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Beginnings: themolecular pathology ofhemoglobin 13

ROS

Hemolysis

Dyserythropoiesis

Hepatosplenomegaly

Bone marrow expansion

Increased Erythropoietin

Iron accumulation

https://t.me/med1917

anemia that can necessitate blood transfusion, often associated with a concurrent illness particularly infection.

The most severe form of α thalassemia is the hemoglobin Bart’s Hydrops fetalis syndrome (BHFS). This most commonly occurs due to the inheritance of no α- globin genes (− −/− −), though it can arise due to the inheritance of two severe non- deletional alleles (αTα/αTα) or a compound heterozygotes for a severe non- deletional allele and an allele bearing no α genes (− −/αTα). In homozygotes for the common − −/− − genotypes, the only oxygen carrying molecule that keeps affected fetuses alive in utero beyond the first few weeks’ gestation is hemoglobin Portland (ζ2γ2), and the condition is marked hematological by severe intrauterine anemia with high levels of hemoglobin Bart’s (γ4) and hemoglobin H (β4) with ~20% hemoglobin Portland. Clinically, the fetuses exhibit prolonged intrauterine edema and resultant signs of cardiac failure including generalized edema, pleural, and pericardial effusions. Until recently, BHFS was considered to be a universally fatal disorder, but with improvements in screening and antenatal care, an early diagnosis followed by intra-

uterine transfusions can keep fetuses alive until term. Thereafter, the clinical course can be quite variable and some infants acquire associated developmental abnormalities in utero. To date, only 100 or so infants with the BHFS have survived and all require regular blood transfusion with chelation therapy or a stem cell transplant.

The β thalassemias

Almost all of the 200 or more different mutations that underlie the β thalassemias result in reduced β- globin chain production. Synthesis of the α- globin chain proceeds normally and hence there is imbalanced globin chain output with an excess of α chains (Figure1.8). Unpaired α chains precipitate

in both red cell precursors and their progeny with the production of inclusion bodies. These interfere with normal red cell maturation and survival in a variety of complex ways. Their attachment to the red cell membrane causes alterations in its structure, and their degradation products, notably heme, hemin (oxidized heme), and iron, result in oxidative damage to the red cell contents and membrane. These interactions result in intramedullary destruction of red cell precursors (dyserythropoiesis) and in shortened survival of such cells as they reach the peripheral blood (hemolysis). The end result is a hypochromic microcytic anemia of varying severity. Carriers of β thalassemia compensate in a variety of ways and are asymptomatic. By contrast, homozygotes and heterozygotes for β- globin mutations cannot fully compensate for the deficiency of β- globin chain production. Anemia causes tissue hypoxia and the production of relatively large amounts of erythropoietin; this leads to an expansion of the ineffective bone marrow, which can result in bone deformity, a hypermetabolic state with wasting and malaise, and bone fragility.

A large proportion of hemoglobin in the blood of β thalassemia patients is of the fetal variety. Normal individuals produce about 1% of Hb F, unevenly distributed among their red cells. In the bone marrow of individuals with β thalassemia, any red cell precursors that synthesize γ chains come under strong selection because they combine with α chains to produce fetal hemoglobin, and therefore the degree of globin chain imbalance is reduced. Furthermore, the likelihood of γ chain produc- tion seems to be increased in a highly stimulated erythroid bone marrow. It seems likely that these two factors combine to increase the relative output of Hb F in this disorder. However, it has a higher oxygen affinity than Hb A, and hence patients with β thalassemia are not able to adapt to low hemoglobin levels as well as those who have adult hemoglobin. The greatly expanded, ineffective erythron leads to an increased rate of iron absorption; this, combined with iron received by blood transfusion,

Figure1.8 The underlying pathophysiology ofβ thalassemia. Unpaired α- globin chains precipitate causing cellular necrosis and apoptosis, resulting in ineffective erythropoiesis and anemia.

Excess

α globin chains

α Genes β Genes

α mRNA

α Globin

HbA

β mRNA

β Globin

Anemia

14 Molecular Hematology

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leads to progressive iron loading of the tissues, with subsequent liver, cardiac, and endocrine damage. The constant bombardment of the spleen with abnormal red cells leads to its hypertrophy. Hence, there is progressive splenomegaly with an increased plasma volume and trapping of part of the circulating red cell mass in the spleen. This leads to worsening of the anemia. All these pathophysiological mechanisms, except for iron loading, can be reversed by regular blood transfusion which, in effect, shuts off the ineffective bone marrow and its consequences. Thus, it is possible to relate nearly all the important features of the severe forms of β thalassemia to the primary defect in globin gene action. However, can we also explain their remarkable clinical diversity?

Phenotypic diversity

While β thalassemia is a classic monogenic autosomal recessive disorder, there is a remarkable degree of phenotypic variability, even though approximate genotype- phenotype predictions can be made. This is primarily due to the types of mutation that are inherited (β+/β+, β0/β+, or β0/β0 thalassemia) giving rise to increasing globin chain imbalance and increasing amounts of excess - globin chains. However, other co- inherited genetic modifiers that act in various ways together with physiological adaptation to anemia also contribute to clinical outcome.

The central pathophysiological driver of β thalassemia is the excess pool of α- globin chains (Figure1.8). The secondary modifiers act to reduce that excess pool. Naturally occurring co- inherited α- globin deletions can significantly ameliorate β thalassemia as while total (α + β) globin levels are reduced, the balance between the chains is improved. The effect is most marked in individuals with Hb E/β thalassemia: individuals who co- inherit the − −/αα or −α/−α genotypes rarely require regular blood transfusion. The converse also applies; extra copies of α- globin can also significantly worsen symptoms. Even carriers of β thalassemia who co- inherit more than four α- globin genes (e.g. ααα/αα) may have a relatively severe form of β thalassemia requiring blood transfusion.

A major route to reducing the α- globin excess is to pair the free chains with γ- globin chains, forming fetal hemoglobin (α2γ2). Patients with β thalassemia who co- inherit any of the mutations causing hereditary persistence of fetal hemoglobin (HPFH) have a relatively mild clinical course.

Other modifiers do not affect globin expression but rather the clinical sequelae of the disease. These include iron regulation and metabolism, response to infection, bone metabolism and the hepatic processing of bilirubin. Various physiological adaptations to anemia may contribute to the overall clinical picture. While these are incompletely understood, well- conducted studies in Hb E/β thalassemia have shown that the erythropoietin response to anemia varies depending on age, with a reduction in the response, with its attendant erythroid expansion and splenomegaly, as patients get older (Table1.2).

Table1.2 Mechanisms forthe phenotypic diversity ofthe

β thalassemias

Genetic modifiers

Primary: alleles of varying severity Secondary: modifiers of globin chain imbalance

α Thalassemia Increased α- globin genes: ααα or αααα Genes involved in unusually high Hb F response

Tertiary: modifiers of complications

Iron absorption, bone disease, jaundice, infection

Adaptation to anemia

Variation in oxygen affinity (P50) of hemoglobin Variation in erythropoietin response to anemia

Environmental

Nutrition Infection Others

There may be genetic variation in the adaptive mechanisms.

Development oftherapies

Many of the advances in our understanding of the basic principles and mechanisms underlying molecular genetics and how this is perturbed in human genetic disease came from careful studies of patients with hemoglobinopathies. Central to these research efforts was the aim of ultimately using this knowledge to improve the management of patients with hemoglobinopathies. We have now reached a point at which both thalassemia and sickle cell disease are potentially curable by gene therapy or gene editing, but the expense would preclude most affected patients being treated in this way (Figure1.9).

Similarly bone marrow transplantation is only available for a minority of cases. Blood transfusion and iron chelation pose a huge burden on the health systems of countries where the hemoglobinopathies are prevalent. Where acceptable, preimplantation selection and pre- natal intrauterine diagnosis have been proven to reduce the numbers of affected individuals to very small numbers (e.g. Cyprus and Sardinia). In reality, the problem is now transforming from a biomedical scientific problem to one of ethics and medical economics. Many of the strategies developed for the hemoglobinopathies are now also being applied to other genetic diseases with very similar attendant issues.

Pharmacological options

Many treatments for the thalassemias have sought to addressthe primary underlying mechanism of the disease; the imbalance in α- and β- globin chain synthesis. Most efforts have aimed to do this by re- activating γ- globin expression to reduce the free pool of α- globin chains by making fetal hemoglobin. While the drug hydroxyurea is

Beginnings: themolecular pathology ofhemoglobin 15

-globin expression

Genome editing

Allogeneic hematopoietic

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to increase fetal globin expression

base editing

or to decrease α-globin expression

Lentiviral gene therapy

cell transplant

Figure1.9 Current and future therapeutic strategies to treat β thalassemia.

widely used in sickle cell disease to increase hemoglobin F, to some degree, its effect in β thalassemia has been more limited. 5- Azacytidine, decitabine, sodium phenylbutyrate, ruxolitinib, histone deacetylase inhibitors and the immunomodulatory agent pomalidomide have all been trialed but with varying and somewhat disappointing results. Unfortunately, these drugs have a wide range of unwanted side- effects, and consequently their clinical use has been restricted.

Blood transfusion

and iron chelation

Pharmacological approach

to increase fetal Hb

Pharmacological approach

to reduce α

major repressor of g- globin, this leads to robust induction of hemoglobin F, as was noted when a short hairpin containing microRNA targeting BCL11A was transduced using a lentiviral- vector into HSCs. These so- called shmiRNAs, under the control of an erythroid enhancer ensuring tissuespecific expression, bind to and functionally inactivate BCL11A RNA in erythroid cells. While effective in these trials, lentiviral- based approaches carry the risks described above.

Gene therapy

The correction of globin chain imbalance by adding wild- type or corrected β- globin variants has been a goal of the field since the 1980s. Researchers developing this approach for hemoglobinopathies have been at the forefront of the developing genetherapy for all types of human genetic diseases, with much work performed to optimize gene expression, improving viral vectors and overcoming significant safety setbacks. Over the last decade, patients have been successfully treated by a number of groups using lentiviral vectors, which integrate their genomic cargo widely across the genome. Since there is no way of controlling where the lentivirus integrates, there are thousands of independent integrants in patients’ stem cells. There is a concern that one or more of the integrants will underlie the development of a malignant clone, which may be selected and cause myelodysplastic syndrome or leukemia: this has occurred with early versions of retroviruses used for gene therapy. Nevertheless, there have also been successes, most prominently reported ina multi- center international phase 3 trial in patients with transfusion- dependent thalassemia and sickle cell disease byintroducing an anti- sickling version of the β- globin gene (T87Q). In the disease- genotypes treated (patients with β0/β0 genotypes were excluded), the results have been encouraging.

Lentiviral- vectors have also been used to downregulate

BCL11A in patients with sickle cell disease. As BCL11A is the

Gene editing

A more precise and more recently developed approach is to use the programmable CRISPR/Cas9 system to perform targeted gene- editing. This involves the targeting of an endonuclease enzyme, cas9, to a specific location within the genome using a homing complementary “guide RNA” sequence. The best- characterized treatment has been to use Cas9 to disrupt an erythroid- specific enhancer of BCL11A to reduce its expression specifically in erythroid tissue, thereby releasing BCL11A’s repression of γ- globin gene expression. This geneediting approach has been tested in clinical trials with excellent early results in patients with β thalassemia and sickle cell disease, with longer- term safety data awaited. Future approaches may make use of recently developed modular additions to CRISPR/Cas9, namely base editors that can programmably deaminate a cytidine to a thymine or an adenine to a guanine. These editors are able to efficiently revert point mutations— which comprise the majority of β- globin variants— without creating double- strand breaks; a key safety improvement. Advanced pre- clinical work has shown that base editors can target the mutations causing sickle cell disease and β thalassemia, as well as editing the erythroid enhancer of BCL11A and sequences within the γ- globin pro- moter that bind repressive proteins, thereby increasing levels of fetal hemoglobin.

16 Molecular Hematology

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Conclusion

Research into the hemoglobinopathies began nearly a hundred years ago, and much of what we know about gene structure, fundamental aspects of mammalian gene regulation and the pathological states that arise when their expression is disrupted comes directly from research into the α- and β- globin loci. While immense strides have been made in our understanding of these genes, and how to manage patients clinically, for the majority of patients, a cure is out of reach. However, past and recent technological advances offer theopportunity to diagnose these conditions prenatally, and significantly alter the course of thalassemia treatment by combining precise genome engineering techniques with the wealth of genetic data gathered by numerous researchers in the field, some of which has been described in this introductory chapter.

Further reading

General background

Peltonen, L. and McKusick, V.A. (2001). Genomics and medicine.

Dissecting human disease in the postgenomic era. Science 291: 1224–1229.

Weatherall, D.J. (2013). The role of the inherited disorders of hemo-

globin, the first “molecular diseases,” in the future of human genetics. Annu. Rev. Genomics Hum. Genet. 14: 1–24.

Weatherall, D.J. and Clegg, J.B. (2001). The Thalassaemia Syndromes,

4e. Oxford: Blackwell Science.

Weatherall, D.J., Schechter, A.N., and Nathan, D.G. (eds.) (2013).

Hemoglobin and Its Diseases. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Hemoglobin genetics andstructural variants

https://globin.bx.psu.edu/hbvar/menu.html. Giardine, B., Borg, J., Viennas, E. etal. (2014). Updates of the HbVar

database of human hemoglobin variants and thalassemia mutations. Nucleic Acids Res. (Database issue): D1063- 9.

Steinberg, M.H., Forget, B.G., Higgs, D.R., and Weatherall, D.J. (eds.)

(2009). Disorders of Hemoglobin, 2e. NewYork: Cambridge University Press.

Hemoglobin switching

Lu, H.Y., Orkin, S.H., and Sankaran, V.G. (2023). Fetal hemoglobin regula-

tion in beta- thalassemia. Hematol. Oncol. Clin. North Am. (2): 301–312.

Sankaran, V.G. and Orkin, S.H. (2013). The switch from fetal to adult

hemoglobin. Cold Spring Harbor Perspect. Med. https://doi. org/10.1101/cshperspect.a011643.

Thein, S.L. and Menzel, S. (2009). Discovering the genetics underlying foe-

tal haemoglobin production in adults. Br. J. Haematol. 145: 455–467.

The β thalassemias

Fucharoen, S. and Weatherall, D.J. (2013). The hemoglobin E thalas-

semias. Cold Spring Haror. Perspect. Med. https://doi.org/10.1101/ cshperspect.a011734.

Nienhuis, A.W. and Nathan, D.G. (2013). Pathophysiology and clinical

manifestations of the beta thalassemias. Cold Spring Harbor Perspect. Med. https://doi.org/10.1101/cshperspect.a011726.

Olivieri, N.F., Muraca, G.M., O’Donnell, A. etal. (2008). Studies in hae-

moglobin E beta-

Thein, S.L. (2008). Genetic modifiers of the beta-

Br. J. Haematol. 141: 357–366.

thalassaemia. Br. J. Haematol. 141: 388–397.

haemoglobinopathies.

The α thalassemias

Higgs, D.R. (2013). The molecular basis of alpha thalassemia. Cold

Spring Harbor Perspect. Med. https://doi.org/10.1101/cshperspect.

a011718.

Higgs, D.R. and Gibbons, R.J. (2010). The molecular basis of alpha-

thalassemia: a model for understanding human molecular genetics. Hematol. Oncol. Clin. North Am. 24: 1033–1054.

Vichinsky, E. (2013). Natural history and clinical manifestations of the

alpha thalassemias. Cold Spring Harbor Perspect. Med. https://doi. org/10.1101/cshperspect.a011742 .

Evolutionary background ofhemoglobin disorders

Penman, B.S., Pybus, O.G., Weatherall, D.J., and Gupta, S. (2009).

Epistatic interactions between genetic disorders of hemoglobin can explain why the sickleProc. Natl. Acad. Sci. U.S.A. 106: 21242–21246.

Weatherall, D.J., Williams, T.N., Allen, S.J., and O’Donnell, A. (2010).

The population genetics and dynamics of the thalassemias. Hematol. Oncol. Clin. North Am. 24: 1021–1031.

Williams, T.N., Mwangi, T.W., Wambua, S. et al. (2005). Negative

epistasis between the malariasemia and the sickle cell trait. Nat. Genet. 37: 1253–1257.

cell gene is uncommon in the Mediterranean.

protective effects of alpha+ − thalas-

Molecular basis ofprevention andmanagement ofhemoglobin disorders

Cao, A. and Kan, Y.W. (2013). The prevention of thalassemia. Cold

Spring Harbor Perspect. Med.

Lo, Y.M. and Chiu, R.W. (2010). Noninvasive approaches to prenatal

diagnosis of hemoglobinopathies using fetal DNA in maternal plasma. Hematol. Oncol. Clin. North Am. 24: 1179–1186.

Nienhuis, A.W. and Persons, D.A. (2013). Development of gene therapy

for thalassemia. Cold Spring Harbor Perspect. Med. https://doi. org/10.1101/cshperspect.a011833.

Orkin, S.H. and Reilly, P. (2016). MEDICINE. Paying for future success

in gene therapy. Science 352: 1059–1061.

Rosanwo, T.O. and Bauer, D.E. (2021). Editing outside the body: Ex vivo

gene- modification for β- hemoglobinopathy cellular therapy. Mol. Ther. (11): 3163–3178

Chapter2

https://t.me/med1917

Stem cells

David T. Scadden

Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University and Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA

Introduction, 17 Stem cell definitions anddistinctions, 17 Hematopoietic stem cell concepts andtheir origin, 18 Molecular regulation ofhematopoiesis, 20

Introduction

The generation of sufficient numbers of blood cells to maintain homeostasis requires sustained production of mature cells. This process, called hematopoiesis, yields approximately 1011 blood cells daily, with the capability for dramatic increases in the number and subsets of cells in response to physiological stress. Hematopoiesis is, therefore, a highly dynamic process dependent upon numerous modulating factors. Its prodigious production capability derives from the sustained presence of a cell type, which is generally quiescent, but the descendants of which proliferate vigorously. This cell is the hematopoietic stem cell (HSC).

Stem cell definitions anddistinctions

Stem cells derive their name from their ability to durably produce daughter cells that progressively mature to fully differentiated cells. Stem cells are defined by a combination of the traits of self- maintenance and the ability to produce differentiated offspring. Putting this in more biological terms, stem cells have the unique and defining characteristics of

self-

renewal and of differentiation into mature cell types. Thus,

with each cell division, there is an inherent asymmetry in stem cells that is generally not found with other cell types.

While their name implies that stem cells have specific intrinsic characteristics, there are multiple different types of stem cells, each defined by their production ability. Totipotent stem cells are capable of generating any type of cell in the body, including those of the extra- embryonic tissues, such as

Trafficking ofprimitive hematopoietic cells, 25 Manipulating hematopoietic stem cells forclinical use, 27 Summary, 31 Further reading, 31

the placental tissues (Figure2.1). Pluripotent stem cells may give rise to any type of cell found in the body except those of the extra- embryonic membranes. They can produce ectoderm, mesoderm, or endoderm cells. It has also become possible to create pluripotent cell by “reprogramming” mature cells. This allows for pluripotent cells to be made from any individual, a powerful tool for basic biology, disease modeling, and possible future cell therapies. These are called induced pluripotent stem cells (iPS). Pluripotent stem cells not made by reprogramming include embryonic stem cells, isolated from the inner cell mass of the blastocyst, embryonic germ cells, isolated from embryonic gonad precursors, and embryonic carcinoma cells, isolated from teratocarcinomas. Pluripotent stem cells may be maintained indefinitely in culture under specialized conditions that prevent differentiation. In particular, embryonic stem cells have been used to generate “knockout” mice, animals harboring targeted gene disruptions via homologous recombination that permit the invivo study of individual gene function. Multipotent stem cells, such as the HSCs of the bone marrow, are capable of giving rise to multiple mature cell types, but only those of a particular tissue, such as blood. They cannot make cells from tissues of another germ layer. For example, mesodermally derived HSC cannot form ectodermally derived neurons or endodermally derived gut cells. Multipotent stem cells are found in adults, perhaps in all tissue, and function to replace dead or damaged tissue. Such stem cells are commonly referred to as “adult” stem cells. Lastly, unipotent stem cells only form one type of cell. Germ cell stem cells are one example and some consider memory cells of B or T cell lineage to be unipotent stem cells.

18 Molecular Hematology

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Inner cell mass

BlastocystTotipotent cellsFertilization

Pluripotent cells

Embryonic stem

(ES) cells

Figure2.1 Sources and types of stem cells.

Source: Adapted with gratitude from the National Institutes of Health Stem Cell Information website.

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 requires 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 (ICs), termed colony- 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.

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 colonyforming 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

forming

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 a 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 (LTC) 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 (CAFCs). These multipotential cell types were considered 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. These groupings are simple models for what is likely to be a continuum of cells with self- renewal and multipotent differentiation capacity. The

Fetal tissues

Adult tissues

Multipotent cells

“Tissue” or “adult”

stem cells

Stem cells 19

Microenvironment

Hematopoiesis

https://t.me/med1917

Figure2.2 Schematic view of hematopoiesis. Seetext for definition of abbreviations. Source: Adapted from Hoffman, R. (2000). Hematology: Basic Principles and Practice, 3e.

CFU-F/RF

long- term repopulating cells also tend to be more quiescent, serving as a deep reserve for blood cell production in times of physiologic stress. More mature progenitors (also called colony- forming cells or 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 2.2), 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, serving, in effect, as unipotent stem cells. Their inability to produce multiple different types of daughter cells distinguishes them from multipotent HSCs.

In summary, the compartment model has given rise to terms that are generally applied to cells of hematopoietic origin. HSCs 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 neutrophil 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

LTC-IC/CAFC

HPP-CFC

Precursor cellsProgenitor cellsStem cells

Short-termLong-term

CFU-S

CFU-C

CFU-Mix

Mature cell

to cytokines, while the stem cells are long- lived, cytokineresistant, 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 governed by epigenetic characteristics established early in development. 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 in relation to external signals combined with levels of transcription factors present at that moment in the cell.

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 granulocyte macrophage colony- stimulating factor (GMCSF) 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

20 Molecular Hematology

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receptors in progenitor cells. Expression of the erythropoietin receptor in a macrophage progenitor results in macrophage colony formation, whereas expression of the macrophage colony- stimulating factor (M- CSF) receptor 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 (G- CSF) receptor 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 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.

More recent studies have been used to assess when hematopoietic progenitors establish a committed cell fate. Cells can be stalled in differentiation at the granulocyteprogenitor (GMP) stage by inducible expression of HoxB8 fused to an estrogen receptor. When estrogen is removed, the cells begin a differentiation program marked by changes in gene expression and acquisition of mature cell functions. Reexposing the cells to estrogen can reverse the differentiation changes up to a particular point (~40 h for mouse cells). After that time, the cells proceed inexorably toward terminal differentiation even with re- introduction of estrogen HoxB8 expression. Therefore, there is a “point of no return” in differentiation that marks full commitment. Molecularly, this is evident in chromatin configuration as beyond the point of no return, there is no transcription factor access to key regulatory regions.

monocyte

Molecular regulation of hematopoiesis

The molecular nature of stem cell regulatory pathways has been determined using a variety of genetic approaches, including genetic loss- of- function and gain- of- function studies. These have provided several important concepts regarding the molecular control of hematopoiesis. First, some genes have binary functions and exert an effect by being above a threshold of expression, rendering their targets “on.” Other genes function in a continuum and have different effects at different levels. Secondly, while perturbations in single genes may have dramatic cellular effects, gene products often function in complexes and, in the case of transcription regulators, are only active if they have access to sites of open chromatin. Finally, signal integration often depends on the assembly of large signaling complexes and the spatial proximity of molecules to facilitate interaction is therefore important.

Cell- intrinsic regulators ofhematopoiesis

Cell cycle control

The quiescent nature of HSCs is supported by their low level of staining with DNA and RNA nucleic acid dyes, which is consistent with low metabolic activity. These studies have indicated a heterogeneity among stem cells with a subgroup that is deeply quiescent. Various studies have sought to determine the cell- intrinsic regulators of hematopoiesis involved in HSC cycle control.

RNA analysis has been used to profile pertinent transcription factors and other molecules in HSCs induced to differentiate along various lineages by the application of cytokines. Elevated levels of cyclin- dependent kinase inhibitors (CDKIs) have been observed, suggesting that CDKIs present in HSCs function to exert a dominant inhibitory tone on HSC cell cycling. The bone marrow of some mouse strains deficient in CDKI p57(CDKN1C), p21(CDKN1A), or p18(CDKN2C) have increased HSC cell cycling, suggesting that these CDKIs function as a dominant negative regulators of HSC proliferation. Other CDKIs, such as p27(CDKN1B), may serve as negative regulators of hematopoietic progenitor cells.

Self- renewal, commitment, andlineage determination

Experimental results involving transcription factors have demonstrated cell- intrinsic roles in both global and lineagespecific hematopoietic development. Mice engineered to lose the function of specific genes have been used to define the impact of those genes on hematopoiesis. Loss- of- function studies involving the transcription factors c- Myb, AML1 (CBF2), SCL (tal- 1), LMO2 (Rbtn2), GATA- 2, and TEL/ ETV6have demonstrated global effects on all hematopoietic lineages. Stem cells in animals deficient in these molecules fail to establish definitive hematopoiesis. Similar methods have indicated that some regulators have different roles at different times in development. For example the gene product of transcription factor, SCL is absolutely required for establishing HSCs. Unexpectedly, there is not a requirement for SCL once the stem cell pool is present in the adult. Rather, SCL is required only for erythroid and megakaryocytic homeostasis. Therefore, transcription factor regulation of the stem cell compartment can be highly dependent on the stage of development of the organism.

of- function studies have also proved useful in identify-

Lossing lineage- specific transcription factors. Mice genetically deficient in the transcription factor Ikaros lack T and B lymphocytes and natural killer cells, but maintain erythropoiesis and myelopoiesis. Notch is required for T lineage induction and shifts differentiation away from B cell development. Further, losing expression of some genes can enable cells to revert back in differentiation. For example the Pax- 5 transcription factor is essential for B cell maturation and loss of it results in cells that

Pro-T

NK cell

Megakaryocyte

https://t.me/med1917

Stem cells 21

HSC

ST-HSCLT-HSC

SCL (++) C/EBPα (±) GATA-2 (++) PU.1 (±) NF-E2 (–) Aiolos (±) GATA-1 (±) GATA-3 (±)

Lymphoid pathway

CLP

IL-7R

–

c-mpl

SCL (–) C/EBPα (–) GATA-2 (–) PU.1 (+) NF-E2 (–) Aiolos (+) GATA-1 (–) GATA-3 (+)

Myeloid pathway

CMP

–

IL-7R

c-mpl

SCL (++) C/EBPα (±) GATA-2 (+) PU.1 (±) NF-E2 (+) Aiolos (±) GATA-1 (+) GATA-3 (–)

IL-7R

SCL (–) C/EBPα (–) GATA-2 (–) PU.1 (–) NF-E2 (–) Aiolos (++) GATA-1 (–) GATA-3 (++)

Pro-B

IL-7R

SCL (–) C/EBPα (–) GATA-2 (–) PU.1 (+) NF-E2 (–) Aiolos (++) GATA-1 (–) GATA-3 (–)

GMP

–

Epo-R

SCL (+) C/EBPα (++) GATA-2 (–) PU.1 (±) NF-E2 (–) Aiolos (–) GATA-1 (–) GATA-3 (–)

MEP

Epo-R

SCL (++) C/EBPα (–) GATA-2 (++) PU.1 (±) NF-E2 (++) Aiolos (–) GATA-1 (++) GATA-3 (–)

T cell

B cell

Monocyte

Granulocyte

Erythrocyte

Figure2.3 Transcription factors active at various stages of hematopoiesis. CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte monocyte progenitor; MEP, megakaryocyte erythrocyte progenitor; NK, natural killer. Source: Adapted from Akashi, K., Traver, D, Miyamoto, T, Weissman, I.L.etal. (2000). A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature. 404: 193–197.

have already rearranged their B cell receptor or immunoglobin locus showing up in the T and NK cell compartment. The critical roles of specific transcription factors in establishing and maintaining cell fate are shown in Figure2.3.

Cell- extrinsic regulators

Ultimately, hematopoietic stem and progenitor cell decisions are regulated by the coordinated action of transcription factors on accessible chromatin sites as modified by extracellular signals. Extracellular signals in the form of hematopoietic growth factors are mediated via cell surface hematopoietic growth factor receptors. Hematopoietic growth factors exert specific effects when acting alone and may have different effects when combined with other cytokines. There are at least six receptor superfamilies, and most growth factors are members of the type I cytokine receptor family. The effects of various cytokines during myelopoiesis are illustrated in Figure2.4.

Type I cytokine receptors

Type I receptors do not possess intrinsic kinase activity but lead to the phosphorylation of cellular substrates by serving as docking sites for adapter molecules with kinase activity. Examples of receptors in this family include leukemia inhibitory factor (LIF), interleukin (IL)- 1, IL- 2, IL- 3, IL- 4, IL- 5, IL- 6, IL- 7, IL- 9, IL- 13, IL- 18, GM- CSF, G- CSF, erythropoietin, prolactin, growth hormone, ciliary neurotrophic factor, and c- mpl. These receptors share several features, including enhanced binding and/or signal transduction when expressed as heterodimers or homodimers, four cysteine residues, and fibronectin type III domains in the extracellular domain, WSXWS ligand- binding sequence in the extracellular cytokine receptor domains, and lack of a known catalytic domain in the cytoplasmic portion. Another shared feature of receptors in this family is the ability to transduce signals that prevent programmed cell death (apoptosis).

22 Molecular Hematology

G-CSF, IL-1, IL-6, IL-10, IL-11, IL-12, IL-13

Mixed progenitor

https://t.me/med1917

cell

FLT3, SCF, IL-3, IL-6, GM-CSF, G-CSF

Myelomonocytic

progenitor

SCF, IL-3, IL-6, G-CSF

GM-CSF, CSF-1

CFU-GEMM

CFU-M

HPP-CFC

CFU-GM

G-CSF

CFU-G

b-FGF, HGF, LIF, SCF/KL, FLT3 Ligand, TPO

IL-3

IL-5

IL-4

IL-5

GM-CSF

CFU-Eos CFU-Baso

SCF

Pluripotent

stem cell

NGF

Figure2.4 Cytokines active at various stages of hematopoiesis. See text for definition of abbreviations. Source: Adapted from Hoffman, R. (ed.). Hematology: Basic Principles and Practice, 3e.

CSF-1, IL-3

GM-CSF

CSF-1

GM-CSF

Monocyte Neutrophil

MCP-1-3, IP-10, Rantes, MIP-1

G-CSF

GM-CSF

IL-4

MyeloblastMonoblast

G-CSF

GM-CSF

MyelocytePromonocyte

G-CSFCSF-1

Chemokines

IL-8, NAP-2, Gro-

IL-5

GM-CSF

IL-5,

GM-CSF

Eosinophil

Eotaxin, MCP-4 IL-8, MCP-1,

IL-10, IL-9,

IL-3, IL-4

NGF

Rantes

CSF

Basophil, mast cell

Type II cytokine receptors

This class includes the receptors for tissue factor, IL- 10, and interferon (IFN)- γ. This family contains a type III fibronec- tin domain in the extracellular domain, like the type I family.

Receptor tyrosine kinases

Receptors with intrinsic kinase activity are of considerable relevance to hematology because their ligands are growth factors, but also because abnormalities of them can result in unregulated activation. For example internal tandem duplications of the Flt3 receptor are associated with AML and activating mutations of c- kit play a key role in systemic

mastocytosis and core binding factor AMLs. Loss of c- fms, the receptor for M- CSF, has been associated with myelodysplasia and a predisposition to AML. The availability of agents targeting tyrosine kinases also makes these receptors of particular interest in hematologic disease. Details of receptorligand pairs are provided in Table2.1.

Protein serine–threonine kinase receptors

This family includes the 30 members of the transforming growth factor (TGF)- β superfamily, which bind to their receptors as homodimers. Members of this family include the three TGF- β receptors: type I (TbRI, 53 kDa), type II (TbRII, 75 kDa), and type III (TbRIII, 200 kDa). Members of

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