All stem cell types have two cardinal functions: self-renewal and differentiation (Fig. 1-1). Stem cells exist to generate, maintain, and repair tissues. They function successfully if they can replace a wide variety of shorter-lived mature cells over prolonged periods. The process of self-renewal (see below) assures that a stem cell population can be sustained over time. Without self-renewal, the stem cell pool would become exhausted and tissue maintenance would not be possible. The process of differentiation leads to production of the effectors of tissue function: mature cells. Without proper differentiation, the integrity of tissue function would be compromised and organ failure or neoplasia would ensue.
Signature characteristics of the stem cell. Stem cells have two essential features: the capacity to differentiate into a variety of mature cell types and the capacity for self-renewal. Intrinsic factors associated with self-renewal include expression of Bmi-1, Gfi-1, PTEN, STAT5, Tel/Atv6, p21, p18, MCL-1, Mel-18, RAE28, and HoxB4. Extrinsic signals for self-renewal include Notch, Wnt, SHH, and Tie2/Ang-1. Based mainly on murine studies, hematopoietic stem cells express the following cell surface molecules: CD34, Thy-1 (CD90), c-Kit receptor (CD117), CD133, CD164, and c-Mpl (CD110, also known as the thrombopoietin receptor).
In the blood, mature cells have variable average life spans, ranging from 7 h for mature neutrophils to a few months for red blood cells to many years for memory lymphocytes. However, the stem cell pool is the central, durable source of all blood and immune cells, maintaining a capacity to produce a broad range of cells from a single cell source, yet keeping itself vigorous over decades of life. As an individual stem cell divides, it has the capacity to accomplish one of three division outcomes: two stem cells, two cells destined for differentiation, or one stem cell and one differentiating cell. The former two outcomes are the result of symmetric cell division, whereas the latter indicates a different outcome for the two daughter cells—an event termed asymmetriccell division. The relative balance for these types of outcomes may change during development and under particular kinds of demands on the stem cell pool.
DEVELOPMENTAL BIOLOGY OF HEMATOPOIETIC STEM CELLS
During development, blood cells are produced at different sites. Initially, the yolk sac provides oxygen-carrying red blood cells, and then the placenta and several sites of intraembryonic blood cell production become involved. These intraembryonic sites engage in sequential order, moving from the genital ridge at a site where the aorta, gonadal tissue, and mesonephros are emerging to the fetal liver and then, in the second trimester, to the bone marrow and spleen. As the location of stem cells changes, the cells they produce also change. The yolk sac provides red cells expressing embryonic hemoglobins while intraembryonic sites of hematopoiesis generate red cells, platelets, and the cells of innate immunity. The production of the cells of adaptive immunity occurs when the bone marrow is colonized and the thymus forms. Stem cell proliferation remains high, even in the bone marrow, until shortly after birth, when it appears to dramatically decline. The cells in the bone marrow are thought to arrive by the bloodborne transit of cells from the fetal liver after calcification of the long bones has begun. The presence of stem cells in the circulation is not unique to a time window in development; however, hematopoietic stem cells appear to circulate throughout life. The time that cells spend freely circulating appears to be brief (measured in minutes in the mouse), but the cells that do circulate are functional and can be used for transplantation. The number of stem cells that circulate can be increased in a number of ways to facilitate harvest and transfer to the same or a different host.
MOBILITY OF HEMATOPOIETIC STEM CELLS
Cells entering and exiting the bone marrow do so through a series of molecular interactions. Circulating stem cells (through CD162 and CD44) engage the lectins (carbohydrate binding proteins) P- and E-selectin on the endothelial surface to slow the movement of the cells to a rolling phenotype. Stem cell integrins are then activated and accomplish firm adhesion between the stem cell and vessel wall, with a particularly important role for stem cell VCAM-1 engaging endothelial VLA-4. The chemokine CXCL12 (SDF1) interacting with stem cell CXCR4 receptors and ionic calcium interacting with the calcium sensing receptor appear to be important in the process of stem cells getting from the circulation to where they engraft in the bone marrow. This is particularly true in the developmental move from fetal liver to bone marrow.
However, the role for CXCR4 in adults appears to be more related to retention of stem cells in the bone marrow rather than the process of getting them there. Interrupting that retention process through either specific molecular blockers of the CXCR4/CXCL12 interaction, cleavage of CXCL12, or downregulation of the CXCR4 receptor can all result in the release of stem cells into the circulation. This process is an increasingly important aspect of recovering stem cells for therapeutic use as it has permitted the harvesting process to be done by leukapheresis rather than bone marrow punctures in the operating room. Granulocyte colony-stimulating factor and plerixafor, a macrocyclic compound that can block CXCR4, are both used clinically to mobilize marrow hematopoietic stem cells for transplant. Refining our knowledge of how stem cells get into and out of the bone marrow may improve our ability to obtain stem cells and make them more efficient at finding their way to the specific sites for blood cell production, the so-called stem cell niche.
HEMATOPOIETIC STEM CELL MICROENVIRONMENT
The concept of a specialized microenvironment, or stem cell niche, was first proposed to explain why cells derived from the bone marrow of one animal could be used in transplantation and again be found in the bone marrow of the recipient. This niche is more than just a housing site for stem cells, however. It is an anatomic location where regulatory signals are provided that allow the stem cells to thrive, to expand if needed, and to provide varying amounts of descendant daughter cells. In addition, unregulated growth of stem cells may be problematic based on their undifferentiated state and self-renewal capacity. Thus, the niche must also regulate the number of stem cells produced. In this manner, the niche has the dual function of serving as a site of nurture but imposing limits for stem cells: in effect, acting as both a nutritive and constraining home.
The niche for blood stem cells changes with each of the sites of blood production during development, but for most of human life it is located in the bone marrow. Within the bone marrow, the perivascular space particularly in regions of trabecular bone serves as a niche. The mesenchymal and endothelial cells of the marrow microvessels produce kit ligand and CXCL12, both known to be important for hematopoietic stem cells. Other cell types, such as sympathetic neurons, nonmyelinating Schwann cells, macrophages, osteoclasts, and osteoblasts, have been shown to regulate stem cells, but it is unclear whether their effects are direct or indirect. Extracellular matrix proteins like osteopontin also affect stem cell function. The endosteal region is particularly important for transplanted cells, suggesting that there may be distinctive features of that region that are yet to be defined that are important mediators of stem cell engraftment. The functioning of the niche as a supportive context for stem cells is of obvious importance for maintaining hematopoiesis and in transplantation. An active area of study involves determining whether the niche is altered in disease and whether drugs can modify niche function to improve transplantation or normal stem cell function in hematologic disease.
EXCESS CAPACITY OF HEMATOPOIETIC STEM CELLS
In the absence of disease, one never runs out of hematopoietic stem cells. Indeed, serial transplantation studies in mice suggest that sufficient stem cells are present to reconstitute several animals in succession, with each animal having normal blood cell production. The fact that allogeneic stem cell transplant recipients also never run out of blood cells in their life span, which can extend for decades, argues that even the limiting numbers of stem cells provided to them are sufficient. How stem cells respond to different conditions to increase or decrease their mature cell production remains poorly understood. Clearly, negative feedback mechanisms affect the level of production of most of the cells, leading to the normal tightly regulated blood cell counts. However, many of the regulatory mechanisms that govern production of more mature progenitor cells do not apply or apply differently to stem cells. Similarly, most of the molecules shown to be able to change the size of the stem cell pool have little effect on more mature blood cells. For example, the growth factor erythropoietin, which stimulates red blood cell production from more mature precursor cells, has no effect on stem cells. Similarly, granulocyte colony-stimulating factor drives the rapid proliferation of granulocyte precursors but has little or no effect on the cell cycling of stem cells. Rather, it changes the location of stem cells by indirect means, altering molecules such as CXCL12 that tether stem cells to their niche. Molecules shown to be important for altering the proliferation, self-renewal, or survival of stem cells, such as cyclin-dependent kinase inhibitors, transcription factors like Bmi-1, or microRNA-processing enzymes like Dicer, have little or different effects on progenitor cells. Hematopoietic stem cells have governing mechanisms that are distinct from the cells they generate.
HEMATOPOIETIC STEM CELL DIFFERENTIATION
Hematopoietic stem cells sit at the base of a branching hierarchy of cells culminating in the many mature cell types that compose the blood and immune system (Fig. 1-2). The maturation steps leading to terminally differentiated and functional blood cells take place both as a consequence of intrinsic changes in gene expression and niche-directed and cytokine-directed changes in the cells. Our knowledge of the details remains incomplete. As stem cells mature to progenitors, precursors, and, finally, mature effector cells, they undergo a series of functional changes. These include the obvious acquisition of functions defining mature blood cells, such as phagocytic capacity or hemoglobin synthesis. They also include the progressive loss of plasticity (i.e., the ability to become other cell types). For example, the myeloid progenitor can make all cells in the myeloid series but none in the lymphoid series. As common myeloid progenitors mature, they become precursors for either monocytes and granulocytes or erythrocytes and megakaryocytes, but not both. Some amount of reversibility of this process may exist early in the differentiation cascade, but that is lost beyond a distinct stage in normal physiologic conditions. With genetic interventions, however, blood cells, like other somatic cells, can be reprogrammed to become a variety of cell types.
Hierarchy of hematopoietic differentiation. Stem cells are multipotent cells that are the source of all descendant cells and have the capacity to provide either long-term (measured in years) or short-term (measured in months) cell production. Progenitor cells have a more limited spectrum of cells they can produce and are generally a short-lived, highly proliferative population also known as transient amplifying cells. Precursor cells are cells committed to a single blood cell lineage but with a continued ability to proliferate; they do not have all the features of a fully mature cell. Mature cells are the terminally differentiated product of the differentiation process and are the effector cells of specific activities of the blood and immune system. Progress through the pathways is mediated by alterations in gene expression. The regulation of the differentiation by soluble factors and cell-cell communications within the bone marrow niche are still being defined. The transcription factors that characterize particular cell transitions are illustrated on the arrows; the soluble factors that contribute to the differentiation process are in blue. This picture is a simplification of the process. Active research is revealing multiple discrete cell types in the maturation of B cells and T cells and has identified cells that are biased toward one lineage or another (rather than uncommitted) in their differentiation. EPO, erythropoietin; RBC, red blood cell; SCF, stem cell factor; TPO, thrombopoietin.
As cells differentiate, they may also lose proliferative capacity (Fig. 1-3). Mature granulocytes are incapable of proliferation and only increase in number by increased production from precursors. The exceptions to the rule are some resident macrophages, which appear capable of proliferation, and lymphoid cells. Lymphoid cells retain the capacity to proliferate but have linked their proliferation to the recognition of particular proteins or peptides by specific antigen receptors on their surface. Like many tissues with short-lived mature cells such as the skin and intestine, blood cell proliferation is largely accomplished by a more immature progenitor population. In general, cells within the highly proliferative progenitor cell compartment are also relatively short-lived, making their way through the differentiation process in a defined molecular program involving the sequential activation of particular sets of genes. For any particular cell type, the differentiation program is difficult to speed up. The time it takes for hematopoietic progenitors to become mature cells is ~10–14 days in humans, evident clinically by the interval between cytotoxic chemotherapy and blood count recovery in patients.
Relative function of cells in the hematopoietic hierarchy. The boxes represent distinct functional features of cells in the myeloid (upper box) versus lymphoid (lower box) lineages.
Although hematopoietic stem cells are generally thought to have the capacity to form all cells of the blood, it is becoming clear that individual stem cells may not be equal in their differentiation potential. That is, some stem cells are “biased” to become mature cells of a particular type. In addition, the general concept of cells having a binary choice of lymphoid or myeloid differentiation is not entirely accurate. A cell population with limited myeloid (monocyte and granulocyte) and lymphoid potential is now added to the commitment steps stem cells may undergo.
The hematopoietic stem cell must balance its three potential fates: apoptosis, self-renewal, and differentiation. The proliferation of cells is generally not associated with the ability to undergo a self-renewing division except among memory T and B cells and among stem cells. Self-renewal capacity gives way to differentiation as the only option after cell division when cells leave the stem cell compartment, until they have the opportunity to become memory lymphocytes. In addition to this self-renewing capacity, stem cells have an additional feature characterizing their proliferation machinery. Stem cells in many mature adult tissues may be heterogeneous with some being deeply quiescent, serving as a deep reserve, whereas others are more proliferative and replenish the short-lived progenitor population. In the hematopoietic system, stem cells are generally cytokine-resistant, remaining dormant even when cytokines drive bone marrow progenitors to proliferation rates measured in hours. Stem cells, in contrast, are thought to divide at far longer intervals, measured in months to years, for the most quiescent cells. This quiescence is difficult to overcome in vitro, limiting the ability to effectively expand human hematopoietic stem cells. The process may be controlled by particularly high levels of cyclin-dependent kinase inhibitors like p57 or CDKN1c that restrict entry of stem cells into the cell cycle, blocking the G1-S transition. Exogenous signals from the niche also appear to enforce quiescence, including the activation of the tyrosine kinase receptor Tie2 on stem cells by angiopoietin 1 on niche cells.
The regulation of stem cell proliferation also appears to change with age. In mice, the cyclin-dependent kinase inhibitor p16INK4a accumulates in stem cells in older animals and is associated with a change in five different stem cell functions, including cell cycling. Lowering expression of p16INK4a in older animals improves stem cell cycling and capacity to reconstitute hematopoiesis in adoptive hosts, making them similar to younger animals. Mature cell numbers are unaffected. Therefore, molecular events governing the specific functions of stem cells are being gradually made clear and offer the potential of new approaches to changing stem cell function for therapy. One critical stem cell function that remains poorly defined is the molecular regulation of self-renewal.
For medicine, self-renewal is perhaps the most important function of stem cells because it is critical in regulating the number of stem cells. Stem cell number is a key limiting parameter for both autologous and allogeneic stem cell transplantation. Were we to have the ability to use fewer stem cells or expand limited numbers of stem cells ex vivo, it might be possible to reduce the morbidity and expense of stem cell harvests and enable use of other stem cell sources. Specifically, umbilical cord blood is a rich source of stem cells. However, the volume of cord blood units is extremely small, and therefore, the total number of hematopoietic stem cells that can be obtained in any single cord blood unit is generally only sufficient to transplant an individual of <40 kg. This limitation restricts what would otherwise be an extremely promising source of stem cells. Two features of cord blood stem cells are particularly important. (1) They are derived from a diversity of individuals that far exceeds the adult donor pool and therefore can overcome the majority of immunologic cross-matching obstacles. (2) Cord blood stem cells have a large number of T cells associated with them, but (paradoxically) they appear to be associated with a lower incidence of graft-versus-host disease when compared with similarly mismatched stem cells from other sources. If stem cell expansion by self-renewal could be achieved, the number of cells available might be sufficient for use in larger adults. An alternative approach to this problem is to improve the efficiency of engraftment of donor stem cells. Graft engineering is exploring methods of adding cell components that may enhance engraftment. Furthermore, at least some data suggest that depletion of host NK (natural killer) cells may lower the number of stem cells necessary to reconstitute hematopoiesis.
Some limited understanding of self-renewal exists and, intriguingly, implicates gene products that are associated with the chromatin state, a high-order organization of chromosomal DNA that influences transcription. These include members of the polycomb family, a group of zinc finger–containing transcriptional regulators that interact with the chromatin structure, contributing to the accessibility of groups of genes for transcription. One member, Bmi-1, is important in enabling hematopoietic stem cell self-renewal through modification of cell cycle regulators such as the cyclin-dependent kinase inhibitors. In the absence of Bmi-1 or of the transcriptional regulator, Gfi-1, hematopoietic stem cells decline in number and function. In contrast, dysregulation of Bmi-1 has been associated with leukemia; it may promote leukemic stem cell self-renewal when it is overexpressed. Other transcription regulators have also been associated with self-renewal, particularly homeobox, or “hox,” genes. These transcription factors are named for their ability to govern large numbers of genes, including those determining body patterning in invertebrates. HoxB4 is capable of inducing extensive self-renewal of stem cells through its DNA-binding motif. Other members of the hox family of genes have been noted to affect normal stem cells, but they are also associated with leukemia. External signals that may influence the relative self-renewal versus differentiation outcomes of stem cell cycling include specific Wnt ligands. Intracellular signal transducing intermediates are also implicated in regulating self-renewal. They include PTEN, an inhibitor of the AKT pathway, and STAT5, both of which are downstream of activated growth factor receptors and necessary for normal stem cell functions including self-renewal, at least in mouse models. The connections between these molecules remain to be defined, and their role in physiologic regulation of stem cell self-renewal is still poorly understood.