Hematopoiesis: Basic Concepts

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Hematopoiesis Basic Concepts

• Blood cell production is highly regulated to
  maintain circulating cell numbers within
  relatively constant levels and to respond
  rapidly to conditions requiring extra cells
• Continuous and prodigious production in the
  adult marrow and lymphoid tissues of new, mature
  blood cells from more primitive precursors
  1011 per day (lt 1010 RBCs/hour, 108- 109
  WBCs/hour)
• Normal hematopoiesis involves an exquisitely
  regulated balance between self-renewal, terminal
  differentiation, migration, and cell death.
• Replacement of hematopoietic cells is achieved
  via differentiation of primitive pluripotent
  stem cells through a series of cell divisions
  (usually the last 3 to 5 cell cycles exhibit
  terminal differentiation programs of most
  lineages)

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Hematopoiesis Basic Concepts

• All arise from a single cell type the
  hematopoietic stem cell (HSC).
• HSCs are pluripotent (can give rise to
  differentiated blood cells of all lineages T
  B NK lymphocytes, erythrocytes, mast cells,
  megakaryocytes platelets, eosinophils,
  basophils, neutrophils, monocytes/macrophages,
  DCs, etc.). Lack the markers of
  differentiated blood
• HSCs are rare, mainly quiescent,
  undifferentiated cells that on occasion produce
  by mitosis 2 kinds of progeny
• more stem cells (self-renewal)
• cells that begin to differentiate along the
  various paths to terminally differentiated
  hematopoietic lineages
• Path of differentiation is generally regulated
  by the need for more of that particular type of
  blood cell, and is controlled by the appropriate
  cytokines and hormones and growth factors,
  colony stimulating factors (CSFs)

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lymphocytes terminally diff. can undergo
further division (i.e. memory cells)
Granulocytes terminally diff. no further
division
anuclear
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Model of Stem Cell Decisions HSC will choose
one of 2 pathways self-renewal (maintains
primitive state) or differentiation (driven
toward a more mature state)
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lymphocytes terminally diff. can undergo
further division (i.e. memory cells)
Granulocytes terminally diff. no further
division
anuclear
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Lineage diagram outline of how hematopoietic
cells are increasingly restricted in the types of
progeny to which they can give rise as
differentiation proceeds
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The Hematopoietic Developmental Hierarchy

• During the life of an individual, 2 separate
  hematopoietic systems
• Both arise during embryonic development but
  only one persists in adult
• PRIMITIVE HEMATOPOIESIS system derived from
  the extra-
• embryonic YOLK SAC consists mainly of
  nucleated erythroid cells
• that carry oxygen to the developing embryonic
  tissues an early
• circulatory system.
• As the embryos size increases, primitive
  system superseded by
• DEFINITIVE HEMATOPOIETIC system, which
  originates in the
• embryo itself and continues throughout adult
  life. Is made up of all
• adult blood cell types including erythrocytes,
  and cells of myeloid
• and lymphoid lineages.

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Sites of hematopoiesis During development,
hemato- poiesis is initiated sequentially in
different tissues
PRIMITIVE HEMATOPOIESIS occurs in the
YOLK SAC at mouse embryonic day 7.5 (E7.5),
and probably starts 4 weeks in humans
Primitive hematopoiesis is characterized by
the production of fetal erythrocytes (nucleated)
and the lack of lymphocytes and myeloid cells
except for macrophages.
For many years the YS was assumed to be the
primary site of formation of the HSCs that
migrate to and colonize the fetal liver and
subsequently the bone marrow.
d. 31-34
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1st DEFINITIVE multipotent hematopoietic stem
cells are generated within the embryonic AGM
region of the para-aortic splanchnopleuric
mesoderm (d. 30-37 human, and late d. 10/early d.
11 mice) DEFINITIVE HEMATOPOIESIS HSCs can
restore long-term multilineage hematopoiesis when
transplanted into adult myeloablated recipients,
and generates enucleated erythrocytes, various
kinds of myeloid and lymphoid cells, and
long-term reconstituting hematopoietic stem cells
(LTR-HSCs) Upon transplantation into NOD-SCID
mice, cultured AGM HSC cells showed lymphomyeloid
reconstitution YS cells were only capable of
contributing to the myeloid lineage (Tavian et
al., 2001).
CD34 cluster of cells (also CD45
hemato-specific)
AGM region aorta-gonad-mesonephros
ventral floor of dorsal aorta (also umbilical
and vitilline arteries _at_ connection with dorsal
aorta)
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Source of these first definitive HSCs??
Shared expression patterns of a number of
molecules by both intra-aortic cluster HSCs and
underlying endothelial cells supports the
existence of a HEMANGIOBLAST or primitive
mesenchymal endothelial-like cell with
hematogenic potential that lies on the ventral
floor of the dorsal aorta AKA hemogenic
endothelium Example VEGF receptor (VEGF R)
neither HSCs nor blood vessel epithelium develop
without the ligand, VEGF. Also, angiopoietin
produced by early blood cells to induce blood
vessels to grow in the vicinity. Recent ID of a
morphologically distinct layer of cells
resembling a stromal layer underlying the ventral
floor of the dorsal aorta in the AGM suggests
that this region could represent a
microenvironment or niche supporting HSC
development (Marshall et al., 1999, Dev. Dyn.
215139)
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The fetal liver is the main organ for
hematopoiesis during fetal life
After about d. 37 (5 weeks), HSCs from the AGM
begin to colonize the fetal liver, and at 6 weeks
hematopoiesis (definitive) takes place in the
fetal liver until bone marrow is
formed Throughout fetal life, the liver is the
chief organ for production of myeloid and
erythroid cells.
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Fetal liver HSCs seed thymus, bone marrow and
spleen
At about 8 wks, liver HSCs differentiate in the
thymus to mature T lymphocytes (10 wks) which
populate fetal lymph nodes, spleen and gut by 12
wks and other peripheral lymphoid tissue by weeks
14-15. Bone marrow (BM) is seeded by liver HSCs
by 8 weeks. B-lymphopoiesis takes place in liver
_at_ 7 wks., then shifts to bone marrow.
Hematopoiesis from fetal BM is mainly myeloid and
contributes only minimally to the blood pool
throughout fetal life. After birth, BM becomes
main hematopoietic organ. Many HPC and HSC in
circulation during fetal life and immediately
after birth 24-48 hours after birth they
disappear due to lodgement in BM.
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Bone marrow site of pre- and peri-natal,
childhood, and adult hematopoiesis
During fetal growth, hematopoiesis takes place
in all bony cavities (axial and appendicular
skeleton) as well as in liver and spleen. Prior
to birth, splenic and hepatic hematopoiesis
disappear, and gradually thereafter hematopoietic
tissue (red marrow) is replaced by adipocytes
(yellow marrow) beginning in the distal bones and
retracting to the adult pattern by age ten.
Yellow marrow can be reactivated by an increased
demand for blood cells, i.e., anemia or blood
loss, but does not normally produce blood
cells In the adult, hematopoietic marrow is
confined to the axial skeleton (sternum,
vertebrae, iliac/pubic bones, ribs) and proximal
portions of the humerus and femur.
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Bone marrow, contd
Bone marrow is specially construed to support the
proliferation, differentiation, and maintenance
of hematopoietic cells Honeycombed latticework
of venous sinuses large, thin-walled
veins Endothelial cells lining the marrow sinuses
are bounded by STROMAL CELLS that generate an
EXTRACELLULAR MATRIX that mechanically support
hematopoietic cells and vasculature provide a
nurturing microenvironment for hematopoiesis
hematopoietic colonies Adjacent cells in marrow
endothelial cells, fibroblasts, adipocytes,
osteoblasts, and macrophages and reticular
connective tissue. Close contact between
hematopoietic cells and these cells, especially
the stroma facilitates transmission of
proliferative signals or diffusion of locally
produced cytokines Maturing blood cells can
enter the circulation through openings in the
vascular sinuses (megakaryocytes erythroblasts
clustered against sinuses) usually go first to
other hematopoietic tissues for further
maturation Distinct microenvironments per
lineage, i.e., erythropoietic, eosinophilic, etc.
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Bone marrow, contd

• BONE MARROW STROMAL CELLS Stroma derived from
  Greek word meaning mattress or bed.
• Large spread-out cells that appear to provide a
  bed for hematopoietic cells
• Derived from mesenchymal stem cells and are not
  of hematopoietic origin.
• Express class I histocompatibility antigens,
  but lack the hematopoietic cell surface marker
  CD45
• Attempts to classify stromal cells by
  morphology have resulted in a multitude of
  descriptive names, including adipocytes,
  pre-adipocytes, smooth-muscle-like,
  fibroblastoid, endotheloid, epitheloid, blanket,
  and reticulum cells.
• So far, there are no definitive markers that
  predict whether a stromal cell line will support
  stem cells. Neither the morphology of stromal
  cells, nor the known cell surface antigens, nor
  the patterns of cytokines production are
  predictive of support function.

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From NIH Stem Cell Primer at http//www.nih.gov/ne
ws/stemcell/scireport.htm
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The hematopoietic stem cell

• All hematopoietic cells arise from a single
  type of cell the hematopoietic stem cell
  (HSC).
• HSCs are pluripotent (can give rise to
  differentiated blood cells of all lineages T
  B NK lymphocytes, erythrocytes, mast cells,
  megakaryocytes platelets, eosinophils,
  basophils, neutrophils, monocytes/macrophages,
  DCs, etc.)
• HSCs are rare (1 in every 105 nucleated cells
  in adult bone marrow)
• Are mainly quiescent, undifferentiated cells
  that on occasion produce by mitosis 2 kinds of
  progeny (asymmetric division)
• more stem cells (HSCs have a limited ability to
  self-renew)
• progenitor cells that can undergo further
  divisions and become progressively more
  differentiated and more restricted in their
  capacity for self renewal

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The hematopoietic stem cell, contd

• Express the CD34 surface marker, but lack
  differentiation markers of more mature cells
  (such as CD38). Often express the receptor for
  vascular endothelial growth factor (VEGF). Also
  express Sca-1.
• Because they are mitotically quiescent, can
  also be selected on their ability to survive in
  presence of 5 fluorouracil (5FU) which kills
  proliferating cells.
• In vivo, HSCs (as well as the full hierarchy
  of hematopoietic progenitor cells) are
  maintained throughout adult life constant, very
  slow state of turnover.
• In culture, in the absence of the appropriate
  regulatory signals, tend to lose ability to
  self-renew and become lineage-restricted
• In vivo, a highly regulated microenvironment
  ensures that pluripotent stem cells are
  generated in sufficient numbers and at the
  appropriate developmental stage to seed
  subsequent hematopoietic tissues

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Functional assay to ID stem cells in vivo is the
cell able to stably generate multiple
hematopoietic lineages in irradiated or SCID
(immunodeficient) mice?

• Have done this with human HSCs, termed SCID
  repopulating cells or SRCs able to repopulate
  human hematopoiesis in immunodeficient mice --
  LTR long-term repopulation
• SRC homes to engrafts murine BM and produces
  immature cells
• Capable of multilineage difftn
• Only found in CD34/38- frxn
• -- purify these to get enriched
• HSC population, and/or select
• for VEGF receptor expression
• After cells are transplanted, a
• CD34/CD38- population of
• cells supports short term
• hematopoiesis, following which
• a CD34-/CD38- population
• supports long-term reconstitution

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Most strict definition of an HSC ability to
serially
reconstitute hematopoiesis (HSC loses ability to
do this after it has self-renewed several times)
HSC home to marrow are quiescent for up to 48
hrs
Serial transplantation to 2ndary host
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Progenitor cells and precursor cells

• Cycling stem cells renew and give rise to more
  mature multipotent progenitor cells, which are
  more restricted in the offspring which they will
  generate. This is associated with tremendous
  amplification in cell number.
• Progenitor cells
• are multipotent
• do not self-renew or have only an extremely
  limited capacity
• respond best to multiple cytokines
• is a compartment of hematopoiesis that expands
  the number of cells dramatically
• are named by the types of colonies they give
  rise to
• The pluripotent HSC gives rise to lymphoid and
  myeloid stem cells, the latter of which gives
  rise to a GEMM progenitor termed CFU-GEMM
• CFU-GEMM is a multipotent cell giving rise to
  granulocyte, erythroid, monocyte, and
  megakaryocyte colonies
• CFU-GM gives rise to both granulocyte and
  monocyte colonies

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Expression of new sets of genes

• Cell cycle status is tightly associated with the
  function of cells at each step of hematopoiesis
• Primitive stem cells (CD34) slow cell cycling
  or dormancy
• Progenitor populations rapid cycling required
  for effective expansion
• Terminally differentiated cells withdrawal from
  cell cycle suitable and sometimes prerequisite
  for functions of mature cells

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Progenitor cells and precursor cells

• Precursor cells (Committed precursor cells)
• blast cells committed to unilinear
  differentiation much more mature than
  progenitor cells
• do not self-renew
• respond best to one or 2 cytokines
• still replicate until near terminal
  differentiation
• progeny increasingly acquire specific
  differentiation markers and functions
• include CFU-G, CFU-M, CFU-E, and CFU-Baso,
  giving rise respectively to granulocytes,
  monocytes, eosinophils, and basophils

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Self renewal
Proliferation
X
Expression of new sets of genes
Differentiation

• Cell cycle status is tightly associated with the
  function of cells at each step of hematopoiesis
• Primitive stem cells (CD34) slow cell cycling
  or dormancy
• Progenitor populations rapid cycling required
  for effective expansion
• Terminally differentiated cells withdrawal from
  cell cycle suitable and sometimes prerequisite
  for functions of mature cells

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Differentiation and lineage commitment

• Path of differentiation is generally regulated
  by the need for more of one particular
  type of blood cell, and is controlled by the
  appropriate cytokines and hormones and growth
  factors, colony stimulating factors (CSFs)
• Differentiation and lineage commitment occur
  under the influence of a complex array of signals
  from the extracellular environment, especially
  cytokines such as stem cell factor (c-Kit
  ligand), IL-3, GM-CSF, and G-CSF binding of
  cytokines to cell surface receptors results in
  the initiation of a cascade of signal
  transduction events within the cell.
• Series of negative regulators amplification
  circuits provide additional control over the
  process of hematopoiesis.

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Neutrophils
Major component of innate immune system 1st
line of defense against infection Surround
microorganisms with pseudopodia Pseudopod fusion
to form phagosome (phagocytosis) Granuole
release into phagosome Secretion of granuole
contents
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Francois Paraf, M.D NEJM 1997
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• A key concept The marrow contains a large
  storage pool of neutrophils which can be reserved
  for release in a setting of stress, and that the
  exponential expansion of progenitor cells can be
  augmented by granulocyte colony stimulating
  factor (G-CSF) under stress conditions. 
• Neutrophils and Host Defense
•     Most are in the marrow--reserve
•     Circulating neutrophils half in vessel,
  half adherent to wall
•     Have peptide receptors for and diapedese in
  response to FMLP (N- formyl-methionyl-leucyl-pheny
  lalanine), chemotaxins
• Steady flow of neutrophils into superficial
  tissue, skin, mucosa, lungs needed to prevent
  infection.
•            Bactericidal mechanisms
•       Degradative enzymes in granules
•       Oxidative killing
•    Superoxide generation by NADPH-dependent
  oxidase
• Involves hexose monophosphate shunt, cytochrome
  b
• Patients with Chronic Granulomatous Disease
  lack cytochrome b cannot generate superoxide,
  and develop repeated infections.
•  

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Example of differentiation from the precursor
stage onward myeloblast to granulocyte
(neutrophil maturation)
Terminal cell division
Increasing development of granuoles
(antibacterial and phagocytic) Increasing
phagocytic function pseudopodia extend around
microorganisms and fuse to form a phagosome into
which granuole contents are released
Expansion of cell number occurs as cells in the
mitotic or proliferative pool replicate The
post-mitotic pool can no longer divide but
continues to mature into terminally
differentiated cells In the setting of
infection or stress, maturation time may be
shortened, divisions may be skipped, and cells
may be released into the bloodstream earlier
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Mobilization of marrow cells (transendothelial
migration)
Mobilization of hematopoietic progenitor cells is
a multistep process common themes in this
process (not in chronological order)
1) Adhesion interactions -- must first be
disrupted for progenitor cell trafficking. In
general adhesion molecules are expressed on
hematopoietic progenitor cells in the marrow but
are downregulated or degraded to facilitate
egress of progenitors from the BM -- selectins,
selectin ligands, integrins, CD44 and PECAM
2) Chemotaxis Transendothelial gradients of
chemokines (cytokines with chemotactic
activity) produced by stromal cells control
direction and efficacy of transendothelial
migration of hematopoietic progenitors mature
leukocytes respond to a variety of chemokines,
but most important so far is SDF-1 stromal
cell-derived factor-1 (made by BM stromal
cells). Significant chemotactic activity in
hematopoietic progenitor and stem
cellssignificantly enhances retention in BM and
homing to BM. The chemokine receptor CXCR-4
is the receptor for the SDF-1 chemokine
similarly upregulated before mobilization and
downregulated after transendothelial migration.
3) Paracrine cytokines may support mobilization
Growth factor stimulated hematopoietic
cells produce cytokines such as vascular
endothelial growth factor (VEGF) that act on
endothelial cells to support migration by
increasing endothelial fenestration and
permeability.
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Mobilization of stem cells for HSCT
Mobilization various molecules administered to
donors can mobilize CD34 stem cells out of
marrow into circulation where can harvest from
peripheral blood granulocyte-colony stimulating
factor (G-CSF), granuloctye-macrophage colony
stimulating factor (GM-CSF), flt-3 ligand, stem
cell factor (SCF), and a variety of cytokines
(IL-7, IL-3, IL-12) and chemokines (IL-8, SDF-1),
as well as chemotherapeutic agents
cyclophosphamide and paclitaxel, with varying
degrees of efficacy G-CSF (Neupogen,
Filgrastim) most commonly used, daily
stimulations of healthy donors, sometimes used
with the chemotherapeutic agent cyclophosphamide
induce proliferation of hematopoietic cells
within the bone marrow Mobilized PBL cells have a
much faster engraftment than do bone marrow
cells, due to the increased cell dose of
transplanted mobilized cells and increased
numbers of committed progenitor cells. BUT
proliferation differentiation potential of
CD34/CD38- stem cells from mobilized PBL is
inferior to that of undifferentiated BM. Cord
blood stem cells Immediately after birth,
relatively high levels of immature CD34
progenitor stem cells circulating (for about 48
hours) umbilical cord blood represents a
promising source of stem cells for
transplantation, but s are too low for
transplant into adults need to find a way to
expand ex vivo (stromal cells).
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Model of mechanisms of stem cell mobilization by
G-CSF disruption of retention in BM proximity
to stromal cells

• 1) Disruption of adhesion interactions btwn. HSC
  and BM stromal microenvironment
• VLA-4 is an integrin on HSCs that binds to
  VCAM-1 (another adhesion molecule) on stromal
  cells -- needs to be downregulated for HSCs to
  egress. HSCs also increase the expression of
  proteolytic enzymes elastase MMPs, and cathepsin
  G during mobilization so that they can cleave
  VCAM-1 from the stromal cells.
• Disruption of chemotactic interactions
• SDF-1 retention signal produced by BM stromal
  cells gets degraded by same proteolytic enzymes
• CXCR4 is upregulated on HSCs in mobilization
  why? Probably important for egress to interact
  with SDF-1 in the blood.
• 3) Degradation of extracellular matrix

Lapidot and Petit, 2002 Exp. Hematology 30973