Mesenchymal Stem / Stromal
Stem Cell Toxicity
Compared to hepatotoxicity, cardiotoxicity, neurotoxicity, renal toxicity and others, toxicity to the blood-forming system (haemo-, hemo-, hematotoxicity) has been considered one of the accepted toxicities, especially for anti-cancer drugs. Toxicity to normal cells is an expected price to pay with many anti-cancer treatments. For virtually all anti-cancer treatments, the differential effect of the drug on cancer cells and normal cells will define the therapeutic index. It should also be pointed out that for most drugs, there is a specific window of time during the circadian rhythm of cells when a drug has the greatest therapeutic index. However, hemo-, hematotoxicity had, for many years, remained at or near the bottom of the list of important toxicities to detect. Hemotoxicity was and still is, part of the general toxicology performed during pre-clinical animal studies. This includes analyzing blood parameters and tissue and organ pathology. Unfortunately, animal hematotoxicity is rarely predictive and often cannot be extrapolated to the human situation. If any in vitro studies are performed, transformed cell lines are usually used and have little bearing on the in vivo human situation.
The nearest type of assay that had been used to detect toxic effects to the blood-forming system was the colony-forming cell (CFC) assay. Indeed, the European Center for the Validation of Alternative Methods (ECVAM) used this assay in many studies. The majority of these studies however, detected the granulocyte-macrophage colony-forming cell or GM-CFC as a means to predict drug-induced neutropenia. There are very few drugs that only affect the myelomonocytic lineage. By focusing just on the GM-CFC progenitor cells without studying effects on other cell lineages and especially the lympho-hematopoietic stem cells, a very restricted viewpoint was obtained. As a result, it might be assumed that only one lineage was affected. In reality, however, more than one lineage is usually affected. This means that the action of the drug is not specific to a particular lineage, but rather on the most important part of the system, namely the stem cell compartment. This produces cells that feed into the different lineages. In other words, the effect of a drug on the cell lineages is secondary to its primary action on the stem cells.
Coupled with the fact that the CFC assay has many flaws with respect to hemotoxicity testing and even other applications, HemoGenix® decided to design, develop and validate a new cell-based, in vitro assay platform that could be highly predictive of the human in vivo lympho-hematopoietic system and could be performed at any stage in the drug development pipeline with high throughput capability. The result was the HALO®-Tox HT Predictive Hemotoxicity Platform composed of both HALO®-96 Tox and HALO®-384 HT formats.Based originally on the "classic" CFC assay, but incorporating both Suspension Expansion Culture (SEC) and Bioluminomics™ patented Technology, the HALO® Predictive Hemotoxicity Platform is the most advanced stem and progenitor cell hemotoxicity screening and testing platform available. The methylcellulose- and the bioluminomics™-based CAMEO™-96 platform and, to a much lesser degree, the subjective CAMEO™-4 "classic" CFC assay should be used primarily to detect effects on differentiation and maturation, rather than proliferation. HALO®-Tox HT has been validated against the Registry of Cytotoxicity Prediction Model and has been shown by the investigators at Hoffmann-La Roche to demonstrate as much as 82% concordance with in vivo data.
HALO®-Tox HT is capable of detecting hemotoxicity on 15 different cell populations of the lympho-hematopoietic system from multiple species namely, human, non-human primate, horse, pig, sheep, dog, rat and mouse. In addition it was designed to multiplex with other add-on assays so that as much information could be obtained from a single sample as possible. HALO®-Tox HT is also part of the HemoGenix® ComparaTOX™ Platform. Specialized HALO® assays are also available to analyze drug-drug interactions (HALO®-384 DDI) as well as residual toxicity and changes in cell sensitivity after drug administration (HALO®-96 PRT).
Available as contract research services or as assay kits for in-house implementation, the HALO® Predictive Hemotoxicity Platform has provided valuable information for small, medium and many of the largest biopharmaceutical companies as well as the National Toxicology Program (NTP).
Although mesenchymal stem/stromal cells (MSC) may not, at first site, appear to be an important target for toxicity testing, they are, like the hematopoietic stem cells, very sensitive to potential toxic agents by virtue of their proliferative activity. Mesenchymal stem/stromal cells provide the progenitors for:
The hematopoietic stroma consists of fibroblasts, adipocytes (fat cells), endothelial cells and reticular cells, which together constitute part of the "stem cell niche". Without the hematopoietic stroma, hematopoiesis does not function or does not function properly. It follows that toxic agents affecting either the MSCs themselves so that they cannot differentiate into components of the hematopoietic stroma or those that actually damage or destroy the hematopoietic stroma, e.g. environmental agents and radioactivity, can have devestating effects.
Toxicity during osteogenesis, chondrogenesis and adipogenesis, not to mention the effects on muscle and neural cells, can also be significant. In addition, recent studies have also demonstrated that MSCs can have a T-cell immunsuppressive effect that might be important in transplantation and other disease treatments. Drug toxicity could therefore have significant effects on how these treatments function. The extent to which toxicity affects the MSCs and their ability to differentiate to produce lineage-specific progeny can be tested with help of the MSCGlo™-Tox HT Platform.
MSCGlo™-Tox HT is the only MSC toxicity assay platform designed for both 96- and 384-well plate formats. Replacing the "classic" and subjective CFU-F assay, MSCGlo™ can be used to detect toxicity to the MSCs themselves and their potential for differentiation. Using proven bioluminomics™ technology, MSCGlo™ is also the only MSC assay system that can be multiplexed with other assays, such as MSC characterization by flow cytometry.
As explained in other sections on this website, all stem cells exhibit the same characteristics and properties:
It follows that any interruption or damage to the stem cells, such that these characteristics and properties cannot be expressed, can result in serious consequences. Regardless of whether the stem cells are part of a continuously proliferating or partially proliferating system, the stem cells are probably the most sensitive and important cells to consider with respect to toxicity. As the cells that are responsible for the continuous production of functionally mature cells from several biological systems as well as those responsible for many regenerative processes, the response capability of stem cells are fundamental and pivotal to biological processes and the success of many clinical treatments.
Besides the primary continuously proliferating systems of the body, lympho-hematopoiesis, the gut, reproductive organs, skin and corneal cells of the eye, many other stem cell systems are now being actively investigated and include, but not limited to:
It should be emphasized that the characteristics and properties that define stem cells may be extremely difficult or impossible to substantiate.
Methodologies used for one biological system may be applied to another. For example, the ability of proliferating cells to form colonies in semi-solid media provides an in vitro surrogate system by which potential stem cells can be characterized. Alternatively, measuring cell potency or proliferation and expansion potential under different conditions and compared to cells of the same tissue or organ can provide valuable information. The ability of the cells to respond to various drugs, perturbations and other agents will also help in the characterization of potential stem cells. Confirming the presence of specific stem cell types also means that a better understanding of potential toxicity to the system can be investigated. Alternatively, it can provide information for targeted drug therapy for cancer and other diseases.
These aspects were taken into account for the development of the STEMAssays™ Platform, in particular, STEMGlo™-Tox HT. Like its counterparts, HALO®-Tox HT and MSCGlo™-Tox HT, STEMGlo™-HT is an in vitro bioluminomics™ toxicity screening and testing platform using 96- and 384-well plate formats. STEMGlo™-HT differs from its counterparts however, in that it provides the flexibility to incorporate different culture reagents and conditions depending upon the cells being tested.
Some specific STEMAssays™ have also been developed. For example, STEMClone™, STEMGlo™-PREP, STEMGlo™-PRT and NeuroGlo™-HT. The former allows stem cells to be grown under clonal conditions in methylcellulose and to assess both proliferation and differentiation ability. STEMGlo-PREP is an assay used to investigate the self-renewal and expansion properties of stem cells. STEMGlo-PRT detects residual stem cells after treatment with a drig or other agent and determines whether the stem cells have changed their sensitivity. NeuroGlo™-HT is an assay that uses ES-derived neural stem and progenitor cells to detect potential toxicity.
Stem cell toxicity testing is a rapidly growing field. Its success will depend on the characterization of the stem cells and the accrual of information on the biological system that will allow it to be used as a tool to predict potential toxicity. The STEMAssay™ provides a tool that will help in defining the reagents and conditions necessary to understand the system and potential toxicity to that system.
Hepatotoxicity is a major component of a drug toxicity profile. The liver is considered the primary site of detoxification. The cytochrome P450 enzymatic system in the liver is primarily responsible for detoxification, but also the effects seen during drug-drug interactions. Therefore potential toxic effects that damage or destroy the liver and hepatocytes can have significant consequences.
With the availability of high quality fresh and cryopreserved hepatocytes from human and other species, it has been possible for HemoGenix® to develop its own contract research services and assays kits specific for hepatotoxicity.
Hepatocytes exhibit a high rate of metabolism and contain high concentrations of intracellular ATP (iATP). Bioluminomics™ assays are designed to measure iATP using a luciferin/luciferase bioluminescence readout. For hepatotoxicity screening and testing, HemoGenix® has designed HepatoGlo™-HT, a 96- or 384-well plate, high throughput toxicity assay platform for fresh or cryopreserved human or animal hepatocytes.
In addition, a cell-based drug-drug interaction assay is also available for hepatocytes that can be combined with cytochrome P450 enzyme assays to provide meaningful cellular information on the effects of inhibition or induction of P450 enzymes.