Hematopoiesis Notes

Hematopoiesis

Introduction

Hematopoiesis (or Hemopoiesis):
- A process that produces all blood cells from Hematopoietic Stem Cells.

Prerequisites:

Myeloid cells:
RBCs, Platelets, Neutrophils, Eosinophils, Basophils, Monocytes
Lymphoid cells:
- B lymphocytes, T lymphocytes, and NK lymphocytes.
Bone marrow:
- Histology and organization, including Red BM, Yellow BM, and BM niche.
Cell phenotype by Flow cytometry:
- Cluster of differentiation (CD).

Why hematopoiesis is vital:

Mature blood cells have a short life span:
- RBCs: 120 days
- Platelets: 7 to 10 days
- WBCs: a few days in the bloodstream
Mature blood cells do not have the capacity for division (except monocytes and lymphocytes).
The number of blood cells is relatively constant.

Important notions about hematopoiesis:

Continuous process:
- Occurs throughout life, from embryo to death.
Regulated and adaptive process:
- Depends on physiological or pathological states
  - Altitude: More RBCs
  - Infection: More WBCs
- Under the control of cytokines and growth factors (proliferation or inhibition).
Most regenerative tissue:
- Production of 10^{11} to 10^{12} cells daily.
  - RBCs: 200x10^9 daily = 2x10^6 /second
  - WBCs: 50 – 100 x 10^9 daily
  - Platelets: 100 x 10^9 daily

Plan for Studying Hematopoiesis

Definition: Define Hematopoiesis and Hematopoietic Stem Cells
Sites of Hematopoiesis: State the different sites.
Process of Hematopoiesis:
- Difference between stem cells, progenitor cells, precursor cells, and mature cells.
Factors Involved: Discuss factors involved in hematopoiesis.
Methods of Studying Hematopoiesis: Define different methods.
Clinical Implications: Understand the clinical implications.

Learning Objectives

Define Hematopoiesis and Hematopoietic Stem Cell
State the different sites of hematopoiesis
State the difference between stem cells, progenitors cells, precursors cells and mature cells
Discuss factors involved in hematopoiesis
Define the different methods of studying hematopoiesis
Understand the clinical implications

Sites of Hematopoiesis

Embryonic Life (3 weeks post-conception):
- Yolk Sac
Fetus:
- Early: Liver, Placenta
- Late: Liver, Spleen, Placenta, Bone Marrow
After Birth:
- Bone Marrow
Throughout post-natal life, hematopoiesis slowly becomes confined to the axial skeleton:
- Skull
- Vertebrae
- Sternum
- Pelvic bones
- Ribs
- Diaphysis of the long bones
Bone Marrow Smear:
- Site of puncture: Manubrium of sternum.
Bone Marrow Biopsy
- Bakkenkam
In some states of medullary insufficiency such as Thalassemia and myelofibrosis:
- Hematopoiesis can revert to its original sites
- Mainly spleen and liver
- That causes hepatomegaly and splenomegaly

The Process of Hematopoiesis

Hematopoietic Stem Cells (HSC’s):

Till and McCulloch experiment (1961):
- Lethal dose of radiation ≈ 30 days
- Death by Aplastic Anemia
- Injection of homozygous Bone Marrow leads to Reconstitution of all blood cells

Hematopoietic hierarchy

HSC’s → Progenitors → Precursors → Mature Cells
Small pool of HSC’s, developing into larger pools of progenitors and precursors, ultimately differentiating into mature cells.
Multistep process

Hematopoietic Stem Cells properties

Multipotency:
- Capacity to differentiate into different cell lines.
Self-renewal:
- Capacity of division into HSCs to maintain a sufficient stock of HSCs.
Capacity of differentiation:
- Once differentiated, produces only one cell lineage.
Low mitotic activity
Cannot be morphologically identified: Looks like a large lymphocyte

Progenitor Cells

Rise from HSCs.
Lineage-committed cells (Myeloid OR Lymphoid lineage).
Classified into two main groups:
- Multilineage progenitor
- Single-lineage progenitor
Two multilineage progenitors:
- CMP: Common Myeloid Progenitor
- CLP: Common Lymphoid Progenitor
Single-lineage progenitors produce 1 or 2 types of mature cells. *Hematopoietic Multipotent Progenitor (MPP)
- Common Myeloid Progenitor (CMP):
  - Mature cells: Erythrocytes and Platelets
- Common Lymphoid Progenitor (CLP):
  - Mature cells: B lymphocytes, T lymphocytes, NK lymphocytes
High mitotic activity.
Less self-renewal potential.
Cannot be morphologically identified.
Common in marrow and in lymphoid organs for lymphoid cells.
*Lymphoid Lineage

Myeloid Lineage

Megakaryocyte-Erythrocyte Progenitor (MEP)
Colony-Forming Unit-Baso (CFU-Baso)
Colony-Forming Unit-Eosinophil (CFU-Eo)
Granulocyte Macrophage Progenitor (GMP)
- Basophils
- Eosinophils
Burst Forming Unit Erythroid
**Colony Forming Unit Erythroid
- Precursor RBCs**
Colony Forming Unit Megakaryocyte: Precursor MK
Colony-Forming Unit-Granulocyte Monocyte (CFU-GM)
*CFU-G
* Precursor G
*CFU-M
* Precursor M

Precursor cells: Blast Cells

Can be morphologically identified.
Unilineage committed: Give rise to one type of mature cell.
Have high mitotic activity.
Loss of self-renewal potential.
Common in marrow and in lymphoid organs for lymphoid cells.

Erythropoiesis:
- Proerythroblasts → Red Blood Cells
Granulopoiesis:
- Myeloblasts → Granulocytes
  - Polynucléaire neutrophile:
    - Aspect morphologique
      12 µm de diamètre environ ;
    - noyau a une chromatine condensée
    - segmenté en 2 à 5 lobes réunis par un filament fin de chromatine ;
    - la majorité des polynucléaires neutrophiles a 3 lobes
    - cytoplasme beige rosé avec de très nombreuses granulations neutrophiles marron beige.
      *Polynucléaires éosinophiles
      *Granulations spécifiques
      *Aspect morphologique
      *Cellule de 12 à 17 µm de diamètre ; le noyau est peu segmenté (classiquement bilobé en bissac) ; la chromatine est condensée. Le cytoplasme contient du glycogène et plusieurs types de granulations des mitochondries et un Golgi plus développés que PNN. Les granulations spécifiques sont de grande taille et apparaissent de couleur orangé après la coloration de MGG.
      *Les polynucléaires basophiles
      *Granulations spécifiques
Thrombopoiesis:
- Megakaryoblast → Platelets
Lymphopoiesis:
- Lymphoblasts → Lymphocytes
Monocytopoiesis:
- Monoblasts → Monocytes

Identification of Hematopoiesis Involved Cells

HSCs and progenitors are not morphologically identified
Can be identified by searching for some surface antigens: Cluster of differentiation (CD)
During the hematopoietic process, cells acquire some Ag and lose others
Identifying cell populations via CDs is done by using flow cytometry
There are more than 370 CDs described
CDs are identified by using antibodies
*Lymphocytes T CD8
*Lymphocytes T CD4

Regulation of Hematopoiesis

The hematopoiesis is a regulated and adaptive process.
Adapted to physiological or pathologic conditions in order to:
- Maintain HSCs population.
- Induce proliferation and differentiation.
- Raise blood production to meet excess demand.
The regulation is a complex process:
- Intercellular signals: HSCs and BM niche interactions.
- Cytokines and Growth factors.

Hematopoietic Growth Factors (HGF)

HGFs are molecules that influence cell growth and proliferation.
Their action is made through interaction with cell surface receptor.
That leads to the activation of intracellular signaling cascade.
HGFs can be divided into 2 groups:
1. HGFs that affect the whole hematopoietic process:
  - Mainly Early stage of hematopoiesis.
  - SCF: Stem Cell Factor (action on HSCs).
  - IL-3: Interleukin 3.
  - GM-CSF: Granulocyte-Monocyte Colony Stimulating Factor.
  - IL-7: Lymphoid lineage.
2. HGFs that act exclusively on specific lineage
  - EPO: Erythropoietin (erythroid lineage).
  - TPO: Thrombopoietin (thrombopoiesis).
  - G-CSF: Granulocyte Colony Stimulating Factor.
  - M-CSF: Monocytes Colony Stimulating Factor.
    Simplified diagram illustrating the key growth factors and cytokines at different stages of the hematopoietic hierarchy.

Exploration of Hematopoiesis

Complete Blood Count (CBC)
Bone Marrow Smear
Bone Marrow Biopsy

Complete Blood Count

Gives the number of different blood cells.
Very easy to perform.
Automated test.

Bone Marrow Smear

Puncture of the bone marrow.
Site of puncture:
- Sternum
- Iliac Bone
Spreading on slide
Coloration
Optic microscopy: Precursors
Indication:
- Bone marrow failure
- Diagnosis of a vast majority of hematologic cancers
  Percentage of Lineage
- Granulocytic lineage: 50 – 70%
- Erythroid lineage: 10 – 30%
- Megakaryocytes: 1%
- Lymphoid lineage: 10 - 30%

Bone Marrow Biopsy

Site of biopsy: Iliac crest
Indication:
- Bone marrow smear non-contributive for the diagnosis:
  - Myelofibrosis, Metastasis of solid tumors
- Staging work-up of some hematological malignancies

In the Clinic

Clinical Applications:

The comprehension of the hematopoietic process has allowed many applications in the clinic:

Hematopoietic Growth Factors:
- Erythropoietin (EPO):
  - Targets erythroid progenitors.
  - Leads to further differentiation and production of more RBCs.
  - Recombinant EPO is used in the treatment of some anemias:
    - Renal failure
    - Myelodysplastic syndrome
    - Cancer-related anemia
    - Patients who refuse transfusion
- Thrombopoietin (TPO):
  - Gives rise to platelets production
  - By proliferation and differentiation of megakaryocytes.
  - In practice, we use TPO receptor agonists.
  - Indication:
    - Immune thrombocytopenia
    - Bone marrow failure
    - Hepatitis C-associated thrombopenia
- Granulocyte-Monocyte Colony Stimulating Factor (GM-CSF) and Granulocyte Colony Stimulating Factor (G-CSF):
  - Activate the proliferation and differentiation of myeloid progenitors and precursors.
  - Production of more neutrophils and monocytes.
  - Indication:
    - Acquired and congenital Neutropenia
    - Mobilization of HSCs to peripheral blood
Mobilization of HSCs:
- The use of G-CSF or GM-CSF mobilizes the HSCs from the bone marrow to the blood.
- The HSCs can be selected by using the specific surface antigen: CD34.
- The HSCs can be harvested and then used in transplantation.

Summary and Key points

Hematopoiesis products blood cells
It’s a well regulated process
The site of hematopoiesis changes during development
HSCs are the key cell of hematopoiesis
Characteristic of HSCs are self-renewal and differentiation
The hematopoiesis starts with HSCs then progenitors, precursors, and finally mature cells
As HSCs differentiate, their CD’s expression changes
Regulatory factors of hematopoiesis are growth factors