Hematopoiesis
Blood
Blood is the life sustaining “river” of the body. Blood provides the oxygen and
nutrients necessary for life while removing waste from the tissues. It remains ever
vigilant in fighting off invaders. It stops the bleeding when the body is injured. It
defines who we are and distinguishes self from non-self.
What is blood?
Blood is a mixture of cellular and fluid elements. The fluid component of blood
is called plasma. In total, the volume of blood averages about 70 mL/kg (or
about 4.9 liters in a 70 kg adult).
The cellular component of blood consists of three major types of cells each with
distinct functions.
1. Red blood cells
2. White blood cells
3. Platelets
Red blood cells’ major function is to transport oxygen to the tissues and remove
carbon dioxide in exchange. This is achieved via a molecule in the red blood cell
cytoplasm called hemoglobin. Hemoglobin consists of four globin proteins
(typically, 2 -globin chains and 2 -globin chains in adult humans) each associated
with heme (a protoporphyrin ring with iron). The red cell has a life span of
approximately 100-120 days.
The white blood cells comprise a heterogeneous group of cells that play a vital role
in our immune system. They identify self from non-self and defend the body against
invaders. These cells were discussed in detail during the immunology course. As
you will recall, the white cells consist of two large categories of cells: the myeloid
and lymphoid lineage cells. The myeloid lineage includes the neutrophils,
monocytes, eosinophils, and basophils. The lymphoid lineage includes Blymphocytes, T-lymphocytes, and natural killer cells. Each plays a distinct role
within the immune system.
Platelets major function is to help stop bleeding at the site of vascular injury. The
platelet has a life span of 7-10 days.
Plasma is the fluid component of blood. The plasma is composed largely of water
along with a variety of nutrients, electrolytes, and proteins. Some of the major
proteins found in plasma include the following: albumin, lipoproteins, transport
proteins, complement proteins, globulins (antibodies), and the coagulation system
proteins.
Where is Blood Made?
Hematopoiesis is the process by which blood cells are produced and involves a
complex balance of cellular proliferation, differentiation, morphogenesis, and
functional maturation. This process must be sustained throughout life in
order for life to be maintained.
From where do the blood cells come? That answer varies during the course of
human development.
Hematopoiesis in utero is divided into overlapping phases as different organ
systems are involved:
 Mesoblastic period (blood island of the yolk sac, 3-10 weeks gestation)
 Hepatic period (liver and spleen, 6 weeks – 7 months gestation)
 Myeloid period (bone marrow, 5 months gestation – birth and beyond)
The bone marrow begins to produce blood cells during the third month and
becomes the predominant site of hematopoiesis during the last two months of
gestation and the only site of hematopoiesis by about three weeks postpartum.
Throughout the remainder of life, hematopoiesis only occurs in the liver, spleen, and
other sites if the bone marrow becomes diseased, a process termed extramedullary
hematopoiesis. Active hematopoiesis takes place in all bones at birth, but is
normally restricted to the centrally located flat bones (red marrow) (ribs,
vertebrae, pelvis, sternum) after puberty. In contrast, adipose cells gradually fill the
marrow spaces of the peripheral bones.
Illustration by: Michal Komorniczak (Poland)
Bone Marrow
As you will hopefully recall from your anatomy and histology courses, bone consists
of a thick outer (cortical) layer of dense (compact) bone, and an inner layer of
sponge-like trabecular (cancellous, spongy) bone. A single layer of endosteal cells
lines the inner cortex and honeycomb network of trabecular bone.
In bones with red marrow, the cavities of the trabecular bone are filled with
clusters of hematopoietic cells (stem cells and their progeny) and a vast
network of vascular channels (marrow sinusoids). Fibroblastoid (reticular)
cells cover the adventitial surface of the sinusoids and their cytoplasmic
processes form a support lattice for hematopoietic cells. The fibroblastoid
cells, vascular endothelial cells, macrophages, extracellular matrix molecules,
adipocytes (fat cells), and other non-hematopoietic cells comprise the bone
marrow stroma. In addition to providing an adhesive framework for the
hematopoietic cells, the stromal cells produce many of the soluble chemical
factors (hematopoietic cytokines) that regulate and control the process of
hematopoiesis and the egress of mature blood cells from the bone marrow
into the bloodstream. This regulatory mechanism assures that the normal
production rate of blood cells is equal to the senescence rate, and that the
production of hematopoietic cells can increase in response to disease states.
The stromal cells, chemical substances, vessels, and other materials that support the
hematopoietic cells are referred to collectively as the hematopoietic
microenvironment.
Hematopoietic Regulation
Thus, there are four major processes of hematopoiesis
1. stem cell survival
2. self-renewal
3. proliferation
4. differentiation
What steps must be undertaken when processing – controlling – managing
hematopoiesis
1. cytokine, hormone, or CSF signaling
2. Binding of a surface receptor
3. Signal transduction pathways, which in the case of the major
hematopoietic CSFs) which are all part of the same superfamily of
receptors thus have much homology
a. JAK-STAT pathways for proliferation
b. MAPK pathway for differentiation and antiapoptosis to promote
cell viability
4. Transcription factor networks
5. Translation
a. microRNA
6. Post-translational modifications
7. Repeat (based on effect of the product generated)
As such, every process of hematopoiesis (stem cell survival, self-renewal,
proliferation, and differentiation) is orchestrated by a combination of growth
factors, cytokines, microRNA, and transcription factors. Cytokines are
hormone-like chemical signals (hematopoietic cytokines) within the
"microenvironment" provided by the stromal cells and extracellular matrix of the
bone marrow. Cytokines include the interleukins (IL), interferons (IFN), tumor
necrosis factor (TNF). CSF and hormones are derived generally from external tissue
and secreted more systemically. Colony stimulating factors include the likes of
erythropoietin (EPO), thrombopoietin (Tpo), colony-stimulating factors for
granulocytes (G- CSF) and granulocyte-macrophages (GM-CSF), among many others.
The cytokine and colony stimulating factor composition of the microenvironment is
dynamic in response to local and systemic factors/needs.
Each cytokine and colony stimulating factor has its target(s), but the response
to these factors is not hard fast. The response varies depending upon the other
cytokines and CSFs present within the local microenvironment at that precise point
in time; the surface receptor types and numbers present on the cell at that point in
time; and the composition of intracellular microRNA and transcription factors
present at that point in time.
Some key factors are noted on the illustration below.
The cellular membrane receptors for the major hematopoietic colony stimulating
factors have been studied and many have been found to be closely related in
structure (from a hematopoietic receptor superfamily). The receptors are
themselves highly regulated with changing numbers during cell differentiation. The
combination of a regulator with its membrane receptor leads to a structural change
in the receptor, the triggering of a complex sequence of biochemical events (signal
transduction). In the case of many of the major hematopoietic colony stimulating
factors’ receptors, the signal pathways employ are both….
a. JAK-STAT pathways for proliferation
b. MAPK pathway for differentiation and antiapoptosis to promote
cell viability
The generation of intracellular substances (signals) in the cytoplasm leads to the
generation/recruitment/activation of transcription factors, which have the capacity
to activate / repress genes that encode proteins influencing cell proliferation,
differentiation, maturation, function and apoptosis (programmed cell death).
The molecular events underlying hematopoiesis are incredibly complex and
continue to be defined.
But let’s stop for a moment to consider the processes by which genes are controlled…
Transcriptional Regulatory Networks
There are approximately 3 billion bp that make up the human genome and the
approximately 20,000 or so human genes.
Two main components leading to gene transcription (decoding):
1. Transcription Factor (TF) proteins
a. Usually contain multiple domains
i. DNA binding domain (usually to only 4-6bp sequences) with
similarities TF proteins can be grouped into distinct families,
which often bind to similar DNA sequences, such as…
1. Homeobox
2. Basic helix-loop-helix
3. Zinc finger
ii. Transcription activation domain
iii. Protein-protein interactions domain
1. Some common interactions shared within and between
TF protein families
2. DNA sequence motifs within regulatory genes to which the TF proteins bind.
a. Random occurrence would have the 4-6bp sequences appear far more
commonly than the number of genes being regulated thus there must
be more than just simple sequence recognition. Appears that
regulatory genes are associated with clusters of TF recognized
binding sequences (up to 5)—in contrast to the randomly occurring
sequences, which are more isolated/solitary.
b. Clusters of binding sites plus protein-protein interactions lead to
assembly of high-order complexes.
i. Data suggests some TF complexes exist in the nucleoplasm
and thus are at least partially pre-formed prior to DNA
binding.
High-order complexes allows for genes to have networks receiving input from
multiple upstream regulators with antagonistic or synergistic interactions (crosstalk).
1. Difficult to model as many different interaction combinations
2. Recent models based on pooled experimental data developed
c. One model based on 11 TF proteins controlling common myeloid
progenitor (CPM) differentiation (PLoS ONE 2011;6(8):e1000771).
i. Encodes the combined logic governing the interactions in
which
1. TF are either “ON” or “OFF”
2. Interactions use logic functions “AND”, “OR”, and
“NOT”
ii. Held up well when predicting consequences of gene knockouts
and over-expression in terms of the 4 terminal cell line
differentiation (neutrophils, erythrocytes, monocytes, and
megakaryocytes)
iii. Was not able to account for means by which the CMP pool
(stem cell/progenitor pool) was sustained
d. A second model based on 10 stem cell TF proteins identified HSC-like
gene expression as a stable attractor state (Bioinformatics
2013;29(13):i80-i88).
i. Focused on cross-regulation between TF proteins via
numerous positive feedback loops
ii. Requires simulation of external triggers allowing for exit
toward more differentiated states
e. Reality is HSC can balance both self-renewal and differentiation so
neither of the above models is completely accounting for the real
complexities
What leads to the production of these transcriptional factor proteins is also of
interest as these are also gene products that would require transcription regulation.
Cooperative TF binding is only the first critical step regulating gene expression
1. Recruitment of
a. Accessory proteins, such as chromatin-modifying enzymes
b. Multiple components of RNA polymerase holocomplex
2. Transcriptional elongation
3. RNA processing
4. Nuclear export
5. Translation
6. Post-translational modifications
Let’s also briefly introduce the concept of micro-RNA
MicroRNA are small non-coding RNA sequences, which (often negatively) regulate
translation of coding mRNA by binding the untranslated 3’ region of the target RNA.
They occur evolutionarily in association with increased organismal complexity. It is
hypothesized that microRNA may be a means by which evolutionary adaptation may
occur more quickly as it is likely easier to develop novel microRNA compared with novel
coding genes. The microRNA genes are often located within coding gene introns or very
near to coding genes.
Hematopoietic Regulation -- Summarizing
Every process of hematopoiesis (stem cell survival, self-renewal, proliferation,
and differentiation) is orchestrated by a combination of growth factors,
cytokines, microRNA, and transcription factors. Cytokines are hormone-like
chemical signals (hematopoietic cytokines) within the "microenvironment"
provided by the stromal cells and extracellular matrix of the bone marrow. The
cytokine and colony stimulating factor composition of the microenvironment is
dynamic in response to local and systemic factors/needs. Each cytokine and colony
stimulating factor has its target(s), but the response to these factors is not hard fast.
The response varies depending upon the other cytokines and CSFs present within
the local microenvironment at that precise point in time; the surface receptor types
and numbers present on the cell at that point in time; and the composition of
intracellular microRNA and transcription factors present at that point in time.
SO now let’s look at how these processes might occur in regulating hematopoiesis,
recognizing that even our discussion will not be able to describe the full complexity but
merely highlight some aspects of the process.
Granulopoiesis
As an example, we will start by looking more closely at the process of
granulopoiesis, which is the differentiation and maturation of the granulocytic cells
and monocytes.
As part of the discussion we will highlight a few key regulatory components, by way
of examples, including two transcription factor proteins, GATA2 and PU.1
As with all of the hematopoietic cells, the granulocytes derive form the pleuripotent
hematopoietic stem cell. We will recall that there is an ongoing balance being
orchestrated at the stem cell level—the balance between maintaining the stem
cell pool (self-renewal) and differentiation (lineage commitment).
Let’s digress for a moment to discuss GATA2…
GATA
GATA is a group of 6 TF proteins, which belong to the zinc-finger family of TF
proteins—these contain 2 zinc finger domains.
GATA2 is located on the long arm of chromosome 3 at position 21.3
1. In embryonic development, GATA2 pivotal in endothelial to
hematopoietic transition that produces the first HSCs. As such,
homozygous knock-out is lethal.
2. In adults, GATA2 is required for HSC survival and self-renewal
What are the targets of GATA2?
1. GATA2 cooperates with six other TF proteins to form a heptad regulatory
unit. These other factors include: TAL1, LYL1, LMO2, ERG, FLI1, and RUNX1).
Targeted genes of the heptad include microRNAs and are lineage-specific.
2. GATA2 also has direct targets including
a. GATA1
b. PU.1
c. CEBPa
3. GATA2 also interacts directly with FOG1, PU.1, and CEBPa
Haplo-insufficiency of GATA2 still allows for cell differentiation, but it also appears to
lead to a depletion of the HSC pool (i.e. appears to lead to impaired HSC self-renewal).
Conversely, over-expression of GATA-2 appears to impair differentiation. Thus,
balancing self-renewal and differentiation appears to be a major component of
GATA2 function.
Clinical syndrome associated with GATA2 deficiency / mutations  autosomal
dominant with >90% penetrance
1. MonoMAC = monocytopenia with increased susceptibility to atypical
mycobacterial infections.
2. Loss of dendritic cells, monocytic B-cells, and NK cells.
3. Familial AML/MDS
4. Pediatric neutropenia with aplastic anemia
Longitudinal study of kindreds suggested a progressive course related to impaired
cellular immunity
1. Increased warts (related to HPV infection susceptibility), herpes viruses, etc..
2. Mononuclear cellular loss over time ultimately severely so
(monocytopenia, DCs, B-cells, and NK cells with a decreased CD4:CD8 ratio
<1)
3. MDS often at younger ages with median 21-33 years (lifetime risk 90%
with hereditary MDS pattern)—often normal to hypocellular with
megakaryocytic atypia and fibrosis on the marrow examination and often
with better preserved Hgb, Plts, and neutrophil counts compared with other
MDS patients. Lymphocyte subset analysis by flow will reveal a profound
depletion of B-cells and NK cells. Often association with progressively
increased FLT3 ligand concentrations.
4. Pulmonary alveolar proteinosis
5. Infection with mycobacterium, fungi, and lymphedema.
6. Increased infection related cancers (related to HPV, EBV, etc…) but also skin
and breast cancers.
7. Increased risk of AML (hereditary AML cases)