Myelosuppression

Myelosuppression can be evaluated using the hematopoietic colony forming assay (or colony-forming cell assay, CFC assay), which has been validated for determining maximum tolerated dose (MTD) by the European Centre for the Validation of Alternative Methods (ECVAM).^1,2

Benefits of the colony forming assay (CFC assay):

• Yields clinically predictive information, allowing for better planning and a reduction in in vivo studies³
• Physiologically relevant primary cells are used
• Simultaneously assesses both proliferation and differentiation
• Provides both quantitative (colony number and size of colonies) and qualitative (cell and colony morphology) data
• Used to determine IC₅₀ and IC₉₀ values

Contract Assay Services has the expertise to assess myelosuppression using custom-designed colony forming assays to meet your needs and goals. Experiments can be performed using human, mouse, rat and canine cells, as different species can show differential hematosensitivities to pharmaceuticals and toxins.

Learn more about Contract Assay Services - download the brochure

How the colony forming assay (CFC assay) works:

• Cells are plated in semi-solid medium
• Cultures are incubated for up to two weeks (depending on formulation of medium)
• Progenitor cells proliferate and differentiate
• The semi-solid medium allows the progeny of each progenitor to remain together to form distinct colonies
• Colonies are enumerated, and the size and morphology of colonies can be evaluated

FIGURE 1: Determination of IC₅₀ Values for 5-Fluorouracil



Dose response curves and IC₅₀ values for both human BM-derived erythroid and myeloid progenitors incubated with 5-Flurouracil.

FIGURE 2: Colony Size Changes in the Presence of an Inhibitory Compound



Table 1. Comparison of Myelotoxicity (CFU-GM) in Different Species

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Immunosuppression

Recommendation for Assessing Immunosuppression

Did you know that you can reduce the amount of immunosuppression testing required by assessing myelotoxicity with the colony forming assay as a first step?

"Compounds that are capable of damaging or destroying the bone marrow will often have a profoundly immunotoxic effect, since the effectors of the immune system itself will no longer be available. Thus, if a compound is myelotoxic, there may be no need to proceed with additional evaluation since the material will be a de facto immunotoxicant...An initial evaluation of myelotoxicity should be performed. If a compound is myelotoxic, there may be no need to proceed with additional evaluation." ⁴

Save time in drug development by assessing myelotoxicity with the colony forming assay before proceeding with extensive immunosuppression testing.

The in vitro colony forming assay (or colony-forming cell assay, CFC assay) has been shown to yield clinically predictive information, allowing for better planning and overall reduction in in vivo studies.³

Contract Assay Services has the expertise to assess myelotoxicity using the colony forming assay.

Learn more about the colony forming assay

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References:

1. Pessina A, et al. Toxicol In Vitro 15: 729-740, 2001
   
2. Pessina A, et al., Toxicol In Vitro 23: 194-200, 2009
   
3. Pessina A, et al., Toxicol Sci 75: 355-367, 2003
   
4. Gennari A, et al., J Immunotoxicol 2: 61-83, 2005
   

Mesenchymal

Mesenchymal stem cells, under the appropriate conditions, can differentiate into cells that make up adipose tissue, cartilage, bone and muscle. Drug candidates destined for the tissue engineering market can be assessed for effects on the differentiation potential, as well as for stimulatory or inhibitory effects on mesenchymal stem and progenitor cells using the CFU-F assay.

FIGURE 1. Human bone marrow-derived MSCs cultured in MesenCult™-XF display multi-lineage differentiation potential.



FIGURE 2. Effects of Doxorubicin on Human CFU-F



Dose response curve and IC₅₀ value for human BM-derived CFU-F incubated with Doxorubicin.

FIGURE 3. Human Bone Marrow-Derived CFU-F Colonies in the Presence of an Inhibitory Compound.



Inhibitory compounds can affect the size and density of the CFU-F colony.

The CFU-F assay can:

• Measure the effects of a test compound on progenitor frequency
• Assess proliferative or expansion potential of progenitors
• Quantitate mesenchymal progenitors in bone marrow

Contract Assay Services can design and perform custom experiments for mesenchymal stem and progenitor cells. New media products for the quantification of mouse CFU-F from compact bone have recently been developed, allowing Contract Assay Services to offer testing on this richer source of mesenchymal and progenitor cells.

Neural

Examine neurotoxic effects of compounds and environmental toxins on primary neural stem and progenitor cells using both proliferation and differentiation assays. Changes in differentiation or proliferation potential can be indicative of neurotoxic effects.

• The neural colony-forming cell (NCFC) assay can assess proliferation potential of neural stem and progenitor cells.
• Differentiation assays can assess differentiation potential of neural stem and progenitor cells into neurons, astrocytes and oligodendrocytes.

Neurospheres are used for both the differentiation assay and the neural colony-forming cell (NCFC) assay. They are three-dimensional floating spheres containing cells from different stages of neural development: neural stem cells, neural progenitor cells and mature neural cells.

Figure 1. Assays For Neural Stem and Progenitor Cells

Overview

In vitro testing using primary cells is recommended wherever possible as it has the advantage of being representative of the human condition.

In vitro testing with primary cells can help you:

• Obtain cost-effective information throughout the drug discovery process
• Reduce animal testing
• Determine toxicity and/or potency of compounds

Use customized assays with primary cells on:

• Hematopoietic stem and progenitor cells
• Mesenchymal stem and progenitor cells
• Neural stem and progenitor cells

Contract Assay Services has the expertise to perform in vitro testing with primary cells and uses industry-leading products, including MethoCult™ for hematopoietic assays, MesenCult™ and NeuroCult™.

Characterization of True Stem Cells

Together, STEMCELL Technologies and Contract Assay Services provide products and expertise required in the characterization of true primary as well as embryonic stem cells.

Sphere Assays

Liquid sphere assays are used to grow and assess certain stem cells. Single cell suspensions are cultured in a specialized medium that allows cells to form clusters of cells, or spheres, which are made up of primitive, undifferentiated cells.

Contract Assay Services has the capability to perform sphere assays using optimized media from STEMCELL Technologies, including NeuroCult™ and MammoCult™. The applications of the sphere assay vary and it is customizable.

Mesenchymal

Mesenchymal stem cells, under the appropriate conditions, can differentiate into cells that make up adipose tissue, cartilage, bone and muscle. Drug candidates destined for the tissue engineering market can be assessed for effects on the differentiation potential, as well as for stimulatory or inhibitory effects, on mesenchymal stem and progenitor cells using the CFU-F assay.

FIGURE 1. Human bone marrow-derived MSCs cultured in MesenCult™-XF display multi-lineage differentiation potential.



FIGURE 2. Effects of Doxorubicin on Human CFU-F



Dose response curve and IC₅₀ value for human BM-derived CFU-F incubated with Doxorubicin.

FIGURE 3. Human Bone Marrow-Derived CFU-F Colonies in the Presence of an Inhibitory Compound.



Inhibitory compounds can affect the size and density of the CFU-F colony.

The CFU-F assay can:

• Measure the effects of a test compound on progenitor frequency
• Assess proliferative or expansion potential of progenitors
• Quantitate mesenchymal progenitors in bone marrow

Contract Assay Services can design and perform custom experiments for mesenchymal stem and progenitor cells. New media products for the quantification of mouse CFU-F from compact bone have recently been developed, allowing Contract Assay Services to offer testing on this richer source of mesenchymal and progenitor cells.

Neural

Examine neurotoxic effects of compounds and environmental toxins on primary neural stem and progenitor cells using both proliferation and differentiation assays. Changes in differentiation or proliferation potential can be indicative of neurotoxic effects.

• The neural colony-forming cell (NCFC) assay can assess proliferation potential of neural stem and progenitor cells.
• Differentiation assays can assess differentiation potential of neural stem and progenitor cells into neurons, astrocytes and oligodendrocytes.

Neurospheres are used for both the differentiation assay and the neural colony-forming cell (NCFC) assay. They are three-dimensional floating spheres containing cells from different stages of neural development: neural stem cells, neural progenitor cells and mature neural cells.

Figure 1. Assays For Neural Stem and Progenitor Cells

Inhibition

Inhibition of proliferation and/or differentiation of hematopoietic progenitors can be determined using the in vitro colony forming assay, or colony-forming cell assay (CFC assay). The colony forming assay can be used to determine whether compounds have hemotoxic (hematotoxic) effects on blood or bone marrow. In addition, with the appropriate medium formulation, specific effects on various progenitor types, for example, erythroid or myeloid, can be examined.

Learn more about the benefits of the colony forming assay and how it works.

FIGURE 1: Determination of IC₅₀ Values for 5-Fluorouracil



Dose response curves and IC₅₀ values for both human BM-derived erythroid and myeloid progenitors incubated with 5-Flurouracil.

FIGURE 2: Colony Size Changes in the Presence of an Inhibitory Compound



In vivo assays can also be used to answer your questions about inhibition of hematopoiesis in mouse models.

Examples include:

• Myelosuppressive or hemotoxic effects of compounds on frequency of CFC in fetal liver, adult bone marrow, peripheral blood or spleen sources following dosing
• Kinetics of recovery of hematopoietic progenitors subsequent to the induction of myelosuppression

Stimulation

Stimulatory activity of a molecule can be tested using the colony forming assay, or colony-forming cell assay (CFC assay). The stimulatory activity is identified by an increase in the number of colonies and/or an increase in colony size because the colony forming assay is both quantitative and qualitative.

Learn more about the benefits of the colony forming assay and how it works.

FIGURE 1. Stimulation by Erythropoietin (EPO)



Stimulation of erythroid progenitor proliferation by EPO is both quantifiable (A) and qualitative (B; colony size increases with increasing EPO). In vivo models can be used to confirm stimulatory activity of molecules.


In vivo assays can also be used to answer your questions about stimulation of hematopoiesis.

Examples include:

• Mobilization of hematopoietic stem cells into the peripheral blood
• Progenitor frequency evaluation following treatment
• Changes in engraftment potential of hematopoietic stem cells in congenic mice recipients
• Stimulatory effects of compounds on human hematopoietic stem cells using the NOD/SCID mouse model

Characterization of Hematopoietic Stem and Progenitor Cells

Contract Assay Services can provide and customize in vivo assays to evaluate the ability of compound-treated cells to successfully engraft mice. This service can be performed to evaluate both mouse and human HSC using the NOD/SCID model, as well as the in vitro LTC-IC assay.

Validation and Recommendation

The colony forming assay, or colony-forming cell assay (CFC assay), for myeloid progenitors has been validated for the determination of maximum tolerated doses (MTD) by the European Centre for the Validation of Alternative Methods (ECVAM).

Assays for colony-forming unit - granulocyte, macrophage (CFU-GM) and colony-forming unit - megakaryocyte (CFU-Mk) have been shown to be predictive of clinical outcomes such as neutropenia^1,2 and thrombocytopenia.³

Assessment of myelotoxicity is recommended as the initial step for performing in vitro testing of immunosuppression.⁴

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References:

1. Pessina A, et al., Toxicol Sci 75: 355-367, 2003
   
2. Casati S, et al., Toxicol In Vitro 17: 69-75, 2003
   
3. Pessina A, et al., Toxicol In Vitro 23: 194-200, 2009
   
4. Gennari A, et al., J Immunotoxicol 2: 61-83, 2005