Hematological Disorders in Children Pathogenesis and

Treatment

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Editor
Eiichi Ishii
Department of Pediatrics
Ehime University Graduate School of Medicine
Toon, Ehime
Japan

ISBN 978-981-10-3885-3    ISBN 978-981-10-3886-0 (eBook)

DOI 10.1007/978-981-10-3886-0

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Preface

In recent years, the pathogenesis of hematological disorders in children has been

clarified, which has led to remarkable progress in the treatment outcome for these
disorders. In particular, molecular targeted therapy has been shown to have an effect
on childhood leukemia refractory to conventional therapy. Further development of
these strategies will enable us to use more effective and less toxic therapies for
hematological disorders in the future. Here we describe the pathogenesis and treat-
ment (present and future) of several representative hematological disorders in chil-
dren, especially focusing on the genetic and molecular aspects.
Most hematological disorders in children arise from intrauterine endogenous or
exogenous exposures, genetic susceptibility, or several factors after birth. A good
example is the case of infant leukemia. Greaves et al. [1] clarified the etiology of
infant leukemia; analysis of MLL gene rearrangements in identical leukemic twins
suggested that the MLL gene undergoes prenatal rearrangement and leukemic cells
are present in the blood of newborns. In utero exposure to some drugs and foods that
possess functional similarity to topoisomerase-II inhibitors and certain environmen-
tal factors may affect MLL gene rearrangements, which contribute to leukemogen-
esis [2]. This conclusion can be expanded to B-cell precursor acute lymphoblastic
leukemia (ALL) with TEL-AML1 [3] or hyperdiploidy [4], acute myeloid leukemia
(AML) with AML1-ETO [5], and acute megakaryocytic leukemia with GATA1
mutations in Down syndrome [6]. Clarification of leukemogenesis after birth is
needed to establish more appropriate treatment modalities in childhood leukemia.
We have recently shown that the MLL-miRNA let7b-oncogene HMGA2 pathway
plays an important role in the proliferation of leukemic cells and could be a possible
molecular target for the therapy of infant leukemia [7].
Congenital bone marrow failure syndrome in children is an inherited disease,
including Fanconi anemia (FA), dyskeratosis congenita, Shwachman–Diamond
syndrome, Diamond–Blackfan syndrome, severe congenital neutropenia, and con-
genital amegakaryocytic thrombocytopenia, which are characterized by cytopenias
in different hematologic lineages and several congenital abnormalities [8]. Early
diagnosis of bone marrow failure syndrome is important for optimizing clinical
management, anticipating possible complications that may develop later in life, and

v
vi Preface

providing appropriate genetic counseling for the family [9]. Many of these syn-
dromes require multidisciplinary and multispecialty medical care for appropriate
surveillance and management. As a representative disease, FA is caused by germ-
line mutations in DNA repair genes with autosomal recessive inheritance. A major
problem of FA is its high risk of cancer, which increases with age. In particular, the
risk of myelodysplastic syndrome or AML is relatively high, indicating that most
patients with FA need hematopoietic stem cell transplantation (HSCT) before the
onset of malignancy [10]. However, the rate of treatment-related side effects and
graft-versus-host disease, as well as the risk of head and neck cancers, is high in FA
patients after HSCT, demonstrating the need for a more appropriate conditioning
regimen [8].
A high risk of malignancy can also be seen in other types of congenital bone
marrow failure syndrome, and the molecular pathology will be similar and some-
times overlap between different subtypes. Genetic advances have already led to
improved diagnosis, particularly in cases wherein the presentation is atypical.
These advances may also lead to new treatments. In the meantime, it is important
to obtain accurate information on the incidence and natural history of each disor-
der in order to provide a more rational basis for the optimal provision of clinical
services [11].
Acquired aplastic anemia (AA) is an uncommon, life-threatening disorder in
childhood. Because of major advances in diagnosis and therapeutic approaches,
nowadays AA is a disease that results in long-term survival in more than 90% of
cases [12]. In recent years, an immune-mediated pathogenesis for AA has been sug-
gested because immunosuppressive therapy is usually effective and bone marrow
lymphocytes from patients can suppress normal marrow cells in vitro [13]. Numerous
studies have established that HSCT, especially bone marrow transplantation, from
HLA-matched donors is highly successful, with five-year survival rates of >90%
[14]. Unfortunately, horse antithymocyte globulin (ATG) is no longer available and
an alternative agent, rabbit ATG, is associated with a lower response rate for AA in
children [15].
Hemophagocytic lymphohistiocytosis (HLH) is characterized by fever and hepa-
tosplenomegaly associated with pancytopenia, hypertriglyceridemia, hypofibrino-
genemia, and infiltration of histiocytes with hemophagocytic activity. HLH can be
classified into two distinct forms, primary and secondary HLH; primary HLH
includes familial hemophagocytic lymphohistiocytosis and several immunodefi-
ciencies, while secondary HLH is usually associated with infections [especially
Epstein–Barr virus (EBV) infection], immune disorders, or malignancy (especially
non-Hodgkin lymphoma). In primary HLH, uncontrolled T lymphocyte activation
by impairing defects of genes, such as perforin (PRF1) and MUNC13-4, results in
large quantities of inflammatory cytokines that promote macrophage infiltration and
formation of the cytokine network [16]. The pathogenesis of secondary HLH, espe-
cially EBV-HLH, is not fully understood. In EBV-HLH, inflammatory cytokines
produced by EBV-infected T cells or natural killer cells are responsible for macro-
phage activation and subsequent development of HLH [17].
The treatment of HLH has been established in recent years. Immunochemotherapy
followed by HSCT can be used for primary HLH, while the clinical course of
Preface vii

­ BV-­HLH varies among patients and treatment that is more appropriate should be
E
organized for secondary HLH. Identification of all instigating mechanisms may
prompt the development of novel approaches, including gene therapy, for this disor-
der in the future.
Langerhans cell histiocytosis (LCH) is a rare clonal disorder characterized by the
proliferation of clonal CD1a-positive LCH cells in the skin, bone, lymph nodes, and
other organs. LCH cells are immature dendritic cells, and the JAG-mediated Notch
signaling pathway may play an important role in maintaining LCH cells in an
immature state [18]. More than half of the LCH patients have the oncogene BRAF
mutation, suggesting that LCH is a neoplastic disorder [19]. The outcome of LCH
varies depending on the extent of organ involvement, and treatment should be
planned according to the clinical subtype. In single-system disease, local corticoste-
roid therapy can be used for patients with single bones affected by lesions with no
CNS risk [20], while chemotherapy is available for those with multiple affected
bones or with CNS-risk lesions. In multisystem disease, on the other hand, chemo-
therapy including vincristine, cytarabine, and corticosteroid has been used with
great success, with a mortality rate of only 10% [21].
Despite their successful treatment, LCH patients often develop long-term
sequelae, including diabetes insipidus, orthopedic problems, hearing loss, neuro-
logical problems, growth-hormone deficiency, pulmonary fibrosis, and biliary cir-
rhosis [22]. The incidence of these sequelae increases with follow-up time. Further
novel therapeutic measures are required to reduce these permanent sequelae.
Hematological disorders in children are usually rare, but clarifying their patho-
genesis has progressed and appropriate treatment has been established in the recent
years. More effective and less toxic therapies for these hematological disorders need
to be developed in the near future.

Ehime, Japan Eiichi Ishii

References

1. Greaves MF. Infant leukemia biology, aetiology and treatment. Leukemia.

1996; 10: 372–7.
2. Greaves MF, Maia AT, Wiemels JL, Ford AM. Leukemia in twins: lessons in
natural history. Blood. 2003; 102: 2321–33.
3. Wiemels JL, Cazzaniga G, Daniotti M, et al. Prenatal origin of acute lympho-
blastic leukaemia in children. Lancet. 1999; 354: 1499–503.
4. Panzer-Grümayer ER, Fasching K, Panzer S, et al. Nondisjunction of chromo-
somes leading to hyperdiploid childhood B-cell precursor acute lymphoblastic
leukemia is an early event during leukemogenesis. Blood. 2002; 100: 347–9.
5. Wiemels JL, Xiao Z, Buffler PA, et al. In utero origin of t(8;21) AML1-ETO
translocations in childhood acute myeloid leukemia. Blood. 2002; 99: 3801–5.
6. Ahmed M, Sternberg A, Hall G, et al. Natural history of GATA1 mutations in
Down syndrome. Blood. 2004; 103: 2480–9.
viii Preface

7. Wu Z, Eguchi-Ishimae M, Yagi C, et al. HMGA2 as a potential molecular target

in KMT2A-AFF1-positive infant acute lymphoblastic leukaemia. Br J
Haematol. 2015; 171: 818–29.
8. Rivers A, Slayton WB. Congenital cytopenias and bone marrow failure syn-
dromes. Semin Perinatol. 2009; 33: 20–8.
9. Khincha PP, Savage SA. Neonatal manifestations of inherited bone marrow
failure syndromes. Semin Fetal Neonatal Med. 2016; 21: 57–65.
10. Alter BP, Giri N, Savage SA, et al. Malignancies and survival patterns in the
National Cancer Institute inherited bone marrow failure syndromes cohort
study. Br J Haematol. 2010; 150: 179–88.
11. Dokal I, Vulliamy T. Inherited bone marrow failure syndromes. Haematologica.
2010; 95: 1236–40.
12. Hartung HD, Olson TS, Bessler M. Acquired aplastic anemia in children.
Pediatr Clin North Am. 2013; 60: 1311–36.
13. Kagan WA, Ascensao JA, Pahwa RN, et al. Aplastic anemia: presence in human
bone marrow of cells that suppress myelopoiesis. Proc Natl Acad Sci U S A.
1976; 73: 2890–4.
14. Samarasinghe S, Steward C, Hiwarkar P, et al. Excellent outcome of matched
unrelated donor transplantation in paediatric aplastic anemia following failure
with immunosuppressive therapy: a United Kingdom multicenter retrospective
experience. Br J Haematol. 2012; 157: 339–46.
15. Yoshimi A, Niemeyer CM, Fuhrer MM, et al. Comparison of the efficacy of
rabbit and horse antithymocyte globulin for the treatment of severe aplastic
anemia in children. Blood. 2013; 121: 860–1.
16. Ishii E, Ueda I, Shirakawa R, et al. Genetic subtypes of familial hemophago-
cytic lymphohistiocytosis: correlations with clinical features and cytotoxic T
lymphocyte/natural killer cell functions. Blood. 2005; 105: 3442–8.
17. Kogawa K, Sato H, Asano T, et al. Prognostic factors of Epstein-Barr virus-­
associated hemophagocytic lymphohistiocytosis in children: Report of the
Japan Histiocytosis Study Group. Pediatr Blood Cancer. 2014; 61: 1257–62.
18. Morimoto A, Oh Y, Shioda Y, et al. Recent advances in Langerhans cell histio-
cytosis. Pediatr Int. 2014; 56: 451–61.
19. Badalian-Very G, Vergilio JA, Degar BA, et al. Recurrent BRAF mutations in
Langerhans cell histiocytosis. Blood. 2010; 116: 1919–23.
20. Morimoto A, Ishida Y, Suzuki N, et al. Nationwide survey of single-system
single site Langerhans cell histiocytosis in Japan. Pediatr Blood Cancer. 2010;
54: 98–102.
21. Morimoto A, Shioda Y, Imamura T, et al. Intensified and prolonged therapy
comprising cytarabine, vincristine and prednisolone improves outcome in
patients with multisystem Langerhans cell histiocytosis: Results of the Japan
Langerhans Cell Histiocytosis Study Group-02 Protocol Study. Int J Hematol.
2016;104:99–109.
22. Haupt R, Nanduri V, Calevo MG et al. Permanent consequences in Langerhans
cell histiocytosis patients: a pilot study from the Histiocyte Society-Late Effects
Study Group. Pediatr Blood Cancer. 2004; 42: 438–44.
Contents

Part I Hematopoiesis

1 Hematopoietic Stem Cells: The Basis of Normal

and Malignant Hematopoiesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   3
Mariko Eguchi, Minenori Eguchi-Ishimae, and Eiichi Ishii

Part II White Blood Cell Disorders

2 Acute Lymphoblastic Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   33

Daisuke Tomizawa and Nobutaka Kiyokawa
3 Acute Myeloid Leukemia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   61
Souichi Adachi, Akitoshi Kinoshita, Daisuke Tomizawa,
Takashi Taga, and Hiroyuki Takahashi
4 Myelodysplastic Syndrome (MDS) and Juvenile
Myelomonocytic Leukemia (JMML) . . . . . . . . . . . . . . . . . . . . . . . . . . .   87
Daisuke Hasegawa and Atsushi Manabe
5 Neutropenia (In Infancy and Childhood). . . . . . . . . . . . . . . . . . . . . . . . 109
Masao Kobayashi, Yoko Mizoguchi, Shuhei Karakawa,
Satoshi Okada, and Hiroshi Kawaguchi

Part III Red Blood Cell Disorders

6 Childhood Aplastic Anemia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Hiroshi Yagasaki
7 Inherited Bone Marrow Failure Syndrome, TAM. . . . . . . . . . . . . . . . . 145
Etsuro Ito, Kiminori Terui, and Tsutomu Toki

ix
x Contents

Part IV Platelet and Coagulation Disorders

8 Immune and Inherited Thrombocytopenia in Children. . . . . . . . . . . . 173

Masue Imaizumi
9 Pathogenesis and Treatment of Hemophilia. . . . . . . . . . . . . . . . . . . . . . 189
Keiji Nogami and Midori Shima
10 Thrombotic Disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Shouichi Ohga and Masataka Ishimura

Part V Histiocytic Disorders

11 Langerhans Cell Histiocytosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Akira Morimoto
12 Primary Hemophagocytic Lymphohistiocytosis. . . . . . . . . . . . . . . . . . . 247
Takahiro Yasumi, Hirofumi Shibata, Saeko Shimodera,
and Toshio Heike
13 Hemophagocytic Lymphohistiocytosis, Secondary . . . . . . . . . . . . . . . . 263
Ryu Yanagisawa and Yozo Nakazawa
 Part I
Hematopoiesis
Chapter 1
Hematopoietic Stem Cells: The Basis
of Normal and Malignant Hematopoiesis

Mariko Eguchi, Minenori Eguchi-Ishimae, and Eiichi Ishii

Abstract Hematopoietic stem cells (HSCs), which are responsible for producing
all blood cell types, first appear in the early stage of embryonic development and
transit through several different tissues, including the yolk sac, aorta-gonad-­
mesonephros (AGM) region, placenta, and fetal liver, before colonizing in the bone
marrow where they reside throughout the individual’s life. HSCs, characterized by
the ability to self-renew and generate all types of blood cells, are supported by their
specific environment called niches and depend on many developmental signaling
pathways, molecules, and cytokines for their generation, maintenance, and expan-
sion. Any disruption in this well-balanced system may cause aberrant HSC produc-
tion, leading to malignant hematopoiesis. Leukemic stem cells (LSCs), originally
identified using xenograft models of acute myeloid leukemia (AML), are a distinct
cell population that can initiate leukemia in immunodeficient mice. LSCs are
thought to emerge from HSCs or hematopoietic progenitors after obtaining multiple
genetic changes that provide aberrant growth advantage and self-renewal ability.
The emergence of LSCs is a multi-step event, including genetic diversification and
clonal selection, resulting in genetic heterogeneity among leukemic cells. LSCs
generally exist in the immature CD34+CD38− leukemic population in most cases of
AML and share some features with normal HSCs. However, recent studies have
shown that in acute lymphoblastic leukemia (ALL), LSCs exist in B-lineage-­
committed progenitors expressing CD19. In contrast to that in AML, in which LSCs
generate leukemic cells in a hierarchical order with LSCs at the top, leukemia prop-
agation in ALL is better explained by a stochastic model.

M. Eguchi (*) • M. Eguchi-Ishimae • E. Ishii

Department of Pediatrics, Ehime University Graduate School of Medicine,
Shitsukawa, Toon 791-0295, Ehime, Japan
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2017 3

E. Ishii (ed.), Hematological Disorders in Children,
DOI 10.1007/978-981-10-3886-0_1
4 M. Eguchi et al.

1.1 Introduction

Maintenance of the hematopoietic system relies on hematopoietic stem cells (HSCs),

which have the ability to produce entire blood cells in a hierarchical manner and are
simultaneously capable of self-renewing. HSCs are a very rare population among bone
marrow cells and usually reside in the stem cell niche, a specific microenvironment that
regulates stem cell fate. Well-regulated hematopoietic regulatory networks maintain
the differentiation system of HSCs and homeostasis of the blood cell production. Any
change that disturbs this balance in the maintenance and function of normal HSCs can
cause aberrant HSC production, resulting in malignant hematopoiesis.
Malignant hematopoiesis that is observed in leukemia is also sustained by the
presence of stem cells called leukemic stem cells (LSCs). Although, like that of
normal HSCs, self-renewing ability is one of the hallmarks of LSCs; the latter pos-
sesses aberrant growth advantage over normal HSCs, rapidly dominating the bone
marrow space.
LSCs were originally identified in acute myeloid leukemia (AML), using xeno-
graft models, as a distinct cell population that can initiate leukemia in immunodefi-
cient mice [1]. LSCs arise from pre-LSCs after obtaining genetic changes that
confer self-renewal ability and some growth advantages. However, these genetic
changes are not sufficient to fully transform pre-LSCs to cause overt leukemia.
Since the existence of LSCs has been proven, identification of additional genetic
changes that transform HSC into pre-LSCs and pre-LSCs into LSCs, and the identi-
ties of LSCs, has been intensively studied because LSCs and pre-LSCs are the most
suitable targets of anticancer therapies [2–5].
In this chapter, recent knowledge on HSCs and LSCs and the role of genetic
changes in transforming normal HSCs into malignant LSCs will be described,
mainly focusing on childhood leukemia.

1.2 Hematopoietic Stem Cells in Normal Hematopoiesis

1.2.1 Shifting Site of Hematopoiesis

Although blood cells can be regenerated throughout the individual’s life, the site for
hematopoiesis, namely, residence of HSCs, shifts during embryonic development
(Fig. 1.1). In mammals, hematopoiesis is recognized initially in the yolk sac at the
very beginning of the fetal development. This initial hematopoiesis is termed as
“primitive hematopoiesis,” giving rise to circulating red blood cells that provide oxy-
gen to tissues, necessary for embryonic growth. This primitive hematopoiesis does
not produce HSCs that have the ability to reconstitute hematopoiesis in the later stage
[6] and is rapidly replaced by “definitive” hematopoiesis, which is responsible for the
continuous production of all the mature blood cells throughout the adult life [7, 8].
Around embryonic day (E) 11 in the mouse fetus, this definitive hematopoiesis com-
mences in the embryo proper in the aorta-gonad-mesonephros (AGM) region, an
area surrounding the dorsal aorta (Fig. 1.1). Clusters of hematopoietic cells appear in
1 Hematopoietic Stem Cells: The Basis of Normal and Malignant Hematopoiesis 5

Birth
Ventral
mesoderm
Yolk sac

AGM

Placenta

Fetal liver
Bone marrow

Mouse

5 10 15 (days)
Human

10 20 30 40 70 (days)

Fig. 1.1 Site of hematopoiesis during fetal development. Hematopoietic stem cells (HSCs) are
thought to derive from the ventral mesoderm. The initial blood production termed as “primitive
hematopoiesis” occurs in the yolk sac, producing red blood cells to supply oxygen for rapidly
growing tissue. This primitive hematopoiesis is rapidly replaced by adult-type hematopoiesis
termed as “definitive hematopoiesis,” which produces HSCs that have the ability to give rise to all
hematopoietic lineages. Definitive hematopoiesis, occurring in aorta-gonad-mesonephros (AGM)
region, is also found in the placenta, where it moves from the fetal liver to the bone marrow to
generate hematopoietic cells throughout life

the ventral wall following the formation of the aorta tube. High frequencies of HSCs
were subsequently detected in the other sites such as umbilical arteries, indicating
that the major arteries of the embryo are important sites for the emergence of defini-
tive HSCs [9]. In addition, placenta was also shown to harbor adult-repopulating
HSCs, beginning at E11 and expanding until E12.5–E13.5 of mouse embryo, con-
taining more HSCs than the AGM [10–12]. Subsequently, accompanying the migra-
tion of HSCs, the site of definitive hematopoiesis moves onto the fetal liver, which
serves as the major hematopoietic organ for the rest of the fetal development. During
the fetal period, the HSC population expands in the fetal liver [13], and, finally, the
site of hematopoiesis shifts to the bone marrow shortly before birth, where blood
cells are produced during the entire life of the individual. Although HSCs in the fetal
liver are proliferative, giving rise to both myeloid and B-lymphoid lineage cells,
HSCs in the bone marrow seem to lose their proliferation tendency, to be a relatively
quiescent HSC population [14–17].

1.2.2 Identification of HSCs

HSCs demonstrate long-lasting self-renewal ability and differentiate into all types
of hematopoietic cells. Self-renewal is the ability of a cell to produce itself by cell
division, whether symmetrical or asymmetrical, and this self-renewal ability is the
essential characteristic of all stem cells, including HSCs.
HSCs are an extremely rare population, existing only one in a million bone mar-
row cells [18]. Owing to stem cell’s self-renewal and reconstruction ability, in vivo
6 M. Eguchi et al.

transplantation into conditioned hosts has been applied for the characterization of
these stem cells. HSCs have shown self-renewal and differentiation capacity even in
a single-cell level [19, 20]. In mice, HSC activity is evaluated and quantified by
transplanting cells into myeloablative (usually by irradiation), syngeneic recipients.
Availability of severe combined immunodeficient (SCID) mice made it possible to
apply this transplantation strategy to human materials such as bone marrow and
cord blood. Development of severe immunodeficient nonobese diabetic/SCID
(NOD/SCID) mice and its subsequent generation of NOD/SCID/IL-2Rg null (NOG
or NSG) mice allowed engraftment of a very small number of cells and greatly
enhanced the isolation of HSCs [21–24]. Cells that generate all lineages but are
capable of engrafting only hematopoietic lineages transiently are defined as short-­
term HSCs (ST-HSCs) or multipotent progenitors (MPPs), whereas HSCs that
repopulate hematopoietic lineages beyond 12 weeks are defined as long-term HSCs
(LT-HSCs) [20].
Lineage and differentiation stage-specific expression of surface proteins is
notable in hematopoietic cells; therefore, HSCs and progenitor cells are defined
by the cell surface phenotype, as described in Fig. 1.2 [20]. Prospective isolation
of highly purified HSCs is made possible using monoclonal antibodies for these
cell surface proteins and by precise fluorescence-activated cell sorting (FACS)
[25]. Mouse HSCs were initially isolated as lineage marker-negative (Lin−),
c-kit+, and Sca-1+ (LSK) population [26, 27]. By extensively studied xenograft
models, it was identified that a proportion in CD34− LSK cells, which show
CD150+CD48−SLAM+ phenotype, possesses LT-HSCs activity in mouse [25, 28].
By contrast, CD34+ cells, which are found in less than 5% of hematopoietic cells,
are enriched for HSCs and progenitors in humans. While phenotype of human
HSCs was described as Lin−CD34+CD38−CD90(Thy1)+CD45RA− [21, 29–31],
Lin−CD34+CD38−CD90−CD45RA−, which has lost CD90 expression, represents
its descendant multipotent progenitors [32]. In addition, a recent study showed
an essential role of CD49f in separation of HSCs [23]. Human HSCs are cur-
rently thought to be phenotypically Lin−CD34+CD38−CD90+CD45RA−CD49f+.
When HSCs give rise to MPPs, first, CD49f expression is lost, and then they dif-
ferentiate into immature progenitors and mature hematopoietic cells with spe-
cific cell surface phenotype under the influence of various transcriptional controls
(Fig. 1.2) [20].
Within the CD34+CD38− population in human, CD90−CD45RA− cells are mul-
tipotent progenitors (MPP) with transient multilineage engraftment capacity [32].
CD90−CD45RA+ cells show differentiation to all lymphoid lineages as well as some
of the myeloid lineage without self-renewal ability, functionally similar to murine
lymphoid-primed multipotent ­progenitors (LMPP) [33] corresponding to multi-
lymphoid progenitor (MLP) in human.
1 Hematopoietic Stem Cells: The Basis of Normal and Malignant Hematopoiesis 7

T
ETP cells

CD45RA+
CD45RA+ CD10+
GATA3
CD45RA+ FLT3+ CD19+
FLT3+ CD10+
CD10+ CD7– B
proB cells
PU.1 E2A PAX5
MLP B/NK
CD45RA– CD45RA– E2A
CD90+ CD90–
CD49f+ CD49f– NK
cells
FLT3+ FLT3+ GATA2

HSC MPP
Dendritic
cells
RUNX1
CD45RA +
TAL1
FLT3+
MLL Mono-
CD10–
ETV6 CD7– cytes
GATA2
BMI1
GMP
GFI1 Granulo-
PU.1 C/EBPα cytes
CMP CD45RA–
FLT3–
GATA1 CD10–
CD45RA- Erythro-
CD7–
FLT3+ cytes
CD10–
CD7– MEP GATA1

Megakaryo-
cytes

– – –
Lin CD34+CD38 Lin CD34+CD38+ Lin+

Fig. 1.2 Hematopoietic hierarchies and their regulators in human. Using specific antibodies and
applying precise fluorescence-activated cell sorting (FACS) technique, HSCs, progenitor cells, and
differentiated hematopoietic cells can be defined by cell surface phenotypes as shown. Transcription
factors and signaling pathway known to be involved in hematopoietic cell differentiation are
depicted. Hematopoietic development is blocked in the absence of many of these factors, as deter-
mined by gene knockout studies. MPPs multipotent progenitors, MLP multi-lymphoid progeni-
tors, ETPs earliest thymic progenitors, CMPs common myeloid progenitors, GMPs
granulocyte-macrophage progenitors, MEP megakaryocyte/erythroid progenitors, Lin cocktail of
cell surface markers for all terminally differentiated hematopoietic cells. Modified from [20]

1.2.3  ranscriptional Control of HSCs: Hematopoietic

T
Hierarchies and Lineage Determination

Blood cells are produced in developmental hierarchy, with multipotent HSCs at the apex
and terminally differentiated cells at the bottom [20]. Key regulators controlling hemato-
poietic cell maintenance and differentiation differ in various stages of hematopoietic cell
development, and many of these regulators are known to be transcription factors that
play a central role in determining cell fate intracellularly. To date, more than 50
8 M. Eguchi et al.

transcription factors have shown to affect HSCs survival and differentiation, forming
transcriptional hierarchies active during hematopoietic differentiation (Fig. 1.2). Through
the analysis of conventional gene knockout mice, some transcription factors have proven
to play a crucial role in each stage of hematopoietic cell development. These crucial
transcription factors have been often identified as targets of leukemia-specific gene
abnormalities such as chromosome translocations, resulting in fusion gene formation.
HSCs and progenitor cells also respond to some external signals by activating a
series of signaling cascades inside the cells, which may also activate other signaling
pathways to form complex networks, finally activating lineage determinant tran-
scription factor(s) [34, 35]. External signals, which work as determinants for stem
cell fate decision, include interactions with surrounding cells through cell adhesion
molecules and secreted molecules such as cytokines. Thus, microenvironments in
the stem cell niche are critical for regulating the fate of HSCs [36, 37].
Growth factor receptor FLK1, as well as its ligand VEGF, and transcription fac-
tor TAL1 (SCL), with its protein partner LMO2, are essential factors for HSC for-
mation and function in the early hematopoietic stage of development such as in the
embryonic yolk sac. TAL1 and its protein partner LMO2 are necessary for the
development of both primitive and definitive hematopoiesis because they specify
blood lineage from the hemangioblast [38]. In AGM and fetal liver stage, AML1
(RUNX1), GATA2, GATA3, LMO2, TEL (ETV6) transcription factors, histone
methyltransferase MLL (KMT2A), and KIT with its ligand SCF (KITLG) are
required for hematopoietic cell survival, proliferation, and differentiation [35, 39–
42]. ETS transcription factor TEL initiates adult hemangioblast program in the dor-
sal lateral plate mesoderm by switching on the FLK1 ligand VEGFA, where ETS
factor FLI1 together with GATA2 ensures the expression of FLK1 [43].
The pluripotent HSCs generate mature hematopoietic cells via multipotent pro-
genitors and committed precursors. Key regulators that control hematopoietic cell
maintenance and differentiation differ in various stages of hematopoietic cell devel-
opment (Fig. 1.2). During fate decision of hematopoietic cells, key lineage-restricted
factors promote their own lineage differentiation and simultaneously act against
factors that drive cells into other linages [35]. GATA1 promotes erythroid, mega-
karyocytic, and eosinophil differentiation, whereas PU.1 (SPI1) promotes myeloid
differentiation in common myeloid progenitors (CMP). This GATA1 and PU.1
antagonism promotes cell differentiation to their specific lineage. This kind of lin-
eage determination is also known between EKLF and FLI1 for erythroid and mega-
karyocytic lineages in megakaryocyte/erythroid progenitors (MEP), GFI1 and PU.1
for n­ eutrophil versus monocyte differentiation in granulocyte/macrophage progeni-
tors (GMP), and C/EBP and FOG1 for eosinophil and multipotent cell fate [35].
For B-cell development, PAX5 is required for B-cell commitment [44], and other
B-cell transcription factors such as E2A and EBF1 specify gene activation toward
the B-lineage. In T-cell development, NOTCH1 commits cells to T-cell lineage,
whereas other transcription factors such as GATA3 support T-cell differentiation
under NOTCH signaling. Further, GATA3 and T-bet (TBX21) antagonize in T-cell
differentiation to promote Th1 and Th2 cells [45]. It is now known that these lin-
eages can be reprogrammed upon expression of critical transcription factors such as
GATA1, C/EBP, and GATA3 [35].
1 Hematopoietic Stem Cells: The Basis of Normal and Malignant Hematopoiesis 9

Notably, in HSCs and progenitor cells, alternative cell fate potentials are pre-
served by keeping simultaneous low expression of key genes for several lineages
[46]. Differentiation of HSCs to certain lineage takes place by selecting and stabiliz-
ing the expression of a subset of genes, which are essential for that direction from a
wide range of expression of lineage-oriented genes and, at the same time, down-
regulating expression of other irrelevant genes under the effects of selected domi-
nantly expressed gene(s). Thus, to maintain a hierarchy of hematopoietic
differentiation from HSCs, precise regulation of transcription factors expression is
critical.
This multiple co-expression of lineage-oriented genes, such as transcription fac-
tors in HSCs and progenitor cells, is essential for maintaining multipotentiality and
flexibility in cell fate decisions. Loss of expression of one lineage determinant tran-
scription factor and upregulation of another lineage-specific gene in already lineage-­
committed cells can induce lineage conversion in normal hematopoiesis [46, 47]
and also in leukemogenesis [48], indicating lineage plasticity.

1.2.4 Microenvironment of HSCs: The Niche

HSCs undergo a wide range of cell fate, such as self-renewal, quiescence, and dif-
ferentiation into all mature blood cells. Fate of HSCs is tightly regulated by external
stimuli provided by the HSC microenvironment, known as niche, and by intercel-
lular regulatory programs. The adult bone marrow niche is best studied among vari-
ous stages of fetal and adult hematopoietic development.
The major components of the niche that support HSC fate in the bone marrow are
the osteoblastic and vascular niches, which lie in close proximity and are thought to
influence HSCs in a cooperative manner (Fig. 1.3) [49].
HSCs exist adjacent to the osteoblasts, which align bone surface of the bone mar-
row, and are under the regulation of bone morphogenetic protein (BMP)—the osteo-
blastic niche. Various studies using mouse models showed that osteoblastic niche
maintains HSCs thorough various signaling molecules, including Notch1, Tie-2,
N-cadherin, and Mpl [49]. HSCs are also found to migrate to blood vessels within the
bone marrow—the vascular niche. LT-HSCs in mice are defined through SLAM
markers (CD150+, CD41−, CD48−, cKit+, Sca-1+, lineage-) and lie adjacent to the
bone marrow sinusoidal endothelial cells that express Notch ligands, Jagged-1 and
Jagged-2, and KIT ligand SCF. The bone marrow also contains other stromal cells
that support HSCs and hematopoietic progenitors by producing cytokines such as
SCF. Cells surrounding the vasculature, CXC chemokine ligand 12 (CXCL12)-
abundant reticular (CAR) cells, and Nestin+ mesenchymal stem cells have been iden-
tified as the independent niche components (the perivascular niche). CAR cells exert
their niche functions through SCF and CXCL12 secretion into the microenvironment
[49–51], regulating the proliferation of HSCs. Nestin+ cells possess the ability of
multilineage differentiation into various mesenchymal cell lineages, including osteo-
blasts. Nestin+ cells highly express chemokine/cytokines such as CXCL12, SCF,
angiopoietin-1, and osteopontin that are involved in the regulation of HSCs with
10 M. Eguchi et al.

l
cel
CXCL12

scu +
lar
r
SCF

per Lep
TPO SCF
TPO

iva

BM sinusoidal endothelial cells

Osteopontin

CXCL12
Jagged1/2
HSC
Osteoblasts

N-Cadherin
CXCL12
SCF
Jagged1/2 Ang-1
TGF-β
Jagged1
Ang-1
CXCL12 CXCL12
CXCL12

cell
Ang-1 SCF
Ang-1

CAR
Nes
t
periv in+ (NG
TGF-β ascu 2+
lar c )
ell

BM arteriole
Schwann cell
endothelial cells
(Sympathetic nerve fiber)

Fig. 1.3 Stem cell niche in adult bone marrow. The major components of the niche that support
HSCs cell fate in the bone marrow are the osteoblastic and vascular niches that lie in close proxim-
ity and are thought to influence HSCs in a conjoined manner. HSCs exist adjacent to the osteo-
blasts, which align bone surface of the bone marrow, and are under the regulation of bone
morphogenetic protein (BMP) (the osteoblastic niche). HSCs are also found to have migrated to
blood vessels within the bone marrow—the vascular niche. Bone marrow also contains other stro-
mal cells that support HSCs and hematopoietic progenitors by producing cytokines such as KIT
ligand SCF. CAR cell CXCL12-abundant reticular cell, Ang-1 angiopoietin-1, OPN osteopontin

other niche components. Bone marrow macrophages, non-myelinating Schwann

cells [52], and extracellular matrix protein such as tenascin-C (TNC) [53] have been
recently reported to possess niche function in the bone marrow.

1.3 Stem Cells in Abnormal Hematopoiesis

1.3.1  isruption of Transcription Network Leads

D
to Hematopoietic Malignancy

The transcription factors that are critical for hematopoiesis are mainly various
classes of DNA-binding proteins, and perturbation of the function of these proteins
is known to cause hematopoietic defect and malignancies. Many of these transcrip-
tion factors involved in hematopoiesis are known to be disrupted by chromosome
translocations, deletions, or mutations, resulting in defective normal cell