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Background - Hematopioetic
 
  • The Blood-Forming System

  • Cell Populations and Properties

  • Relationship between 

    CFU and HALO

The Blood-Forming System

 

 

 

The blood-forming system (hematopoiesis) can be compared to a tree

 

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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. This is why it is necessary to separate clonal, methylcellulose, differentiation assays from non-clonal, non-methylcellulose, proliferation assays. Differences between these different assay formats provide a better understand of the properties and characteristics of the cells being detected. Some of these properties and characteristics are shown in the diagram. 

 

 

Hematopoietic stem cells

 

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Hematopoietic 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.

ColonyGro™, 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.

It should be emphasized that:

  • ColonyGro™ and CAMEO™-4, like all other colony-forming cell assays, detects differentiation ability or potential. ColonyGro™ and CAMEO™-4 are NOT cell proliferation assays.
  • To measure stem cell proliferation, use CAMEO™-96, HALO®, HemoFLUOR™ or HemoLIGHT™.
  • To detect stem cell differentiation and measure proliferation in the same assay, use CAMEO™-96.
  • A note on cell population designation.
  • The following tables showing the cell populations that can be detected with different HemoGenix® assays, distinguish between those cell populations detected using the clonal, methylcellulose colony-forming cell assays (ColonyGro™, CAMEO™-4 and CAMEO™-96) and those that are non-clonal, non-methylcelluose assays (HALO®, HemoFLUOR™ and HemoLIGHT™). Since no colonies are formed in the non-methylcellulose assays since these incorporate Suspension Expansion Culture™ (SEC™) Technology, the cell population detected cannot be designated as a colony-forming cell or CFC. Instead, all stem cell populations detected have been designated with the prefic "SC" and all progenitor cell populations detected have been designated with the prefix "P".  

 

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

Methylcellulose/

Non-Methylcellulose

Designation

Stem Cell Type Growth Factor/Cytokine Cocktail

ColonyGro™/

CAMEO™-4

CAMEO™-96

HALO®/

HemoFLUOR/

HemoLIGHT

HPP-SP X /

SC-HPP X

Quiescent, primitive lympho-hematopoietic stem cell: High Proliferative Potential - Stem and Progenitor Cell

IL-3, IL-6, SCF,
Flt3-L
No No Yes (only for human cells)

HPP-SP XT /

SC-HPP XT

Quiescent, primitive lympho-hematopoietic stem cell: High Proliferative Potential - Stem and Progenitor Cell

IL-3, IL-6, SCF,
Flt3-L+CD3+CD28
No No Yes (only for human cells)

HPP-SP 1 /

SC-HPP 1

Quiescent, primitive lympho-hematopoietic stem cell: High Proliferative Potential - Stem and Progenitor Cell

IL-3, IL-6, SCF, TPO, Flt3-L Yes Yes Yes

HPP-SP 1T /

SC-HPP 1T

Quiescent, primitive lympho-hematopoietic stem cell: High Proliferative Potential - Stem and Progenitor Cell

IL-3, IL-6, SCF, Flt3-L+IL-2+CD3+CD28 No No Yes (only for human cells)

HPP-SP 2 /

SC-HPP 2

Induced, primitive lympho-hematopoietic stem cell: 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 /

SC-GEMM 1

Primitive hematopoietic stem cell equivalent to the Colony-Forming Cell - Granulocyte, Erythroid, Macrophage, Megakaryocyte EPO, GM-CFC, IL-3, IL-6, SCF, TPO, Flt3-L Yes Yes Yes

CFC-GEMM 2 /

SC-GEMM 2

Primitive hematopoietic stem cell equivalent to the Colony-Forming Cell - Granulocyte, Erythroid, Macrophage, Megakaryocyte EPO, GM-CFC, IL-3, IL-6, SCF, TPO Yes Yes Yes

CFC-GEMM 3 / 

SC-GEMM 3

Primitive hematopoietic stem cell equivalent to the Colony-Forming Cell - Granulocyte, Erythroid, Macrophage, Megakaryocyte

EPO, GM-CFC, G-CSF, IL-3, IL-6, SCF + TPO

(similar to MethoCult™ H4435)

Yes Yes Yes

CFC-GEM 1 /

SC-GEM 1

Mature hematopoietic stem cell equivalent to the Colony-Forming Cell - Granulocyte, Erythroid, Macrophage

EPO, GM-CFC, IL-3, IL-6, SCF

Yes Yes Yes

CFC-GEM 2 /

SC-GEM 2

Mature hematopoietic stem cell equivalent to the Colony-Forming Cell - Granulocyte, Erythroid, Macrophage

EPO, GM-CFC, IL-3, SCF

(similar to MethoCult™ H4434)

Yes Yes Yes

CFC-GEM 3 /

SC-GEM 3

Most mature hematopoietic stem cell equivalent to the Colony-Forming Cell - Granulocyte, Erythroid, Macrophage

EPO, GM-CFC, G-CSF, IL-3, SCF

(similar to MethoCult™ H4034)

Yes Yes Yes

 

 

 

Hematopoietic progenitor and precursor cells

 

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Hematopoietic 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 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

Methylcellulose/
Non-Methylcellulose
Designation
Progenitor Cell Type Growth Factor/Cytokine Cocktail CAMEO™-4 CAMEO™-96

HALO®/

HemoFLUOR/

HemoLIGHT

BFU-E 1 /

P-BFU 1

Primitive erythropoietic progenitor cell equivalent to the Burst-Forming Unit - Erythroid EPO, IL-3, SCF Yes Yes Yes

BFU-E 2 /

P-BFU 2

Primitive erythropoietic progenitor cell equivalent to the Burst-Forming Unit - Erythroid EPO alone Yes Yes Yes

GM-CFC 1 /

P-GM 1

Primitive granulocyte-macrophage progenitor cell equivalent to the Granulocyte-Macrophage - Colony-Forming Cell GM-CSF, IL-3, SCF (similar to H4534) Yes Yes Yes

GM-CFC 2 /

P-GM 2

Primitive granulocyte-macrophage progenitor cell equivalent to the Granulocyte-Macrophage - Colony-Forming Cell

GM-CSF, G-CSF, IL-3, SCF

(similar to H4035)

Yes Yes Yes

GM-CFC 3 /

P-GM 3

Primitive granulocyte-macrophage progenitor cell equivalent to the Granulocyte-Macrophage - Colony-Forming Cell

GM-CSF alone

Yes Yes Yes

Mk-CFC 1 /

P-Mk 1

Primitive megakaryocyte progenitor cell equivalent to the Megakaryocyte - Colony-Forming Cell

TPO, IL-3, SCF

Yes Yes Yes

Mk-CFC 2 /

P-Mk 2

Primitive megakaryocyte progenitor cell equivalent to the Megakaryocyte - Colony-Forming Cell

TPO alone

Yes Yes Yes
T-CFC / P-Tcell Primitive T-lymphocyte progenitor cell equivalent to the T-Colony-Forming Cell

IL-2

Yes Yes Yes
B-CFC / P-Bcell Primitive B-lymphocyte progenitor cell equivalent to the Megakaryocyte - Colony-Forming Cell

IL-7

Yes Yes Yes

 

Hematopoietic 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 Type Growth Factor/Cytokine Cocktail CAMEO™-4 CAMEO™-96 HALO®
CFU-E Erythropoietic precursor cell: Colony-Forming Unit - Erythroid EPO alone Yes Yes NA
G-CFC Granulocyte precuGranulocyte - Colony-Forming Cell G-CSF Yes Yes NA
M-CFC Macrophage - Colony-Forming Cell M-CSF Yes Yes NA

 

The Relationship Between the CFU Assay, CAMEO™-96, HALO®, HemoFLUOR™ and HemoLIGHT™

The colony-forming unit (CFU) or colony-forming cell (CFC) assay was introduced in 1966 independently by Bradley and Metcalf in Melbourne, Australia and Pluznik and Sachs in Rehovot, Israel. Since that time, it has been a maintainstay for basic hematopoietic stem and progenitor cell research. In the early 1970s, the assay was modified to detect human cells. With the use of methylcellulose as the semi-solid medium used for clonal growth, instead of agar or a plasma clot, the assay that requires the stimulation of primitive hematopoietic cells by different combinantions of growth factors and/or cytokines to produce colonies, became not only a widespread research tool, but the only assay that could detect stem and progenitor cell functionality. This is because primitive hematopoietic stem and progenitor cells are morphologically unidentifiable. It is their ability to proliferate and produce a coloniy of functional mature cells that can be identified under the microscope that allows the presence of these rare cell populations to be detected. This is why the CFU assay is a "functional" assay.

In 2001, Hemogenix® receive a SBIR grant from the National Cancer Institute to develop a high throughput stem cell hemotoxicity assay. Assay development started in January 2002 and, what is now CAMEO™-96, was launched in March 2002 at the Society of Toxicology meeting in San Francisco.

To develop CAMEO™-96, the CFU assay was rebuilt from the ground up. CAMEO™-96 is, like the traditional CFU assay, a methylcellulose, clonal assay, with two major differences. First, it is performed in a 96-well plate. Second, instead of counting colonies, CAMEO™-96 is an instrument-based, quantitative and highly sensitive assay incorporating ATP bioluminescence signal detection readout. The miniaturization of the assay was developed from a previous modification of the traditional CFU assay that is now called CAMEO™-4.

By detecting changes in the intracellular ATP concentration using bioluminescence, the traditional CFU assay was transformed from a differentiation assay into a cell proliferation assay for hematopoietic cells. Since proliferation occurs prior to differentiation, this meant that CAMEO™-96 could be completed in just 7 days, instead of 14 days for the CFU assay.  

For high throughput toxicity testing, however, methylcellulose cannot be used because it cannot be dispensed by commercially available liquid handlers (robots). This meant that methylcellulose had to be removed from the assay and replaced with a culture system that used normal pipettes and pipettes tips. This step led to the development of Suspension Expansion Culture™ (SEC™) Technology and was instrumental in turning a 96-well plate assay into a 384-well plate assay for high throughput screening. CAMEO™-96 developed into the Hematopoietic Assays via Luminescence Readout or HALO® Platform. All HALO® assays incorporate SEC™ Technology. And the ATP biolumninescence readout is the only readout available for primitive hematopoietic stem and progenitor cell populations that incorporates the ability to fully calibrate and standardize the assay as well as providing an internal proficiency test prior to measuring samples. It is also the only hematopoietic assay that can be validated according to regulatory requirements. In addition, HALO® is also the most rapid hematopoietic assay availble; STEMpredict™ can be performed in just 3 days, while many HALO® applications required just 5 days.

HALO® has now been joined by two other non-clonal, instrument-based, proliferation assays for lympho-hematopoietic cells namely, HemoFLUOR™ and HemoLIGHT™, which have a fluorescence or absorbance readout, respectively.

The information below demonstrates how the traditional CFU assay reagents (also available as ColonyGro™) correlate with CAMEO™-4, CAMEO™-96 and HALO®. The data show that unless there is a special need for a to detect hematopoietic cells under clonal conditions, the colony-forming assay can be completely replaced by HALO® 

 

Different ways of counting colonies in a colony-forming unit (CFU) assay

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The CFU Assay and Counting Colonies

To count colonies, an inverted microscope is required. Colonies derived from human bone marrow (shown in the diagram opposite), mobilized peripheral blood or umbilical cord blood, can be counted in two ways as shown in the diagram.

  1. The colonies on the top row are all counted individually, each as a single colony. This is the "traditional" way of counting colonies. It does not take into account that within each of these colonies, several smaller colonies have grown or aggregated together to form one large colony.
  2. The lower row shows how each large colony consists of smaller colonies that have grown or aggregated together. By counting each of the smaller colonies or "Proliferation Units" (PU), a better estimation of the number of original colony-forming cells (CFC-GEMM, BFU-E and GM-CFC) can be obtained. Note that only a colony that is spherical and has a single center is derived from a single cell. The colonies shown in the lower row are all derived from several colony-forming cells resulting in the colony being irregular in shape.

 

 

Standardization of the CFU assay: How measuring ATP bioluminescence can predict colony formation

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Correlation between Differentiated Colony Counts and Proliferation and Standardization of the CFU/CFC Assay

When the more demanding, but more accurate method of counting Proliferation Units is employed, the number of Proliferation Units counted correlates, in a cell dose-dependent manner, directly with proliferation of the cells producing the Proliferation Units when measured using an ATP bioluminescence assay such as CAMEO™-96. The diagram shows that, when Relative Luminescence Units (RLU), as a measure of intracellular ATP (iATP) concentration is detected on day 7 of culture for human bone marrow cells, there is a correlation with the number of Proliferation Units counted either on day 10 or day 14 of culture. This means that measuring iATP on day 7, predicts the outcome on days 10 or 14.

 

Correlation between Differentiated Colony Counts and Proliferation and Standardization of the CFU/CFC Assay

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Another way of viewing this correlation is also shown to the left. In this diagram, the results from CAMEO™-4 correlate directly with CAMEO™-96 as a function of cell dose. This correlation, like the one above, is to be expected. The cells first have to proliferate. This proliferation is detected by CAMEO™-96. The same proliferating cells then differentiate and mature, which is detected using CAMEO™-4. 

These results demonstrate that CAMEO™-96 and CAMEO™-4 are detecting the same cells, but using two different readouts. Therefore, CAMEO™-96 can replace CAMEO™-4.

Another important implication of the correlation between measuring proliferation using ATP bioluminescence and colony counting is that the straight-line graph produced between these two parameters allows the total colony counts to be expressed as ATP concentration equivalent units. This is because the ATP bioluminescence assay is calibrated and standardized against an external ATP standard and controls. By using CAMEO™-96 to count differentiated colonies and measure proliferation in the same assay using the same reagents and under the same conditions, the CFU/CFC assays can be standardized against a second assay. CAMEO™-96 is the only assay available that can standardize the CFU/CFC assay. 

 

 

Replacement of the CFU assay with HALO: The Relationship Between CAMEO™-4, CAMEO™-96 and HALO®

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What is the Relationship Between CAMEO™-4, CAMEO™-96 and HALO®?

An important question often asked is, "what is the relationship between the in vitro assays for lympho-hematopoietic cells"? Since both CAMEO™-96 and HALO® were both derived from the "classic" colony-forming cell assay, it would be expected that a relationship exists between all three. This is precisely the case. The graphs in the diagram shown on the left demonstrate that there is a direct correlation between CAMEO™-4, CAMEO™-96, HALO®-96 and HALO®-384 HT. This correlation means that the results from one assay will predict results from one of the other assays. In addition, the correlations demonstrates that one assay can replace the other. As mentioned above, unless there is a need for a clonal assay, all of the methylcellulose assays can be replaced by HALO®.