myelodysplastic syndromes (MDSs) demonstrate reduced expression of VLA-4; these patients have hypercellular bone marrow but a paucity of circulating hematopoietic cells, indicating impaired egress from the marrow and increased intramedullary apoptosis (118). However, like the conditional deletion of the β1-integrin subunit gene, conditional deletion of the VCAM-1 gene (119,120) does not disrupt bone marrow retention of HSCs or hematopoiesis, although it affects migration, specifically of T cells, to the bone marrow (119). Thus, these VLA-4 and VCAM-1 experiments indicate that molecules critical for homing in the transplant model or in mobilization by growth factors or drugs may not be essential for maintaining adult hematopoiesis because of overlapping functions of adhesion receptors. Yet, individual cytoadhesion molecules may play very specific roles such as that of VCAM-1 in T-lymphocyte homing and megakaryopoiesis (121).
The selectin family of cytoadhesion molecules has functions in the lymphocyte homing to lymphoid tissues and in leukocyte rolling and adhesion to activated endothelial cells. The selectin family members are designated E, P, and L. HSCs have receptors for the selectins, and they can exhibit the rolling phenomenon similar to that of leukocytes (122,123). Some data also indicate that the selectins might be important for stem cell transmigration across endothelium (124) and homing. Mice in which genes for two or all three selectins have been deleted are viable, although several aspects of their marrow hematopoiesis are abnormal (125). These mice also are defective in hematopoietic progenitor cell homing to the bone marrow but not the spleen after transplantation (125,126).
The adhesion molecules of all families are transmembrane proteins, and many can act as receptors that activate specific intracellular signaling pathways. These adhesion molecules/receptors, in turn, may be regulated by other intracellular signaling pathways (107,127,128). Thus, the interactions of the hematopoietic cells with stromal cells and matrix can be highly modulated by the adhesion receptors, both in transmitting signals from the microenvironment into the cell and in translating the state of intracellular signaling pathways into changes in the number and affinities of adhesion molecules. Activation of Kit by its ligand (KL) modulates adhesion functions that are mediated by integrins α4β1 (VLA-4) and α5β1 (VLA-5) (115,129,130). Another example is the chemotactic cytokine (chemokine) receptor CXCR4 and its ligand stromal-derived factor-1α (SDF-1). SDF-1, secreted by bone marrow stroma, is the only known chemokine that elicits directed chemotactic response in HSCs via interactions with CXCR4 on their cell surface (131,132). Mice lacking SDF-1 or CXCR4 have defective hematopoiesis in fetal bone marrow, due to a decreased ability of HSCs to home from the fetal liver to the marrow cavity (133,134). Antibodies against CXCR4 block engraftment of severe combined immunodeficiency (SCID) mouse bone marrow by transplanted CD34-enriched HSCs and hematopoietic progenitor cells (135,136). SDF-1 is expressed on bone marrow endothelium and appears to induce binding of circulating progenitors to the vascular endothelium by activating the integrins VLA-4, VLA-5, and lymphocyte function–associated antigen (137,138,139), as well as CD44 and hyaluronic acid (140). The complement component C3a, which is secreted by bone marrow stromal cells and increased during physiologic stress, increases binding of CXCR4 to SDF-1, and thus promotes homing of HSCs to the marrow, as well as egress of progenitor cells from the marrow into circulation (141).
Several of the adhesion molecules on hematopoietic cells specifically bind to sites on particular matrix macromolecules. For example, HSCs and more mature progenitors bind to fibronectin, primarily through interaction with the integrin receptors α4β1 and α5β1 (59,105,107,108,142). Another cytoadhesion molecule that interacts with several matrix macromolecules is CD44, which binds with glycosaminoglycans (hyaluronic acid being the major CD44 ligand) (143). The proteoglycans, proteins with extensive sulfation such as heparan sulfate and chondroitin sulfate, are extracellular matrix proteins that may contribute to adhesion between the stroma and the hematopoietic progenitor cells (69,77,78,79,80). The proteoglycans can also concentrate soluble growth factors. For example, GM-CSF binds to heparan sulfate in the marrow matrix (144,145). Interactions of KL and Kit, SDF-1 and CXCR4, or VCAM-1 and VLA-4 or VLA-5 all stimulate Rac GTPases as a downstream mechanism to promote adhesion and migration of HSCs (146).
Hematopoietic Stem Cells
Age of Morphologists
Fascinating accounts of the history of experimental efforts in hematopoiesis are presented in Blood, Pure and Eloquent, edited by M.M. Wintrobe (147,148). One milestone in understanding the origins and development of blood cells was the recognition by Neumann and Bizzozero in the mid-19th century that the bone marrow is a site of red blood cell production throughout postnatal life. Another major advance made in the late 19th century by Paul Erlich, Artur Pappenheim, and others was the application of synthetic dyes and various staining/fixing techniques that led to precise morphologic characterization and classification of blood and marrow cells. A third milestone was the development of the idea of a pluripotent stem (ancestral) cell that gives rise to all of the mature blood cell types through extensive proliferation and differentiation. By use of refined staining methods, Pappenheim observed various transitional cells and organized them into a relational scheme—a tree whose various branches when traced backward converged to a mononuclear cell that had none of the distinct features of the end-stage blood cells or the transitional stages. He proposed the notion that this cell was so morphologically primitive that it could be the common ancestor of all blood cells. Although most morphologists between 1900 and 1940 accepted the idea of ancestral cells in a hematopoietic series leading to progressively more mature types, there was much debate about how many ancestral cell types there were. Many workers believed that lymphocytes had a separate origin from myeloblasts and thus that there were dual or perhaps plural ancestral cells. Reviews of the conflicting concepts of the origin of hematopoietic cells as of the late 1930s are presented in detail in Handbook of Hematology (149).
Advent of Hematopoietic Progenitor Transplantation
In the late 1940s and the 1950s, several new approaches were developed to study hematopoiesis. Among them were radiation exposure followed by grafting of hematopoietic tissue, use of chromosome cytogenetics, and use of radioactive materials. Lorenz et al. (150) showed that mice and guinea pigs may be protected against otherwise lethal whole body irradiation by injections of bone marrow from other animals of their respective species. Ford et al. (151) used bone marrow from donor mice that had a morphologically identifiable chromosomal marker to show that hematopoiesis in the irradiated recipient mice was reconstituted by cells from the donor marrow—that is, the protected animals were chimeras with respect to their hematopoietic tissues. These experiments did not settle the question about how many types of ancestral cells there were, but experiments generating radiation chimeras have since been used with great power to study the nature of stem cells and their progeny.
Till and McCulloch (152) used radiation/grafting experiments to prove directly the existence of an ancestral cell with multilineage potential. In spleens of mice at one week after transplantation, they found growth of macroscopic colonies containing cells of multiple hematopoietic lineages. These colonies were the progeny of individual transplanted cells that were called colony-forming units–spleen. Because the cells in these spleen colonies could, in turn, be injected into secondary, irradiated mice and give rise to spleen colonies, the CFUs-S apparently replicated themselves within the colonies. When the observation time for CFU-S assays was extended from 1 week to 2 weeks after transplantation, a series of evanescent colonies was found, and those appearing on later days had greater self-replication and multilineage differentiation capacities (153,154). Early studies could not demonstrate lymphoid cells in spleen colonies (155,156), but more recent studies indicate that CFU-S colonies contain lymphoid progenitors as well as myeloid progenitors (157). However, several studies showed that cells with the capacity for long-term hematopoietic reconstitution of irradiated mice can be separated from most CFUs-S by size and density (158). Thus, many CFUs-S, although multipotent, do not have long-term repopulating capacity.
Hematopoietic Repopulating Unit
The term long-term hematopoietic repopulating unit is operationally defined as a cell or group of cells that provide long-term hematopoietic reconstitution of ablated animals, including repopulation of all myeloid and lymphoid cell lineages. This function, if it can be accomplished by a single cell, is the operational definition of a pluripotent HSC. The identification of hematopoietic repopulating units requires hematopoietic cell transplantation into an ablated host. Although the most suitable experimental animal is the mouse, several studies have indicated significant differences between the hematopoietic systems and the responses to hematologic stress in rodents and those of large animals including humans (100,159). However, the analyzed cells are not spleen colonies but rather the reconstituted hematopoietic tissues after an extended period of time after transplantation.
Definitive Evidence for a Pluripotent Stem Cell
Animal reconstitution experiments with hematopoietic cells that were individually marked genetically have verified the existence of HSCs and demonstrated their capacity for extensive self-renewal (160,161,162). In these marking experiments, hematopoietic cells were infected in vitro with a recombinant retrovirus that was able to integrate its DNA (provirus) into a cell but could not replicate and spread to other cells. The one-time, random integration of the provirus into the DNA of an individual cell provides a specific marker for the progeny of that cell that develop in an animal after transplantation. Random integration ensures that each provirus has unique flanking sequences of DNA and thus has a high probability of yielding a DNA fragment of a distinguishable size after cutting with a restriction enzyme that does not cut the provirus. Several months after transplantation of the genetically marked cells and establishment of hematopoiesis, it is typically observed that all types of cells in the blood and lymphoid organs contain progeny of an individually marked cell, proving that it was pluripotent. Often, these clones of marked cells continue to contribute to all of the hematopoietic lineages in the animal for an extended period. Also, when these primary recipient animals are subsequently used as donors for secondary recipient animals, frequently the same clones of HSCs are apparent in these secondary recipients. This persistence can even be demonstrated in tertiary recipients (160,161). Thus, clearly many HSCs reproduce themselves (self-renew) over a long period. Yet not all clones of HSCs are so long lived; some produce progeny for varying periods and then apparently become extinct. Finally, marked clones have been observed to begin contributing to hematopoiesis after some period of posttransplantation latency, indicating that dormancy is possible. Thus, these studies have demonstrated that, after transplantation, some HSCs contribute continuously to hematopoiesis for a long time—in mice, apparently for the whole lifetime of the animal. Other HSCs contribute and then become extinct, and finally, some may remain dormant for some period and then contribute. Additional transplantation studies of marked HSCs in mice (163) have suggested that polyclonal hematopoiesis is more common and that long-term contribution by individual stem cells is more rare than the earlier studies indicated (160,161). Studies using retroviral insertion site analyses for larger animals, particularly nonhuman primates, have provided some evidence of polyclonal hematopoiesis (164,165,166). To what extent these possible behaviors are manifest in normal, nontransplanted mice or larger animals is not clear.
Enrichment of Hematopoietic Stem Cells
There are approximately 1 to 4 HSCs per 100,000 nucleated cells in hematopoietic tissues, and it has not been possible to isolate a pure population of HSCs. The identification of relatively immature HSCs from more committed progenitor cells on the basis of various physical properties, immunophenotypic markers, and functional attributes has greatly advanced the field of hematopoiesis (167). Table 5.1shows reagents and procedures commonly used to enrich the HSCs in a hematopoietic cell population. However, prospective isolation of single, human HSCs still remains elusive. HSC markers that are expressed from fetal stages through adult life include CD34, CD31 (PECAM1), and Kit, but these markers can also be identified in endothelial cells (168). In humans, CD34+, CD90 (Thy-1)+, Kit+ cells that are negative for lineage markers (CD10, CD14, CD15, CD16, CD19, and CD20) are considered a relatively purified population enriched for in vivo repopulating HSCs (167). In mice, the Sca-1(Ly-6A/E)+, CD90 (Thy-1)+, Kit+ cells that are negative for lineage markers (CD3, CD4, CD8, B220, Mac-1, Gr-1, and Ter119) are considered a relatively purified population enriched for in vivo repopulating HSCs (167). Isolation of candidate HSCs based on phenotypic markers expressed on the cell surface was first tested in congenic mouse transplantation models and subsequently purified HSCs were successfully transplanted in a xenogeneic immunodeficient mouse model (167). The successes in mouse models led to human trials using purified HSCs based on immunophenotypic marker enrichment (167). Recently, signaling lymphocyte and activation molecule (SLAM) markers including CD150, CD244, and CD48 were found to be expressed differentially allowing identification of functionally distinct enriched murine HSCs capable of in vivo marrow repopulation (44). The most immature HSCs with in vivo hematopoietic repopulation potential are detectable within the CD150+/CD244-/CD48- population. Whether combining SLAM markers and known phenotypic markers will permit prospective isolation of single, in vivo repopulating HSCs remains to be examined. With the advances in technology, procedures have been developed to enrich greatly the proportion of HSCs in isolated cell populations from mouse, human, and other sources. Despite variations by different investigators, each procedure consists of a series of steps that discriminate and select cells based on experimentally determined properties of stem cells (169,170,171,172,173,174,175,176). In general, the steps consist of combinations of the following:
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Table 5.1 Summary of Reagents and Procedures that Discriminate Cell Populations Enriched in Stem Cells |
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1. Density gradient isolation of low-density mononuclear cells (excludes erythrocytes and mature granulocytes).
2. Fractionation of cells based on sedimentation rate by elutriation.
3. Flow cytometry sorting using forward and perpendicular light scatter windows to exclude many large blasts, monocytes, and granulocytes. In addition, flow cytometry–based sorting has been used to isolate more immature HSCs based on their immunophenotypic markers as well as functional attributes. Examples are flow cytometric sorting of HSCs based on minimal retention of rhodamine 123 or Hoechst dye 33342 (SP cells) or on binding of fluorescence-labeled wheat germ agglutinin, antibodies to Thy-1 (human), or HLA-DR. HSCs retain low levels of rhodamine 123 and Hoechst 33342, while most express Thy-1 but little HLA-DR.
4. Incubation of cells with a panel of antibodies to specific antigens on differentiated cell types and subsequent removal of the cells bearing those antigens. After removal of the antigen-bearing cells, the remaining population is referred to as lineage negative (Lin-).
5. Isolation of cells that display on their surfaces an antigen associated with immature HSCs but not widely expressed on committed, maturing hematopoietic cells. In humans, CD34 and CD133 have been commonly used. In some strains of mice, the Sca antigen (Ly-6A/E) is selectively expressed on stem cells. Kit is highly expressed on stem cells of both mice and humans but is widely distributed on other cell types.
6. Isolation of immature HSCs based on functional properties instead of complete dependence on immunophenotypic markers is an emerging alternative strategy. An advantage of functional properties is their relative lack of fluctuation compared to phenotypic markers, which may vary depending upon the ontogenic stage or the cellular context of the HSC. Immature HSCs also express more intracellular aldehyde dehydrogenase (ALDH) when compared to their more differentiated progeny, and their isolation using flow cytometry has been based on this increased expression (177). The principles of some of these new cell isolation techniques are discussed in following sections.
Side Population Cells
A flow cytometry–based isolation method has recently been used to isolate immature cells possessing HSC characteristics from several species including human (178,179,180,181). This technique relies on the differential ability of HSCs to efflux the DNA binding dye Hoechst 33342 (178). Hoechst low cells are termed side population (SP) cells due to their typical flow cytometry profiles in Hoechst red versus Hoechst blue bivariate dot plots (Fig. 5.2). Thus, detection relies on HSC function rather than phenotype in that SP cells pump out Hoechst dye by using multidrug resistance (MDR)–like proteins such as BCRP1 that are expressed on the cell membrane (182). Interestingly, stem cells from nonhematopoietic tissues have been shown to display similar SP phenotype (183).
Whether SP cells detected in nonhematopoietic tissues are derived from bone marrow and lodge in various tissues during embryonic development remains to be determined. Transplantable human HSC activity in human fetal liver cells, detectable in nonobese diabetic (NOD/SCID) immunodeficient mice, is associated exclusively with the SP cells, whereas a large majority of long-term culture initiating cells (LTC-ICs) in the fetal liver lack the SP phenotype (184). In bone marrow and liver of adult mice and some other studied species, LTC-ICs are highly enriched in SP cells, but even in those tissue sources, a smaller majority of LTC-ICs are in the non-SP fraction (179,185).
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Figure 5.2. Side population (SP) of unfractionated mouse bone marrow cells. Scattergram of cells analyzed by flow cytometry after staining with Hoechst dye 33342 and detection by flow cytometry with both red (horizontal axis) and blue (vertical axis) Hoechst filters. Side population cells are shown in area enclosed by pink lines. Courtesy of Claudio Mosse, Laura Ford, and Mary Zutter. |
Aldehyde Dehydrogenase
The surface expression of CD34 by human HSCs and other hematopoietic progenitors has been widely used as a surrogate marker for cells that yield an optimal graft in clinical transplantation (177). However, CD34+ cells are very heterogenous, and a subpopulation such as CD34+, CD90+cells or CD34+, CD38- cells are further enriched for HSCs with in vivo repopulating activity. Recent evidence showed that many HSCs in normal bone marrow of adult animals do not express CD34 (186). However, CD34 is induced in virtually all HSCs mobilized into the blood by growth factors. Furthermore, CD34 is expressed on HSCs of human and murine fetal livers (184,187) and on HSCs from all fetal and neonatal tissues, including umbilical cord blood (186). Therefore, an alternative strategy is to isolate HSCs based on functional characteristics rather than depending solely on surface immunophenotype. Aldehyde dehydrogenase (ALHD) is involved in retinoid metabolism, and it is relatively abundant in immature HSCs, where it is believed to protect HSCs from alkylating agents, such as cyclophosphamide, which are administered in patients diagnosed with hematologic malignancies (188). Flow cytometry can be used to isolate high ALDH-expressing HSCs (177,189), but such isolations for clinical applications will require further refinement.
Hematopoietic Stem Cell Assays
There are many scientific questions about hematopoietic progenitor cells that can only be pursued with cell culture–based manipulations of such cells. Also, with the advent of hematopoietic tissue transplantation in humans, there is great need for an in vitro assay that identifies human cells capable of long-term hematopoiesis in patients. To infer that any in vitro assay measures HSCs, one must correlate the properties of the cells analyzed in vitro with those of repopulating units tested in vivo. Such comparisons can presently only be done for mouse cells because in vivo assays of repopulating units transplanted into hosts of the same species as the test cells are not feasible or are not fully proven for other species. Progress in the development of murine xenograft models to assay human stem cells may lead to satisfactory quantification in the future. In vitro systems have been developed for culturing murine and human hematopoietic cells, and the assayed human cells are presumed to represent the same stages in the hematopoietic hierarchy as their mouse cell counterparts. Culture of Hematopoietic Cells (190) describes many of the cell culture methods for assaying hematopoietic progenitor cells. In vitro assays have their limitations in that short-term assays such as colony-forming cell (CFC) assays identify the relatively mature progenitor cells of a population while long-term repopulating capacity that is limited to more immature populations of HSCs cannot be assayed in vitro.
The strength and drawbacks of HSC functional assays have recently been summarized (191). Variables that influence the interpretation of results of HSC assays include absence of absolute correlations between the phenotype and function of specific cell populations, functional heterogeneity of cell populations, and variation in proliferative capacity of immature HSC populations. HSC assays can be classified under three categories: (a) immunophenotyping, (b) in vitro clonogenic assays, and (c) in vivo transplantation assays. The ultimate purpose of these assays is to be able to determine HSC activity prospectively.
Immunophenotyping
Monoclonal antibodies and flow cytometry techniques have resulted in significant progress in the immunophenotype-based cell isolations. Various immunophenotype combinations have been used to isolate immature HSCs with in vivo marrow repopulation potential, but at present the consensus is that HSCs having a set of specific phenotypic markers capable of in vivo hematopoietic repopulation are still lacking. Successful hematopoietic engraftment in clinical transplantation can be predicted by numbers of CD34+ cells in a hematopoietic graft (167). Although CD34+ cell enumeration can be considered as a surrogate HSC assay, commonly used immunophenotypic markers (CD34, CD133, CD90, Kit, etc.) are not indicators of HSC function (192).
In Vitro Clonogenic Assays
Hematopoietic cells are organized in a hierarchical manner and can be compartmentalized based on their functional capacities. The history of each mature blood cell can be traced back to its precursor-progenitor cells and eventually to a single HSC. To detect the full functional potential of a given population of cells, an in vitro environment must permit survival and growth of all potential lineages of blood cells. In vitro clonogenic assays like the colony-forming assays can determine the numbers of various progenitors present in a population of hematopoietic cells. In addition to the numbers of progenitors, these assays can estimate the proliferative potential of a given population by measuring the sizes of individual colonies. Lastly, colony-forming assays can determine the differentiation capacity of a hematopoietic cell population by analyzing the types of various lineages generated in the colonies under specific conditions.
Short-term Assays
Several types of colonies can be detected in an optimal culture condition based on cellular morphology and the colony size. An atlas of various types of short-term hematopoietic colonies grown in vitro is available (193). Several types of short-term in vitro colonies detectable in semisolid media are described in Table 5.2 and the relationships among the respective progenitors are shown in Figure 5.3. Pure erythroid, megakaryocytic, monocyte-macrophage, and granulocytic colonies can be identified. The size and time required for growth of these pure colonies varies depending upon the differentiation status of the committed progenitors in the cell population.
For example, among erythroid colonies, the colony-forming units–erythroid (CFUs-E) form smaller erythroblast colonies than the more immature burst-forming units–erythroid (BFUs-E). Examples of relatively immature committed progenitor cells are those that give rise to colonies composed of erythroid, megakaryocytic, granulocyte, and macrophage lineages (mixed lineage colony-forming units [CFUs-mix]) and colonies of only granulocyte and monocyte-macrophages (granulocyte-macrophage colony-forming units [CFUs-GM]). The mixed lineage colonies are largest because they arise from the most immature progenitors that possess the highest proliferative potential. However, even the in vitro assays that detect these immature CFU-mix progenitors cannot detect more immature quiescent HSCs, which are likely to be responsible for long-term sustained hematopoietic reconstitution following transplantation.
Long-term In Vitro Assays
In humans and mice, two types of progenitor cells called long-term culture initiating cells (LTC-ICs) and cobblestone area–forming cells (CAFCs) can be detected using long-term assays. Since these assays extend beyond the 2 to 3 weeks when most committed progenitors proliferate and differentiate, these committed progenitors are lost from the cultures after 3 weeks. The relationships of these long-term progenitors to short-term progenitors are shown in Table 5.2 and Figure 5.3. However, by 5 weeks or more of long-term culture, more immature progenitors that are dormant during the initial weeks but which possess extensive proliferating capacity have continued to proliferate. One type of long-term culture assay detects early-stage hematopoietic progenitors that are capable of initiating long-term hematopoiesis in culture after seeding them onto irradiated stromal cell monolayers (human [194,195]; mouse [171,172]). These LTC-ICs (194) sustain production of multilineage progenitors for 4 to 6 weeks. In some instances, these cultures have been extended for more than 10 to 12 weeks (196,197). This continued production of hematopoietic progenitors of multiple lineages in individual cultures is measured after several weeks by harvesting the cultured cells and doing secondary assays for various types of lineage-committed progenitors. Long-term cultures require a supporting stromal monolayer that is commonly generated from bone marrow–derived mesenchymal or fibroblast cells. The stromal layer supports the proliferation and differentiation of seeded hematopoietic progenitor cells, but at later times it sloughs from the culture dish and fails to sustain continuation of the culture. In CAFC assays, islands or colonies of hematopoietic cells can be recognized morphologically in situ (171). These cobblestone colonies integrate within the supporting stromal layer, forming clusters of flattened, optically dense, morphologically homogenous-appearing cells tightly adherent with the stromal layer (198,199). CAFC assays are one-step cultures in contrast to LTC-IC assays, which require plating of fresh hematopoietic cells on established stromal layers. Using limiting dilution and Poisson statistics, the frequency of LTC-ICs or CAFCs in a test population or following culture can be determined (171,172,195,197,200).
Assays of murine bone marrow cells for LTC-ICs and for day 28 CAFCs yield estimates of 1 to 4 LTC-ICs or CAFCs per 105 marrow mononuclear cells—a value comparable to that obtained for HSCs in repopulation assays (201,202). A modification of the mouse LTC-IC assay (203,204) has led to a demonstration that some LTC-ICs form lymphoid as well as myeloid progenitors in vitro. However, evidence obtained more recently indicates that LTC-ICs do not correspond in a 1:1 ratio to hematopoietic repopulating units. For example, several studies have shown that ex vivo expansion of hematopoietic cell populations with growth factors in culture leads to a loss of in vivo repopulating cells (205,206), although measured LTC-ICs do not decrease in parallel. Another limitation of in vitro assays is their inability to measure homing capacity. Homing capacity is likely to be an important attribute of HSCs, and HSCs lose homing capacity during culture (207). Several mechanisms may contribute to the loss of HSC homing capacity during culture including down-regulation of specific adhesion molecules (206), induction of FAS/CD95-mediated apoptosis (207), and perturbations of normal cell cycle (208).
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Figure 5.3. Diagram of hematopoietic cell differentiation. Hematopoietic stem cells can duplicate themselves during cell division (self-replicate), as indicated by the curved arrow. Most descendants of the stem cells are committed to differentiate. This commitment process occurs through a series of steps or stages, each of which leads to further restriction of lineage choice, until finally, the descendent cells are limited to a single lineage. After lineage commitment, the progenitor cells continue to differentiate and mature into the terminally differentiated cells found in the blood. The arrow is dashed between LTC-IC and the common lymphoid progenitor because this pathway has not been directly observed in this assay. The diagram shows only the steps of commitment and does not depict the proliferation of cells that occurs throughout the process. The amplification of cell numbers accompanying differentiation is very large. The progenitors and the respective assays for them are described in the text and/or Table 5.2. BFU-E, erythroid burst-forming unit; CFU-E, erythroid colony-forming unit; CFU-G, granulocyte colony-forming unit; CFU-GM, granulocyte-macrophage colony-forming unit; CFU-M, macrophage colony-forming unit; CFU-mix (GEMM), granulocyte-erythroid-macrophage-megakaryocytic colony-forming unit; CFU-MK, megakaryocyte colony-forming unit; CFU-NK, natural killer cell colony-forming unit; CFU-preB, pre–B-lymphocyte colony-forming unit; CRU, competitive repopulating unit; d8 CFU-S, day 8 spleen colony-forming unit; d12 CFU-S, day 12 spleen colony-forming unit; d28–35 CAFC, day 28 to 35 cobblestone area–forming cell; LTC-IC, long-term culture-initiating cell; LTRC, long-term repopulating cell; SRU, severe combined immunodeficiency mouse repopulating unit. |
In Vivo Hematopoietic Assays
In vivo assays can measure various features of HSCs including homing, survival, proliferation, and differentiation into hematopoietic lineages. Homing and subsequent development of donor-derived blood cells is termed hematopoietic engraftment. To sustain life-long hematopoiesis in the host, the transplanted HSCs must self-renew and re-establish an HSC pool. Because in vivo assays can be monitored for a prolonged period for the survival, proliferation, and differentiation of transplanted HSCs and, ultimately, the establishment of donor-derived hematopoiesis, they remain the gold standard for measuring the true functional potential of HSC grafts.
Traditionally, the contribution of donor cells in vivo is determined by chimerism studies in host bone marrow or peripheral blood samples using donor genetic or immunophenotypic markers. Donor-derived cells serve as a good indicator for transplanted HSC activity (sometimes termed long-term repopulating cells [LTRCs]), but a chimerism assay is not capable of determining the number of active, functional clones present in the graft. Engraftment capacity in a transplanted cell population can be enhanced by recruitment of a dormant clone, increased proliferation of existing clones, or a combination of both processes. The number of in vivo repopulating cells in a population can be determined by limiting dilution assays using surrogate in vivo syngeneic or xenogeneic mouse models. A competitive repopulation unit (CRU) assay is most commonly used to determine the frequency of in vivo repopulating clones in a cell population. To quantify hematopoietic repopulating units by transplantation, it is necessary to transplant serial dilutions of a test cell population into a group of animals. Even if repopulating units are present in the transplanted cell population, ablated animals receiving no short-term sustaining progenitor cells would die with a lack of hematopoiesis before the transplanted cells could reconstitute the animal. Therefore, competitive repopulation assays (163,201,202) are used in which the recipient animals survive in the short term because they contain or are given an additional source of progenitor cells that can be distinguished from the donor test population. CRU assays can be defined as in vivo assays in which donor hematopoietic cells are allowed to compete with cells from a second donor or endogenous host cells for hematopoietic repopulation of the host. A constant number of competitor marrow cells when added with a purified population of enriched HSCs eliminates the chance of potential replicative stress on the small number of HSCs following transplantation (202). Generally, donor cells with distinct genetic/phenotypic markers are mixed with a constant number of competitor marrow cells from a distinguishable (genetic/phenotypic) second donor. In one form of this assay, one mixes various dilutions of the test population of hematopoietic cells bearing a distinguishable genetic marker with a constant number of hematopoietic cells (source of short-term reconstituting support cells) from a congenic mouse strain. One then transplants the mixtures into ablated host mice of the congenic strain used to procure the supportive cells. The reconstituted mice are hematopoietic chimeras with progenitor cells derived from both of the hematopoietic cell sources in the transplant. Using the genetic marker, one can determine the fraction of hematopoietic cells derived from each cell genotype in the chimeric animals at various time points after transplantation. From these ratios and the known dilutions of test cells given in the transplants, one can calculate the number of hematopoietic repopulating units in the test population by binomial correlation and covariance methods (202) or by using limiting dilution analysis and Poisson statistics (163,201). A variation of this assay uses limiting dilutions of genotypically distinct donor cells to transplant into stem cell–deficient W/Wv mice that can be used as hosts rather than hematopoietically ablated mice (209). A second variation uses, as hosts, mice that have been transplanted previously and thus have a reduced or weakened endogenous stem cell competition capacity (201).
Using these competitive repopulation assays in mice, it has been shown that the hematopoietic repopulating unit has an incidence in murine marrow of 1 per 104 (201) to 1 per 105 nucleated cells (202). As discussed in the section Definitive Evidence for a Pluripotent Stem Cell, the minimal hematopoietic repopulating unit has been proved to be a single HSC. Thus, the competitive repopulation assays can be used to measure enrichment of murine HSCs after purification schemes (210) and also to analyze possible replication of HSCs during in vitro culture. Because a single HSC is a hematopoietic repopulation unit, CRU assays can be used to detect the degree of enrichment of an HSC purification procedure and to determine the absolute increase in the number of CRUs following an ex vivo expansion. Some discrepancies between competitive and noncompetitive assays have been noted and are due to the use of defective competitors (211). CRU assays may underestimate the true frequency of SCID repopulating units (SRUs), if only 5 to 20% of a purified population is able to seed and initiate hematopoietic repopulation (98). However, CRU assays would be more accurate if engraftment efficiency is nearly absolute (96,97).
Human Hematopoiesis in Animal Hosts after Xenografts
Two murine models have been explored that can support xenografts of human hematopoietic cells and support multilineage, long-term human hematopoiesis. Murine models that are commonly used are derivatives of the NOD/SCID strain (212,213), strains deficient in the RAG1 or RAG2 genes necessary for T- and B-cell receptor rearrangements (214,215), and a fetal ovine system (216,217). NOD/SCID/β2-microglobulinnull mice support proliferation and differentiation of immature human hematopoietic progenitors (212,218,219,220). Residual natural killer (NK) cell activity of NOD/SCID mice has also been inhibited by genetic manipulation to create γcnull strains (221) administration of monoclonal antibodies against interleukin (IL)-2Rβ (222) have promoted human T-cell differentiation in vivo. The bone marrow, peripheral blood, or spleen of NOD/SCID mice can be assessed at 6 to 12 weeks after transplantation to determine the degree of chimerism represented by infused human hematopoietic cells present in the mouse host. The hematopoietic repopulation ability of transplanted human cells in a sublethally irradiated mouse is termed SCID mouse repopulating cells (SRCs), the frequency of which can also be determined by limiting dilution analysis (223). One issue remains problematic in determining whether SRCs are the equivalent of human LTRCs. Multilineage hematopoiesis in these mice can only be evaluated for 4 to 5 months at most, because the animals later develop thymic lymphomas. This limited period for evaluation of engraftment may result in some progenitors being scored as stem cells that cannot self-renew or support long-term engraftment in humans.
Hematopoiesis in Large Animal Models
Humans vary from mice in many aspects, including their lifespan, HSC turnover rates, and daily demand for hematopoietic cell production (100,166,205). Abkowitz et al. have identified important differences between the kinetics and behavior of HSCs in large animals and rodents (224). The production of blood cells for the whole lifespan of a mouse is equivalent to blood cell production of a human in a single day. This limited replication demand due to the relatively short murine lifespan poses a significant challenge to determine the long-term sustained hematopoietic activity of transplanted human hematopoietic cell populations. To overcome this limitation, serial transplantation of human cells in immune-deficient mouse models has been attempted (225) and human cells have been found to persist for several years after transplantation in a preimmune in utero fetal sheep model (226). Several large animal models are available for HSC studies including feline, canine, ovine, and nonhuman primates (227), but the genetic and biologic similarity between humans and nonhuman primates suggests that the nonhuman primate model is probably the best available model to study human hematopoiesis (100,166). Another advantage of using nonhuman primates is that their relatively long lifespan (up to 30 years) compared to rodents (up to 3 years) allows long-term monitoring after transplantation, irradiation, cytokine therapy, chemotherapy, etc. Simultaneous transplantation of genetically marked autologous cells in lethally irradiated nonhuman primates and immune-deficient mice demonstrated that the reconstituting cells in primates and in mice are distinct, suggesting a lack of overlap between these two cell populations (159).
Stem Cells in Culture
Repopulation studies in irradiated mice, as well as experience with bone marrow transplantation in humans, provide strong evidence that HSCs can replicate and expand extensively in vivo (self-renewal). A very significant advance for clinical medicine would be the in vitro expansion of transplantable HSCs. However, mouse HSCs generally decline substantially relative to input numbers over a period of 1 to 4 weeks in culture (174,228,229), even though clonal analysis indicates that some HSC clones proliferate (228). Also, for unknown reasons, repopulating activity is lost with the entrance of cultured HSCs into active cell cycle (208). Similarly, homing of actively cycling HSCs is reduced by decreased expression of several molecules on the cell surface (206,230). Despite this inability to expand transplantable HSCs in vitro, more mature types of progenitors, including those with multilineage or single-lineage potential, can be greatly expanded in vitro. Thus, cell expansion technology may be useful to obtain high numbers of hematopoietic progenitor cells that may support patients in the short term after high-dose chemotherapy or marrow transplantation.
In principle, a successful ex vivo expansion strategy must, therefore, preserve HSC function and permit HSCs to self-renew in order to maintain or expand the number of transplantable HSCs during the culture. Because HSCs self-renew in vivo and establish the HSC pool during fetal life and re-establish it following transplantation, HSC self-renewal in culture requires a microenvironment in vitro that is similar to the hematopoietic microenvironment in vivo. Such culture conditions have not been achieved (231). Most previous studies of ex vivo expansion of HSCs have used either known growth factor combinations or unknown signals provided by stromal feeder layers in a coculture system (232), but these attempts to expand HSCs in vitro have resulted in progressive loss of HSCs and generation of large numbers of relatively committed progenitor cells (232). These results indicate that stromal support or culture conditions in vitro were inadequate to promote the self-renewal type of HSC division, but they likely induced differentiation of HSCs that lacked self-renewal capacity.
Human umbilical cord blood (CB) has been established as an important alternative source of transplantable HSCs instead of bone marrow or peripheral blood stem cells in children, but the limited number of HSCs present in a single unit of CB poses a significant risk for its use in adult patients, who require greater numbers of input HSCs (231). Successful ex vivo expansion of HSCs in CB is clinically significant, but several decades of research have failed to show any significant progress in enhancing the kinetics of hematopoietic reconstitution following transplantation of ex vivo expanded CB grafts (231). These studies demonstrated loss of immature HSCs during ex vivo expansion while committed mature progenitor cells expanded, ultimately resulting in marrow failure following infusion of such CB grafts.
Several genetic regulatory molecules have been implicated in HSC self-renewal (233,234). Induction of these putative self-renewal genes and transduction of cells with genes (e.g., HoxB4) encoding these intrinsic factors using retroviral vectors or recombinant proteins in culture have been attempted to promote self-renewal of HSCs. Also, intrinsic factors that may regulate HSC fate can be targeted by changing the chromatin structure of genome by epigenetic mechanisms (235,236). Recent studies suggest that ex vivo conditions previously used in attempts to expand HSCs may silence genes that are crucial to the self-renewal type of HSC divisions (237). These investigators have shown that treatment of cultured CB cells with chromatin modifying agents is associated with a significant expansion of in vivo repopulating cells in the cultures as demonstrated by transplantation in immunodeficient xenogeneic mouse models (237).
Committed Hematopoietic Progenitor Cells
Committed hematopoietic progenitor cells are progeny of HSCs that have begun to differentiate and can no longer convey long-term reconstitution of all hematopoietic lineages in ablated animals—that is, committed cells cannot give rise to HSCs. The term commitment is also used to denote other irreversible differentiation events that lead to further limitation of potential for development into terminal cell types. The latter idea is consistent with the observation that cell colonies grown in vitro from individual progenitor cells (or spleen colonies grown in vivo) contain a single mature cell type or limited combinations of mature cell types. Except for the HSCs, which can self-replicate as shown by the reflexive arrow, Figure 5.3 depicts recognized stages of committed hematopoietic lineages. Each progenitor cell stage represents an individual cell (the term unit was originally used because progenitors had not been shown to be individual cells) that gives rise to a definite, limited repertoire of mature cells as observed in various in vitro and in vivo assays. Although space in Figure 5.3 does not permit the representation of cell proliferation, the cells undergo multiple cell divisions between the stages shown. Thus, small numbers of HSCs give rise to greater numbers of the earlier committed progenitors that, in turn, are amplified through cell division at each subsequent point in the differentiation process. According to this scheme, each successive stage has a more restricted differentiation potential, and there is a succession of commitment steps. Just as the molecular processes in cells that determine whether a stem cell undergoes self-renewal or commitment to differentiate are not understood, neither are the molecular events that lead to subsequent commitment steps.
Multilineage Progenitors
When HSCs give rise to progeny that are committed to differentiate, early generations of these progeny have the potential to give rise to descendants representing multiple lineages. The majority of the murine CFUs-S cannot provide long-term reconstitution of ablated animals, but they are multipotential because they grow into a spleen colony containing multiple cell lineages in vivo. Under culture conditions in semisolid medium with adequate supportive growth factors, these progenitor cells can form colonies of multiple cell lineages in vitro (238,239). Similar multilineage colonies can also be demonstrated in vitro in human hematopoietic cell populations (240). When the hematopoietic cells reach maturity, the lineage composition of the colonies can be determined by picking out the colonies and spreading the cells on microscope slides followed by conventional staining techniques or by immunostaining using lineage marker antibodies. Not all multilineage colonies that appear in in vitro or in vivo assays contain all cell lineages; several combinations of cell lineages are characteristic. For example, some colonies contain granulocytes, erythrocytes, macrophages, and megakaryocytes (mixed colonies), other colonies contain granulocytes and macrophages (GM colonies), and so forth. Table 5.2 describes a variety of hematopoietic progenitor stages that are defined by in vitro assays and Figure 5.3demonstrates their hierarchical relationships. Table 5.3 lists the names and abbreviations of growth factors and their receptors that are discussed in this chapter.
The occurrence of colonies with various combinations of lineages has been interpreted in several ways (models) in order to explain how cells are committed to become a particular type of blood cell (241). The data favor the idea that there are multiple commitment steps and that these steps lead to loss of specific lineage potential in a definite order. The first lineage commitment step separates lymphoid from myeloid potential, then granulocyte/ macrophage potential is separated from erythroid/megakaryocyte potential, and so on, until finally, a descendant cell has only one lineage capability. This idea of successive commitment steps is embodied in Figure 5.3. Although this idea is probably generally correct, there are variant models that differ somewhat in their interpretation (241). Also, it must be remembered that in vitro growth conditions may not be permissive for all possible lineages to appear in a colony. Thus, caution must be exercised in interpreting the exact lineage commitment pathways. Multilineage progenitors, CFUs-S, and in vitro–derived multilineage colonies had been thought to be incapable of generating lymphoid cells. However, several relatively recent studies have shown that lymphoid cells are produced by several such progenitors, but that they were not observed previously because growth factor support for colony development was not permissive for descendent lymphoid cells (157,203,242).
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Table 5.3 Abbreviations for Growth Factors and Their Receptors |
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Single-lineage Progenitors
The descendants of the multilineage colony-forming cells are ultimately restricted to a single-lineage potential. The more mature, single-lineage–committed progenitor cells are assayed in vitro by their ability to form colonies. These progenitor cells include CFU-G, CFU-M, CFU-E, CFU-MK (243,244,245), CFU-preB (203,246), and CFU-DL (247,248) for colony-forming unit–granulocyte, –macrophage, –erythrocyte, –megakaryocyte, –B lymphocyte, and –dendritic/ Langerhans, respectively (Table 5.2 and Fig. 5.3). In some lin-eages, it is possible to observe stages of maturity within the lineage-committed progenitors. For example, the single-lineage–producing BFU-E is a more immature erythroid progenitor than the CFU-E, forming colonies of many more mature erythroid cells after a longer period of time than the CFU-E.
Terminal Phases of Differentiation
Cells in the final stages of hematopoiesis that are sufficiently differentiated can be identified by morphology using light micro-scopy with preparations of hematopoietic tissue. These cells are erythroblasts, myelocytes, monocytes, and megakaryocytes, and, because of the vastly amplifying cell divisions that occur by the time the final stages are reached, these cells are by far the most prevalent cells seen in hematopoietic tissues. They are only capable of a few cell divisions, on the order of one to four, yet they are undergoing dramatic specialized changes associated with terminal phases of differentiation/maturation. The erythroblasts rapidly accumulate hemoglobin and begin to assemble a unique membrane skeleton that later maintains the shape and deformation properties of the mature erythrocytes. The nucleus of the erythro-blast becomes condensed and is extruded from the cell, leaving an irregular, organelle-containing reticulocyte. Subsequently, over the course of a few days, extensive remodeling occurs within the reticulocyte that eliminates the internal organelles and changes the membrane so that the biconcave erythrocyte is formed. This remodeling process involves selective proteolysis and is not yet well understood. In myelocytes, granules that contain specific proteolytic enzymes are formed in the cytoplasm. The nuclei undergo a condensation process that ultimately results in a multilobular nucleus that is retained in the mature cell. Maturing monocyte precursors undergo similar changes. The terminal-stage megakaryocytes replicate their DNA and undergo several nuclear divisions without cytokinesis; thus, they become polyploid. Dense granules and α-granules form in the cytoplasm, and the cytoplasm becomes highly compartmentalized by demarcation membranes. Platelets form as small portions of the demarcated megakaryocyte cytoplasm separate from the whole cell.
Theories of Commitment
Extrinsic Regulation versus Stochastic Mechanisms
There are two contrasting ideas about the general nature of the commitment process: (a) extrinsic regulation, in which external factors initiate cell signaling pathways and determine the cell’s fate; and (b) that intrinsic, irreversible gene expression changes in cells that establish the alternative differentiation events occur stochastically. In this latter process, the alternative initial changes are probabilistic, and the probabilities for the alternative changes are unaffected by the cellular environment. Evidence for and against each of these two views has been reviewed by Metcalf (249) and Enver et al. (250).
The extrinsic regulation theory holds that each HSC or progenitor cell has the unlimited and equivalent capacity to differentiate into any of its potential progeny, and that specific signals from the cell’s environment determine to which pathway that individual cell commits. These deterministic signals are initiated by growth factors produced in the marrow stroma, cytokines in the cell’s environment, or direct cell–cell interactions. Specific receptors on the HSC or progenitor cell are activated by the extrinsic factor and, in turn, generate signals internally that change gene expression. The gene expression changes that are activated characterize the subsequent differentiation state, and the hematopoietic cell develops along a predefined program of changes in that specific lineage until, at some point, new signals are received that redirect another sequence of gene expression changes. Further signals are required at subsequent differentiation branch points to activate further commitment steps. Traditionally, support for such extrinsic regulation came from experiments in which populations of undifferentiated, multipotential hematopoietic cells (usually an immortal cell line) are cultured in the presence of one or another mixture of test growth factors. After a time period of several days, a cell population emerges that is composed of a high percentage of cells recognizable as one or several particular lineages, and the lineage composition of the emergent population depends on the particular growth factors used in the culture (251,252). In such experiments, the initial population of undifferentiated hematopoietic cells is undergoing cell division and cell death at unknown comparative rates. Because a differentiated population takes several days to become predominant, it is difficult to determine if the cells were induced to differentiate by the test growth factors or were rather selected for survival or growth by permissive conditions. More recent support for the extrinsic regulation theory of commitment comes from experiments using reciprocal bone marrow chimeras. Labrie et al. showed that B-cell generation in young or aged mice following sublethal irradiation followed by bone marrow transplant was dependent upon the stromal environment of the recipient mouse rather than the source of transplanted marrow (253). The transcription factors necessary for B-cell lineage commitment are up- or down-regulated based on the microenvironment; the marrow of older mice has fewer osteoblasts and increased adipocytes compared to younger mice, resulting in differential expression of cytokines and chemokines. This change in environment results in decreased rag2 expression, and thus a decrease in B-cell progenitors (253).
The competing theory of commitment is the stochastic or cell-autonomous model, in which commitment is cell intrinsic. This model is probabilistic, and the cellular environment does not change the probabilities that each cell will commit to a particular lineage. A stochastic model for commitment to differentiation versus self-renewal of stem cells was first proposed by Till et al. (254). Later, Ogawa et al. (255) proposed that commitment of multipotential hematopoietic progenitors to individual myeloid lineages is also a stochastic process. The role proposed for hematopoietic growth factors in the stochastic model is that of providing a permissive or selective environment for survival and/or proliferation of the progenitor cells that bear the cognate surface receptors for the growth factors. Support for the stochastic model of commitment comes from experiments in which hematopoietic progenitor cells are plated such that each culture initially contains a single cell. The two daughter cells after an initial cell division are separated and replated individually into cultures with the same or different growth factors. Several studies of this type have been done (241,256,257,258,259), and it has been shown that a significant fraction of paired daughter cells each gives rise to subsequent progeny representing different combinations of lineages. The type of progeny of these asymmetric divisions does not appear to be influenced by cytokines (259). Thus, so far, experiments manipulating single cells have yielded data consistent with the stochastic model of lineage commitment. Yet, such experiments require analysis of the fate of hundreds of cell pairs under various conditions, and experiments have not yet been done that have completely resolved the issue. Whether cytokines can have any direct influence on differentiation is not clear. It is clear that in vitro, many cells do commit to differentiation into particular lineages and do differentiate without a detectable influence of cytokines. This latter conclusion was supported by a study that showed that clones of a multipotential cell line, when engineered to express a gene that protects the cells from apoptosis (bcl-2), can undergo spontaneous differentiation into multiple lineages in the absence of any added growth factors (260).
In vivo repopulation experiments have also been used to analyze deterministic and stochastic models of the commitment to self-renewal of HSCs and the commitment to differentiation of HSCs and their progeny. Analyzing short-term and long-term multilineage hematopoietic progenitors that are detected following transplantation of purified HSCs isolated on the basis of immunophenotypic markers, Morrison et al. have concluded that HSC repopulation in the mice is mainly a deterministic process (261). Experiments in which a single cell that can rescue a lethally radiated, transplanted mice (262) have been interpreted as being consistent with this deterministic conclusion in that the self-renewal ability of highly enriched, purified HSCs is predictable (261). On the other hand, mathematical models designed by Abkowitz et al. (263) to explain the behavior of HSCs in vivo in autologous bone marrow transplantation of glucose-6-phosphate dehydrogenase (G6PD) heterozygous female Sfari cats indicated that stochastic events could explain all forms of clonal contribution to hematopoiesis in these experimental animals. Likewise, with a mathematical model based on data from mouse chimeras in a study of competitive clonal hematopoiesis, Roeder et al. (264) developed a “flexible, self-organized” but stochastic model that described HSC activities in chimeric mice.
Molecular Mechanisms of Differentiation
From a molecular mechanistic standpoint, based on current ideas about gene transcription, it is easier to propose molecular schemes to explain a deterministic model of commitment than a stochastic one. One can suppose that a receptor signal activates a specific transcription factor (among several possible ones), and that specific factor turns on or represses a gene, which, in turn, activates or suppresses other specific genes. Irreversible sequences of gene expression can be hypothesized that are fixed in daughter cells of the original, affected cell (265). One is less accustomed to thinking about how transcription of alternative genes might be stochastic and how such alternatives might result, then, in different sequences of gene transcriptional changes that are passed on to progeny cells. Two familiar examples are the stochastic gene rearrangements that occur in lymphocytes to form the T-cell and B-cell antigen receptors. No such rearrangements of genetic material are known to occur in connection with commitment of other hematopoietic progenitors, but the possibility does exist for as yet unidentified genes. Another way in which expression of critical genes might be stochastic is through competition of transcription factors for the genetic elements that control the expression of the genes or through competition at some critical time between nuclear matrix-associated proteins for sites on the DNA in the vicinity of various genes. Recent reviews have analyzed the role of transcription factors and their associated cofactors in the modifications of chromatin that induce or inhibit transcription of specific genes involved in the fate of hematopoietic progenitor cells (266,267). Aside from the binding of transcription factors, there are modifications of DNA and chromatin structure that regulate gene expression either reversibly or irreversibly. These modifications include acetylation, phosphorylation, ubiquitination, and methylation of the histones, the major components of chromatin, as well as methylation of DNA. How the specificity of these processes for particular genetic regions is controlled has yet to be determined.
Role of Particular Transcription Factors
Some specific transcription factors exhibit hematopoietic lineage-restricted expression, and some are known to be essential for the complete differentiation of individual lineages. Among the transcription factors that have been shown to influence lineage decisions are several members of the homeobox (HOX) family and the Ikaros proteins (268,269). Also, the mammalian Polycomb group of genes that regulates expression of various HOX genes probably plays roles in lineage decisions (270). Two examples of transcription factors whose lineage associations are more fully understood are GATA-1, which is essential for terminal erythrocyte and megakaryocyte differentiation, and PU.1, which is essential for B-lymphocyte as well as macrophage development (271,272). Specific factors not only play a direct role in the expression of lineage-specific genes but, in some cases, appear to antagonize transcription factors important for other lineages; thus, they can repress the expression of genes characteristic of other lineages. For example, GATA-1 can suppress PU.1 activity and PU.1 can suppress GATA-1 activity by direct protein interactions that block the function of each other (272). PU.1 and GATA-1 play positive roles in the transcription of their own genes (autoregulatory loops) (273,274). Thus, hypothetically, an excess of GATA-1 over PU.1 could down-regulate PU.1 expression at the level of transcription, and excess PU.1 could likewise down-regulate GATA-1. In multipotent cells, it is known that there is expression at low levels of sets of genes characteristic of multiple hematopoietic lineages (275). Thus, commitment appears to occur not only by up-regulation of a single-lineage program of gene expression, but also by the irreversible suppression of competing differentiation programs. Because of observations such as those described above for GATA-1 and PU.1 and because the forced overexpression of particular transcription factors can cause lineage switches in certain in vitro cell systems, some investigators have proposed that the transcription factor profile (stoichiometry relationships) of multipotent cells directs their lineage commitment decisions through cross-antagonism mechanisms (272,276). How variations in transcription factor stoichiometry occur could be either by extrinsic signals or by stochastic mechanisms. A transcription factor network has been proposed in which combinations of specific lineage-instructive transcription factors at various stages of hematopoietic differentiation from HSCs through the lineage-specific progenitor cells play roles in cell fate decisions (267).
Hematopoietic Growth Factors
In the course of development of in vitro assays for the various multilineage and single-lineage hematopoietic colony-forming cells, it became clear that several different growth factors exist, each capable of supporting a particular spectrum of hematopoietic cell types. Initially, most of these factors were discovered to exist in “conditioned media” that had been used to culture specific cell lines or specific primary cell types. Such conditioned media were necessary or greatly stimulatory for hematopoietic cell colony growth. An example of a conditioned medium source is medium from pokeweed mitogen-stimulated murine spleen cells (190). The number of known hematopoietic growth factors has now expanded greatly and will likely increase. The known growth factors have been purified, and the genes that encode them have been cloned. These growth factors, which are glycoproteins, are now available as pure, recombinant factors due to the successful cloning and expression of their genes at high levels in modified eukaryotic cells. The availability in abundance of pure growth factors has led to the identification and cloning of genes for their specific cell receptors. Conversely, in some cases, discovery of a putative receptor molecule through molecular cloning has led to the identification of previously unidentified growth factors.
Hematopoietic Growth Factor Receptors
Based on certain structural and functional features of the receptors for hematopoietic growth factors, two families of ligands/ receptors have been recognized: the cytokine receptor family and the tyrosine kinase receptor family (Table 5.4). It was noted by Bazan (277), through modeling of the secondary structures of receptors for several hormones and hematopoietic growth factors, that predicted structural similarities existed among a particular receptor group. This group is called the cytokine family of receptors. These receptors have an approximately 200–amino acid extracellular binding segment composed of two discrete folded domains of approximately 100 amino acids. These domains share significant sequence and similar predicted secondary structure. Predicted secondary structure of these domains includes seven β strands folded into antiparallel β sandwiches with a similar topology. Bazan (277) proposed a model of cytokine binding to these receptors in which the cytokine fits into a generic structural framework formed by the relatively conserved topology of the β-sheet faces and the connecting loops between the β strands. Because the member receptors of the cytokine group have these structural similarities, it was expected that the binding domains of their ligands, the cognate growth factors, would have some common structural features, which is the case. Several of the cytokine growth factors have had their structures determined by x-ray crystallography: growth hormone, IL-2, macrophage colony-stimulating factor (CSF-1), GM-CSF, interferon-γ, G-CSF, IL-10, IL-5, IL-4, and murine leukemia inhibitory factor. The determined structure in each case includes a topologic feature consisting of a “bundle” of α-helices (278,279,280,281,282). There are variations in how these structures are formed from portions of the peptide chains, but they are, nevertheless, similar. For reviews of this cytokine growth factor/receptor group, see references (277,283,284,285). The receptor protein chains have a single membrane-spanning domain, and their intracellular domains have no areas of amino acid homology to tyrosine kinases or guanosine triphosphate–binding domains that are found in other classes of signal-transducing receptors.
Most of the functional receptors for the cytokine growth factors consist of complexes of two or more protein chains of the cytokine receptor family (Table 5.4), and functional receptors for different cytokine growth factors sometimes contain a common cytokine receptor peptide chain. For example, IL-3, GM-CSF, and IL-5 receptors consist of a ligand-specific α-chain and a common gp140 βcsubunit. Similarly, the so-called IL-6 family of cytokines (Table 5.4) uses receptor complexes consisting of one or two common gp130 chains plus an additional ligand-specific subunit (286). Although receptors of several subunits are common among the cytokine receptor group, several members including those for EPO, thrombopoietin (TPO), and G-CSF consist of a single cytokine family protein chain. These receptors are each displayed as homodimers on the surface of the respective lineage-specific progenitor cells (287).
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Table 5.4 Classification of Hematopoietic Factors Based on Their Receptor Types |
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The signal transduction mechanisms for the cytokine family of receptors have been investigated by many groups (283,284,285). In general, each of these receptors interacts with several intracellular proteins. When their ligand binds, they activate one or more members of the Janus kinase (JAK) family of protein tyrosine kinases. The JAK proteins are physically associated with the cytoplasmic portion of each receptor protein and they act as chaperones for the migration of the receptors from the endoplasmic reticulum and Golgi to the progenitor cell surface (288,289). The JAK proteins phosphorylate themselves as well as the cytoplasmic portions of the receptors. These phosphorylations of the receptor– JAK complexes lead to the docking of other signal transduction molecules, which are, in turn, also activated by phosphorylation (290,291,292). Among the signaling pathways known to be activated by these hematopoietic growth factor–bound receptors are phosphoinositol 3-OH kinase/protein kinase B (PI 3-kinase/Akt), the STAT (signal transducer and activator of transcription) family proteins that move from the cytoplasm to the nucleus and act as transcription factors through sequence-specific binding to DNA, and mitogen-associated protein kinase/extracellular signal-related kinase (MAPK/ERK). It is not yet fully understood what the roles of each of these receptor functions are in causing the final cellular effects, but the role of JAK activation is clearly central to the action of the cytokine receptors in that murine knockout mutations of JAK result in hematopoietic failure (293), while gain-of-function mutations lead to multilineage expansion in myeloproliferative disorders in humans (287) and in a murine model (294). In addition to stimulating various signal transduction pathways that lead ultimately to increased production of the lineage-specific blood cells, the activated receptors activate pathways that limit the signaling function. These limiting mechanisms involve the internalization and degradation of the receptors, the activation of phosphatases within the progenitor cell, and the induction of specific suppressors of cytokine signaling (SOCS) (287).
The second group of known hematopoietic growth factors consists of members whose receptors are themselves protein tyrosine kinases, the receptor tyrosine kinases (RTKs) (295) (Table 5.4). The factors of this group that are best understood in hematopoiesis are KL and CSF-1. The respective receptors are products of the c-kit and c-fms proto-oncogenes. One widely distributed receptor tyrosine kinase/ligand system that is important in hematopoiesis is insulinlike growth factor (IGF)-1 and its receptor IGF-1R. IGF-1 has specifically been shown to be important for erythroid cell differentiation in vitro in culture systems without added serum (296), although it is probably important for other lineages as well. The receptor tyrosine kinase FLK-2/FLT3 (mouse) or STK-1 (human) has been identified on early hematopoietic progenitors (297,298,299), and its cognate ligand, FL (300), has multiple effects in hematopoiesis (88a,299,301,302,303). In chickens, the Erb-B receptor, through binding of transforming growth factor-α, has a profound effect on expansion of early erythroid progenitors (304). Finally, there are reports of expression of additional receptor tyrosine kinases on some hematopoietic cells, but their significance in hematopoiesis has not been extensively studied. These include FLK-1 (305,306), MRK (307), and TIE and TEK (308,309).
The RTKs comprise a large group of receptors that have been subdivided into types based on structure (295,310) (Table 5.4). Kit, CSF-1R, and FLK-2/FLT3 are receptors of a subfamily of the platelet-derived growth factor receptor family (or type IV [310]) that are characterized by five immunoglobulinlike domains in the extracellular portion. FLK-1 is a member of the other subfamily of the platelet-derived growth factor receptor family (or type V [310]) that has seven such immunoglobulin domains. The genes for the related type III and type V receptors are clustered together in three locations in the human genome (310). IGF-1R is a member of the insulin family of receptor tyrosine kinases.
The intracellular mechanisms by which the RTKs trigger their biologic effects are, like those of cytokine receptors, under intense investigation (311,312). Interestingly, many of the same intracellular pathways appear to be stimulated by both types of receptors. It remains to be elucidated whether or how these various receptors can initiate specific intracellular functions not common to all of the other receptors.
Factors That Act on Multilineage Progenitors
In vitro cultures of hematopoietic colony-forming cells have continued to be very useful in defining growth factor effects on various lineages of cells (88a,313,314,315,316,317). Many of the hematopoietic growth factors exhibit positive growth effects on HSCs or progenitors with multilineage potential, or both. These include KL, GM-CSF, G-CSF, CSF-1, IL-3, IL-4, IL-6, IL-11, IL-12, FL, leukemia inhibitory factor, oncostatin M, and TPO (318,319,320,321). In addition, some members of this same group can support differentiation of certain cell types to late stages or even to full maturity. For example, G-CSF, GM-CSF, CSF-1, IL-3, KL, and IL-6 can all support formation of small neutrophilic granulocytic colonies, and CSF-1 and GM-CSF can also support macrophage colonies and mixed granulocyte/macrophage colonies (318). Other members of this group do not support formation of colonies of mature hematopoietic cells alone but exert their effects in combinations with other factors. They may affect only early-stage progenitors, and their partner factors support the later differentiation, or they may act simultaneously with other factors on cells at any development stage that yields enhanced cell production as a result of a combination of different stimulatory signals. Such enhancement or potentiating effects are not restricted to those factors that have no effect alone but are rather observed with particular combinations of all members of the group listed above.
Potentiation of hematopoietic cell production in in vitro assays by combinations of growth factors can occur in two basic ways. A combination of growth factors may allow proliferation and differentiation of individual cells that would otherwise die or remain dormant in the presence of a single factor. Second, potentiation can occur by enhanced proliferation in the presence of the combined factors. The latter effect appears to apply to the examples of the combined effect of KL with G-CSF, GM-CSF, IL-3, IL-6, or EPO on expansion of populations of progenitors (322,323,324). The numbers of colonies formed in the presence of the combinations are not increased greatly, but there is a large increase in the size of the colonies. The proliferation of HSCs, however, appears to be an example of a requirement of a combination of factors for recruitment of dormant cells into proliferation and differentiation (241,318,325,326,327).
When growth factors with effects on multilineage progenitors act alone or in combination, the result of early rounds of proliferation and differentiation is the generation of progeny that become committed individually to form different lineages of mature cells. For some lineages, the resultant single-lineage progenitors cannot complete differentiation and maturation without lineage-specific factors; late committed erythroid progenitors (CFU-E) require EPO, or they die. Likewise, appearance of lymphoid cells requires IL-7, and maturation of megakaryocytes and formation of platelets is greatly enhanced by TPO. Thus, the full development of hematopoietic cells from stem cells or early-stage progenitors requires the action of growth factors (alone or in combination) that support the multilineage progenitors and, in addition, growth factors that support terminal differentiation of committed single-lineage progenitors.
Granulocyte and Macrophage Growth Factors
In addition to affecting multilineage progenitors, several growth factors also support terminal differentiation of granulocytes and macrophages. GM-CSF supports growth and development of both granulocytes and monocytes/macrophages (243,328,329). G-CSF, CSF-1, and IL-5 selectively support differentiation of neutrophilic granulocytes, monocytes/macrophage (243,329), and eosinophilic granulocytes (330,331), respectively. Dendritic/Langerhans cells arise from progenitor cells that also give rise to monocytes. In vitro culture of these progenitors in the presence of tumor necrosis factor-α plus GM-CSF or IL-3 favors generation of dendritic cells (247,248,332,333,334,335). Mast cell differentiation is supported by KL (330,336,337,338,339).
G-CSF, GM-CSF, and CSF-1 not only support differentiation of late-stage progenitors, but also can activate the resulting mature blood cells, stimulating such functions as phagocytosis (340,341,342,343). KL also activates mature mast cells, causing them to release histamine (339).
IL-5 is produced by T lymphocytes, whereas GM-CSF, G-CSF, KL, and CSF-1 are produced by multiple cell types including fibroblasts, endothelial cells, and macrophages. Thus, all of the cell types that produce the granulocyte and macrophage growth factors are distributed throughout the body, and T cells and macrophages are concentrated at the site of inflammation. Circulating late-stage granulocyte and macrophage progenitors as well as mature granulocytes and macrophages could thus be exposed to these factors at areas of inflammation as well as in the bone marrow stroma. It is plausible that these growth factors may play important roles in activation of granulocytes and macrophages as well as supporting final maturation stages at locations of inflammation. Production of granulocytes or macrophages does not appear to be regulated systemically through a feedback mechanism that senses the mature cell numbers in the body and then modifies granulocyte or monocyte production in the bone marrow.
Megakaryocyte Growth Factors
In vitro colony assays have been developed for quantifying megakaryocyte progenitor cells, termed colony-forming units– megakaryocyte (CFUs-MK). As in the case of other early committed progenitors, the growth of such colonies is augmented by several of the CSFs with multilineage activity such as IL-3, IL-6, GM-CSF, KL, and IL-11 (245,344,345). Unlike granulocytes and monocytes/macrophages, bone marrow production of platelets is regulated by the number of platelets in the blood. Reduction of platelet numbers in rodents by antiplatelet antibodies or by exchange transfusion of platelet-poor blood causes an increase in the number of megakaryocytes in the hematopoietic tissues as well as an increase in their size and ploidy; conversely, platelet transfusion decreases these parameters (245). However, such manipulations did not affect CFU-MK numbers in the hematopoietic tissues (346), leading to the speculation that megakaryocyte differentiation and platelet production are controlled by a thrombopoietic factor that is induced by thrombocytopenia (245,347).
A growth factor has been identified that has some properties of a physiologic regulator of platelet production. This factor, TPO, exerts its effect through the activation of a cytokine receptor termed Mpl (348,349,350). Mpl was identified earlier as the viral oncogene product of the mouse retrovirus, myeloproliferative leukemia virus (351). Recombinant TPO increases megakaryocyte and platelet numbers in vivo and stimulates CFU-MK growth in vitro (352,353). Mice bearing homozygous, nonfunctional alleles of c-mpl are viable with greatly diminished platelet numbers (319), indicating that although TPO is not essential for platelet production, it is a strong in vivo regulator of the process. TPO production has been shown to be regulated by blood platelet numbers (353), and platelet numbers regulate the mRNA for TPO in the marrow and spleen but not in the liver and kidney (354). It is not yet clear that the modulation of TPO mRNA in these organs is responsible for the regulation of overall TPO protein levels. Several studies indicate that TPO is constitutively synthesized in the liver and that its level in blood is determined by its removal from circulation by binding to the c-Mpl receptor on platelets and bone marrow megakaryocytes (348).
Growth Factors for B Lymphocytes
Methods for culture of B lymphocytes and their progenitor cells were originally described by Whitlock and Witte (355). Subsequently, a colony assay for B-cell progenitors, CFU-preB, was described, in which it was found that IL-7 is a very potent growth stimulatory factor for such progenitors (246). The role of IL-7 in lymphoid cell development in vivo was demonstrated by generating mice in which the genes for the IL-7 receptor are nonfunctional. Such mice have a profound reduction in thymic and peripheral lymphoid cellularity with defects in both B- and T-cell development (356). Because of the realization of the importance of IL-7 in B-cell growth in vitro, it has been incorporated into culture media when examining the lineage potential of early multilineage progenitors (203,204,242). Progenitors with both myeloid and lymphoid potential can be observed and quantified and, although loss of this dual potential had been thought to occur at stages significantly beyond the commitment of the HSCs to differentiate, recent studies suggest that commitment to lymphoid or myeloid differentiation is among the first decisions made when HSCs differentiate (267).
Growth Factors for Erythroid Cells
The physiologic regulator of erythrocyte production is EPO, and this regulation is very precise, keeping the red cell mass within narrow limits (357). EPO acts on committed erythroid progenitors to support the later phases of erythroid differentiation (357). The regulation is achieved by EPO’s action to modulate apoptosis of these progenitors. The production of EPO is regulated by the O2 activity in the vicinity of specialized EPO-producing cells in the kidney. These cells are peritubular cells, located in the renal cortex (358,359,360). By sensing O2 activity, they essentially measure the oxygen delivery capacity of the blood, and they adjust EPO production to achieve the number of erythrocytes needed for normal tissue O2 tension. The liver also contains specialized cells that can produce EPO in an oxygen-dependent manner, although in adult animals, the contribution of the liver to total EPO production is much less than that of the kidney. In the specialized kidney and liver cells, the transcription of the EPO gene is controlled by an oxygen-dependent transcription factor, hypoxia-inducible factor (HIF), that interacts with DNA sequences corresponding to the 3′ untranslated sequence of the mRNA and also with sequences in the EPO promoter region (361). HIF ubiquitination and subsequent proteasomal degradation are dependent upon the hydroxylation of two specific prolines (362,363,364) and an asparagine of HIF (365,366) by nonheme, iron-containing hydroxylases that use molecular oxygen as a substrate for the reactions. With normoxia, HIF is rapidly hydroxylated and degraded. With hypoxia, HIF is not hydroxylated and degraded, but it forms part of a transcription complex that binds the 3′ enhancer sequence and induces EPO gene transcription. In addition, tissue specificity of expression in the kidney requires specific, cis-acting DNA sequences far upstream (between 6 kilobase pairs and 14 kilobase pairs) of the coding sequence (367,368). EPO is secreted rapidly into the circulation, and in the bone marrow it binds to EPO receptors on erythroid progenitor cells in the CFU-E through early erythroblast stages. The EPO–EPO receptor interaction not only triggers signal transduction, but also leads to endocytosis and degradation of both the EPO and EPO receptor (369). This erythroid progenitor-mediated consumption of EPO appears to be a major determinant of the metabolic fate of EPO both in vitro (370) and in vivo (371).
KL is also specifically required for erythroid cell development as shown by its requirement for growth of human BFU-E in vitro under serum-free conditions (372). The Kit receptor is present on multilineage progenitors and on the BFU-E, and it persists on erythroid progenitors up to the proerythroblast stage. KL thus has a stimulatory effect on erythroid progenitors throughout most early stages, including those of the CFU-E and proerythroblast, when EPO stimulation becomes essential for further development. IGF-1 also appears to have a specific role in erythroid development, as it also appears necessary for proper erythroid differentiation in serum-free cultures (296,373). Other multilineage growth factors, such as IL-3 and GM-CSF, have a stimulatory effect on BFU-E growth in vitro, although there does not appear to be a specific requirement for these factors.
In Vivo Significance of Hematopoietic Growth Factors
With the exception of EPO, the growth factors were initially discovered and studied because of their support of hematopoietic cells in vitro. Loss of function of EPO or the EPO receptor in knockout mice leads to embryonic death at approximately day 13 of gestation due to failure of production of definitive erythrocytes (374,375). An important question is, what is the physiologic role in vivo of those factors that were discovered in vitro? This question has been studied by several approaches, but perhaps the most revealing is to understand the phenotype of animals resulting from loss of function mutations in the genes for the growth factors or their specific receptors. Mice bearing spontaneous mutations in some of the growth factor genes have been identified. In additional cases, mutant animals were created by using homologous recombination-directed muta-genesis (gene knockout) in murine embryonic stem cells and using these cells to generate mouse strains bearing the mutation (328).
Table 5.5 summarizes the phenotypes of mice bearing loss of function mutations for several growth factors. Of the factors with effects on multilineage cells, the significance of KL is the most clear. Spontaneous mutations at the W locus and the Sl locus affect Kit and its ligand, respectively, and complete loss of either is lethal at an early embryonic stage. Certain partially functional alleles in these genes are compatible with life, but animals bearing them have hematopoietic deficits in several cell lineages. The gene encoding CSF-1 is defective in the spontaneous mutant op/op mice. These young mice lack teeth and develop osteopetrosis due to failure to develop osteoclasts derived from macrophages (376). They also have great deficiencies in macrophage populations in some but not all tissues. Interestingly, with age, the op/op mice undergo significant correction of their macrophage deficiency. GM-CSF–deficient mice appear to have normal production of granulocytes and monocytes, but they have alveolar proteinosis because of defective functioning of alveolar macrophages (377,378). In summary, CSF-1 appears to be important for normal production of some macrophage types, whereas GM-CSF is important for the function of alveolar macrophages. Nevertheless, production of some macrophages, even in the absence of function of both genes (379), indicates that unknown regulators are important for monocyte/macrophage production and function. G-CSF is important for maintenance of normal neutrophil numbers and granulocyte/macrophage progenitors in the bone marrow. Only 20 to 30% of normal neutrophil numbers are present in mice lacking G-CSF; they have reduced capacity to mobilize neutrophils on demand; and deficient mice are more susceptible to some bacterial infections (380). Although IL-3 is one of the most potent stimulators of hematopoietic colony growth in vitro, several mouse strains lack functional IL-3 α-receptor subunits and no IL-3 binding or demonstrable function in the cells of the animals. However, such animals are hematologically normal (381). Mice deficient in the Flk-2/FLT3 receptor develop into healthy animals with largely normal mature cell populations derived from hematopoietic tissues. They do have some specific deficiencies in primitive B-cell progenitors and in the transplantability of their stem cells (382). Mice deficient in the ligand for FLT3 have a more severe phenotype than those deficient in the receptor itself (383). The physiologic role of other multilineage factors remains to be clarified. Mutant mice have been generated that lack function of factors affecting more restricted lineages. Mice lacking IL-7 exhibit a profound reduction in B- and T-cell development (356), and those lacking TPO only have approximately 10% of the normal number of platelets (319).
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Table 5.5 Phenotypes Caused by Nonfunctional Mutations in Genes for Hematopoietic Growth Factors or Their Receptors |
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Thus, with some exceptions, the hematopoietic growth factors discovered because of their effects in vitro appear to be important in vivo in maintaining some aspects of hematopoiesis or in the function of some subclasses of mature hematopoietic cells. The roles of some of the factors that affect progenitors with multilineage potential have not yet been fully evaluated in vivo. The evidence from mutant mice also indicates that there are as yet undiscovered factors—in particular, factors that regulate production and function of macrophages and related cells.
Mechanisms of Action of Hematopoietic Growth Factors
The roles of growth factors in colony development and differentiation into mature cells in vitro are unknown. Because colony formation requires cell proliferation and differentiation, “stimulation” of colony formation has been inferred to be synonymous with initiation of cell cycling or induction of a differentiation program in progenitor cells, or both. However, promotion of cell survival by prevention of programmed cell death (apoptosis) has been recognized as a definite, specific function of hematopoietic growth factors and hormones. Thus, it is possible that in some instances, growth factors cause proliferation in a particular type of progenitor, whereas in another instance, a growth factor acts to prevent apoptosis. The developmental stage of a cell may determine its response to a growth factor. Finally, combinations of factors may be required for a particular cell response.
Induction of Differentiation
With the exception of the Indian hedgehog and BMP4 inductions of hematopoiesis in embryonic development, little evidence exists that growth factors are directly responsible for inducing the gene expression changes that are critical for the commitment of cells to a particular lineage pathway. Although some hematopoietic growth factors are known to activate certain transcription factors, no data have yet proved that those specific transcription factors directly control a differentiation step.
Mitogenesis
It is difficult to determine whether a particular growth factor is a mitogen for a specific type of normal hematopoietic progenitor cell—that is, to determine whether it causes the cell to enter an active cell cycle from an initially quiescent state. To make such a determination requires that one has a nearly pure population of the cells in question, that the cells remain alive for at least 24 hours without the growth factor (to distinguish proliferation effects from survival effects), and that culture conditions can be found such that the growth factor can be assessed in the absence of other factors or such that each factor can be assessed independently. These conditions can occasionally be met using purified populations of explanted, late-stage hematopoietic progenitors (384), but they have not yet been achieved with stem cells or early multilineage progenitors. In lieu of normal progenitors, numerous studies have used transformed hematopoietic cell lines that are dependent on a hematopoietic growth factor for continuous growth. One can often put such cells into a quiescent cell cycle state without initiating apoptosis by transient growth factor depletion. Thus, although such lines can meet the above criteria, they may be abnormal in that their transformed phenotype may result from selection for mutations in genes that control the cell cycle or apoptosis.
The notion that a growth factor acts as a mitogen implies that the target cell exists in a quiescent state of cell cycle state without the factor. Such a state appears to apply to some hematopoietic progenitors in vivo but not to others. For the majority of progenitor cell types, their ability to exist in such an inactive state is not known. Many HSCs appear to be quiescent in vivo, or when freshly isolated, because most of them are resistant to 5-fluorouracil, an agent that selectively kills cells that are undergoing DNA synthesis (100,203,385,386,387). On the other hand, most committed progenitors do not appear to be quiescent. In mice, they are sensitive to killing by 5-fluorouracil treatment over a 2-day period (203,385). In the committed erythroid lineage, early progenitor populations (BFU-E) have a smaller fraction of cells in DNA synthesis at a given time (approximately 30%) than the later-stage CFU-E (approximately 70%), suggesting that some BFUs-E may spend time in a quiescent state (388,389,390,391). Some evidence suggested that exposure of BFUs-E to EPO can increase their fraction in DNA synthesis (390,391), but other studies indicated that EPO has no effect on cell cycle status of BFUs-E (388,389). In the case of CFUs-E, the fraction of cells in DNA synthesis is unmodified by EPO exposure. However, EPO is absolutely necessary for the survival of CFUs-E and for progression of CFUs-E into mature erythrocytes.
One case in which mitogenic effects of a growth factor have been clearly demonstrated on primary normal progenitors is that of CSF-1 action on macrophage progenitors from mouse bone marrow (384). These cells require CSF-1 for survival, but at low levels of CSF-1, they remain alive but are quiescent in regard to cell cycle. Higher doses of CSF-1 induce these quiescent progenitors to re-enter the cell cycle and proliferate. Thus, these two effects of CSF-1 are separable, and the observed effect on cells depends on the dose of growth factor. Potentially, each of these effects could come into play in vivo, depending on the concentration of CSF-1 in the target cell’s environment.
Prevention of Apoptosis
A large body of evidence suggests that most or all normal hematopoietic progenitor cells require specific growth factors to prevent apoptosis (392,393,394,395,396,397,398). Growth factors can prevent apoptosis by several mechanisms, and more than one of these mechanisms can be supported simultaneously by a single growth factor operating on a cell. One general mechanism of apoptosis suppression is the increased synthesis of antiapoptotic proteins encoded by the members of the bcl-2 gene family (399,400,401,402,403,404,405,406,407,408,409,410,411,412). In neutrophils and macrophages, the antiapoptotic proteins Mcl-1 and A1 are regulated (403,404,405,406,413). In progenitors of macrophages, megakaryocytes, erythrocytes, eosinophils, and B and T lymphocytes, the antiapoptotic protein Bcl-x plays a key role in survival at particular developmental stages (399,400,401,402,407,409,410,411,412,413). A second mechanism of apoptosis suppression by growth factors is the activation of Akt/protein kinase B through the activation of phosphoinositol 3-OH kinase (414). It is not fully understood how Akt/protein kinase B prevents apoptosis, but one possibility is through phosphorylation and inactivation of BAD, an apoptosis-promoting member of the Bcl-2 protein family (414). Still other mechanisms undoubtedly exist by which growth factor receptors send antiapoptotic signals.
In control of erythrocyte production, much evidence suggests that the physiologically relevant function of EPO is regulation of apoptosis in late-stage progenitors, not regulation of cell cycling (393,415,416,417). In vivo, most CFUs-E and proerythroblasts are in DNA synthesis (i.e., the majority of these progenitors are in the S phase of the cell cycle) regardless of the EPO levels in the animals from which they were obtained (388,389). In other words, CFUs-E and proerythroblasts, which are the cells with the greatest number of EPO receptors and are highly dependent on EPO, are in active cell cycle under all conditions; therefore, EPO does not induce them into cell cycle. CFUs-E and proerythroblasts of mice and humans undergo prompt apoptosis in the absence of EPO (393,415,416,417). In a proerythroblast population, the fraction of cells that undergoes apoptosis is dependent on the concentration of EPO over an extensive range of concentrations (416). Studies using cultures of purified human CFU-E under serum-free conditions with added combinations of pure growth factors indicated that the number of cell divisions that occur during late erythroid maturation depends on the concentration of KL, but that EPO by itself can keep the cells alive (373). Finally, studies of mice in which either the EPO gene or the EPO receptor gene has been knocked out show that the homozygous fetuses die because of failure of definitive erythropoiesis. In the livers of these fetuses, CFUs-E develop, but there is evidence of extensive apoptosis of erythroid cells (374,375).
EPO increases transcription of the bcl-x gene during late stages of erythroid differentiation, and it also stimulates the Akt/protein B kinase pathway. How EPO or other hematopoietic growth factors induce transcription of bcl-x is not yet completely understood, although roles for several transcription factors have been suggested (418,419,420,421,422). However, studies with conditionally bcl-x null mice demonstrate that the antiapoptotic effect of EPO on CFUs-E and early-stage erythroblasts is not mediated by Bcl-x (423).
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Figure 5.4. Model of erythropoiesis based on erythropoietin (EPO) suppression of programmed cell death (apoptosis). Erythroid progenitor cells enter a period of development in which they are dependent on EPO for survival (EPO-dependence). Surviving viable cells are indicated by intact nuclei, while cells undergoing programmed cell death caused by insufficient EPO are indicated by fragmented nuclei. Before entering the EPO-dependent period, the progenitors can survive without EPO (pre-EPO-dependence). Cells surviving transit through the EPO-dependent period (post-EPO-dependence) can complete maturation into erythrocytes (bottom rows of cells) without EPO. The number of cell generations encompassed by the EPO-dependent period is uncertain, but the stages of colony-forming unit erythrocyte and proerythroblast are in the EPO-dependent period. The EPO-dependent cells are heterogeneous in their requirements for EPO, with the most dependent cells on the right side of the diagram and least dependent cells on the left, as shown. A. During normal erythropoiesis, a minority of the potential EPO-dependent cells supply the daily needs for red blood cell turnover. B. Following hemorrhage or hemolysis, increased EPO allows erythroid cells with less EPO-dependence to survive, increasing erythrocyte production. C. In renal disease, decreased EPO results in apoptosis of many erythroid cells that would survive during normal erythropoiesis. As a result, only the least EPO-dependent cells survive, and erythrocyte production is decreased. |
A model of erythropoiesis has been proposed based on EPO preventing apoptosis in late-stage erythroid progenitors (Fig. 5.4) (393,424). In this model, EPO is required to prevent apoptosis of CFUs-E and subsequent proerythroblast stages and, possibly, the immediate predecessors of the CFU-E, the mature BFU-E. The model also requires that individual progenitors within the EPO-dependent population exhibit a range of sensitivities to EPO such that there is an extended dose range of EPO over which individual progenitors may survive and continue proliferation and differentiation. Thus, the level of EPO ultimately controls erythrocyte production by regulating the number of dependent progenitors that survive or die.
The two examples discussed here, CSF-1/macrophage progenitors and EPO/erythroid progenitors, indicate that different cellular mechanisms may be important in the regulation of particular types of progenitors. Also, different growth factors or combinations may activate different cellular mechanisms in a given cell type (e.g., KL plus EPO supports survival and proliferation of human CFUs-E, whereas EPO by itself supports survival without proliferation) (373). For most progenitor types and growth factor combinations, we do not yet know which cellular functions are regulated because pure cell systems are not readily available. It is also not known how the cellular processes of proliferation, apoptosis, and differentiation are related mechanistically or if apoptosis regulation by growth factors is achieved by a mechanism common to all cell types and all factors or if there are several different mechanisms.
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