Hematopoiesis

- process of blood cell production, differentiation and development.

- Consists: Bone marrow, thymus, spleen, liver, lymph node
ORIGIN:
Hematopoietic stem cells (HSCs)
- foundation of the adult hematopoietic system
- Ultimately responsible for the continuous daily production of all mature blood lineages
- Most thoroughly characterized type of adult stem cell
- Defined at the single-cell level by their dual capacity for self-renewal and mutipotential
differentiation
Embryo
- produces the first adult repopulating HSCs
Type of human stem cells:
Totipotential stem cells
Pluripotential stem cells
Multipotential stem cells
Totipotential stem cells
- present in the first few hours after ovum is fertilized
- Most versatile type of stem cell
- Can develop into any human cell type even embryo into fetus
Pluripotential stem cells
- present several days after fetilization
- Can develop into any human cell type except fetus
Multipotential stem cells
- derived from pluripotent stem cells
- Found in adults but limited to specific type of cells to form tissues
- Ex: Bone marrow stem cells - can produce all types of blood cells, bone cartilage, and
adipose (fat) cells
Stem cell populations
- essential for lifelong maintenace of organ function
Signaling pathways
- important control devices of HSC fate
Stem cell therapy
- focuses on (embryonic, fetal, cord, adult blood) stem cell therapy

EARLY DEVELOPMENT OF BLOOD CELLS

Embryonic blood cells (except lymphocyte type of white blood cell)
- originate from the mesenchymal tissue arises from mesoderm (embryonic germ layer)
Both produces, respectively, potent HSCs and mutipotent progenitor cells before their
apperance in the yolk sac
Aorta-gonad-mesonephros region- mesodermally derived intraembryonic region
Paraaortic splanchnopleure - slighty earlier developmental stage
Mammalian embryo
 - contains atleast two spatially separated sources of hematopoietic cells

ANATOMICAL SITES OF BLOOD CELL DEVELOPMENT

Yolk sac
- Where first hematopoietic cells are generated
Erythroblasts
- primitive red blood cells
- First blood cells
- Formed during first 2 to 8 weeks
Mesenchymal stem/progenitor cells and HSCs
- circulate together in the peripheral blood during the first trimester to the second
ontogenic sites of hematopoeisis:
1. Liver
2. Bone marrow
Liver
- will become the site of blood cell development
- By second month of gestation: becomes the major site of hematopoeisis
- Predominates from about the second to fifth month of the fetus
Bone marrow
- will begin to function in the production of blood cells in fourth month of gestation
- After the fifth fetal month, it will begins to assume the ultimate role as the primary site of
hematopoiesis (medullary hematopoiesis)
Three distinct clumps or colonies:
1. Paraxial mesoderm- tracks the path of the notochord
2. Intermediate mesoderm - hovers just beside it for a short stretch of the embryos length
3. Lateral plate mesoderm- fills the rest of the space
- Forms an important contact with the ectoderm, endoderm and
extraembryonic shell to the outside

BONE MARROW SITES AND FUNCTIONS

Bone marrow
- forms within the cavities of all bones and may be present in two forms:
Yellow marrow
Red marrow
- one of the bodys largest organ
- Represents approximately 3.5% to 6% total body weight
- Average 1,500g in adult
- Hematopoietic marrow organized around bone vasulature
- Consists of the following:
> hematopoietic cells (erythroid, myeloid, lymphoid, megakaryocyte)
> fat tissue
> osteoblasts
> osteoclasts
 > stroma
Yellow marrow
- normally inactive and composed mostly of fats (adipose)
Red marrow
- normally active in the production of the most types of leukocytes, erythrocytes and
thrombocytes
- Young persons: found in both appendicular and axial skeleton
- Adults: found in axial skeleton and proximal ends of long bones
- Age 18: only found in vertebrae, ribs, sternum, skull bones, pelvis and some extent the
proximal epiphyses of femur and humerus
Extramedullary hematopoiesis
- Spleen , liver and lymphnodes revert back to producing immature blood cells
Cases of enlargement of spleen and liver in physical examination:
1. When bone marrow becomes dysfunctional in ces such as aplastic anemia, infiltration by
malignant cells, or overproliferation of a cell line (ex: leukemia)
2. When the bone marrow is unable to meet the demands placed on it, as in hemolytic
anemias

CELLULAR ELEMENTS OF BONE MARROW

Progenitor Blood Cells
Pluripotent stem cell
- first in a sequence of steps of hemtopoietic cell generation and maturation
Multipotential hematopoietic stem cell
- progenitor of all blood cells
Stem cells
- carry out the ultimate burden of generating multilineage mature blood cells over the
lifetime of the organism
- Have the capacity for self-renewal as well as proliferation and differentiation into
progenitor cells
Stem cell plasticity
- concept referred in which blood, brain, and many other regions of the body have their
own specialized stem cells thar capable of making of making replacement cell
Multipotent adult progenitor cells
- aka master cells
- adults carry a reservoir of this
- Capable of rebuilding almost any damage tissue
- expresses an enzyme (telomerase) that keeps cells from aging.
- In vitro: can be coaxed into becoming muscle, cartilage, bone, liver or different types of
neurons and brain cells
Three phases according to cell maturity:
1. Primitive, multipotential cells
2. Intermediate cells
3. Mature cells
Primitive, multipotential cells
- most immature group capable of self-renewal and differentiation into all blood cell lines
- Progenitor of two major ancestral cell lines:
> Lymphocytic stem cells
> Nonlymphocytic stem cells

Intermediate cells
- Group consists of progenitor cells destined to develop into distinct cell lines
Mature cells
- most developed grouped with specific function
Lymphocytic stem cells
- Aka lymphoid stem cell
- Precursor of either mature T cells or B cells/plasma cells
Nonlymphocytic (myeloid) stem cell
- Progresses to the progenitor of CFU-GEMM
Colony forming unit (CFU)
- colony forming unit
- Used as a prefix to record the number of colony-forming units of different progenitor cells
that are identified through in vitro clonal assays
- each can produce a colony of one hematopoietic lineage under appropriate growth
conditions
Colony forming unit, granulocyte-erythrocyte-monocyte-megakaryocyte
- CFU-GEMM
- In eythropoiesis: it differentiate into burst-forming unit-erythroid (BFU-E)
- can lead to formation of the following:
> Colony forming unit granulocyte macrophage/monocyte (CFU-GM)
> Colony forming unit eosinophil (CFU-Eo)
> Colony forming unit basophil (CFU-B)
> Colony forming unit megakaryocyte (CFU-Meg)
Growth factors, inhibitors and microenvironment
- They control the formation and development of mature blood cells from the bone marrow
multipotential stem cells
Microenvironment (locale)
- Influences the behavior and controls the proliferation of multipotential cells
Bone
- seems to provide the microenvironment the mist appropriate for proliferation and
maturation of cells
Hematopoietic progenitor cells (HPCs)
- can mobilized from the bone marrow to the blood by a wide variety of stimuli, including
hematopoietic growth factors and and chemokines
- In bone marrow: exist in highly organized, three-dimensional microenvironment
composed of diverse population of stromal cells and an extracellular matrix rich in
fibronectin, collages, and various proteoglycans
 - Can be found in umbilical cord blood (UCB)
Individual hematopoietic cytokines
- can be lineage specific or can regulate cells in multiple lineages
Umbilical cord blood hematopoietic cells
- have been employed successfully as a therapeutic source of autologous and allorgenic
transplants for more than 20 years
Cyrptopreservation
- prolongs the storage time of UCB

Multilineage:
Common Lymphoid/Myeloid Progenitor (CLP or CMP)
Two lineage
T cells and Natural Killer cells (TNKs)
Granulocytes and macrophages (GMs)
Megakaryocytes and erythroid cells (MEPs)
Unilineage
B cells (BCPs)
NK cells (NKPs)
T cells (TCPs)
Granulocytes (GPs)
Monocytes (MPs)
Erythrocytes (EPs)
Megakaryocytes (MkPs)

Erythropoiesis
- occurs in distinct anatomical sites called erythropoietic islands
- Erythroid cells account for 5% to 38% of nucleated cells in normal bones
- Erythropoietic islands: specialized niches in which erythroid preecursors proliferate,
differentiate and enucleate
- Each island consists of a macrophage surrounded by cluster of erythroblasts
- Within erythroid niches these functions occur:
> cell-cell
> cell-extracellular matrix adhesion
> positive and negative regulatory feedback
> central macrophage
Granulopoiesis
- recognized as a maturational unit
- Early cells located at cords and bone trabeculae
- Neutrophils resides at proliferating pool (3-6 days for mature cells) and maturation
storage pool (if needed, 6-10 hours)
Lymphopoiesis
- lymphocytes and plasma cells are produced at lymphoid follicles
- Lymphocytes: randomly dispered throughout the cords
 - Lymphoid follicles: can be observed at the of 50
- Lymphoid cells: account for 1% to 5%
Megakaryopoiesis
- takes place adjacent to the sinus endothelium
- Megakaryocytes: protrude through the vascular wall as small cytoplasmic process to
deliver platelets into the sinusoidal blood
- Develop into platelets approx. 5 days
Marrow stromal cells
- Meshwork is composed of: reticulum cells, histiocytes, endothelial cells, adipose cells
- It is where the hematopoietic cells are suspended in a delicate semifluid state
- Produce an extracellular matrix composed of collagen and proteins (ex:glycoproteins
and proteoglycans)
- Extracellular matrix: critical for the maintenance of normal renewal and differentiation of
bone marrow cells
Mast cells
- a connective tissue cell of mesenchymal origin normally observed at bone marrow
- Abundant blue-purple granules that usually obscure the round or oval reticular nucleus
contain: heparin, histamine, serotonin, proteolytic enzymes
- Increase number: abnormal condition (chronic lymphoproliferative disorders or chronic
infections)
Macrophages
- aka reticulum cells or histiocytes
- Large cells at bone marrow
- Siderophages: macrophages containing iron-rich hemosiderin and ferritin
- Gaucher cells: macrophages filled with uncatabolize glucocerebrosides
Bone cells
- Osteoblasts: bone matrix-synthesizing cells
- resembles plasma cells (usually observed in groups)
- Usually seen in adult
- Increased number from children and patients with metabolic disease
- Osteoclasts: bone-remodeling
- Resembles megakaryocyte

INTERLEUKINS
- protein molecules that work in conjunction with hematopoietic growth factors to stimulate
proliferation and differentiation of specific cell lines
- Cytokines that act independently or in conjunction with other interleukins to encourage
hematopoietic growth
- Cell signaling molecules
- Part of cytokine super family of signaling molecules
- Inter: between
- Leuk: leukocytes (WBC)
- Method of immune cross-talk and communication
 - Primary messengers and directors of immune system
- 35 well known interleukins
- Have limited role in hematopoietic homeostasis but major role in host responses to
infection and antigenic challenge
- Can cause: cellular proliferation, cell activation, inflammation, physiology changes (fever,
pain, allergies as with histamine release and growth
Hematopoietic growth factors
- each is encoded by single gene
- Major role is to regulate proliferation and differentiation of HPCs and regulate survival
and function of mature blood cells
- Being used and tested in clinical trials for treatment of a variety of hematological
disorders
- Capable of mobilizing HPCs
- Interact with blood cells at different levels in the cascade of cell differentiation from the
multipotential progenitor to the circulating mature cell
- Chromosome 7: gene for erythropoietin
- Chromosome 5 (long arm): gene for granulocyte-macrophage CSF (GM-CSF),
interleukin-3 (IL-3), monocyte CSF (M-CSF)
- Chromosome 17: for granulocyte CSF (G-CSF)
- Recombinant DNA technology: erythropoietin, GM-CSF, G-CSF, M-CSF and IL-3 are
factors that have been identified, cloned and produced through this
- Specific factors: used as adjunct therapy in a wide variety of disease
- G-CSF and GM-CSF: affect the myeloid cells
- IL-7: stimulates T and B lymphocytes
- IL-12: targets natural killer cells
- VLA-4, VCAM-1 and hyaluronan receptors: molecules that are important for mobilization
EXAMINATION OF MATURING BLOOD CELLS
Complete Blood Cell Count (CBC)
- routinely performed in hematology laboratory
- WBCs are examined, identifed and counted
- RBCs and platelets are also carefully examined during this procedure
2 characteristic for cell identification
Overall cell size
Nuclear-Cytoplasmic ratio (N:C)
Overall cell size
- usually compared with the size of mature erythrocytes
- Erythrocytes and leukocytes decrease in overall size as maturation progresses
Nuclear-Cytoplasmic ratio
- amount of space occupied by the nucleus in relationship to the space occupied by the
cytoplasm
- Size of nucleus decreases as cell matures
- 4:1 2:1 or 1:1: Blasts forms of erythrocytes, leukocytes and megakaryocytes except
thrombocytes and mature eythrocytes and lymphocyte
 - Anuclear: lacks nucleus (thrombocytes and erythrocytes)
- 4:1 3:1: mature lymphocytes
Nuclear characteristics
Chromatin Pattern
Nuclear Shape
Presence of Nucleoli
Chromatin pattern
- demonstrates characteristics pattern (most distincetive nuclear feature of a cell in terms
of maturity and cell type recognition
- Loose-looking arrangement to more clumped pattern as a cell matures
- Pyknotic: dense or compact
Lymphocytes smooth or homogeneous pattern
throughout development until mature
stage, when clumped heterochromatin is
more obvious

Granulocytes fine to highly clumped pattern

Monocytes lacy pattern which becomes finer as the

cell matures

Erythrocytes continue to develop a more clumped

pattern as maturation progresses, until
extremely dense (pyknotic) nucleus is lost
(extruded) fromthe mature cell

Nuclear Shape
- round or oval: young cells
- Monocytes may have slightly folded nuclear shape
Lymphocytes round or oval nucleus
Some have small cleft

Monocytes kidney bean-shaped

Folded or horseshoe are common

Mature Neutrophils, Eosinophils, segmented nuclei attached to one another

Basophils by fine filaments
Lobe ranges from 2-5 depending on the
cell type
Presence of Nucleoli
- as cell mature, nucleoli are usually not visible
- Three cell lines of erythrocytes, leukocytes and megakaryocytes have nucleoli in the
earliest stages
Lymphoblasts 1 or 2 nucleoli
 Myeloblasts 1 to 5 nucleoli

Monoblasts 1 or 2 but may have 3 or 4 nucleoli

Erythroblasts may not have any nucleoli

May have up to 2 nucleoli with darker
stain than other types of blast cells

Megakaryoblasts 1 to 5 nucleoli
Cytoplasmic Characteristic
staining or color intensity
granulation
Shape
Quantity of cytoplasm
Vacuolization
Inclusion bodies

Staining of color Intensity

- progresses from darker blue (active protein synthesis) to lighter bluu, blue-gray or pink
- Immature erythrocytes: dark blue cytoplasm to paler and gray looking as cell synthesizes
hemoglobin
- Lumphocytrs: pale skyblue cytoplasmic color
Granulation
- progresses from no granules to nonspecific to specific granules
- In Size: very fine to coarse
- In Color: red (azurophilic), blue (basophilic), orange (eosinophilic)
- In Amount of granulation per cell
Cytoplasmic shape
- most distinctive variation occurse in some blasts forms, monocytes and megakaryocytes
- Pseudopods: may be observed in mature monocytes and in some luekocyte blast forms
- Megakaryocytes: developed more irregular outline as cell matures
Quantity of Cytoplasm
- increases with age
- Abnormalities of lymphocytes ~ increase amounts of cytoplasm
Vacuolization
- increases with age
- Commonly seen in older cells and abnormal conditions except monocytes
- Monocytes: have vacuoles throughout their life cycle and under normal conditions
- Anticoagulant: can produce vacuoles as artifacts if the blood stored for a
longer-than-acceptable period
Inclusion bodies
- They aid in identification of cell types
- Myelocytic or Monocytic blast: Auer bodies or Auer rods
 - Eythrocytic and Leukocytic inclusions: indicative of specific disease

Type Nuclear Chromatin Cytoplasmic Granules Color of Percentage

Shape color granules
Average

Segmented Lobulated Very Pink Many Pink, a few 56

neutrophil clumped blue

Band form Curved Moderately Blue/Pink Many Pink 3

neutrophil clumped

Lymphocyt Round Smooth Light blue Few or Red 34

e absent

Monocyte Indetend/T Lacy Gray-blue Many Dusty blue 4

wisted

Eosinophil Lobulated Very Granulated Many Orange 2.7

clumped

Basophil Lobulated Very Granulated Many Blue 0.3

clumped