Hematopoiesis • Hepatic period

• Myeloid period
BLOOD
- Red liquid that is circulated by the heart and flows in veins, arteries and MESOBLASTIC PERIOD
capillaries - Begins during embryonic development
Components: - Starts 19 to 20 days until 8-12th week of gestation
• RBC - The fetus is about 2.25 to 5mm in size
• WBC - Yolk sac is the chief site of hematopoiesis
• Platelets - The yolk sac is an ovoid structure joined to the embryo by a stalk.
PHYSICAL CHARACTERISTICS - It contains mesoderm derived cells
- Fluid In-vivo - “haemangioblasts”
- Coagulates 5-10 mins In-vitro - which differentiate to form (nucleated) red blood cells and endothelial cells
- Red due to Hb which generate a capillary system (“plexus”) within the yolk sac.
- pH 7.4 - At the same time the heart and aorta start to form: these join up with the
- Thick and viscous (3-4 x thicker than water) capillary plexus and the erythrocytes start to circulate.
- Makes up 7-8% (75-85ml/Kg) of the total body component
approximately 5 – 6 liters
- Approximately 20g of solid/100 ml of blood
Composition
• Liquid portion
◦ Plasma – with fibrinogen
◦ Serum – no fibrinogen, factor II, V, VIII
• Solid portion
◦ RBC (erythrocytes)
◦ WBC (leukocytes)
◦ Platelets (thrombocytes)

HEMATOPOIESIS
- Production of blood and its constituent elements
- Processes of blood cell derivation and maturation
Includes:
• cellular proliferation
• cell differentiation
• morphogenesis
• functional maturation
• death

THREE PERIODS OF HEMATOPOIESIS

• Mesoblastic period

MESOBLASTIC PERIOD
- Responsible for erythropoiesis; minimal leukopoiesis and megakaryopoiesis
- PE formed 3rd month of gestation.
- Definitive normoblasts replaced PE when dies out.
- (Bear in mind that cells of the immune system undergo further proliferation
and differentiation in the periphery – especially in secondary lymphoid tissue –
during immune responses: the purpose of haematopoiesis is to generate cells
which are capable of responding to pathogens.)
HEMATOPOISES

HEPATIC PERIOD
- Begins during the 5th to 6th week of gestation
- Liver is the primary until the 6th week and may continue until 1st or 2nd week
after birth
- Erythropoiesis – mainly RBC’s are formed (RBC with fetal hemoglobin – 2
alpha and 2 gamma globin chains)(HbF, HbA and HbA2)
- Granulopoiesis and lymphopoiesis are minimal
- Although hepatic, there are significant contributions by the:
• Spleen(B cells together with the Kidneys),
• Thymus(T cells ;first organ to be fully developed) and
• lymph nodes.

MYELOID PERIOD
- Bone and bone marrow
- A start from 5 months of gestation and increase during the last trimester and at
birth, the marrow is the chief site of normal hematopoiesis
- Adult Hb is produced with 2 alpha and 2 beta globin chains.
- In children, it is situated in the flat bones of skull, clavicle, sternum, ribs,
vertebrae, pelvis also in the long bones of legs and arms.
- In 18 years old and above, the red bone marrow confined to the flat bones
only: skull, clavicle, sternum, ribs, vertebrae, and proximal ends of bones
(femur and humerus)
- The remaining marrow is replaced by fat cells (yellow marrow)
- The BM is in effect a highly specialised tissue comprising a range of cells
- some of which (haematopoietic cells) form blood cells
- others (stromal cells) provide a support functions for the
haematopoietic cells, providing the specialised environment needed for
haematopoiesis to occur.
- Yet other cells (osteoblasts and osteoclasts) are concerned with ADULT HEMATOPOIETIC TISSUE
producing the bone itself. 18 MYELOID PERIOD (Bear in mind that • Bone Marrow – cavities of cortical bones;Red and Yellow;
cells of the immune system undergo further proliferation and Retrogression
differentiation in the periphery – especially in secondary lymphoid • Red Marrow-extravascular chords with developing cells
tissue – during immune responses: the purpose of haematopoiesis is to
generate cells which are capable of responding to pathogens.)
 • Fibronectin, Laminin, Collagen, Hemonectin and Thrombospondin for
adhesion of HSCs to the ECM
LIVER
- Hematopoiesis of the 2nd Trimester
- Proteins, DNA, RNA, Bilirubin conjugation
- Kupffer cells and EC
- Hepatocytes

SPLEEN
- Largest lymphoid organ
- White pulp, Red pulp and Marginal zone
- Culling – phagocytosed with subsq degradation
- Pitting – splenic macrophages removes inclusions
- 30% of platelets are sequestered in the spleen

MARROW CIRCULATION LYMPH NODES

- Nutrient and Periosteal Arteries (foramina) - Bean Shaped Structure
- NA A/D Central LongVein(bone canal) Arterioles in the inner lining of the - Cortex – B Cell Proliferation(germinal centers)
CB(endosteum)=sinusoids(endost eal beds) periosteal cap PA OB - Paracortex – T Cells and Macrophages
- Periosteal cap venous sinuses Central LongtVein Foramina - Medulla – B Cells and Plasma Cells

THYMUS
- Cortex – waiting zone for progenitors T cells
- Medulla – contains 5 % mature T cells

HEMATOPOIETIC MICROENVIRONMENT
- Semifluid matrix
- Stromal cells (EC, Adipocytes, Macrophages, Osteoblast, Osteoclasts,
Reticular cells (Fibroblasts))
- Extracellular matrix
• Proteoglycans for progenitor binding
 - HSCs are self-renewing cells
- The other daughters of HSCs (myeloid and lymphoid progenitor cells), cannot
self-renew.

• Totipotential - develops into a whole organisms; placenta and amniotic

sac
• Pluripotential - Can’t produce placenta and amniotic sac
• Multipotent - produces all cell types in one organ system Oligopotent -
HEMATOPOIETIC STEM CELLS lymphoid tissues
- Hematopoietic stem cells (HSCs) reside in the medulla of the bone (bone
marrow) • Uni/Monopotent - One Cell Type

PRINCIPLES OF NORMAL CELL MATURATION

1. Cytoplasmic differentiation:
• Loss of basophilia
• Cytoplasmic granules
• Elaboration of hemoglobin for RBC
2. Nuclear maturation
• A. Structure and cytochemistry
- round or oval nucleus – younger cells
- large nucleus/cytoplasmic ration – younger cells
• B. Changes in shape -more lobulations mean mature cells
3. Reduction in cell size
• the smaller the size, the more mature the cell is.

HOW CELLS ARE RELEASED FROM THE BONE MARROW INTO THE
CIRCULATION
• RBC – by hypoxia and erythropoietin
• WBC – by the presence of chemotaxins
• Platelets – by shedding of megakaryocytic cytoplasm

The haematopoietic stem cell

- source of all blood cells
• Haematopoietic stem cells (HSCs) are the parental cell type for ALL
blood cells
◦ and probably also endothelium.
• HSCs are (usually) present only in the bone marrow
◦ also present in cord blood and in (adult) peripheral blood after
treatment with “mobilising” cytokines.
• The HSC is (probably) able to divide indefinitely, or at least a very
large number of times
◦ experiments in 1960s in which mice were irradiated or treated with
drugs to destroy their bone marrow showed that transplantation of
very small numbers of bone marrow (BM) cells could
“reconstitute” the mouse
◦ serial transplant could be done.
• Experiments in the 1970s showed that all blood cell types could be
derived from single BM cells.
• These depended on irradiating the BM cells used to reconstitute the
mice with SMALL doses of radiation, not enough to kill the cells but
enough to cause minor chromosome alterations (“chromosomal
markers”) in rare cells
◦ each marker unique;
◦ all descendents of a marked cell (a CLONE) have the same maker.
• In some individual transplanted mice there could be found cells of all
leukocyte types bearing the same marker
• hence a single pluripotent stem cell present in the transplant was
capable of giving rise to all lineages.
◦ (Probably not totipotent i.e. cannot make all body cells.) 42 The
haematopoietic stem cell
• In other mice, ALL myeloid but NOT lymphoid cells had the same
marker
• And in yet others ALL lymphoid but NOT myeloid cells had the same
marker. 43 The haematopoietic stem cell
• This implied that there were also stem cells of more restricted potency
◦ capable of producing EITHER myeloid OR lymphoid cells
◦ these are now known as common lymphoid or myeloid progenitor
cells
◦ CLP & CMP
• Stem cell self renewal:
◦ evidently since mature leukocytes die the HSC must be able to
proliferate at least over the lifetime of the individual.
◦ This proliferation must be assymetric to generate a progenitor cell
(CMP or CLP) which has more limited differentiation capacity
AND replace the stem cell.
• CMPs and CLPs are transient amplifying (TA) cells & proliferate
PRIOR TO further differentiation (i.e. before they become granulocytes
or whatever)
◦ this is a limited proliferation, increasing cell numbers.
What is the HSC?
- A rare (<0.1%) cell present in bone marrow expressing the antigen CD34.
- We now know that CD34+ cells are the essential cell type for BONE
MARROW TRANSPLANTATION.
- Other cells express CD34 (e.g. endothelial cells) so it us not a unique marker
for HSCs.
- It is argued that stem cells require a specialised environment to maintain the
“stem-ness” of the stem cell:
- the stem cell niche.
- HSCs are found in two locations in BM:
- the endosteum (boundary between solid bone & marrow) associated
with osteoblasts;
- the perivascular region around vascular sinusoids (large blood vessels
with thin-walled comprised of fenestrated endothelium). 50 The
haematopoietic stem cell
- Hence “endosteal” and “vascular” niches.
Do they have different roles?
- It has been suggested that the endosteal niche contains long-term,
slowly dividing HSCs which maintain the vascular niche HSCs.
 - In the vascular niche the HSCs are more actively dividing and - Evidently the progenitor cell is now in a different environment.
progenitor cells are produced. - Growth factors drive its growth and then differentiation.
- (Perhaps the job of the stem cell niche is to slow cell division and
block differentiation.)
- The different phases of differentiation and the growth factors required
have been largely worked out by cell culture experiments.
- In vitro culture of bone marrow cells
- culture of BM cells is often done suspended in semi-solid media
so that the progeny of a single cell can be seen as a colony.

- It is not clear what molecules are involved in defining the stem cell niches
- presumably adhesion molecules & diffusible factors produced by the
non-HSC (“stromal”) cells – osteoblasts, endothelial cells etc – within
the niche.

- Alternatively, cells can be grown in liquid culture

- this requires the presence both of haematopoietic cells and BM
stromal cells to recreate something like the stem cell niche
- even so cell growth is very inefficient.
- In both cases added growth factors & cytokines are needed to drive both
proliferation and differentiation of cells.
- There are many of these, some more-or-less specific for different cell types
GROWTH AND DIFFERENTIATION - e.g. Granulocyte/monocyte colony colony stimulating factor (GM-
Progenitor cell growth and differentiation CSF), erythropoeitin (EPO)
- NB the term CSF.
- Others with much broader activities
- e.g. IL-3 which will stimulate growth of most haematopoietic cell types.

- Growth factors/cytokines act both in a paracrine fashion
- they diffuse from the producer cell to the responder cell
- and in a juxtacrine fashion
- they remain on the cell surface of the producer cell and so the
responder cell must be juxtaposed – touching.
- And they are produced both within the BM and from sources outside the BM
- e.g. GM-CSF from lymphocytes, EPO from kidney.
- Infection can massively increase the amount of growth factors present because
of activation of a range of leukocytes at the site of infection
- hence increased haematopoiesis.
- Adhesion interactions especially via integrins are also important in
haematopoiesis
- not just to drive growth & differentiation but also to prevent apoptosis.
ERYTH
ROCY
TES
AND
PLATE
LETES
- The
early
stages
for production of both these cells pass through a common pathway generating a
“megakaryocyte/erthrocyte precursor” (MEP) from the CMP: they then split.
- erythropoietin (EPO: aka epoietin) produced by kidneys specifically
stimulates erythropoiesis
• EPO production stimulated by hypoxia
• EPO binds to erythrocytes so haemorrhage or anaemia results in
increased levels of EPO
• these lead to stimulation of erythropoiesis.
• (Other non-specific GFs/cytokines needed.)
Erythrocyte clearance:
- removed by liver and spleen after 120 days
- as the erythrocyte ages surface proteins particularly “band 3” are
progressively oxidised
- this provides a target for phagocytosis by macrophages lining liver & spleen
sinuses.
Generation of platelets (thrombopoiesis):
- stimulated by thrombopoietin (TPO) & other non-specific growth factors.
- TPO produced constitutively mostly by liver.
- Inflammation can double production by liver via cytokine IL-6.
- In thrombocytopenia (reduced platelets) BM stromal cells also produce TPO.
- Platelets have TPO receptors and so remove TPO from circulation (-ve
feedback).

Platelet clearance:
- removed by liver and spleen after ~ 7 days

GRANULOCYTES AND MONOCYTES

- Granulocytes (basophils, mast cells, eosinophils & neutrophils) and
monocytes share early steps in differentiation via “granulocyte/monocyte
precursor” (GMP) derived from the CMP.
- Formation of GMP from CMP is driven by granulocyte/monocyte colony
stimulating factor (GM-CSF) (etc). LYMPHOCYTES
- Formation of specific cell types is driven by other growth factors, all of which Two pathways:
are produced constitutively and are induced e.g. by inflammation. • T cells
- Most of these cells are relatively short lived and are lost by apoptosis • B cells and NK cells
- the fragments of the dead cells are phagocytosed by cells lining sinuses of - both from CLP
spleen & BM - Initial stages in BM: B & NK cells complete maturation in BM, immature T
- or, in the tissues, by epithelial cells. cells migrtae to thymus and mature there.
 • CD19 +ve = expressing standard B cell marker
• H chain, L chain re-arrangement = formation of mature Ig genes
Lymphocytes
- The process of lymphopoiesis is driven initially by the growth factor IL-7
- hence the importance of its receptor.
- How the different lymphoid lineages are established & what factors influence
this is complex and ill-understood
- by me at any
rate.
- Lymphocytes are long lived cells unless they are activated (by cognate
antigen).
- They then become immune effector cells
- most of these will die after a few days by apoptosis
- some will become memory cells which are very long lived (decades).

lymphopoiesis abbreviations
• DN = negative for both CD4, 8; DP = positive for both; SP = positive
for one or other
• IL-7R = IL-7 receptor
• CD4/8 +ve = expressing one, other or both of these T cell markers
Simplified haematopoiesis schema
• CD25 +ve = expressing IL-2 receptor • MANY INTERMEDIATE STEPS OMITTED.
• α chain, β chain re-arrangement = formation of T cell recepto • GROWTH FACTORS OMITTED.
• Dendritic cells omitted
• IgM =ve = synthesising IgM
◦ probably arise from both both CLP & CMP.
• LGL/NK cells omitted - parallel B cells.
• Periphery = circulation and all tissues except 1ry lymphoid.
• Communication between compartments via circulation.
• Meg = megakaryocyte; DN = double negative.