Stem Cells and Clonal

Haematopoiesis
Learning Objectives

Describe different types of stem cells involved in

Describe haematopoiesis

Understand Understand the processes of differentiation, maturation, and

proliferation in the blood and lymphoreticular system (B&LS)

Explain the concept of clonal haematopoiesis and its clinical

Explain significance
What is hematopoiesis?
• Process of new blood cell formation

• Red cells

• White cells

• Platelets
Sites of Hematopoiesis

• In the first few weeks of gestation the yolk sac is a transient

site of haemopoiesis.
• However, definitive haemopoiesis derives from a population of
stem cells first observed on the AGM
(aorta‐gonads‐mesonephros) region.
• These common precursors of endothelial and haemopoietic
cells (haemangioblasts) are believed to seed the liver, spleen
and bone marrow
Sites of Hematopoiesis…
• From 6 weeks until 6 – 7 months of foetal life, the liver and
spleen are the major haemopoietic organs and continue to
produce blood cells until about 2 weeks after birth.

• The liver and spleen can resume their foetal haemopoietic role
(‘extramedullary haemopoiesis’).

• The placenta also contributes to foetal haemopoiesis.

Bone marrow
• The bone marrow is the most important site from 6 – 7 months of foetal
life.

• During normal childhood and adult life the marrow is the only source of
new blood cells.

• In infancy all the bone marrow is haemopoietic but during childhood

there is progressive fatty replacement of marrow throughout the long
bones so that in adult life haemopoietic marrow is confined to the central
skeleton and proximal ends of the femurs and humeri
Growth factors

• The growth factors may cause cell proliferation but can also
stimulate differentiation, maturation, prevent apoptosis and
affect the function of mature cells
• They share a number of common properties and act at
different stages of haemopoiesis .
• Stromal cells are the major source of growth factors except for
erythropoietin, 90% of which is synthesized in the kidney, and
thrombopoietin, made largely in the liver
Growth factors attach to specific cell
receptors and produce a cascade of
phosphorylation events to the cell
nucleus.
Transcription factors carry the message
to those genes that are to be ‘switched
on’, to stimulate cell division,
differentiation, functional activity or
suppress apoptosis.
Erythropoietin
• Erythropoiesis is regulated by the hormone erythropoietin

• Erythropoietin is a heavily glycosylated polypeptide

• Normally, 90% of the hormone is produced in the peritubular

interstitial cells of the kidney and 10% in the liver and elsewhere

• There are no preformed stores and the stimulus to erythropoietin

production is the oxygen (O2 ) tension in the tissues of the kidney
Erythropoietin…
• Under normoxic conditions, little or no EPO mRNA is
detectable in the kidneys; hypoxia results in the rapid
induction of its transcription

• Effects of EPO on red cell production are mediated by both

increasing proliferation and reducing apoptosis of erythroid
precursors can be elevated 1000 fold by severe anaemia.

• Abnormalities in receptor may cause polycythaemia

Hematopoietic stem cells
• HSCs are foundation of the adult blood system and sustain the lifelong production of all blood
lineages.
• HSCs have the ability to differentiate and generate hundreds of millions of erythrocytes and
leucocytes needed each day
• Present in Marrow in Small Numbers
• Reside Primarily in Marrow
• Very small numbers circulate in blood
• Surface Antigen -CD34+ ("Cluster of Differentiation 34")
• Tight regulation of HSC self-renewal ensures the appropriate balance of blood cell production.
• Disturbance of this regulation and unchecked growth of HSCs and/or immature blood cells results
in leukaemia
Cell Potency
• Totipotent
• Pluripotent
• Multipotent
• Unipotent
Totipotent

• These cells have the ability to differentiate into any cell type,
including both embryonic cells (like all cells in the body) and
extra-embryonic cells (such as the placenta)

• Example: A zygote (the fertilized egg) is totipotent because it

can give rise to a complete organism and support tissues like
the placenta.
Pluripotent
• These cells can differentiate into almost all cell types, but not
extra-embryonic tissues like the placenta.

• Example: Embryonic stem cells, which are derived from the

inner cell mass of the blastocyst, are pluripotent as they can
develop into cells of any of the three germ layers (ectoderm,
mesoderm, endoderm) but not into placental cells.
Multipotent
• These cells can differentiate into multiple, but limited cell
types, usually within a specific tissue or organ lineage

• Example: Hematopoietic stem cells are multipotent because

they can give rise to various types of blood cells (red blood
cells, white blood cells, platelets) but not to other cell types
outside the blood system
Unipotent
• These cells can differentiate into only one cell type, but they
still have the capacity for self-renewal.

• Example: Muscle stem cells (myoblasts) are unipotent

because they can only give rise to muscle cells
Haemopoiesis
• Haemopoiesis starts with stem cell division in which one cell
replaces the stem cell (self‐renewal) and the other is
committed to differentiation
• Haemopoiesis arises from stem cells in the bone marrow.
• Stem cells give rise to progenitor cells which, after cell
divisions and differentiation, form red cells, granulocytes
(neutrophils, eosinophils and basophils), monocytes, platelets
and B and T lymphocytes.
• Hematopoietic Stem Cells (HSCs)
• Pluripotent cells that give rise to all blood cell types.
• Capable of self-renewal and multilineage differentiation.

• Progenitor Cells
• Intermediate stage between stem cells and fully differentiated cells.
• Lose self-renewal ability but are lineage-committed.
• Myeloid lineage: Gives rise to erythrocytes, platelets, monocytes, and
granulocytes.

• Lymphoid lineage: Gives rise to B cells, T cells, and natural killer (NK)
cells.
Differentiation, Maturation, and
Proliferation
• Differentiation
• HSCs differentiate into either lymphoid or myeloid progenitors

• Maturation
• Progenitor cells undergo maturation in the bone marrow, influenced by
transcription factors

• Proliferation
• Continuous cell division in response to cytokine signals ensures
sufficient production of blood cells.
Erythropoiesis
Pronormoblast
• The Pronormoblast is a large cell with dark blue cytoplasm, a
central nucleus with nucleoli and slightly clumped chromatin .

• It gives rise to a series of progressively smaller normoblasts

by a number of cell divisions
Normoblast
• They also contain progressively more Haemoglobin in the
cytoplasm
• The cytoplasm stains paler blue as it loses its RNA and
protein synthetic apparatus while nuclear chromatin becomes
more condensed.
• 3 stages
• Early
• Intermediate
• Late
Normoblast…
• Nucleated red cells (normoblasts) are not present in normal
human peripheral blood

• They appear in the blood if erythropoiesis is occurring outside

the marrow (extramedullary erythropoiesis) and also with
some marrow diseases
Reticulocytes
• The nucleus is finally extruded from the late normoblast within the
marrow and a reticulocyte results, which still contains some
ribosomal RNA and is still able to synthesize haemoglobin
• This cell is slightly larger than a mature red cell and circulates in
the peripheral blood for 1–2 days before maturing, when RNA is
completely lost.
• Gives polychromasia with Romanowsky staining
• Reduced in marrow failure
• Increased in haemolytic anaemias
Mature Red cell

A completely pink‐staining mature

erythrocyte results which is a
non‐nucleated biconcave in shape
Granulopoiesis
Granulopoiesis
• Granulopoiesis is driven by hematopoietic growth factors .
• They are synthesized by a variety of cells, including
fibroblasts and endothelial cells of the bone marrow
• Hematopoietic growth factors such as interleukin-3 (IL-3), GM-
CSF, G-CSF bind to target cells through specific receptors.
• G-CSF is a potent cytokine that influences the proliferation,
survival, maturation, and functional activation of cells
neutrophil-granulocyte lineage.
Hematopoietic Growth Factors
• Granulocyte-Monocyte Colony Stimulating Factor (GM-CSF)
• Secreted by lymphocytes, endothelial cells, stromal cells in marrow
• Stimulates production of granulocytes, monocytes
• Commercially available
• Granulocyte Colony Stimulating Factor (G-CSF)
• Secreted mainly by marrow stromal cells
• Mainly, but not exclusively, stimulates production and function of
granulocytes
• Synthesis rises in response to infections
Hematopoietic Growth Factors…
• Interleukins (IL1, IL2, IL3, ... IL13)
• Mediate multiple, highly complex communications between various
classes of white blood cells
• Levels rise in response to infections.
Platelets – Megakaryopoiesis
• Megakaryoblast

• Megakaryocyte
• Polyploid nucleus
• Platelet release via cytoplasmic fragmentation
Lymphocytes – Lymphopoiesis
• Initial lymphopoiesis takes place in bone marrow
• Maturation takes places in lymph nodes, thymus

• Second cycle of differentiation and proliferation in response to

antigen exposure
• Memory Phase: Follows antigen exposure May live for years
Clonal Hematopoiesis
• Definition
• Clonal expansion of a single hematopoietic stem or progenitor cell.
• Leads to the development of blood cells from a genetically identical
clone.
• Common in Ageing
• Occurs frequently in older individuals, associated with mutations in
genes like DNMT3A, TET2, and ASXL1.
Clinical Significance of Clonal
Haematopoiesis
• Preleukaemic Conditions
• Clonal hematopoiesis increases the risk of hematological malignancies,
such as leukemia.

• Can precede myelodysplastic syndromes (MDS) and acute myeloid

leukemia (AML).
Mechanisms of Clonal Expansion
• Mutations in Key Genes
• DNMT3A and TET2 mutations disrupt DNA methylation, leading to
aberrant cell proliferation.
• JAK2 mutations cause abnormal signaling through the JAK/STAT
pathway, promoting clonal growth

• Cellular Advantage
• Mutated clones may gain a survival or proliferative advantage, leading
to expansion in the bone marrow.
Diagnostic Methods
• Next-Generation Sequencing (NGS)
• Detects somatic mutations in hematopoietic cells

• Flow Cytometry
• Assesses clonal populations based on surface markers

• Bone Marrow Biopsy

• Histopathological examination to detect abnormal hematopoiesis.
Questions