HEMATOPOESIS

Presenter: Dr.Hussen (R1)

Moderetor: Dr.Aliyi ( Assistant Professor of Int.medicine )
Date 23 November 2023
 Outline
• Introduction to hematopoiesis
• Hematopoietic tissues and BM microenvironment
• Developmental biology of hematopoiesis
• Stem cells and growth factors
• Erythropoiesis
• Granulopoiesis
• Monopoiesis
• Thrombopoiesis
• Lymphopoiesis
 Introduction

• Hematopoiesis refers to the process of formation, devel’t, and differentiation of

the formed elements of blood i.e. RBC’S, WBC’S, and platelets.

• In hematopoetic organ or tissues ( bone marrow, liver, spleen ).

• Complex mechanisms regulate hematopoiesis :

in order to maintain homeostasis

to adapt to physiological stress for the entire life of an organism

 Con’t…
• Organized process that depends on 3 important components:
The bone marrow stroma(local control)
The hematopoitic stem and progenitor cells
The hematopoitic growth factors(hormonal control)

• All of mature blood cells and some cell in every tissue of the body are
derived from hematopoietic stem cell
 Con’t…

• Because the half-life of blood cells is short, the physiologic

requirements of hematopoiesis must generate billions of blood cells
daily.

• Must be continuesly replaced by stem cells.

• Stem cells produce in HEMATOPOIETIC ORGAN

 Con’t…
• In adults hematopoiesis occurs in the bone marrow.
• In the newborn it take place at different site depending on gestation
age.
• The first hemopoietic cells are produced in yolk sac of embryo in third
week of embryonic development and these cells are primitive RBCs
 Hematopoietic microenvironment(niche)

• It is an anatomic location which is housing site for stem cells and where
regulatory signals are provided that allow the stem cells to thrive, to expand if
needed, and to provide varying amounts of descendant daughter cells.

• Niche acts as both a nutritive and constraining home.

• The niche for blood stem cells changes with each of the site during
development, but for most of human life, it is located in the bone marrow.
Interaction of stromal cells, growth factors
and haemopoietic cells
Developmental biology of hematopoiesis

Primitive hematopoisis

• 3 weeks- 6 weeks: formation of blood islands in the extraembryonic

yolk sac (Mesoblastic period)

Definitive hematopoiesis

• 6 weeks- 22 weeks: liver and spleen ( Hepatic period)

• 22 weeks- life long: bone marrow (Medullary period)

 Cont….
 Active Hemopoietic marrow is In Adults active hemopoietic •
found, in children throughout :marrow is found only in
the:  The axial skeleton
 Axial skeleton:
 Cranium  The proximal ends of the
 Ribs appendicular skeleton.
 Sternum
 Vertebrae
 Pelvis
 Appendicular skeleton:
 Bones of the Upper & Lower

limbs.
 Bone marrow
• Found in the medullary canal of long bones and in the trabecular cavities of
cancellous bones
• Two types
1. Red marrow – the area of hematopoiesis
2. Yellow marrow- adipose tissue
In the newborn all the marrow is red and becomes substituted by yellow
marrow as age increases.
Fat is 50% of red marrow in the adult
Fatty replacement of the red marrow continues slowly with aging, but
hematopoiesis can be expanded when demand for blood cells is increased.
 Lifespan of blood cells
• RBC ; 120 days
• Platelet; 10 days
• Granulocytes; circ : 9 hours ,tissue : days
• Lymphocyte; circ : variable (hours to years), tissue : weeks to years
 Hematopoietic Response
• Hypoxia RBC
• Infection granulocyte/monocyte
• Antigen lymphocyte
• Hemorrhage platelet
Stem cells and Differentiation
• All blood elements develop from one origin cell- stem cell
• Stem cells are those cells that have the capability of self-renewal and
differentiation.

• First identified in the hematopoietic system, they are likely to be

present in many other tissues.
• Stem cells are hypothesized to be able to divide symmetrically

(in which both daughter cells are either stem cells or differentiated
cells) or asymmetrically

(yielding both a stem cell and a more differentiated cell)

• Self-renewal and differentiation are tightly regulated processes which
ensure homeostasis.

• Deregulation of these processes will inevitably manifest as

myeloproliferative diseases (such as acute myeloid leukemia) or as
bone marrow failure syndromes (such as aplastic anemia).
Progenitor cells of the BM
• As HSCs divide, they generate differentiated progenies, which
gradually display an limited capacity to self-renew and restricted
differentiation options

• The intermediate-stage cells are very much like the pluripotential

stem cells, even though they have already become committed to a
particular line

of cells and are called committed stem cells.

Progenitor cells of the BM
• Form colonies of cells in semisolid media in vitro – described as
colony forming units (CFU).

• CFU-GEMM (granulocytic, erythrocytic,

monocytic,megakaryocytic,CFU-GM,CFU-Mk, etc. )

• Survival and differentiation of progenitor cells influenced by growth

regulatory glycoproteins, called cytokines – include interleukins,
colony stimulating factors
Order of blood cells formation
 Hematopoietic growth factors

• Glycoprotien hormones or cytokines

• Regulate :- proliferation, differentiation and survival of hematopoietic cells

• Produced by bone marrow stromal, T lymphocytes, monocytes and

marcrophage cells except

erythropoietin….90% kidnes, 10 % liver

thrombopoietin….Liver
 Growth factors
• Early acting ( multilineage) • Late acting ( lineage restricted)
• Stem cell factor and Flt3 ligand • G-CSF
• IL-3 and GM-CSF • M-CSF
• IL-6 and IL-11 • EPO
• TPO
• IL-5
• IL- 2,4,7,1012,13,14,15
Cell hierarchy
 Assessment of hemopoiesis
• Hemopoiesis can be assessed clinically by;
• 1- (CBC= complete blood count) on peripheral blood.
• 2- Bone marrow Aspiration also allows assessment of the later stages of
maturation of hemopoietic cells.
• 3- Bone marrow Trephine Biopsy provides a core of bone and bone
marrow to show architecture.
Bone marrow Aspiration
Bone marrow aspiration
 Hypercellular

Normocellular

Hypocellular
 The M:E ratio
• Myeloid to Erythroid ratio in the bone marrow
• Is called M:E ratio
• Normally it is 3-4:1
Normal Blood Cells
 Erythropoiesis

• Erythropoiesis is the process of formation of erythrocytes (RBCs). This

process occurs in the bone marrow (myeloid tissue).
• Begins with the differentiation of small pool of pluripotent stem cells
in to early erythroid progenitor cells.

• These progenitors develop in to recognizable erythroids precursors,

which subsecuently differentiate in to mature erythrocytes.
 Erythropoiesis

• Erythroid progenitor cells:not

morphologically identifiable. Erythroid burst
forming unit (BFU-E) : is the most primitive single
lineage committed erythroid progenitor cell.

• .Erythroid colony forming unit (CFU-E)

• Erythroid precursor cells: morphologically

identifiable. represent about 1/3rd of the

marrow cell population. Proerythroblasts are

the earliest recognizable erythroid precursors.

Erythrocyte development….
 Regulation of erythropoiesis

• Tissue oxygenation- most

important regulator
• Erythropoietin
• Transcription factors
(GATA1,FOG1)
• Iron
• Vitamins- B12 and folic acid
• Other factors ( SCF, IL-3, GM-
CFU…) and nutrients
 Granulopoiesis
• Leukocytes
• Normal count- 4,500- 11, 000cell/mm3
• Divided
• Granulocytes- Neutrophils, Eosinophils and Basophils
• Agranulocytes- Monocytes, Lymphocytes

• WBC differentiation
• Neutrophils- 40-80%
• Lymphocyte- 20-40%
• Monocytes- 2-10%
• Eosinophils- 1-6%
• Basophils- <1-2%
Growth factors in Granulopoiesis
• SCF: stem cell factor
• GM-CSF: granulocyte-macrophage colony-stimulating factor
• G-CSF: granulocyte colony-stimulating factor
• M-CSF: monocyte colony-stimulating factor
• IL-5: Interleukin
• TPO: thrombopoietin
• EPO: erythropoietin
o Highly phagocytic and
bactericidal
o Regulated primarily by G-CSF
o Neutrophilia Causes-
o Infections- Bacterial
o Malignancy- MPD( CML, CNL)
o Drugs/Toxins- Steroids, Li,
TTC, G- CSF
o Trauma- Acute Hemorrhage
o Stress, Exercise, Fear
o Allergy, Autoimmune diseas
o Have acidophilic granules
o There granules contain MPO,
lysozyme and basic protein

o Eosinophilia Causes-
o Infections- Parasites
o Malignancy- CEL, Underlying HD/
Tcell Lymphoma
/carcinoma
o Drugs
o Allergy ( Asthma)/Autoimmune
( CTD)
o Trauma, Idiopathic
 Basophilia Causes-
• Infections- TB ( sometimes)

• Malignancy- MPD ( CML)

• Drugs- Hypersensitivity reactions

o Large purplish-black granules
o There granule contain large amounts of heparin , • Autoimmune/ Allergic
histamine & MBP
o They lack significant amounts of antibacterial or
lysozyme enzymes. • At onset of mensus
Mast cell • Idiopathic
o Functionally & biochemically similar with basophils
o Mast cells exit the marrow as immature precursors &
terminally differentiate in tissues.
• Congenital
o Involved in a phenomenon called IgE-mediated mast cell
degranulation.
MORPHOLOGY OF DEVELOPING NEUTROPHILS

• 6 stages of PMN development.

• These criteria are: relative size,

nuclear volume, chromatin density,
prominence of nucleoli, granule
number, and cytoplasmic staining.
Granulocyte maturation
 Con’t…
• Bone marrow compartments
• The life of granulocytes is spent in 3 environments: Marrow, Blood and Tissues.
1. Marrow pool:
• The mitotic compartment - (2.1 x 10 9 cells per kg)
• containing replicating myeloblasts, promyelocytes, and myelocytes, and
• The maturation-storage compartment = (5.6 x 10 9 cells per kg)
• containing non-replicating maturing metamyelocytes, band forms and PMN.
• Stored neutrophils are called mature neutrophil reserve.

• The transit times of developing myeloid cells is around 10days.

• The replicating phase 6 days.
• Maturation requires an additional 4 to 5 days.
• 3 or 4 days in the storage compartment before emerging into the circulation
2) Blood pool

Stays in circulation for approximately 7 hours

Circulating pool- neutrophils are free in the circulation
Marginated pool- roll along the endothelium of small vessels or are temporarily
sequestered in the alveolar capillaries of the lung.
Cells in the two pools are freely exchangeable.
3) Tissue pool
• Neutrophils enter the tissues, they do not normally return to the blood.
• The flow of cells is unidirectional.
• The life span of neutrophils can be 24 to 48 hours.
• Apoptosis accounts for significant removal of tissue neutrophils through phagocytosis by
macrophages.
Monopoiesis

Largest cell in the peripheral blood

 The monocyte has an irregular nucleus with
grayish, blue cytoplasm that has few granules
 Antigen processing & presentation
 Destruction of microbes and tumor cells

Causes of Monocytosis
 Infections- Chronic infections- E.g TB, Syphilis, CMV
 Malignancy- CMML
 Drugs/Toxins
 Autoimmune/ Allergic
 Idiopathic
Monocyte Maturation

 Pro monocytes
o12-18 micrometer in diameter
oPeroxidase & non specific esterase positive
oVariable number of larger azurophilic granulations like in monocyte
Macrophages
• Mononuclear phagocytes
• Mature from Monocytes in tissues responding
to inflammation and chemotactic stimuli
• Have greater phagocytic capacity and
with Increased content of hydrolytic enzymes.
• Roles
• Phagocytosis, killing, and digestion of MO,
particulate material, or tissue debris;
• Secretion of chemical mediators and
regulators of the inflammatory response;
• interaction (as dendritic cells) with antigen and
lymphocytes in the generation of the immune
response;
• Cytotoxicity, such as killing of some tumor
cells.
 Megakaryopoiesis
• Platelets production: their regulatory cytokines, EPO and TPO
• Stage of maturation : based on cytoplasm & nucleus; 4 stages
o Stage 1/megakaryoblasts
o 8 to 24 μm in spherical diameter
o Large indented nucleus with loose chromatin &
o Prominent nucleoli
o Scanty basophilic cytoplasm ,few alpha granule

o Proliferate by endomitosis
 Con’t
• Endomitosis, a unique form of
mitosis in which the DNA is
repeatedly replicated in the
absence of nuclear or
cytoplasmic division.
 Lymphopoiesis
• The major lymphocyte subsets are B and T cells.

• NK (natural killer) cells are a specialized lymphoid population.

• All cells arise in the bone marrow, but T

cells mature in the thymus, and B cells mature in the lymph

nodes, spleen, or other lymphoid tissues.

• Imminosurface markers are used to classify lymphocytes.

• B cells are identified by CD19 and CD20.

• T cells are identified by CD3, CD4,or CD8.

• NK cells comprise 10% of circulating lymphocytes

and are identified by the CD3–CD56+ phenotype.

The T cells mediate cellular immunity and two major types are
recognised:

1. CD4 positive Helper Cells and

2. CD8 positive Suppressor Cells.

The B cells mediate humoral immunity

• HSCs give rise to MPPs

• Some MPPs become committed to the

B cell lineage
 Transcription factors
• (eg,Ikaros, PU.1, E2A, EBf and Pax5
are essential
• for normal B-cell differentiation.) and
 Suppression of Notch signaling

• Some to T- cell lineage

 Early thymic progenitors, or ETPs)
migrate to the thymus which activate
Notch signaling,
 For lineage specification (GATA-3,
 B cell development
• B cell originate in BM and start maturing in BM and complete maturation in
secondary lymphoid tissue.

• The hallmark characteristic of a mature B cell circulating in blood or residing in

secondary lymphoid tissue is the expression of cell surface immunoglobulin (Ig).

• The cell-surface Ig consists of μ, δ, γ, α, or ε

heavy chains disulfide-linked to κ or λ light
chains

• Its referred to as the B cell receptor (BCR)

 B cell development
Two phases of development
1. Antigen independent -in fetal liver and fetal and adult marrow,
2. Antigen dependent - in secondary lymphoid tissue (LN, spleen)
B Cell developmental stages
• Progenitor (pro-) B cells ( Early and Late)
• Precursor (pre-) B cells (Large and small)
• Immature B cell;

Several different types of antigen-activated B cell

• Plasma cell; and
• Memory B cell.
• Ig a /Ig b mediate signal transduction following BCR cross-linking
 T Cell Development
T cell production in Bone marrow
Maturation takes place in thymus & become active in lymph node.
 CLP cells in marrow migrate in to thymus, get matured and
released in to circulation
 T Cell Receptor Gene Rearrangement and Expression

• TCR genes encode polypeptides designated α,β, δ andϒ.

Stages

• Double negative

• Double positive

• Single positive
T Lineage-Associated Surface Antigens
 Double Negative Stage
 4 stages of DN based on CD-25 & CD -44

o DN-1
o CD -44
o DN-2
o CD 25 & 44
o beta , delta and gamma rearrangement
o DN-3 ( ealry pre t cell )
o Rearrangement completed
o CD 25
o If beta productive rearrangement precede delta -TCR-2,
o if no productive beta rearrangement apoptosis
o DN-4 ( late pre t cell )
o Alpha rearrangement completed
o Pre-TCR complex associated with the CD3 & pseudoalpha
o Transmits signals that drive proliferation and promote cell survival
Double Positive Stage

• Positive Selection
• Occurs in the cortex
• To differentiate to either CD4 + or CD8 T+ cell.
• Ability to interact with MHC cell
• With MHC 1- CD 8 T cell ( cytotoxic)
• With MHC 2- CD 4 TH cell( Helper cell)
• If cant interact, it doesn’t receive survival signals so it undergoes apoptosis.

• Negative Selection
• Occurs in the Medulla
• To rule out Self- reactivity
• Based on TCR binding strength to an antigen
• Strong binding strength- leads to apoptosis
• Weak Binding strength- Survives

Then it leaves the thymus and moves to Lymph node

Single Positive Stage
Mature T cell leave thymus and enter 2ndary Lymphoid tissue.

 CD 8+ cell – Cytotoxic T cell

 For cell mediated immunity
 Binds to MHC 1- which takes intracellular antigens/ proteins and presents on its surface
 MHC 1 is found in almost every cell
 check whether antigen is pathogen and kill it.

CD4 + cell – T Helper cell

 Binds to MHC 2- is found on antigen presenting cells like dendritic cells
 It takes extracellular antigens and presents it on its surface

 TH 1 cell- differentiates through IL 12- helps pump up macrophages

 TH2 cell – IL4- Helps B cell to make antibodies for humeral immunity.
Regulation of T Cell Precursor/Thymocyte Development

• The developmental fate of thymocytes is regulated through physical

interaction with thymic stromal cells (i.e., TEC, fibroblasts, and
dendritic/interdigitating cells) and cytokines produced by thymic
stromal cells.

• TEC synthesize and secrete a complex array of cytokines, including IL-

1, IL-3, IL-6, IL-7, stem cell factor, leukemia inhibitory factor, CXCL12,
and TGF-
NK cells
 Large cytotoxic granular lymphocytes

 Role in innate immune response to viral infection & tumors

 Human NK cells develop from CD34+ hematopoietic stem
cells.
 5 to 10 % of peripheral blood lymphocyte

 Characterized by CD16/56+
Origin & maturation of Nk
Earliest progenitor is bipotential

Arise from CLP or thymic progenitor

Marrow is primarily site of production in adults .

 IL -15 & Flt3l are primarily cytokine

Development
Not clearly known due to limitation in surface markers

Not undergo rearrangement of Ig or TCR genes ,not require for their

devt unlike to other lymphocyte
 References
• Williams hematology 9th Edition
• Concise Guide to hematology 2nd edition
• Harrison’s principles of internal medicine 21st Edition
• Uptodate 2023.