Hematopoiesis

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Hematopoiesis

Chapter · December 2015 DOI: 10.1016/B978-0-12-801238-3.05054-6

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Yusuke Shiozawa Wake Forest School of Medicine

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Introduction 1 Methods to Study Hematopoiesis 2 Hematopoietic Stem Cell Markers 2 Hematopoiesis During Development 2 Hematopoietic Stem Cell Niche 3 Hematopoietic Stem Cell Plasticity 3 References 4

Introduction

Hematopoiesis is the process by which the entire repertoire of blood cell lineages is generated from hematopoietic stem cells (Figure 1). The term hematopoiesis is all-encompassing and includes erythropoiesis, leukopoiesis, and thrombopoiesis, all of which are involved in the production of erythrocytes (red blood cells). In addition, leukopoiesis includes lymphopoiesis, which relates to the generation of lymphocytes and granulocyte-macrophage lineages of myelopoiesis (myelos is Greek for marrow). Adult blood cells types can be separated by sedimentation or by centrifugation of blood samples. On examination of the sample, a red cell layer that is composed of hemoglobin-containing erythrocytes (erthrosis Greek for red) comprises 45% of the total blood volume. Erythrocytes facilitate the transport of oxygen and carbon dioxide between the lungs and tissues. On top of the red cell layer is a thin, white, buffy coat that contains white blood cells or leukocytes (leukos is Greek for white). Leukocytes, which occupy 1% of the blood volume, are composed of granulocytes (leukocytes with granules), monocytes, and lymphocytes. Different types of granulocytes are distinguished using acidic or basic dyes. Whereas eosinophils stain readily with eosin, an acidic dye, basophils stain readily with basic dyes, and neutrophils lack affinity for either acidic or basic dyes. Sixty percent of the leukocytes are neutrophils. Together, leukocytes function as components of the immune system. Examination of a blood clot reveals aggregates of thrombocytes or platelets. Platelets, like adult erythrocytes, do not have a nucleus. Their function is to adhere and aggregate at the site of an injury to form the primary hemostatic plug to prevent further blood loss.

Figure 1 Major differentiation pathways of hematopoiesis. Mature blood cells differentiate from pluripotent hematopoietic stem cells. The differentiation of mature blood cells from hematopoietic stem cells represents a continuous process that involves discrete changes triggered by the surrounding microenvironment and cumulative signals from soluble glycoprotein factors. The signals that stimulate mature blood cell production and signals that act to prevent the overproduction of blood cells are carefully balanced to supply the quantity of blood cells necessary for life. Not all of the regulatory processes are fully understood. Early progenitor cells, such as the colony-forming-unit granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM), are able to differentiate into multiple lineages but are unable to reconstitute the entire hematopoietic system when transplanted into an irradiated host.

☆ Change History: December 2014. Y Shiozawa made small edits in the text, added Keywords, Abstract, ‘Hematopoietic stem cell markers’ section, ‘Hematopoietic stem cell niche’ section, three references.

Reference Module in Biomedical Research http://dx.doi.org/10.1016/B978-0-12-801238-3.05054-6 1 2 Hematopoiesis

Methods to Study Hematopoiesis

Microscopic examination after staining with dyes is an important procedure for studying fully differentiated blood cells and their immediate precursors. Differentiated blood cells and precursors have a defined morphological appearance and can be distinguished readily from each other by direct observation. Precursor cells have limited proliferation potential and differentiate into mature blood cells with finite life spans. The progenitor cell compartment is composed of cells committed to develop into one or more blood lineages. Multilineage progenitor cells are presumed to be more primitive than progenitor cells committed to a single lineage. Cells in this compartment display greater plasticity and proliferative potential as compared to the precursor cell compartment. Progenitor cells are not morphologically distinguishable by microscopic examination. Surface markers and functional assays are used to distinguish progenitor cells from each other. To examine cell-surface markers, multiple monoclonal antibodies that recognize a common cell-surface antigen are joined together to form clusters of differentiation (CD). The clusters are numbered sequentially with respect to when they were discovered and defined. The cell-surface reactivity of monoclonal antibodies to each CD antigen is detected by flow cytometry. The presence and abundance of cell-surface antigens are a distinguishing characteristic of the different progenitor populations and other cells that comprise the hematopoietic system. Selected CD antigens are used as lineage-specific markers. Common cell-surface markers include CD34 for stem cells, Mac-1 for macrophages, Gr-1 for granulocytes, ter-119 for reticulocytes, À CD3 and CD8 for T-cells, and B220 for B-lymphocytes. Stem cells are lineage marker negative (Lin ) because they do not express surface markers used to identify mature blood cells. The colony-forming assay is an in vitro functional assay used to define the process of hematopoiesis. Cells to be tested are harvested and resuspended in methylcellulose or agarose with culture media and growth factors. Over the next few days, responding cells proliferate and differentiate to form discrete colonies. Larger colonies contain highly proliferative cells with multilineage potential. Smaller colonies with lower proliferative potential give rise to mature blood cells within a shorter period of time. Highly proliferative progenitors are also detected in blast-forming assays, a variant of the colony-forming assay. In the blast- forming assay, primary colonies are replated and analyzed for their ability to form secondary colonies. A colony-forming assay capable of definitively identifying hematopoietic stem cells has yet to be developed. The standard for the presence of hematopoietic stem cells is the ability of the cells to regenerate the entire hematopoietic system when transplanted into an irradiated host. Hematopoietic stem cells, which can be concentrated by selecting for cells that express the CD34 cell-surface antigen, are rare cells that can self-renew and differentiate into highly proliferative progenitor cells of all blood lineages. A second side population of CD34 negative hematopoietic stem cells has been identified that may represent an earlier stage in stem cell maturation Goodell et al (1997). In addition to hematopoietic stem cells, the bone marrow contains mesenchymal stem cells that are able to differentiate into bone, muscle, cartilage, and fat, but not blood cells.

Hematopoietic Stem Cell Markers

Since hematopoietic stem cells are an extremely rare population in the marrow (approximately 1/10000 bone marrow cells), the identification of hematopoietic stem cells in the marrow is still challenging. Historically, the functional assays, including in vitro colony-forming assay and in vivo serial dilution transplantation assay, are used to determine stem cell activity. It has been recently shown that the combination of several cell-surface markers can be used to identify hematopoietic stem cells. For human stem cells, CD34+CD38-CD90+CD45R-Lin- cells (Stem cell frequency 1/10 cells) are defined as hematopoietic stem cells Majeti et al, 2007. For mouse, CD150+CD244-CD48-Sca-1+c-Kit+Lin- cells (Stem cell frequency 1/2 cells) are used to identify hematopoietic stem cells Kiel et al, 2005.

Hematopoiesis During Development

The early stages of mammalian hematopoiesis, which begins in the blood islands of the yolk sac, may be divided into two developmental processes (Figure 2). In mice, primitive hematopoiesis begins at embryonic day 7 and is sustained for a relatively short period of time. A common precursor, the hemangioblast, gives rise to both endothelial cells and the primitive hematopoietic cells of the early circulatory system. Primitive hematopoiesis is characterized by the production of large nucleated erythrocytes that express embryonic globins. There are a several primitive monocytes/macrophages and megakaryocytes that are also produced. The switch from primitive hematopoiesis to definitive hematopoiesis coincides with the switch of the principal site of hematopoiesis from the yolk sac to the fetal liver. This switch occurs between embryonic days 10 and 11 in the mouse. Definitive hematopoiesis is distinguished from primitive hematopoiesis by the presence of enucleated adult-type erythrocytes that produce fetal globin in humans and adult globin in the mouse. Long-term repopulating stem cells (LTRSC) capable of reconstituting the hematopoietic system of a lethally irradiated adult host are found within the definitive hematopoietic system. The origin of the cells that seed the fetal liver is controversial. Initial findings suggested that the migration of stem cells from the yolk sac to the fetal liver was necessary to initiate hematopoiesis. Chicken/quail and amphibian grafting experiments suggested the Hematopoiesis 3

Figure 2 Development of the Hematopoietic System. Primitive hematopoiesis is followed by definitive hematopoiesis in mice and humans. Embryonic hematopoiesis begins in the yolk sac and changes to definitive hematopoiesis in the fetal liver. The bone marrow becomes the principal site of hematopoiesis late in gestation.

existence of two independent sources of cells for hematopoiesis. In addition to the yolk sack, para-aortic splanchnopleura (PAS, days 7–8) and the aorta-gonad-mesonephros (AGM, days 9–11) have been identified as intraembryonic sites of definitive hematopoiesis. Transplantation of cells cultured from the AGM demonstrates a high level of LTRSC activity. Cells from the mature yolk sac also show LTRSC activity when transplanted into the livers of newborn pups. Thus, both the yolk sac as well as the intraembyonic PAS/AGM may both serve as sources of cells with LTRSC activity that migrate to populate the fetal liver. After birth, and during early childhood, hematopoiesis occurs in the red marrow of the bone. With age, hematopoiesis becomes restricted to the skull, sternum, ribs, vertebrae, and pelvis. Yellow marrow, comprised of fat cells, replaces the red marrow and limits its potential for hematopoiesis. However, under stress, the yellow marrow can revert to producing blood cells.

Hematopoietic Stem Cell Niche

In the marrow, the fate of hematopoietic stem cells, such as homing, self-renewal, quiescence, and differentiation, depends on their specific microenvironment, which is referred to as the ‘hematopoietic stem cell niche’. The niche protects the functions of hematopoietic stem cells through direct cell-to-cell contact and/or by secretion of cytokines or growth factors to maintain a sufficient blood supply. Furthermore, the dysfunction of the niche can lead to hematopoietic malignancies. Two major niches are known to exist in the marrow, one is the osteoblastic niche and the other is the endothelial niche. The osteoblastic niche is believed to be associated with the regulation of deeply quiescent hematopoietic stem cells, while the endothelial niche is thought to facilitate the activities of more active hematopoietic stem cells. More recently, other components of the bone marrow, including CXCL12 highly expressing reticular cells, adipocytes, and mesenchymal stem cells, have been revealed to serve as the hematopoietic stem cell niche. However, little is known about the functions of these niche cells and the detailed mechanisms whereby the niche controls the activities of hematopoietic stem cells Shiozawa and Taichman (2012).

Hematopoietic Stem Cell Plasticity

The ability of hematopoietic stem cells to differentiate into cells of tissues other than blood has stimulated interest in cell plasticity and promoted the search for other types of stem cells within various organs. Hematopoietic stem cells have been reported to be able to transdifferentiate into cells that comprise the liver, brain, heart, pancreas, and muscle. Alternatively, muscle and neural stem cells are able to transdifferentiate into hematopoietic cells. The finding that stem cells may assume different fates depending on the microenvironmental signals presented to them has broad implications with regard to regenerative medicine. In general, in transdifferentiation studies stem cells are marked with the green fluorescent protein, beta-galactosidase, or by the presence of a Y chromosome. The marked stem cells are followed as they change into cells other than blood. The evidence that these marked stem cells express proteins specific for the transdifferentiated tissues supports the transdifferentiation hypothesis. However, most studies show low frequencies of transdifferentiation with, in some cases, cell fusion being a contributing mechanism. 4 Hematopoiesis

References

Goodell MA, Rosenzweig M, Kim H, Marks DF, DeMaria M, Paradis G, Grupp SA, Sieff CA, Mulligan RC, and Johnson RP (1997) Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nature Medicine 3(12): 1337–1345. Kiel MJ, Yilmaz OH, Iwashita T, Terhorst C, and Morrison SJ (2005) SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121(7): 1109–1121. Majeti R, Park CY, and Weissman IL (2007) Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood. Cell Stem Cell 1(6): 635–645. Shiozawa Y and Taichman RS (2012) Getting blood from bone: An emerging understanding of the role that osteoblasts play in regulating hematopoietic stem cells within their niche. Experimental Hematology 40(9): 685–694.