Stem cell

Normal bone marrow and blood

Tipparat Penglong, Ph.D.
Division of Hematology, Department of Pathology
Faculty of Medicine, Prince of Songkhla University

Learning objectives
After todays lecture, you should be able to explain:
1. The unique properties, types, and sources of stem cells
2. The potential applications and limitations of stem cells in
medicine
3. Structure and function of normal bone marrow
4. Developmental stages and functions of cells in bone marrow
5. Normal morphologies and functions of hematopoietic cells

What are stem cells and their

unique properties?
Definition: Stem cells are undifferentiated biological cells that
can differentiate into specialized cells and can divide to produce
more stem cells.
1) Self-renewal

Stem cell
2) Differentiation potential

Catherin Twomey , the National Academies, Understanding Stem Cells

Stem cells can be discriminated based on

their differentiation potential:
Totipotent stem cells

can give rise to an entire viable organism

Pluripotent stem cells

can give rise to every type of cell derived from

the three germ layers except trophoblast

Multipotent stem cells

can only produce a limited number of cell types

such as hematopoietic stem cell

Unipotent stem cells

cant produce any cell type other than itself

Developmental potential and

reprogramming of stem cells
Egg cell

1) Somatic cell nuclear transfer

(Cloning)

2) Induce pluripotent stem cells

Self-renewal
Mitalipov and Wolf, Adv Biochem Eng Biotechnol, 2009

Types and sources of stem cells

1) Embryonic stem cells

2) Adult stem cells

(pluripotent stem cells)

(multipotent and unipotent stem cells)

The inner cell mass of blastocysts

Found in various tissues such as

-

ESCs

Fat tissue
Bone marrow
Brain
Muscle
Etc.

Multiply by cell division to

replenish dying cells
-

Skin renewal at 20 days

Heart remodeling at 5 years
All bones rebuilt at 10 years
Brain tissue remodelled at 20 years
Leeb C et al., Stem Cell Reviews and Reports, 2010

The potential applications of stem cells

in medicine
Cell therapy
Models of disease for new drug testing
Models of disease for basic knowledge

Therapeutic applicability of stem cells

1) Embryonic stem cells

2) Adult stem cells

(pluripotent stem cells)

(multipotent and unipotent stem cells)

Up to date, there is no single

approved medical treatment
derived from ESCs

Current clinic uses

- Skin grafts
- Hematopoietic stem cells
transplantation

In clinical trial
- Spinal cord injury
- Diabetes
- Severe myopia
- Etc.

In clinical trials
-Spinal cord injury
- Corneal regeneration
- Heart damage
- Etc.

Limitation of stem cells therapies

1) Embryonic stem cells

2) Adult stem cells

(pluripotent stem cells)

(multipotent and unipotent stem cells)

1. Tumors formation
2. Incompatible with the immune
system of patients
3. Ethically problematic cell source
Pluripotent stem cells can develop teratoma

1. Reduce the problem of tumors

formation
2. Reduce the problem of severe
immune reactivity by autologous
treatment.
3. Reduce the problem of ethically
controversial
4. Difficulties in isolation and
purification
5. Limit in proliferation capability

The therapeutic applications of

What is hematopoietic stem cell?
hematopoietic stem cells

Resemble lymphocyte

Answer: HSCs is multipotent stem cell that give rise to all red and
white blood cells and platelets.

Development of hematopoiesis

Micha Komorniczak (Poland)

Sources of hematopoietic stem cells for

therapeutic applications
1) Bone marrow
pelvis

sternum

2) Mobilized
peripheral blood

3) Umbilical cord

Sources of hematopoietic stem cells for

therapeutic applications
1) Bone marrow

2) Mobilized
peripheral blood

3) Umbilical cord

The main source of

HSCs in adult

Require granulocyte
colony-stimulating factorinduced peripheral stem
cell mobilization

Limited number of cells

available from a single
unit

Invasive

Less-invasive

Non-invasive

Graft Versus Host

Disease (GVHD) risk : ++

Graft Versus Host

Disease (GVHD) risk : +++

Graft Versus Host

Disease (GVHD) risk : +

Hematopoietic stem cell therapies

Hematopoietic stem cell transplantation is the
intravenous infusion of hematopoietic stem
cells to establish marrow and immune function
in patients with a variety of acquired and
inherited malignant and nonmalignant
disorders.
Allogeneic stem cell transplantation
-Transplantation using stem cells from
a matched donor carrying the normal gene
Autologous stem cell transplantation
-Transplantation using the patients own stem
cells
-Transplantation using the patients own
modified stem cells

Enrichment and purification methods for

hematopoietic stem cells
Magnetic enrichment
CD34+ cells

CD34+ cells

CD34+ cells
Density-gradient centrifugation

Fluorescence Activated Cell Sorting (FACS)

plasma
mononuclear cell
Ficoll 1.077
Packed red cell +
polymorphonuclear cell

Domen J et al., Chapter 2: Bone marrow (hematopoietic) stem cells. National Institutes of Health, 2006

Question
What do you think about stem cell beauty treatments?

Bone marrow
BM is a highly
cellular structure
present within the
hollow cavities of
hard bone tissue.

pelvis

sternum

Normal bone marrow

Children: bone marrow in all bones is red bone marrow.
Adulthood: bone marrow cells in long bones of hand and
leg become non-functional and are replaced by fat cells to
form yellow bone marrow. (100-age = % cellularity)
The only bones to carry red bone marrow throughout life
are the vertebrae, sternum, rib, hip bone, and skull bones.
In cases of severe blood loss, the body can convert yellow
marrow back to red marrow to increase blood cell
production.
On average, bone marrow constitutes 4% of the total body
mass of humans.

Normal cytologic pattern in BM

Normal cytologic pattern in BM

2015 Sysmex Middle East

Hematopoietic cells and nonhematopoietic cells in bone marrow

2001 Terese Winslow (assisted by Lydia Kibiuk)

Hematopoietic cells: Erythroid, Myeloid,

Lymphoid, and Megakaryocytic series
Pronormoblast

Myeloblast

Lymphoblast

Megakaryoblast

Basophilic Normoblast Promyelocyte Prolymphocyte Megakaryocyte

Polychromatophilic N.

Myelocyte

Orthochromatophilic N. Metamyelocyte
Reticulocyte
(Polychromasia)
Rbc

Band form

Neutro, Eo, Ba

Lymphocyte

Platelet
(Thrombocyte)

Non-hematopoietic cells in bone marrow

Non-hematopoietic cells (BM stromal cells) are connective
tissue cells that support the function of parenchymal cells
(hematopoietic cells)
BM stromal cells release colony stimulating factors, provide
hematopoietic environment that facilitate hematopoiesis

Adipocyte (storing energy as fat)

Osteoblast (synthesize bone)
Osteoclast (resorb bone)
Fibroblast (maintain the structural integrity of connective tissues)
Endothelial cell (create the thin-walled endothelium that lines the
inner surface of blood vessels and lymphatic vessels)
Macrophage (deliver iron for hemoglobin production)

BM stromal cells
Adipocyte

Fibroblast

Macrophage

Osteoblast

Endothelial cell

Osteoclast

Bone remodeling

Bone remodeling

https://www.youtube.com/watch?v=78RBpWSOl08#

Indication for BM examination

ICSH guidelines for the standardization of bone marrow specimens and reports

Disease of bone marrow

Aplastic anemia
Myelodysplastic syndrome
Multiple myeloma
Infections-tuberculosis
Various forms of leukemia

Bone marrow aspiration and biopsy

Aplastic anemia
Myelodysplastic syndrome
Multiple myeloma
Infections-tuberculosis
Various forms of leukemia

Blood
Composition of blood

Blood transports materials through

out the body inside blood vessels.
Average blood volume is 4-6 litters
Hematocrit

Plasma

Liquid part of blood (90% Water)

Helps to regulate body temperature
Contains electrolytes
Plasma transports blood cells, products of digestion and
hormones throughout the body.

Blood cells

Erythropoiesis
Early erythropoiesis

HSC

CMP

MEP

BFU-e

CFU-e

Late erythropoiesis

Basophilic
Orthochromatic
Erythrocyte
erythroblast Polychromatic erythroblast
Proerythroblast
Reticulocyte
erythroblast

Proerythroblast = Pronormoblast
Biggest in erythroid lineage
~ 20-25 m
Large central nucleus with
nucleoli
Fine chromatin
Basophilic cytoplasm
No granule

Basophilic erythroblast =
Basophilic normoblast
Smaller than proerythroblast ~
16-18 m
Heterochromatin
Intense basophilia (lots of
ribosomes!)
No granule

Polychromatophilic erythroblast =
Polychromatophilic normoblast
Smaller than basophilic
erythroblast ~ 9-12 m
Smaller intensely heterochromatic
nucleus
Purple/lilac cytoplasm mix of
basophilia from ribosomes and
growing eosinophilia from
hemoglobin
Last mitotic stage!

Orthochromatic erythroblast =
orthochromatic normoblast
Smaller than
polychromatophilic
erythroblast ~ 7-10 m
Small, compact, intensely
staining nucleus that is getting
ready to be extruded
Eosinophilic cytoplasm due to
abundant hemoglobin

Polychromasia=Reticulocyte
Wright stain

Immature rbc that contained

polyribosomes
Appear as polychromatophilic
erythrocyte on blood smear
when stained with a special
(supravital) stain 0.5% New
methylene blue
Normal range = 0-2%

Polychromasia

Methylene blue stain

Reticulocyte

Erythrocyte (Red blood cell)

Size: 7-7.5 uM
Morphology: biconcave
Composition: hemoglobin
Function: transport oxygen
Normal value: 4-6x106/uL

Life span: ~120 days

http://www.slideshare.net/raghuveer

Normal rbc count in blood

Remains remarkably constant although there are some
variations.
MALE :

5.2 0.3 x 106 /uL

FEMALE :

4.7 0.3 x 106 /uL

Abnormal red blood cells

Hematology Outlines: HH Rashidi MD & JC Nguyen MD

Abnormal red blood cells

Hematology Outlines: HH Rashidi MD & JC Nguyen MD

Abnormal red blood cell sizes

Normochromic normocytic rbc

Microcytic rbc

Macrocytic rbc

Anisocytosis = increased variation of rbc sizes

Example
-Anisocytosis 2+ with microcyte 2+
-Anisocytosis 3+ with microcyte 2+ macrocyte 1+

Abnormal red blood cell staining

Polychromasia
Number of
Polychromasia
per OPF

Grad

1-4

Few

5-10

1+

11-15

2+

16-20

3+

>20

4+

Hypochromic rbc

Abnormal shape of red blood cell

Normal rbc

Ovalocyte

Spherocyte

Tear drop

Target cell

Crenated cell

Abnormal shape of red blood cell

Bite cell

Blister cell

Keratocyte

schistocyte

Poikilocytosis = increased variation of rbc shapes

Inclusion in red blood cell

Hewell-Jolly bodies

Basophilic stippling

-Clusters of
DNA
-Found in
splenectomy,
hemolytic
anemia

-Clusters of RNA
-Found in
hemolytic
anemia, lead
poinsoning

Cabots ring

-Microtubules of
mitotic spindle
-Found in severe
anemia

Pappenheimer bodies

-Abnormal
granules of iron
-Found in
sideroblastic
anemia,

Abnormal red blood cell distribution

SDL

https://www.studyblue.com/#flashcard/view/7036582

Leukocytes (white blood cells)

Granulocytes

40-60%

0-6%

Agranulocytes
0-1%

20-40%

2-6%

Normal value of WBC in peripheral blood : 4.5-10x103/uL

Maturation of granulocytes

no granules

G-CSF

GM-CSF

azurophilic granules

specific granules

UMN Hematography Plus, Labeled by J. Levine

Granulocytes: Neutrophil

Diameter: 10-12 m
Polymorphonuclear or PMN cells (3-5 lobes)
Most numerous WBC in blood (40-60%)
Granules:
Azurophilic granules
Specific granules
Tertiary granules
The contents of these granules have
antimicrobial properties, and help combat
infection.
Function
1st wave of cells in acute inflammation;
can phagocytose bacteria
Life span ~ 5.4 days

Granulocytes: Eosinophil

Diameter: 10-12 m
Bilobed nucleus
Bright red/pink granules contain
Arginine rich major basic protein,
peroxidase, histaminase, arylsulfatase
0-6% in blood
Function:
Important in allergic rxns, parasitic
infections
Phagocytosis of Ab-Ag complexes, and a
variety of organisms, including
complement-coated Candida albicans
and bacteria

Life span 812 days

Granulocytes: Basophil
A small percentage (0.5%) of the cells in
circulation
Lobulated nucleus often obscured by
granules
Dark Blue Granules contain
Heparin: anticoagulant
Histamine: vasodilator
0-1% in blood
Function
Play a role in inducing and
maintaining allergic reactions
Role in hypersensitivity and
anaphylaxis
Life span ~ 60-70 hours

Mast cell
Very similar in both appearance
and function to the basophil
Mast cell Tissue basophil

NIH Publication No. 035423, September 2003

Maturation of agranulocytes

GM-CSF

Interleukines

UMN Hematography Plus, Labeled by J. Levine

Agranulocytes: Monocyte
Monoblast

Promonocyte

Immature
monocyte

Monocyte

Migrate through blood to the tissues; once in tissues they differentiate into phagocytes
(macrophages)
2009 by Ferrata Storti Foundation Jean E. Goasguen et al. Haematologica 2009;94:994-997

Macrophage or histiocyte
Macrophage or histiocyte is a type of immune cell that
destroys foreign substances in an effort to protect the body

from infection.
Phagocytes

Erythroblastic island

eat-me signals

Charles N. Serhan et al. Nature review immunology 2008

Agranulocytes: Lymphocytes
Main functional cells of immune system
T (Thymus) lymphocyte is involved in cell-mediated immunity. (80%)
B (Bone marrow or Bursa derived) lymphocyte is primarily responsible
for humoral immunity (relating to antibodies). (15%)
Natural killer cell plays a major role in defending the host from both
tumors and virally infected cells. (<5%)

Plasma cell
A plasma cell is a mature B
lymphocyte that is
specialized for antibody
production.

Eccentric nucleus ,
perinuclear halo cytopalsm
Rarely found in the
peripheral blood. (0.2%2.8% of the bone marrow
white cell count.

Lymphocyte markers

Thrombocyte (Platelet)
23 m in diameter
Derived from megakaryocyte in bone marrow formed from
small bits of megakaryocyte cytoplasm
Function in blood clotting
Life span 5-9 days
Platelets normally circulate at concentration of
150400 103/uL
Megakaryocyte

Platelet

Thrombopoiesis

Platelet examination
Platelet count: 150-400 x103/uL

Mean platelet volume (MPV): 7.5-11.5 fL

platelet number

platelet size

granule content

platelet distribution

Platelet examination
<5/OPF=Decrease

5-25/OPF=Adequate

Thrombocytopenia
Giant platelet

>25/OPF=Increase

Thrombocytosis
Pale stained platelet

Platelet aggregation

Thank you for your attention

Post test
1. What is this cell type?
A. Neutrophil
B. Basophil
C. Eosinophil
D. Lymphocyte

E. Monocyte
F. Band form neutrophil
G. Red blood cell
H. Platelet

F6
G7

5
3

Post test
2. What is this cell type?
A. Neutrophil
B. Basophil
C. Eosinophil
D. Lymphocyte

E. Monocyte
F. Band form neutrophil
G. Red blood cell
H. Platelet

F6
G7

5
3

Post test
3. What is this cell type?
A. Neutrophil
B. Basophil
C. Eosinophil
D. Lymphocyte

E. Monocyte
F. Band form neutrophil
G. Red blood cell
H. Platelet

F6
G7

5
3

Post test
4. What is this cell type?
A. Neutrophil
B. Basophil
C. Eosinophil
D. Lymphocyte

E. Monocyte
F. Band form neutrophil
G. Red blood cell
H. Platelet

F6
G7

5
3

Post test
5. What is this cell type?
A. Neutrophil
B. Basophil
C. Eosinophil
D. Lymphocyte

E. Monocyte
F. Band form neutrophil
G. Red blood cell
H. Platelet

F6
G7

5
3

Post test
6. What is this cell type?
A. Neutrophil
B. Basophil
C. Eosinophil
D. Lymphocyte

E. Monocyte
F. Band form neutrophil
G. Red blood cell
H. Platelet

F6
G7

5
3

Post test
7. What is this cell type?
A. Neutrophil
B. Basophil
C. Eosinophil
D. Lymphocyte

E. Monocyte
F. Band form neutrophil
G. Red blood cell
H. Platelet

F6
G7

5
3

Post test
8. Which of the following cell type would be expected to be increased in
number with a bacterial infection?

C
G

Post test
9. Which of the following cell type would be expected to be increased in
number with a viral infection?

C
G

Post test
10. Which of the following cell type would be expected to be increased in
number with a parasitic infection?

C
G

Post test
11. Please report red cell morphology of this blood smear.

A.
B.
C.
D.
E.
F.

Normochromic normocytic rbc

Hyperchromic normocytic rbc
Hypochromic normocytic rbc
Normochromic microcytic rbc
Hyperchromic microcytic rbc
Hypochromic microcytic rbc

Post test
12. Please report this platelet smear.

A.
B.
C.
D.
E.
F.

Increase
Adequate
Decrease
Increase with clumping
Adequate with pale stain
Decrease with giant platelet