Hematopoietic stem cell concepts and their origin

The cellular compartment model

The short-lived nature of most blood cells was first deduced in the 1960s using thymidine labeling of reinfused blood. These studies demonstrated that the maintenance of normal numbers of blood cells in the adult required a process with the capacity to briskly generate large numbers of mature cells along multiple blood lineages. The early history of HSC research was largely shaped by cellular biology and animal transplantation experiments. It was advanced by experiments in the early 1960s demonstrating that injection of marrow cells could generate large hematopoietic colonies in the spleen of irradiated mice. Such colonies were the clonal progeny of single initiating cells, termed 'colony-forming units-spleen' (CFU-S), and contained hematopoietic populations of multiple lineages. CFU-S were further transplantable, demonstrating the self-renewing nature of CFU-S. HSCs are a minor component of marrow cells, able both to generate large numbers of progeny differentiated along multiple lines and to renew themselves.

Inner cell mass

Inner cell mass

Fig. 3.1 Sources and types of stem cells. Adapted with gratitude from the National Institute of Health Stem Cell Infomation web site

The field was further advanced by the use of in vitro cell culture techniques; in particular, solid-state cultures of marrow and spleen cells furthered understanding of the colony-forming capacity of individual hematopoietic cells. The original technique demonstrated clonal colonies of granulocytes and/or macrophages, termed 'in vitro colony-forming cells' (CFCs), which are now considered lineage-committed progenitor cells. These cells could be separated from whole marrow cells and from CFU-S, were more numerous than CFU-S and could be detected in splenic colonies as the progeny of CFU-S. These observations gave rise to the concept of the three-compartment model of hematopoiesis, the compartments being stem cells, progenitor cells, and dividing mature cells in increasing numbers; each compartment consists of the amplified progeny of cells in the preceding compartment.

Subsequent analyses have added further complexity to the compartment model of hematopoiesis. The term 'CFU-S' describes at least two groups of precursor cells. One group, arising from committed progenitors with little capacity for self-renewal, gives rise to colonies that peak in size by day 8, while a second, arising from a more primitive cell that is capable of self-renewal, yields colonies that peak in size at day 12. To further highlight the complexity of the hematopoietic hierarchy, a rarer population of hematopoietic cells provides longer-term repopulation of an irradiated host than CFU-S. These long-term repopulating cells have the capacity for sustained self-renewal and were considered the true adult stem cells. The presence of stromal cells in the cultures is important for the long-term culture of CFU-S and repopulating cells. Cells capable of long-term survival in culture on stroma were termed 'long-term culture-initiating cells' (LTC-ICs) and 'cobblestone area-forming cells'. These multipotential cell types were con sidered more primitive than lineage-committed progenitor cells but more mature than long-term repopulating cells.

Thus, a more complex version of the compartmental model has emerged. This provides a model with two populations of stem cells, the most immature group consisting of long-term repopulating cells and a more mature group of short-term repopulating cells. An intermediate group consisting of pre-progenitor cells (blast colony-forming cells) follows, leading to a larger population of lineage-committed progenitor cells. This large group of committed progenitors is stratified on the basis of the number of progeny they are able to generate. The immediate progeny of progenitor cells, cluster-forming cells, have less proliferative capacity. Subsequent progenitors (CFCs) have the capacity to give rise to colonies of clonal origin in semisolid media containing fully mature cells, permitting their analysis. A more mature set of precursor cells constitutes the bulk of bone marrow cells and has unique, identifiable features by light microscopy. Rapid division of precursor cells culminates in the production of mature cells. Although hematopoiesis proceeds according to this orderly scheme (Figure 3.2), special consideration must be given to the development of T and B lymphocytes. These cells are generated in the thymus and bone marrow, respectively, by a similar hierarchical process. Mature T and B lymphocytes enter peripheral lymphoid organs, where they encounter relevant antigens, leading to the production of new cells from reactivated mature cells. This process amplifies the de novo bone marrow formation of T and B lymphocytes. In addition, some members of this type of cell, memory T or B lymphocytes, are capable of sustained self-renewal. Their inability to produce multiple different types of daughter cells distinguishes them from stem cells.

Hematopoiesis

Stem cells

Progenitor cells

Precursor cells

Mature cells

Fig. 3.2 Schematic view of hematopoiesis. Modified from Figure 12.1 in Hematology: Basic Principles and Practice, 3rd Edition, Ronald Hoffman, ed., 2000, with permission from Elsevier

Stem cells

Progenitor cells

Precursor cells

Mature cells

Fig. 3.2 Schematic view of hematopoiesis. Modified from Figure 12.1 in Hematology: Basic Principles and Practice, 3rd Edition, Ronald Hoffman, ed., 2000, with permission from Elsevier

In summary, the compartment model has given rise to terms that are generally applied to cells of hematopoietic origin. Stem cells are those that are multipotent and self-renewing. Progenitor cells have limited ability to self-renew and are likely to be unipotential or of very limited multipotential. Precursor cells are restricted to a single lineage, such as neu-trophil precursors, and are the immediate precursors of the mature cells found in the blood. The mature cells are generally short-lived and preprogrammed to be highly responsive to cytokines, while the stem cells are long-lived, cytokine-resist-ant and generally quiescent.

Models of lineage commitment

Several theories have emerged to describe the manner by which HSCs undergo lineage commitment and differentiate. Some studies support a deterministic theory whereby the stem cell compartment encompasses a series of closely related cells maturing in a stepwise process. Other studies suggest that hematopoiesis is a random, stochastic process. The stochastic theory is based on in vitro observations that multilineage colonies develop variable combinations of lineages and that such lineage choices occur independently of external influences.

Similar controversy exists regarding the role of cytokines in cell lineage determination. An instructive model suggests that cytokine signaling forces the commitment of primitive cells along a particular lineage. Ectopic expression of the granu locyte macrophage colony-stimulating factor (GM-CSF) receptor in a common lymphoid progenitor (CLP) population was capable of converting the cells from a lymphoid to a myeloid lineage. The influence of the GM-CSF receptor was sufficiently dominant to change the entire differentiation program of cells, but only the CLP stage of development. A permissive model postulates that decisions about cell fate occur independently of extracellular signals. This model suggests that cytokines serve only to allow certain lineages to survive and proliferate. Evidence supporting this model is provided by the ectopic expression of growth receptors in progenitor cells. Expression of the erythropoietin receptor in a macrophage progenitor results in macrophage colony formation, whereas macrophage colony-stimulating receptor (M-CSF) in an erythroid progenitor results in erythroid rather than macrophage colony formation. Replacing the thrombopoietin receptor (c-mpl) with a chimeric receptor consisting of the extracellular domain of c-mpl with the cytoplasmic domain of the granulocyte colony-stimulating factor receptor (G-CSFR) results in normal platelet counts in homozygous knock-in mice. Therefore, the instructive and permissive models may both be correct, but at different stages of hematopoietic differentiation. Cells at earlier points in the differentiation cascade may be more plastic and susceptible to fate-altering stimuli, while more committed cells may be irreversibly determined, with only proliferation, cell death or the rate of differentiation susceptible to influence by external signals.

Stem cell plasticity and transdifferentiation

'Plasticity' refers to the concept that HSC development is not limited to hematopoietic cells but may also include cells of other tissue types. Studies have suggested that bone marrow-derived cells may develop into neural cells, skeletal muscle, cardiac muscle and hepatic cells in addition to epithelia of the gut, skin, lung and kidney. The possibility that HSCs have undergone 'transdifferentiation' serves as one explanation for these phenomena. However, it has been shown that hematopoietic cells may fuse with somatic cells and this may account for the finding ascribed to transdifferentiation and the issue of HSC plasticity remains controversial. It is possible that contaminating stem cells other than HSCs provide the other tissue types. Indeed, it is well established that mesenchymal stem cells reside in the bone marrow and have a broad range of capability, producing cartilage, bone, muscle or adipose cells upon proper stimulation.

Studies in which single cells were transferred have produced compelling evidence that cells of multiple tissue types do emerge. These events are very infrequent and are highly dependent on the method of cell selection before transplantation, and there are data indicating that a truly pluripotent stem cell may indeed exist in low abundance within the adult. Whether either fusion or transdifferentiation can ever be induced to occur at sufficient frequency to yield a therapeutic benefit is unclear, but is the subject of intense study.

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