The Blood-Forming System

 

Structure and Organization of the Blood-Forming (Hematopoietic) System

The blood-forming or hematopoietic (hemo, haemo, hemato meaning blood and poiesis, poietic meaning production) system has been studied for centuries. Indeed, it is one of the most studied biological systems of the body. Studies that have led to our present knowledge of the structure, organization and regulation of the system started in the 1940s and 1950s and eventually led to the first quantitative assay in 1961 by Till and McCullock for what we now know to be the hematopoietic stem cell. These mouse transplantation assays eventually led to the first in vitro assays to detect early cells that produce granulocytes and macrophages. These assays were developed and published independently by Bradley and Metcalf in Melbourne, Australia and Pluznik and Sachs in Rehovot, Israel in 1966. In both the in vivo and in vitro assays, the functionality of cells was detected by virtue of their ability to produce colonies of differentiated and mature cells. It is this functionality, the process of proliferation, differentiation and maturation, that identified the colonies as originating from cells that could not be morphologically observed or identified. At the time, it was not known whether one or more cells were responsible for producing colonies. As a result, the term colony-forming unit or CFU was used. From numerous studies, it was found that both the in vivo and in vitro colonies were derived from single cells and the term colony-forming cell or CFC, was coined. As a consequence, the different in vitro cell populations that have been discovered since 1966 have usually been designated either CFU or CFC.

With the development of the in vitro colony-forming unit (CFU) or cell (CFC) assays came the need to identify the humoral or growth factors that stimulated the cells. In late 1970s and early 1980s the use of recombinant gene technology laid the groundwork for producing the first recombinant protein, erythropoietin or EPO. Today, numerous growth factors and cytokines are available in recombinant form and are used in many applications, including assays produced by HemoGenix®. The effect of growth factors and cytokines, added either individually or in combination as cocktails, has led to the discovery of many different cell populations that are shown in the diagram on this page.

In addition, artificially perturbating the system, the use of drugs and other agents such as a radiation, has shown that the blood-forming system, although complex, is organized in a structured manner. It should be emphasized that the blood-forming system is a continuum; the individual cell populations that have been identified over the years have allowed investigators to understand the system by introducing the concept of cell compartments. As a result, one might consider the blood-forming system as being divided into 4 major compartments:

• Stem cell compartment
• Amplification compartment
• Differentiation compartment
• Maturation compartment

Within each of these compartments, it is then possible to consider cell populations as being:

• Stem cells
• Progenitor cells
• Precursor cells
• Mature cells

When a hierarchy of cells, from primitive to mature, is considered within each compartment, it is then easy to visualize the blood-forming system as a tree.

• The primitive stem cells are the roots
• The mature stem cells represent the trunk
• The progenitor cells in the amplification compartment are the main branches
• The precursor cells in the differentiating compartment are represented by the small branches and twigs
• The leaves are the mature cells in the circulation

This type of organization and hierarchy is typically found in several stem cell systems that are responsible for continuously producing cells. These include:

• The lympho-hematopoietic system
• The cells of the gut
• The reproductive organs
• The skin
• Cells in the cornea of the eye

Other stem cell systems that do not continuously produce cells would be expected to be organized in a similar manner. But, the blood-forming system is unique because it is the only system:

• That produces the greatest number of cells
• Whose ontogeny includes sequential organ transition (yolk sac, liver, (spleen), bone marrow)
• That can retrace its path of ontogeny, if required
• That produces multiple mature functional cell types from a single stem cell pool
• Produces mature cells whose functional location is different from the production site

 

Lympho-Hematopoietic Cell Populations and Properties

 

The correct interpretation and conclusions drawn from experimental data are dependent upon using the correct assay for the goal of the study. It is for this reason why HemoGenix® has focused on separating assays that detect proliferation, from those that detect differentiation, and why it is important to understand the properties and characteristics of the cells being detected. Some of these properties and characteristics are shown in the diagram. 

Blood-Forming Stem Cells

The blood-forming stem cells are contained within the stem cell compartment. Some of the properties of stem cells are:

• Their ability to self-renew
• Their undifferentiated state
• Their extensive proliferation capacity
• Their ability to proliferate
• Their ability to form lineage-specific cells

In the diagram, the most primitive stem cells are at the bottom of the diagram and, as described on the previous page, may be considered the roots of the "blood-forming tree". These primitive stem cells are quiescent; they are not in cell cycle and are therefore not proliferating, although they possess the greatest proliferation capacity of all of the cells of the blood-forming system.Within the stem cell compartment, there is a gradation of primitiveness or "stemness". By using different cocktails of growth factors and cytokines, stem cell populations at different stages of "stemness" can be identified and measured using assays that detects the cell's:

• proliferation status or ability, otherwise known as "quality" ,or
• its proliferation potential, also known as stem cell potency

Stem cell "quality" and stem cell potency are discussed in more detail in Background: Cellular Therapy.

CAMEO™-4, CAMEO™-96 and HALO® Assays for the Stem Cell Compartment

Although all three assay platforms can be used to help enumerate the Long-Term Culture - Initiating Cell (LTC-IC), the most primitive in vitro stem cell population that can be detected, HemoGenix® does not produce an assay for the LTC-IC although it does provide contract research services to detect this population. Rather than detecting LTC-IC, which can take 5-7 weeks to perform, HemoGenix® has developed an assay for the slightly more mature stem cell population, HPP-SP or High Proliferative Potential Stem and Progenitor Cell. This population has the capability of producing both lymphopietic and hematopoietic stem and lineage-specific cells. All other stem cell populations detected by CAMEO™-4, CAMEO™-96 and HALO® are derived from the HPP-SP stem cell population.

Please note that:

• CAMEO™-4, like all other colony-forming cell assays, detects stem cell differentiation ability or potential. CAMEO™-4 does NOT measure stem cell proliferation
• To measure stem cell proliferation, use CAMEO™-96 or HALO®
• To detect stem cell differentiation and measure proliferation in the same assay, use CAMEO™-96 

 

CAMEO™-4, CAMEO™-96 and HALO® Assays for Stem Cell Populations

Designation	Stem Cell	Growth Factor/Cytokine Cocktail	CAMEO™-4	CAMEO™-96	HALO®
HPP-SP 1	High Proliferative Potential - Stem and Progenitor Cell	IL-3, IL-6, SCF, TPO, Flt3-L	Yes	Yes	Yes
HPP-SP 2	High Proliferative Potential - Stem and Progenitor Cell	EPO, GM-CFC, IL-2, IL-3, IL-6, IL-7, SCF, TPO, Flt3-L	Yes	Yes	Yes
CFC-GEMM 1	Colony-Forming Cell - Granulocyte, Erythroid, Macrophage, Megakaryocyte	EPO, GM-CFC, IL-3, IL-6, SCF, TPO, Flt3-L	Yes	Yes	Yes
CFC-GEMM 2	Colony-Forming Cell - Granulocyte, Erythroid, Macrophage, Megakaryocyte	EPO, GM-CFC, IL-3, IL-6, SCF, TPO	Yes	Yes	Yes
CFC-GEM 1	Colony-Forming Cell - Granulocyte, Erythroid, Macrophage	EPO, GM-CFC, IL-3, IL-6, SCF	Yes	Yes	Yes
CFC-GEM 2	Colony-Forming Cell - Granulocyte, Erythroid, Macrophage	EPO, GM-CFC, IL-3, SCF (similar to H4434)	Yes	Yes	Yes
CFC-GEM 3	Colony-Forming Cell - Granulocyte, Erythroid, Macrophage	EPO, GM-CFC, G-CSF, IL-3, SCF (similar to H4034)	Yes	Yes	Yes

 

Blood-Forming Progenitor Cells

As stem cells mature, they eventually come to a point of no return. This is called "stem cell determination", where a stem cell enters one or other lineage to become a functionally mature blood cell. A larger proportion of progenitor cells are in cell cycle than the most mature stem cells. This means that progenitor cells exhibit greater proliferation than the stem cells. This is the reason why the progenitor cells are responsible for most of the amplification seen in the blood-forming system; they constitute the amplification compartment. However, although, progenitor cells show increased proliferation, they have lost the ability to self-renew and actually exhibit lower proliferation potential or potency than the stem cells. From a cellular therapeutic viewpoint, it follows that transplanting progenitor cells or measuring the proliferation potential or potency of progenitor cells will not provide any information regarding the stem cells that are transplanted.

Please note that:

• CAMEO™-4, like all other colony-forming cell assays, detects stem cell differentiation ability or potential. CAMEO™-4 does NOT measure stem cell proliferation
• To measure stem cell proliferation, use CAMEO™-96 or HALO®
• To detect stem cell differentiation and measure proliferation in the same assay, use CAMEO™-96

 

CAMEO™-4, CAMEO™-96 and HALO® Assays for the Progenitor Cell Compartment

CAMEO™-4, CAMEO™-96 and HALO® Assays for Progenitor Cell Populations

Designation	Progenitor Cell	Growth Factor/Cytokine Cocktail	CAMEO™-4	CAMEO™-96	HALO®
BFU-E 1	Burst-Forming Unit - Erythroid	EPO, IL-3, SCF	Yes	Yes	Yes
BFU-E 2	Burst-Forming Unit - Erythroid	EPO alone	Yes	Yes	Yes
GM-CFC 1	Granulocyte-Macrophage - Colony-Forming Cell	GM-CSF, IL-3, SCF (similar to H4534)	Yes	Yes	Yes
GM-CFC 2	Granulocyte-Macrophage - Colony-Forming Cell	GM-CSF, G-CSF, IL-3, SCF (similar to H4035)	Yes	Yes	Yes
GM-CFC 3	Granulocyte-Macrophage - Colony-Forming Cell	GM-CSF alone	Yes	Yes	Yes
Mk-CFC 1	Megakaryocyte - Colony-Forming Cell	TPO, IL-3, SCF	Yes	Yes	Yes
Mk-CFC 2	Megakaryocyte - Colony-Forming Cell	TPO alone	Yes	Yes	Yes

 

Blood-Forming Precursor Cells

The precursor cells may be considered the first morphologically identifiable cells that can be viewed on a microscope slide. The precursor cells exhibit greater differentiation than proliferation. In fact, with greater differentiation, the ability to proliferate eventually ceases completely. The number of cell divisions is reduced to 6 or less. The precursor cells have the lowest proliferation potential. As a result, the ability to detect precursor cell proliferation is quite difficult. For this reason, there are no HALO® proliferation assays for the progenitor cells.

 

CAMEO™-4 and CAMEO™-96 Assays for the Progenitor Cell or Differentiation Compartment 

CAMEO™-4, CAMEO™-96 and HALO® Assays for Precursor Cell Populations

Designation	Progenitor Cell	Growth Factor/Cytokine Cocktail	CAMEO™-4	CAMEO™-96	HALO®
CFU-E	Colony-Forming Unit - Erythroid	EPO alone	Yes	Yes	No
G-CFC	Granulocyte - Colony-Forming Cell	G-CSF	Yes	Yes	No
M-CFC	Macrophage - Colony-Forming Cell	M-CSF	Yes	Yes	No