It discusses the process of red blood cell formation as well as its regulation. It was presented at a seminar in department of Haemtology and Blood Transfusion ABU, Zaria. Nigeria.
2. Synopsis
• Introduction
• Site of Erythropoiesis
• Phases of Erythroid Differentiation
• Erythroblastic Islands
• Haemoglobin synthesis
• Regulation of erythropoiesis
– Growth factors
– Hypoxia, Stress, and HIF-Signaling Pathway
– GF receptors,Transcriptional factors, mRNA
• Erythropoiesis in disease condition
• Conclusion
• References
2
3. Introduction
• Erythropoiesis is the generation of RBCs carrying the
respiratory pigment haemoglobin, for the transport of
oxygen to the tissues
• Is a tightly-regulated and complex process originating in
the bone marrow from a multipotent stem cell and
terminating in a mature, enucleated erythrocyte
• It maintains the steady state of an individual’s red cell
mass, producing 1011–1012 new cells per day
• Understanding the basic biology of this process provides
a logical basis for the diagnosis and treatment of
erythroid disorders 3
4. Site of Erythropoiesis
• Fetus
– 0 – 2 months (yolk sac)
– 2 – 7 months (liver, spleen)
– 5 – 9 months (bone
marrow)
• Infants
– Bone marrow (practically
all bones)
• Adults
– Bone marrow of vertebrae,
ribs, sternum, skull, sacrum
and pelvis, proximal ends
of femur
figure/Ontogeny-of-hematopoiesis-in-humans-Changes-in-the-anatomical-location-of-
hematopoiesis_fig1_8677440/download 4
5. Phases of Erythroid Differentiation
1
2
Division and
DD of erythroid
precursors
Generation of
erythroid
committed blasts
Terminal
maturation
3
engagement phase
second phase
final phase
CFUGEMM --- BFUE --- CFUE
proerythroblasts
basophilic
polychromatophilic
orthochromatic
reticulocytes
erythrocytes
5
8. Proerythroblast
• First cell drived from CFU-E
• Large cells (15-20μm in
diameter)
• Irregularly rounded or
slightly oval
• Large nucleous
• Contains fine chromatin
delicately distributed in
small clumps.
• One/several well defined
nucleoli are present
• It has no haemoglobin
• Multiplies several times to
form basophilic erythroblast
8
9. Basophilic Erythroblasts
• Also called Early Normoblasts
• Smaller than proerythroblasts
• Measures 12-16μm in
diameter
• Nucleus occupies 75% of cell
area
• The nucleus is composed of
heterochromatin and
euchromatin linked by irregular
strands(“Wheel spikes” OR
“Clockface appearance”)
• Polyribosomes are still
present, giving rise to
cytoplasmic basophilia
9
10. Polychromatic Erythroblasts
• Also called Intermediate
normoblast.
• Smaller cells measuring
approximately 10-12μm
in diameter.
• Nucleus occupies less
than 50% of the red cell
area
10
11. Orthochromatic Erythroblasts
• This results after the final mitotic
division
• Smallest of the series, measuring
about 8-10μm in diameter
• Nucleus occupies approximately 25%
of the cell and is eccentric
• Still has some residual
monoribosomes and polyribosomes
(thus is still somewhat
polychromatophilic)
• Concentration of Haemoglobin rises
within the erythroblast, thus it stains
like a mature erythrocyte more than
its precursors 11
12. Reticulocyte
• This is an orthochromatic
erythroblast post enucleation
• Small cell 8-16μm in diameter
• Retains mitochondria,small
number of ribosomes, centrioles
and remnants of golgi
apparatus
• On supravital staining, these
aggregates stain deep blue and
are arranged in RETICULAR
strands, hence the name
RETICULOCYTE. 12
13. Erythrocytes
• Typical human erythrocyte has
a disc diameter of 6-8 μm and
a thickness of 2μm
• Volume is about 90fl
• Surface area is about 136μm2.
• Adult humans have about 2-3 x
1013 (20-30 trillion)RBCs at
any given time
• This comprises ¼ of the total
human body cell number
13
15. Erythroblastic Islands
• anatomic niches
• central macrophage
• erythroid cells at
varying degrees of
red cell maturation
• the site of hemoglobin
synthesis
www.researchgate.net/figure/The-erythroblastic-island-From-the-CFU-E-stage-to-the-
formation-of-the-reticulocyte_fig1_282042503/download
15
16. Erythroblastic Islands
• Functions of the central
macrophage
• Anchor erythroblasts and provide
the cellular interactions
• Phagocytose the extruded nucleus
• Direct the transfer of iron to
erythroid progenitors for heme
synthesis
• Regulate the rate of erythropoiesis
via positive and negative feedback
mechanisms
16
21. Erythropoietin
- A heavily glycosylated 34KDa protein
- Normally, 90% is by kidneys and 10% in
the liver
- No preformed stores
- Has a short plasma half life of 6-9 hours
- Serum levels :25-50mμ/ml of cord blood
10-30mμ/ml in adults
21
22. Erythropoietin
• Works in 2 ways:
- Increasing the number of progenitor cells
- Shortens the cell cycle and maturation time
• Late BFU-E and CFU-E are the key targets for
erythropoietin activity .
• CFU-E rapidly responds by proliferating and
differentiating
• It also helps in reducing the rate of apoptosis of these
progenitors
22
23. Hypoxia and HIF-Signaling
Pathway
• Tissue hypoxia leads to activation of the
Epo-EpoR pathway
• Achieves the above via a transcription
factor called HIF
• Also stimulates angiogenesis and
metabolic changes
• Upregulates transferrin receptors
23
24. Hypoxia, Stress, and HIF-
Signaling Pathway
https://www.researchgate.net/figure/Overview-of-hypoxia-inducible-factor-HIF-
regulation-of-erythropoiesis-Reprinted-from_fig1_350185321/download
24
26. GF Receptors,Transcriptional
Factors, mRNA
Regulate gene expression
• Those for stem cells(SCL,GATA-2),NOTCH-1)
• GATA-1 and FOG-1:Erythropoietic and
megakaryocytic differentiation
• Those that enhance committed cells to proceed
through terminal differentiation: GATA-1,FOG-1
• Others include Erythroid Kruppel – like factor
(EKL-F).
26
27. Erythropoiesis in Disease
conditions
• Hypoproliferative erythropoiesis
– Sideropenic eg iron deficiency
– non-sideropenic eg CKD, ACD
• Ineffective erythropoiesis
– megaloblastic anaemia, aplastic anaemia,
thalassaemia, abnormal haemoglobins and
iron deficiency
• Hyperproliferative erythropoiesis
– haemolytic anaemias
27
28. Assessment of Erythropoiesis
Total erythropoiesis and the amount that is
effective can be assessed by:
• History and physical exermination
• FBC and Differentials
• Reticulocyte assessment
• Bone marrow examination
28
29. Conclusion
• Erythropoiesis is a finely regulated complex series of
events that result in the formation of erythrocytes which
carry the function of gaseous exchange.
• The multilevel regulation of erythropoiesis reveals
several molecular targets that can be exploited
therapeutically for treatment and monitoring of either
anemias, erythroleukemias, and other hematological
malignancies
29
30. References
• A. V. Hoffbrand, P. A. H. Moss, Essential Haematology, A John Wiley &
Sons, Ltd., Publication 6th edition
• A Victor Hoffbrand , Douglas R Higgs, David M Keeling, Atul B Mehta,
Postgraduate Haematology, A John Wiley & Sons, Ltd., Publication 7th
edition
• H.M. Waters,L.H. Seal, A systematic approach to the assessment of
erythropoiesis,Clinical & Laboratory Haematology,Volume 23, Issue 5 p.
271-283
• Nagai, A.; Nakagawa, E.; Choi, H.B.; Hatori, K.; Kobayashi, S.; Kim, S.U.
Erythropoietin and Erythropoietin Receptors in Human CNS Neurons,
Astrocytes, Microglia, and Oligodendrocytes Grown in Culture. J.
Neuropathol. Exp. Neurol. 2001, 60, 386–392
• Zon, L.I.; Youssoufian, H.; Mather, C.; Lodish, H.F.; Orkin, S.H. Activation of
the erythropoietin receptor promoter by transcription factor GATA-1. Proc.
Natl. Acad. Sci. USA 1991, 88, 10638–10641.
• Muench, Marcus & Bárcena, Alicia. (2004). Stem Cell Transplantation in the
30
Editor's Notes
Erythropoiesis is a tightly-regulated and complex process originating in the bone marrow from a multipotent stem cell and terminating in a mature, enucleated erythrocyte. Altered red cell production can result from the direct impairment of medullary erythropoiesis, as seen in the thalassemia syndromes, inherited bone marrow failure as well as in the anemia of chronic disease.
At baseline, erythropoiesis occurs at a steady, but low basal rate with approximately 1% of circulating erythrocytes cleared and replaced by new cells daily
Erythropoiesis at steady state- Every second, the human body generates 2 million red blood cells, through the process of erythropoiesis.
In the process of maturation pass through 4 stages:
Reduction in size of cells:25 to 7.2mm
Disappearance of nuclei and nucleus
Appearance of haemoglobin
Changes in the staining properties of cytoplasm
The remaining fatty marrow is capable of reversion to haemopoiesis
and in many diseases there is also expansion of haemopoiesis down the long bones. Moreover, the liver and spleen can resume their fetal haemopoietic role ( ‘ extramedullary haemopoiesis ’ ).
steps of erythroid differentiation
engagement phase, in which HSCs differentiate into more committed erythroid progenitors
common myeloid progenitor the megakaryocytic-erythroid progenitor
burst-forming unit- erythroid (BFU-E)-first progenitor cells committed
the colony forming unit-erythroid (CFU-E)
terminal differentiation
The second phase of erythroid maturation
involves the differentiation of the nucleated precursors from proerythroblasts to basophilic, polychromatophilic and orthochromatic erythroblasts
Its charecterised by the gradual accumulation of hemoglobin, progressive decrease in cell size and nuclear condensation ultimately resulting in enucleation
The final phase involves the maturation of the reticulocyte into erythrocytes
It is during this stage that the erythrocyte acquires its biconcave shape through extensive membrane remodeling
Terminal erythroid differentiation occurs in anatomic niches known as erythroblastic islands
Erythroblastic islands are unique to mammalian erythropoiesis
consist of a central macrophage surrounded by up to 30 erythroid cells at varying degrees of red cell maturation
The cells range from CFU-Es to enucleating erythroblasts and are the site of hemoglobin synthesis by terminally differentiating erythroblasts
The central macrophage functions to anchor erythroblasts within the island and provide the cellular interactions necessary to drive erythroid differentiation and proliferation. Furthermore, the central macrophage has also been shown to phagocytose the extruded nucleus from terminally differentiating erythroblasts and direct the transfer of iron to erythroid progenitors for heme synthesis
also help regulate the rate of erythropoiesis via positive and negative feedback mechanisms. Macrophages secrete cytokines such as insulin-like growth factor-1 that promote erythroid proliferation and maturation
Other functions for the central macrophage are still being investigated
Haemoglobin synthesis in the developing red cell. The mitochondria are the main sites of protoporphyrin synthesis, iron (Fe) is supplied from circulating transferrin; globin chains are synthesized on ribosomes. δ - ALA, δ – aminolaevulinic acid; CoA, coenzyme A.
Haem synthesis occurs largely in the mitochondria by a series of biochemical reactions commencing with the condensation of glycine and succinyl coenzyme A under the action of the key rate limiting
enzyme δ - aminolaevulinic acid (ALA) synthase (Fig. 2.6 ). Pyridoxal phosphate (vitamin B 6 ) is a
coenzyme for this reaction which is stimulated by erythropoietin. Ultimately, protoporphyrin combines
with iron in the ferrous (Fe 2 + ) state to form haem (Fig. 2.7 ), each molecule of which combines
with a globin chain made on the polyribosomes (Fig. 2.6 ). A tetramer of four globin chains each
with its own haem group in a ‘ pocket ’ is then formed to make up a haemoglobin molecule
Erythropoiesis is controlled by cell-cell/cell-matrix interactions along with several cytokines and growth factors including IL-3, IL-6, erythropoietin (EPO) (the main erythropoietic stimulating hormone), EPO-receptor, members of the transforming growth factor-β (TGF-β), activin A, activin receptor-II, Flt3 ligand (Flt3-L), vascular endothelial growth factor (VEGF), stem cell factor (SCF), and thrombopoietin (TPO)
Regulation of erythropoiesis by growth factors: Differentiation of common myeloid progenitor CFU-GEMM into erythroid lineage is governed by IL3, GM-CSF, and SCF. First cell in erythroid lineage is burst forming unit-erythroid (BFU-E) which is EPO responsive and thereafter EPO regulates the maturation of erythroblasts into mature erythrocytes
In response to hypoxia or HIF-prolyl-hydroxylase inhibitors (PHIs), HIF-2 stimulates erythropoietin (EPO) production in the kidneys and liver. This promotes erythropoiesis and leads to increased iron demand in the bone marrow. HIF coordinates erythropoisis with iron metabolism, as it regulates genes involved in iron uptake, release from internal stores, and transport (highlighted in red). Absorbed and stored iron is released into the circulation via ferroportin (FPN) and complexed with transferrin (TF) for transport to liver, bone marrow, reticulocyte endothelial system (RES), and other organs. FPN surface expression is regulated by hepcidin, whereas HIF-2 participates in the transcriptional regulation of FPN. Erythroferrone (ERFE) mediates suppression of hepcidin production in the liver under conditions of accelerated erythropoiesis. Ceruloplasmin (CP) is an HIF-regulated copper-carrying ferroxidase that catalyzes the oxidation of ferrous (Fe 2þ ) to ferric (Fe 3þ ) iron. DCYTB, duodenal cytochrome B (cytochrome b reductase 1); DMT1, divalent metal transporter 1; Hb, hemoglobin
Epo signaling pathways. The three main Epo signaling pathways are highlighted. Positive signaling is associated with STAT5 binding to Mu Y343 and Y401, STAT3 binding to Y431, activation of the MAPK pathway following association of the adaptor protein Grb2 withY464 and binding of the p85 PI3K subunit to Y479. Negative regulation occurs via SHP-1 binding to Y429 and Y431 and CIS and SOCS3 competition for STAT5 binding at Y401. SOCS1 and SOC3 can directly bind to JAK2
GATA-1 triggers erythropoiesis by regulating the transcription of several erythroid differentiation-related genes, including genes involved in heme and/or globin synthesis, glycophorins, anti-apoptotic genes of the BH-3 family, genes involved in cell cycle regulation, and the gene for the erythropoietin receptor (EPOR).
Major molecular players in these networks include, among others, classical hormones (thyroid hormones, androgens, corticosteroids, activin/inhibin and others), vitamins (e.g. vitamin B12 and folic acid), iron, regulators of iron metabolism such as the transferrin receptors-1 and -2, and early acting hematopoietic growth factors such as stem cell factor and interleukin-3
Dysplastic erythroid precursors may exhibit asynchronous nuclear-cytoplasmic development (so-called megaloblastoid features), nuclear abnormalities such as nuclear lobulation, irregular nuclear contours, karyorrhexis, and atypical multinucleation
Dyserythropoiesis may also manifest as ring sideroblasts, which are defined as erythroid precursors with five or more iron granules encircling at least one third of the nuclear circumference
. Dysplastic erythroblasts may show cytoplasmic vacuoles that stain positively for periodic acid-Schiff in a globular or coarsely punctate fashion
