Stem cell

Normal bone marrow and blood

Tipparat Penglong, Ph.D.

Division of Hematology, Department of Pathology

Faculty of Medicine, Prince of Songkhla University

After today’s 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

Learning objectives

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

2) Differentiation potential

Stem cell

Catherin Twomey , the National Academies, Understanding Stem Cells

Stem cells can be discriminated based on their differentiation potential:

• Totipotent stem cells

• Pluripotent stem cells

• Multipotent stem cells

• Unipotent stem cells

can give rise to an entire viable organism

can give rise to every type of cell derived from the three germ layers except trophoblast

can only produce a limited number of cell types such as hematopoietic stem cell

can’t produce any cell type other than itself

Self-renewal

1) Somatic cell nuclear transfer(Cloning)

2) Induce pluripotent stem cells

Mitalipov and Wolf, Adv Biochem Eng Biotechnol, 2009

Egg cell

Developmental potential and reprogramming of stem cells

ESCs

Types and sources of stem cells

1) Embryonic stem cells(pluripotent stem cells)

• The inner cell mass of blastocysts

2) Adult stem cells(multipotent and unipotent stem cells)

• Found in various tissues such as

- 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

• Cell therapy

• Models of disease for new drug testing

• Models of disease for basic knowledge

The potential applications of stem cellsin medicine

Therapeutic applicability of stem cells

1) Embryonic stem cells(pluripotent stem cells)

• Up to date, there is no single approved medical treatment derived from ESCs

• In clinical trial

- Spinal cord injury

- Diabetes

- Severe myopia

- Etc.

2) Adult stem cells(multipotent and unipotent stem cells)

• Current clinic uses

- Skin grafts

- Hematopoietic stem cells transplantation

• In clinical trials

-Spinal cord injury

- Corneal regeneration

- Heart damage

- Etc.

Limitation of stem cells therapies

1) Embryonic stem cells(pluripotent stem cells)

1. Tumors formation

2. Incompatible with the immune system of patients

3. Ethically problematic cell source

2) Adult stem cells(multipotent and unipotent stem cells)

1. Reduce the problem of tumors formation

2. Reduce the problem of severe

immune reactivity by autologoustreatment.

3. Reduce the problem of ethically controversial

4. Difficulties in isolation and purification

5. Limit in proliferation capability

Pluripotent stem cells can develop teratoma

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

The therapeutic applications of hematopoietic stem cells

What is hematopoietic stem cell?

Resemble lymphocyte

Michał Komorniczak (Poland)

Development of hematopoiesis

Sources of hematopoietic stem cells for therapeutic applications

3) Umbilical cord1) Bone marrow 2) Mobilizedperipheral blood

pelvis

sternum

Sources of hematopoietic stem cells for therapeutic applications

3) Umbilical cord1) Bone marrow 2) Mobilized peripheral blood

• Limited number of cells

available from a single unit

• Non-invasive

• Graft Versus Host Disease (GVHD) risk : +

• The main source of HSCs in adult

• Invasive

• Graft Versus Host Disease (GVHD) risk : ++

• Require granulocyte colony-stimulating factor-induced peripheral stem cell mobilization

• Less-invasive

• Graft Versus Host Disease (GVHD) risk : +++

Hematopoietic stem cell therapies

Autologous stem cell transplantation

-Transplantation using the patient’s own stem cells

-Transplantation using the patient’s own modified stem cells

Allogeneic stem cell transplantation

-Transplantation using stem cells from

a matched donor carrying the normal gene

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.

Enrichment and purification methods for hematopoietic stem cells

Magnetic enrichment

Fluorescence Activated Cell Sorting (FACS)

plasma

mononuclear cell

Ficoll 1.077

Packed red cell +

polymorphonuclear cell

Density-gradient centrifugation

CD34+ cells

CD34+ cells

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

CD34+ cells

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 non-hematopoietic cells in bone marrow

2001 Terese Winslow (assisted by Lydia Kibiuk)

Hematopoietic cells: Erythroid, Myeloid, Lymphoid, and Megakaryocytic series

Pronormoblast Myeloblast Megakaryoblast

Basophilic Normoblast

Polychromatophilic N.

Orthochromatophilic N.

Reticulocyte(Polychromasia)

Promyelocyte

Myelocyte

Metamyelocyte

Band form

Neutro, Eo, Ba

Megakaryocyte

Lymphoblast

Prolymphocyte

Rbc Platelet(Thrombocyte)

Lymphocyte

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

Osteoblast Osteoclast

Adipocyte Fibroblast Endothelial cell

Macrophage

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

Composition of blood

• Blood transports materials through out the body inside blood vessels.

• Average blood volume is 4-6 litters

Blood

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 Late erythropoiesis

HSC CMP MEP BFU-e CFU-eProerythroblast

Orthochromaticerythroblast

Reticulocyte

ErythrocytePolychromaticerythroblast

Basophilicerythroblast

Proerythroblast = Pronormoblast

• Biggest in erythroid lineage

~ 20-25 µm

• Large central nucleus with nucleoli

• Fine chromatin

• Basophilic cytoplasm

• No granule

Basophilic erythroblast = Basophilic normoblast

• Smaller than proerythroblast ~ 16-18 µm

• Heterochromatin

• Intense basophilia (lots of ribosomes!)

• No granule

Polychromatophilic erythroblast = Polychromatophilic normoblast

• Smaller than basophilic erythroblast ~ 9-12 µm

• 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 polychromatophilicerythroblast ~ 7-10 µm

• Small, compact, intensely staining nucleus that is getting ready to be extruded

• Eosinophilic cytoplasm due to abundant hemoglobin

Polychromasia=Reticulocyte

• Immature rbc that contained polyribosomes

• Appear as polychromatophilicerythrocyte on blood smearwhen stained with a special (supravital) stain 0.5% New methylene blue

Normal range = 0-2%

Wright stain

Methylene blue stain

Polychromasia

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

Hypochromic rbcPolychromasiaNumber of Polychromasiaper OPF

Grad

1-4 Few

5-10 1+

11-15 2+

16-20 3+

>20 4+

Abnormal shape of red blood cell

Normal rbc Ovalocyte Spherocyte

Crenated cellTear drop Target cell

Abnormal shape of red blood cell

Keratocyte

Bite cell Blister cell

schistocyte

Poikilocytosis = increased variation of rbc shapes

Inclusion in red blood cell

Hewell-Jolly bodies Basophilic stippling

Pappenheimer bodiesCabot’s ring

-Microtubules of mitotic spindle-Found in severe anemia

-Clusters of DNA-Found in splenectomy, hemolytic anemia

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

-Abnormal granules of iron-Found in sideroblasticanemia,

Abnormal red blood cell distribution

SDL

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

Leukocytes (white blood cells)

Granulocytes Agranulocytes

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

40-60% 0-6% 0-1% 20-40% 2-6%

Maturation of granulocytes

GM-CSFG-CSF

UMN Hematography Plus, Labeled by J. Levine

specific granules

azurophilic granules

no granules

Granulocytes: Neutrophil

• Diameter: 10-12 μm• 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 μm• 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 albicansand bacteria

• Life span 8–12 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. 03–5423, September 2003

Maturation of agranulocytes

GM-CSF

UMN Hematography Plus, Labeled by J. Levine

Interleukines

Jean E. Goasguen et al. Haematologica 2009;94:994-997©2009 by Ferrata Storti Foundation

Agranulocytes: Monocyte

Monoblast Promonocyte MonocyteImmature monocyte

Migrate through blood to the tissues; once in tissues they differentiate into phagocytes (macrophages)

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.

Erythroblastic islandPhagocytes

“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)

• 2–3 µm 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

150–400 × 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

Thrombocytopenia

Giant platelet Pale stained platelet Platelet aggregation

5-25/OPF=Adequate >25/OPF=Increase<5/OPF=Decrease

Thrombocytosis

Thank you for your attention

Post test1. What is this cell type?

F

G

A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte

E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet

11

2

3

4

5

6

7

Post test2. What is this cell type?

F

G

A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte

E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet

1

2

1

2

3

4

5

6

7

Post test3. What is this cell type?

F

G

A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte

E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet

1

2

3

4

5

6

7

Post test4. What is this cell type?

F

G

A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte

E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet

1

2

3

4

5

6

7

Post test5. What is this cell type?

F

G

A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte

E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet

1

2

3

4

5

6

7

Post test6. What is this cell type?

F

G

A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte

E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet

1

2

3

4

5

6

7

Post test7. What is this cell type?

F

G

A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte

E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet

1

2

3

4

5

6

7

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.

Post test

A. Normochromic normocytic rbcB. Hyperchromic normocytic rbcC. Hypochromic normocytic rbcD. Normochromic microcytic rbcE. Hyperchromic microcytic rbcF. Hypochromic microcytic rbc

12. Please report this platelet smear.

Post test

A. IncreaseB. AdequateC. DecreaseD. Increase with clumpingE. Adequate with pale stainF. Decrease with giant platelet