Desk Reference for hematology - part 5 pdf

382 HEMATOPOIESIS adult recipients are seen both in the yolk sac and in the liver. For many years, investigators have believed that yolk sac blood islands contained HSCs capable of primitive hemato- poiesis and of migration to the developing liver to initiate definitive hematopoiesis. Chal- lenging the idea of a singular origin of hematopoiesis in the yolk sac, it has been proposed that there is a more potent intraembryonic HSC site in the AGM region. HSCs arise for the first time in the AGM region and migrate to the yolk sac and fetal liver, the main source of hematopoietic cells in fetal life. Around the time of birth, HSCs migrate from the liver to the bone marrow, to be responsible for adult hematopoiesis (see Figure 52). However, this sequential migration has not been directly demonstrated in vivo in mam- malian species. Instead, evidence for this view is based on the quantitative temporal measurement of hematopoietic activity in the yolk sac and liver (using adult recipients) following establishment in the AGM region. Migration is inferred because at this stage precursors become detectable in significant numbers in the blood stream. In this model, the presence of an HSC pool in the yolk sac, with ability to engraft in neonatal hosts before the onset of the AGM region, is not taken into consideration. Figure 50 and Figure 51 summarize the current view of embryonic hematopoiesis. It is possible that HSCs arise from at least two independent sites and that endothelial cells of both the dorsal aorta and of the yolk sac can be the direct precursors of hemato- poietic cells. Indeed, close juxtaposition and temporally parallel onset of the endothelial and hematopoietic lineages are seen in the yolk sac and AGM regions. The idea of a common precursor, the “hemangioblast,” for the endothelial and the hematopoietic lineage first emerged some years ago, and it has been revived in the last decade. Evidence that emphasizes the close relationship between those two lineages includes the observation that genes affecting primitive hematopoiesis encode receptor tyrosine kinases, such as FIGURE 50 Temporal appearance of different types of assayable hematopoietic cells: colony forming units-culture (CFU-C), multipotent progenitors, colony forming units-spleen (CFU-S), and hematopoietic stem cells (HSC) in the yolk sac (top), para-aortic splanchnopleure/aorta-gonad-mesonephros (PAS/AGM) region (middle), and fetal liver (bottom). Note that the more-differentiated progenitors can be detected at earlier stages than the hematopoietic stem cells. The lineage relationships between these cells remains to be established. Also, note that multipotential progenitors and HSCs are detected 1 day earlier in the PAS/AGM than in the yolk sac. (From Dzerziak, E. and Oostendorp, R., Hematopoietic stem cell development in mammals, in Hematopoiesis: A Developmental Approach, Zon, L.I., Ed., Oxford University Press, Oxford, 2001, p. 211. With permission.) 3393_C006.fm Page 382 Monday, November 19, 2007 1:59 PM HEMATOPOIESIS 383 Flk-1, that are involved directly in endothelial-cell proliferation and angiogenesis. Fur- thermore, these cells express markers that are common to both endothelial and hemato- poietic stem cells, e.g., CD31, CD34, c-kit, and VE-cadherin endothelial/hematopoietic cluster marker. Recent studies have used sorted cell populations with endothelial markers from murine yolk sac and AGM. Those cells formed blood cells in culture in the presence of stromal cells. This has been confirmed in humans, and vascular endothelial cells isolated from fetal liver and fetal bone marrow have been shown to also be capable of multilineage hematopoiesis. Considerable attention has been focused on the mechanisms that regulate the induction, differentiation, and maintenance of the hematopoietic system during development (see Figure 52), but there are similarities and important differences with adult hematopoiesis. Hematopoietic cytokines and their associated cell signal transduction pathways have been well studied, but less is known about hematopoietic functions of other classes of signaling molecules. The Notch, Wnt, and Hedgehog pathways play important roles in a variety of FIGURE 51 Migration of progenitors that leads to definitive hematopoiesis. FIGURE 52 Schematic view of transcription factors and hematopoiesis. Yolk sac AGM region Liver Bone marro w BFU-E CFU-E RBC Erythroblast Pluripotent Stem Cell CFC-GEMM CFC-mix CFC-Mk Megakaryocyte Platelets Mast cell Basophil Eosinophil Neutrophil Macrophage CFC-GM B cell T cell Stem-cell Committed progenitor Mature cells AML1 LMO2 TEL GATA-2 GATA-1 GATA-2 SCL NF-E2 GATA-1 FOG-1 SCL GATA-1 FOG-1 Pu-1 Pu.1 E2A EBF CEBP- 3393_C006.fm Page 383 Monday, November 19, 2007 1:59 PM 384 HEMATOPOIESIS developmental processes and have also been shown to control self-renewal or differenti- ation of HSCs and more-committed progenitors. Another group of molecules important for the development of hematopoiesis are the on-bone morphogenetic protein (BMP) members. These are part of the transforming growth factor-β (TGF-β) superfamily of extracellular signaling molecules. Although BMPs were originally discovered on the basis of their ability to induce ectopic bone formation, it rapidly became evident that these peptides have much broader functions during devel- opment. Genetic studies have demonstrated that BMP-4, in particular, plays critical roles in formation and patterning of mesoderm. Hematopoietic specification of ventral meso- derm is sensitive to the concentration of BMP-4, with embryonic globin expression occur- ring within a narrow range. BMP-4 synergizes with vascular endothelial growth factor (VEGF) in the generation of HSCs from embryoid bodies, pointing to a possible role for it at the level of hemangioblast specification. b-Fibroblast growth factor (b-FGF) has been implicated in commitment to hemangioblast development. Thrombopoietin, recently shown to play an important role in maintenance and prolif- eration of the HSC and in yolk sac hematopoiesis, synergizes with, and can actually replace VEGF, indicative of an important role in hemangioblast development. Other molecules involved in fetal hematopoietic commitment decisions include the Wnt pathway. By analyzing embryonic stem (ES) cell lines carrying various mutations and using colony assays to determine the growth factor requirements of ES cells as they differentiate from a pluripotent to differentiated state, it has been possible to dissect some aspects of the genetic regulation of hematopoietic commitment. The basic helix-loop-helix transcription factor stem cell leukemia gene SCL is expressed in all embryonic hematopoietic sites. It is absolutely required for embryonic hematopoiesis; in addition, it is expressed in the embryonic vasculature and is required for proper vascular development, being critical for the development of the hemangioblast. Flk-1 is a receptor tyrosine kinase that is activated by VEGF. Loss of Flk-1 blocks endothelial development and day-8.5 yolk sac hematopoietic development. While SCL is important for the specification of hemangioblasts, Flk-1 enables migration of the heman- gioblasts to sites that would allow their survival and proliferation. The precise relationship and cross talks between SCL and Flk-1 remain to be elucidated. Another genetic regulator, Runx1, also known as Cbfa2 or AML1, is strongly expressed at all hematopoietic sites of the day-8.5 embryo, but its expression is maintained strongly only in intraembryonic sites. Mutation of Runx1 blocks definitive but not primitive hematopoiesis, leading to embryonic death by day 12.5. In serum-free, chemically defined medium, activin A and BMP-4 are able to induce dorsal or ventral mesoderm formation in ES cells, respectively. Recently, BMP-4 has been shown to enhance the self-renewal of the earliest hematopoietic progenitors. A heman- gioblast colony assay has not yet been documented for differentiating human ES cells. Colony forming unit-granulocytes/macrophages (CFU-GM) is detected as early as 5 weeks of gestation. Erythroid progenitors are also present in the yolk sac at this stage. Initially, only nucleated erythroid cells are morphologically identifiable within the yolk sac, but lymphoid cells and megakaryocytes appear later at this stage. Yolk sac erythro- poiesis has ceased by week 10 of gestation. Fetal Hematopoiesis Hematopoiesis changes throughout an individual’s life — from embryonic through fetal life and childhood before finally reaching adult maturation — with regard to both its site and cellular composition (Figure 53). In erythropoiesis, there is also a specific change in globin-chain synthesis from embryonic life onward (Figure 54). 3393_C006.fm Page 384 Monday, November 19, 2007 1:59 PM HEMATOPOIESIS 385 Fetal Liver and Spleen Hematopoiesis At about week 6 of gestation, erythropoiesis begins in the fetal liver extravascularly, with mature cells entering the fetal circulation. Erythropoiesis is also detectable in the spleen by week 12, this remaining the primary site of erythropoiesis until week 24. Circulating platelets are detectable at 8 to 9 weeks. FIGURE 53 Stages of hematopoiesis in the embryo and fetus, indicating the comparative participation of the chief centers of hematopoiesis and the approximate times at which the different types of cells make their appearance. (From Rothstein, G., in Wintrobe’s Clinical Hematology, Lee, G.R. et al., Eds., Williams & Wilkins, Baltimore, 1993, p. 80. With permission.) FIGURE 54 Proportions of the various human hemoglobin polypeptide chains through early life. The hemoglobin electro- phoretic pattern (Gower 2, Gower 1, HbF, HbA, HbA2) typical for each period is also shown. (From Pearson, H.A., J. Pediatr., 69, 473, 1966. With permission.) Primitive Definitive Erythroblasts Megakaryocytes Granulocytes Lymphocytes Monocytes Yolk sac Liver Hepatic period Mesoblastic period Myeloid period Bone Marrow Spleen Lymph Nodes 1 23 4 5 6 7 8 910 Lunar Months 3393_C006.fm Page 385 Monday, November 19, 2007 1:59 PM 386 HEMATOPOIESIS Fetal Bone Marrow Hematopoiesis This commences at around week 16 to 18, as fetal liver hematopoiesis is challenged by hepatocyte proliferation, and assumes the primary role from week 24 onward. Mature neutrophils first appear in the peripheral blood at this time. Control of Fetal Erythropoiesis Fetal primitive multipotent progenitors (murine CFU-S and CFU-GEMM) proliferate more rapidly than newborn or adult. Fetal liver CFU-Ss have greater marrow repopulating ability than adult CFU-Ss. Committed fetal progenitors show a similar proliferative pat- tern, both in the myeloid and erythroid lineages, although CFU-GM show reduced sen- sitivity to GM-CSF and are reduced in number compared with adults. Erythroid progenitors predominate in fetal marrow in contrast to adults. Fetal BFU-E produces larger colonies and shows increased erythropoietin (EPO) sensitivity and earlier maximal colony growth in culture than newborn or adult human BFU-E. The proportional synthesis of γ- globin within the BFU-E colonies is also twofold greater in fetus than newborn. Maximal BFU-E numbers are seen early in the second trimester, and at mid-gestation the BFU-E number is three times that of newborns and ten times that of adults. EPO production is low up to 20 weeks, then remains constant throughout gestation, with values well below adult serum concentrations (mean 1.6 ± 2.5 mIU/ml). Hematopoietic ontogenic control mechanisms have been little studied in humans, 232 but early murine embryonic data have demonstrated expression of erythroid control genes (EPO-receptor, GATA-1, and α-globin) in day-6.5-postconception embryos (mesodermal tissue) prior to the morphological identification of erythroid cells. EPO and EPO-r are also expressed in embryonic stem cells in vitro, derived from the blastocyst. EPO expression is not detected in vivo until the yolk sac stage. The homeobox genes HOX-2.2 and HOX- 2.3 are also expressed at day 6.5 postconception and are part of a family of genes encoding DNA-binding proteins with a major role in embryogenesis. HOX-2.2 and HOX-2.3 are particularly associated with erythroid development and differentiation. Globin Switching (See Figure 55 and Table 71.) An orderly sequence of production of different globin proteins occurs during fetal development in response to changes in requirements for red blood cell oxygen-carrying capacity. The earliest globin chains detectable in the embryo yolk sac are zeta (ζ), an α-type chain with locus on chromosome 16, and epsilon (ε), a β-type chain with locus on chromosome 11. The earliest fetal hemoglobin is thus HbGower1 (ζ2ε2), and it is the major form at 5 to 6 weeks. HbGower2 (α2ε2) is present from 4 to 13 weeks of gestation. HbPortland (ζ2γ2) also persists from 4 to 13 weeks but is found in infants with homozygous α-thalassemia. Synthesis of ζ- and ε-chains ceases at the time the liver takes over from the yolk sac as the site of erythropoiesis. At that time, α- and γ-chains become dominant. HbF (α2γ2) is the major fetal form and accounts for 90 to 95% of the total hemoglobin until 34 to 36 weeks gestation. Adult hemoglobin (HbA; α2/β2) is TABLE 71 Temporal Expression of Globin Chains ζ2ε2 Hb Gower 1 embryonic α2ε2 Hb Gower 2 embryonic ζ2γ2 Hb Portland embryonic α2γ2 HbF fetal α2δ2 HbA2 adult α2β2 HbA adult 3393_C006.fm Page 386 Monday, November 19, 2007 1:59 PM HEMATOPOIESIS 387 detectable from week 11 of gestation, after which time the proportion of HbA increases as HbF declines. The amount of HbF in neonates varies from 50 to 90%, but thereafter declines at a rate of 3% per week and is generally less than 2 to 3% by the age of 6 months. Gene switching at the β-globin locus is accomplished at the transcriptional level and is regulated at the level of chromatin structural changes, exposing DNAse I-hypersensitive sites within the long-terminal-repeat regions (LTRs) located 5′ to the ε-globin gene-pro- moter regions. Silencing of γ-globin transcription is accomplished by transacting factors and not, as originally thought, by direct competition for the β-globin gene. These silencing factors progressively silence the influence of the locus control region (LCR) upon e, Gg, and Ag gene transcription as human embryonic erythropoiesis develops from the yolk sac through fetal liver to bone marrow. Increased proportions of HbF occur in infants small for gestational age who have experienced severe fetal anoxia, who have trisomy 13, and in infants dying from sudden infant death syndrome. Persistence of embryonic hemoglobins has been reported in some infants with developmental abnormalities. Decreased levels are found in trisomy 21. Fetal Blood Groups These are the same as those of adults, apart from the I blood group, where “i” antigen predominates on fetal red cells to be replaced by “I” antigen on adult red cells. There are no blood group antibodies in the absence of immunoglobulin formation. Fetal Blood Cell Values Hemoglobin concentration rises from a mean of 11.7 g/dl at 18 weeks to 13.6 g/dl at >30 weeks, with a steady rise in hematocrit (0.37 l/l to 0.43 l/l) and concomitant fall in mean cell volume (131 fl to 114 fl). Circulating normoblasts constitute 45% of nucleated cells at 18 weeks, falling to 17% at >30 weeks. Lymphocyte percentage falls from 88% to 68%, with neutrophils only rising significantly after 30 weeks (8% at 26 to 29 weeks to 23% at >30 weeks). Eosinophil, monocyte, and basophil percentages remain reasonably constant throughout. Platelet concentration also remains constant. Adult Hematopoiesis Adult hematopoiesis is the process by which HSCs divide and differentiate to maintain a supply of mature blood cells so as not to exhaust the HSC compartment within the lifetime of the individual. HSCs are pluripotent cells able to give rise to at least ten different functional cell types (neutrophil, monocytes/histiocytes (macrophages), basophils, eosi- nophils, erythrocytes (red blood cells), platelets, mast cells, dendritic reticulum cells, and B and T-lymphocytes). Two types of stem cells have been defined. The long-term repopulating cells (LTRC) are capable of producing all blood cell types for the entire life span of the individual and of generating progeny that display similar potentiality on secondary transplant. The short-term repopulating cells (STRCs) reconstitute myeloid and lymphoid compartments for a short period of time. The process by which stem cells give rise to terminally differentiated cells occurs through a variety of committed progenitor cells, often overlapping in their hematopoietic capacity. During commitment, cells can undergo extensive proliferation and sequential differentiation, accompanied by a decrease in self-renewal capability to produce mature cells. The primary function of this transit population is to increase the number of mature cells produced by each stem cell division. The clonal succession model proposed by Kay in 1965 suggests that one or a small number of HSC clones give rise to mature blood cells as needed, and the remaining HSCs remain quiescent and do not contribute to hematopoiesis until the proliferative capacity 3393_C006.fm Page 387 Monday, November 19, 2007 1:59 PM 388 HEMATOPOIESIS of the already engaged stem cells has been exhausted. This hypothesis has been supported by data from retrovirally marked donor-transplant studies in lethally irradiated mice, which have indicated that only one or very few clones contribute to hematopoiesis at any given time. Furthermore, the data from clonal studies of human hematopoiesis in the elderly would support this evaluation. On the other hand, studies that have analyzed steady-state hematopoiesis in an alternative model using bromo-2′-deoxyuridine (BrdU) incorporation kinetics suggest that up to 8% of HSCs enter the cell cycle per day, and although at any given time over 75% of HSCs are in G0, all HSCs are recruited into the cell cycle on average every 57 days. Hematopoiesis is regulated by a complex interaction of secreted cytokines, stromal cell interactions, which in turn regulate transcription factors and the cell cycle machinery. The HSCs divide to contribute to hematopoiesis either in a stochastic process or are directed by microenvironmental cues to differentiate down particular lineages. Hematopoietic stem cells are defined by four distinguishing features: Self-renewal, defined as the production of exact duplicates with the maintenance of all attributes of the original Pluripotentiality, enabling differentiation into all mature hematopoietic lineages Quiescence, such that at a given time point the majority of stem cells will be in G0 Expression of p glycoprotein-like pumps that extrude dyes such as rhodamine-123 In normal steady-state hematopoiesis, the size of the stem cell population is maintained at a constant level by the balance of stem cell production by cell division and stem cell loss via differentiation. This is a tightly regulated process of controlled self-renewal together with the provision of differentiated cells to meet demand, but with considerable capacity to expand the stem cell population when necessary, for example following myelo- suppressive chemotherapy or infections. The concept of an undifferentiated stem cell giving rise to the spectrum of blood cells via an intermediate state of progenitor cells was postulated by Pappenheim as early as 1917. In the 1950s, Miklem and coworkers demonstrated the existence of the hematopoietic stem cell in the bone marrow by the rescue of irradiated recipients by bone marrow injection in mice. Till and McCulloch later characterized such stem cells following the discovery that murine bone marrow contained single cells that could give rise to myelo- erythroid colonies in the spleen of a transplant recipient. In these experiments, random chromosome markers were produced by irradiating donor bone marrow. Following trans- plantation into conditioned recipients, colonies of daughter cells, each derived from a single clonogenic precursor, were found in the spleen. These colonies were shown to contain differentiated myeloerythroid cells together with more primitive cells that could themselves both self-renew and differentiate. They had the ability to produce more spleen colonies and to reconstitute hematopoiesis in lethally irradiated secondary recipients. This observation formed the basis of the widely used “colony forming unit-spleen” (CFU-S) quantitative assay of stem cell activity. The teams of Bradley and Metcalf and of Pluznik and Sachs independently performed experiments seeding adult murine spleen cells onto a soft agar medium in the presence of a feeder layer. These produced clones of cells constituting two types of hematopoietic colonies that could be analyzed morphologically as the colony forming units. Further research into hematopoietic stem cell biology required the development of techniques for cell purification and the refinement of hematopoietic stem cell assays capable of investigating properties of multilineage differentiation as well as self-renewal. It became apparent that day-8 CFU-Ss were not in fact formed by HSC but by more-mature 3393_C006.fm Page 388 Monday, November 19, 2007 1:59 PM HEMATOPOIESIS 389 committed progenitors, and more-primitive day-12 CFU-Ss were described, with the abil- ity to rescue irradiated recipients. In vitro clonogenic assays were also refined. It was demonstrated that the colony forming cells (CFCs) could be subdivided into different classes according to the differentiated progeny they produced in response to various known growth factors. These included the multipotent CFC-Mix, which could produce all of the different types of myeloid cells but not T- and B-lymphocytes. In turn, these underwent differentiation to produce bipotent and unipotent progenitors, such as the granulocyte and macrophage CFCs, eosinophil CFCs, erythroid progenitors called burst- forming units-erythroid (BFUs-E), and the more mature colony forming units-erythroid (CFUs-E) that were able to respond to erythropoietin. This hierarchy of hematopoietic progenitors is shown in Figure 55. Though stem cell assays provided a means of identifying the hematopoietic progenitor subpopulations, their purification and detailed characterization were greatly facilitated by the development of the fluorescence-activated cell sorter (FACS). This provided a rapid means of subdividing cellular populations according to their innate size and granularity profile, together with their expression of specific cell-surface markers. (See Ineffective erythropoiesis; Normoblast; Reticulocyte.) Measurement of Erythropoiesis Normal red blood cell production is extremely effective, and most red blood cells live, or have the potential to live, a normal life span. Under certain conditions, however, a fraction of red blood cell production is ineffective, with destruction of nonviable red blood cells either within the marrow or shortly after the cells reach the blood. Effective erythropoiesis is most simply estimated by determining the reticulocyte count. This count is usually expressed as the percentage of red blood cells that are reticulocytes, but it can also be expressed as the total number of circulating reticulocytes per liter of blood (reticulocyte % × RBC per l). An elevated reticulocyte count may give an erroneous impression of the actual rate of red cell production because of premature release of reticulocytes into the circulation. To correct for this premature release, some workers calculate a reticulocyte index to compensate (see Reticulocyte count). FIGURE 55 Proposed hierarchy of hematopoietic colony forming potential. Pluripotent stem cell Blast CFC CFC-Mix GM-CFC G-CFC M-CFC Eo-CFC Mk-CFC BFU-E Lymphoid stem cell CFU-E Lymphocytes Platelets Eosinophils Erythrocytes Monocytes Neutrophils 3393_C006.fm Page 389 Monday, November 19, 2007 1:59 PM 390 HEMATOPOIETIC REGULATION Ineffective Red Blood Cell Production Ineffective erythropoiesis is suspected when the reticulocyte count is low or is normal or only slightly increased in the presence of erythroid hyperplasia in the bone marrow. In certain disorders, such as Addisonian pernicious anemia, thalassemia, and sideroblastic anemia, ineffective erythropoiesis is a major component of total erythropoiesis. This can be quantified by ferrokinetics. Using ferrokinetic methods, ineffective erythropoiesis is calculated as the difference between total plasma iron turnover and erythrocyte iron turnover plus storage iron turnover. Total Erythropoiesis This is the sum of effective and ineffective red cell production and can be estimated from marrow examination by first determining the relative content of fat and hematopoietic tissue. The myeloid/erythroid ratio is then determined. These, taken in conjunction with the red cell count and the reticulocyte count, will usually provide quantitative information on the rate and effectiveness of red blood cell production. Erythropoiesis can be demonstrated by imaging marrow, liver, and spleen with 99m Tc sulfur colloid or 111 indium, even though these isotopes primarily label the monocyte- macrophage system. Their uptake is similar to 59 Fe, and they can be used to demonstrate erythroid tissue, but accurate quantitation of total erythropoiesis is made by measuring the rate of red blood cell production (see Ferrokinetics). The identification and cloning of an array of growth factors whose activities in vivo and in vitro have marked effects on the growth and function of hematopoietic progenitors was enabled by the combination of hematopoietic assays of protein purification from condi- tioned media and application of cDNA cloning. HEMATOPOIETIC REGULATION The maintenance of hematopoiesis in steady state by a balance of negative and positive cytokine regulators (Table 72). A variety of the cytokines that include the hematopoietic TABLE 72 Hematopoietic Growth Factors Factor Major Target Cell or Precursor EPO erythrocytes G-CSF neutrophil, also precursors of myeloid lineage GM-CSF erythrocyte, neutrophil, eosinophil, basophil, monocyte, megakaryocyte IL-1a and b primitive precursor cells — lymphocyte activating factor IL-2 T-cells IL-3 neutrophil, eosinophil, basophil, monocyte IL-4 T-cells, B-cells cofactor in granulopoiesis IL-5 B-cells, eosinophils IL-6 B-cells and precursors, neutrophils, monocytes, megakaryocytes, early precursor cells IL-7 pre-B-cells, pre-T-cells IL-9 erythroid precursors IL-11 B-cells, megakaryocytes, mast cells IL-12 early precursors, NK lymphocytes M-CSF monocytes SCF stem cells TPO megakaryocytes Note: See “Abbreviations” in the front of this volume; for synonyms, see Cytokines. 3393_C006.fm Page 390 Monday, November 19, 2007 1:59 PM HEMATOPOIETIC REGULATION 391 growth factors (HGFs) (see Table 73), stem cell factor (SCF), flt3 ligand (FL), thrombopoi- etin (TPO), interleukin-3 (IL-3), granulocyte/macrophage colony stimulating factor (GM- CSF), and IL-6 have been shown, in various combinations, to promote the growth and differentiation of hematopoietic stem cells (HSCs) (see Table 73 and Table 74). The role of these cytokines is largely as survival factors for HSCs, and their role in the in vitro self- renewal of HSCs remains controversial. This has important clinical implications, since it is unlikely that the current repertoire of cytokines will result in stem cell expansion for therapeutic purposes. In contrast, TGF-β1 inhibits growth of HSCs. This 25-kDa protein is produced by the BM stroma as well as progenitors and therefore regulates HSCs in a paracrine/autocrine manner. It has been shown that antisense or antibody inhibition of TGF-β1 releases stem cells from quiescence. TGF-β1 induces quiescence through the p21/ 27 pathways. HSC proliferation is intimately linked to the stromal cells and extracellular matrix (ECM) in distinct microenvironmental niches (see Bone marrow). The ECM is composed of a variety of molecules, including fibronectin, laminin, collagens, and proteoglycans. Some components of the ECM bind to cytokines produced by the stroma, immobilizing them within the microenvironmental niches and thus creating zone in which HSCs and cyto- kines can coalesce. More recently, it has been shown that mouse HSC cells (specifically long-term HSC cells) are tethered to N-cadherin-expressing, spindle-shaped osteoblastic cells lining trabecular bone. Consequently, increasing trabecular bone surfaces increases the number of niches and HSC cells to fill them. Osteoblast activity and trabecular bone is regulated by parathyroid hormone (PTH), which enlarges the HSC pool by: Increasing the amount of trabecular bone and with it the available niche space Stimulating bone-lining cells to make large amounts of a ligand called Jagged1, which activates the Notch receptors on the attached HSC cells Directly stimulating the HSC/CFU-S cells to start replicating DNA Without endogenous PTH, the HSC/CFU-S pool shrinks as the cells terminally differ- entiate and hematopoiesis declines. There is also evidence that key regulators of angio- genesis also regulate the bone marrow (BM) microenvironmental niche. HSCs expressing the receptor tyrosine kinase Tie2 adhere to osteoblasts in the BM niche. The interaction of Tie2 and its ligand angiopoietin-1 (Ang-1) leads to tight adhesion of HSCs to stromal cells, resulting in maintenance of long-term repopulating activity of HSCs. Thus, Tie2/ Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the BM niche (see Figure 56). Other signaling pathways are also intimately linked with HSC self renewal (see Cell signal transduction). TABLE 73 Hematopoietic Regulators Cell Lineage Transcriptional Regulators Cytokine Regulators s Pu.1; CEBP-u.1; CEBP-α; CEBP-ε G-CSF; GM-CSF; IL-3; M-CSF; SCF; IL-6 Macrophage Pu.1 GM-CSF; IL-3; M-CSF Eosinophil Pu.1; GATA-1; Fog-1 IL-5; GM-CSF; IL-3 Mast cell Pu.1; GATA-1; Fog-1 SCF; IL-3; IL-9; TPO Megakaryocyte GATA-1; Fog-1; GATA-2; SCL; NF-E2 TPO; IL-3; LIF; SCF; IL-6; IL-11; EPO Erythroid GATA-1; Fog-1; GATA-2; SCL EPO; IL-3; SCF T-cell Ikaros; Ets1; GATA-3; NFATc; TCF1; LEF1; sox4; NF-kB; LKLF IL-2 SCF; IL-7; IL-12; FL B-cell Ikaros; EBF; E2A; RAG1; RAG2; Pax-5; Vav IL-7 SCF; IL-5; IL-12; FL 3393_C006.fm Page 391 Monday, November 19, 2007 1:59 PM [...]... status of the cells For example, cells from chemotherapy-treated mice or progenitors mobilized into the peripheral blood by G-CSF proved to be primarily CD34+CD38− 3393_C006.fm Page 3 95 Monday, November 19, 2007 1 :59 PM HEMATOPOIETIC STEM CELL ASSAYS 3 95 MOUSE HUMAN c-kit+ Thy-1 lo Negative for: CD2 CD3 CD4 CD5 CD8 c-kit+ Sc a-1 + CD4 5R GR -1 Mac-1 Ter119 Thy-1 lo CD34 + AC133 + Negative for: CD2 CD3 CD1... oxygen-dissociation curve makes more oxygen available 3393_C006.fm Page 404 Monday, November 19, 2007 1 :59 PM 404 HEMOGLOBIN TABLE 75 Reference Ranges in Health of Hemoglobin in Peripheral Blood Men Women Infants (full term) Children (1 year) Children (2–6 years) Children (6–12 years) 13.0–17.0 g/dl 12.0– 15. 0 g/dl 17.9–21 .5 g/dl 10 .5 13 .5 g/dl 12 .5 13 .5 g/dl 11 .5 13 .5 g/dl Source: Derived from Reference. .. inositol-linked immunoglobulin superfamily molecules, refines further for HSC In mice, the Sca-1+Lin−Thy-1lo cell subset is enriched for all clonogenic assays and for radioprotective cells, while the Sca-1− subset was enriched for day-8 CFU-S, which represented more-committed myeloid progenitors The total population of Sca-1+Lin−Thy-1lo cells represents approximately 1 per 2000 cells in the bone marrow... In α-chain variants (Hb M-Boston, Hb M-Iwate), the dusky coloration is present at birth In β-chain variants (Hb M-Hyde Park, Hb M-Milwaukee, Hb M-Saskatoon), features appear at the age of 6 months The chief effect of both varieties is cosmetic HEMOGLOBIN O-ARAB (Hb O-Arab) A hereditary structural variant of hemoglobin due to substitution of lysine for glutamic acid at the 121 position of the β-chain... receptor c-kit is expressed on primitive HSC and progenitor cells, providing an additional marker In combination with lineage depletion and Sca-1 expression, such cells possessed day-12 CFU-S and pre-CFUS activity, could form colonies on stromal layer culture, and had the capability to rescue irradiated transplant recipients, giving rise to long-term multilineage repopulation The c-kit+Thy-1loLin−/loSca-1+... occur in thalassemia and in the β-unstable hemoglobinopathies Again, some overlap occurs between normal subjects and those with β-thalassemia trait The reference range in health is 1 .5% to 3 .5% β-Thalassemia subjects have HbA2 levels of 4.0 to 7.0%, except for δβ-thalassemia subjects, in which levels are low Patients with b-unstable hemoglobinopathies have the same reference range as the thalassemia... 0. 05% of murine bone marrow cells, is now widely used to define bone marrow HSC in mice However, they may be less useful in the setting of G-CSF-induced mobilization of progenitors, as ckit expression has been found to be selectively reduced on Lin−Sca-1+c-kit+ cells, but not on Lin−Sca-1−c-kit+ cells in the bone marrow following G-CSF treatment Rhodamine efflux capability may also be added to divide c-kit+Lin−Sca-1+... c-kit+Lin−Sca-1+ cells into moreprimitive Rholo and more-mature Rhohi subsets Although both populations confer similar levels of radioprotection and have similar content of day-12 CFU-S, most if not all longterm repopulating cells are found in the Rholoc-kit+Lin−Sca-1+ fraction Rhohic-kit+Lin−Sca1+ cells displayed in vivo repopulation kinetics resemble those of the short-term repopulating cells The c-kit+Lin−Sca-1−... enrichment for progenitor cells Using a cocktail of antibodies detecting these lineage markers, followed by lineage-positive cell removal by bead adsorption or flow cytometric sorting, murine bone marrow can be effectively divided into lineage-positive (Lin+) and lineage-negative (Lin−) fractions Adding analysis for the expression of Thy-1 or Sca-1, which are cell-surface glycophosphatidyl inositol-linked... - and β-polypeptide chains that migrate as an α-globulin upon serum protein electrophoresis The plasma concentration of haptoglobin is expressed as its hemoglobin-carrying capacity (reference range 0.8 to 2.7 g/l by the radial immunodiffusion method; 0.3 to 2.0 g/l by the hemoglobin-binding-capacity method) With active intravascular hemolysis, the level of haptoglobin falls rapidly The haptoglobin-hemoglobin . CEBP-u.1; CEBP-α; CEBP-ε G-CSF; GM-CSF; IL-3; M-CSF; SCF; IL-6 Macrophage Pu.1 GM-CSF; IL-3; M-CSF Eosinophil Pu.1; GATA-1; Fog-1 IL -5 ; GM-CSF; IL-3 Mast cell Pu.1; GATA-1; Fog-1 SCF; IL-3; IL-9;. GATA-1; Fog-1; GATA-2; SCL; NF-E2 TPO; IL-3; LIF; SCF; IL-6; IL-11; EPO Erythroid GATA-1; Fog-1; GATA-2; SCL EPO; IL-3; SCF T-cell Ikaros; Ets1; GATA-3; NFATc; TCF1; LEF1; sox4; NF-kB; LKLF IL-2. lymphocyte activating factor IL-2 T-cells IL-3 neutrophil, eosinophil, basophil, monocyte IL-4 T-cells, B-cells cofactor in granulopoiesis IL -5 B-cells, eosinophils IL-6 B-cells and precursors, neutrophils,