in-vitro hematological toxicity prediction by colony-forming cell assays

Navneet Kumar Yadav1, Pooja Shukla1,Ankur Omer1 & Rama Kant Singh1

1Division of Toxicology CSIR-Central Drug Research Institute,Lucknow-226031Correspondence and requests for materials should be addressedto R. K. Singh ([email protected], [email protected])

Received 3 July 2013 / Received in revised form 16 October 2013Accepted 17 December 2013DOI 10.1007/s13530-013-0172-7©The Korean Society of Environmental Risk Assessment and Health Science and Springer 2013

Abstract

Hematotoxicology is concerned with the adverse eff-ects of xenobiotics on hematopoietic processes in aliving system. In vitro tests for hematotoxicity havebeen applied in toxicity assessment of chemicals,drugs, food supplements and environment relatedstudies. Hematopoietic Progenitor Colony-FormingCell (CFC) assays and Stromal Cell Assays (also call-ed as Non-hematopoietic Progenitor Assays) are al-ready being used to detect hematological toxicitiesinduced by different contaminants. These in vitrotests have been very useful in reducing the numberof animals required for hematotoxicity testing. In thisreview, applications, limitations and future prospec-tive of in vitro tests for hematotoxicity with emphasison the techniques involved in the Colony forming unitculture systems are described.

Keywords: Hematotoxicology, Hematopoiesis, Clonogenicassays, Stromal cell assay, Hematopoietic progenitor colony-forming cell (CFC) assays, Bone marrow mesenchymal stemcells (BMMSCs)

Introduction

Hematopoietic stem cells (HSC’s) are capable of bothself-renewal and differentiation. They make a balancebetween self-renewal and differentiation to providesufficient primitive cells to sustain hematopoiesis,while generating more mature cells with specializedcapabilities (Figure 1). The enhanced self-renewal cap-acity of primitive HSCs enable their ability to sustain

hematopoiesis through out decades of life and theirability to repopulate a host when used therapeutically1.An optimum peripheral blood count is maintained inthe living system by constant degeneration and regen-eration of blood cells2,3. Immature progenitors produceblood cells from more than one lineage whereas moremature progenitors with lower proliferative potentialto produce cells is restricted to single blood cell lineage:neutrophils, platelets, erythrocytes, monocytes or lym-phocytes. The precursors of neutrophils and mono-cytes are termed as: granulocyte-macrophage colony-forming cells (CFU-GM). Similarly, the erythroid burst-forming units (BFU-E) and megakaryocyte colony-forming units (CFU-Mk) are the counter parts of CFU-GM in the erythroid and megakaryocytic lineages re-spectively. Many hematopoietic progenitors produceclonal colonies in vitro when appropriately stimulatedwith: cytokines, combinations of cytokines, or cytokinecocktails, such as IL-11, Flt-3-ligand (Flt-3-L), Steelfactor, SCF, Tpo, Flt-3-L with either IL-11 or IL-34-6

hence the name ‘colony-forming unit’ or CFU wasgiven. The type of blood cells found in each clonalcolony determines the name of that specific colony.

In addition to hematopoietic stem cells, bone marrowalso contains stem-like cells that are precursors ofnon-hematopoietic tissues7,8. Initially, the precursorsof non-hematopoietic tissues were termed as plastic-adherent cells or colony-forming-units fibroblasts(CFUs) because they readily adhered to culture dishesand formed fibroblast-like colonies9,10. The cells werealso referred to as mesenchymal stem cells or mesen-chymal progenitor cells11 because of their ability todifferentiate into a variety of non-hematopoietic cells.They support the growth and differentiation of hema-topoietic cells and influence the differentiation of hema-topoietic stem cells by providing a hematopoietic-in-ducing microenvironment (HIM) consisting of a cel-lular matrix and factors that promote growth and dif-ferentiation8. MSCs provide circulating progenitorsfor the repopulation of non-hematopoietic tissues12,13.

In vitro Hematotoxicology

The major objectives of toxicity studies are concern-ed with the identification of potentially dangerous tox-icants in order to prevent or control human exposure

In-Vitro Hematological Toxicity Prediction by Colony-FormingCell Assays

and providing drug information relevant for undertak-ing risk-benefit analyses and further conducting clini-cal trials. Safety evaluation in the pharmaceutical in-dustry is a multi-step process in which each step pro-vides more clarity about specific toxicity or more cer-tainty on manifestation of toxicity. During this process,toxicities often are identified only after a significantdosing, duration and consumption of resources, whichthen delays or bars the process of drug development.Identification of safety issues at earlier stages ultimate-ly will reduce late-stage attrition by selecting com-pounds with a lower likelihood of failure14.

Hematotoxicology is concerned with the adverseeffects of xenobiotics and pharmacological levels ofendogenous substances and drugs on the numbers andhomeostatic functions of blood cells. Hematopoietictoxicity observed at relevant pharmacological concen-trations in either preclinical toxicological studies orclinical trials, can hinder and ultimately halt the pro-cess of drug development. Hematological toxicity is acommon side effect of chemotherapy and often a majorlimitation to performing full treatment protocols ofantitumor agents15,16. For non-oncology indications,hematological toxicity may not be a tolerable sideeffect and back-up strategies to identify drug candi-dates without such toxicity can be very costly and timeconsuming. The ability to screen for potential hema-topoietic toxicity prior to in vivo testing could reduce

attrition downstream in the course of drug development.In vitro hematotoxicity assays can play a key role in

bridging the gap between preclinical toxicology stud-ies in animal models and clinical investigations. Theycan aid in the development of new therapeutic agentsand can help in predicting the risk assessments asso-ciated with food additives, other chemicals and pro-ducts. Furthermore, these tests have the potential toreduce the number of animals required for hematotox-icity testing and to permit the refinement of the animalstudies17,18. Many of these tests have been used fordetermining the relative sensitivities of various animalspecies to hematotoxic effects and for studying thesynergistic and antagonistic effects of several com-pounds19.

In-Vitro Test to PredictHematological Toxicity

Hematopoietic Progenitor Colony-FormingCell (CFC) Assays

Clonogenic assays that detect that the cells are capa-ble of producing multi-lineage colonies (granulocyte,erythroid, monocyte, megakaryocyte colony-formingcells, CFU-GEMM and CFU-Mix) or extremely largecolonies (high proliferative potential colony-formingcell, HPP-CFC). The colony-forming cell (CFC) assays

170 Toxicol. Environ. Health. Sci. Vol. 5(4), 169-176, 2013

Figure 1. Hematopoietic and stromal cell differentiation. Credit: 2001 Terese Winslow (assisted by Lydia Kibiuk).

Bone

Natural killer(NK) cell

Tlymphocytes

Blymphocyte

Neutrophil

Basophil

Eosinophil

Red blood cells

Bone (or cartilage)

Osteoblast

Lining cell

OsteocytePre-osteoblast

Skeletal muscle stem cell?

Hepatocyte stem cell?

Platelets

Monocyte/macrophage

Lymphoidprogenitor

cell

Hematopoieticstem cell

Stromalstem cell

Hematopoieticstem cell

Marrowadipocyte

Hematopoieticsupportive stroma

Multipotentialstem cell

Myeloidprogenitor

cell

measure progenitor cells in a given population usingsemisolid agar or well-defined methylcellulose-basedculture media which is more commonly used (Figure2). The majority of CFCs consist of lineage-restrictedcolonies: erythroid restricted burst-forming units-ery-throid (BFU-E) which are more immature than thecolony-forming units erythroid (CFU-E); megakaryo-cyte-restricted CFU-Mk; colony-forming units-granu-locytes (CFU-G), colony-forming units monocytes/macrophages (CFU-M) and colony forming units-gran-ulocytes/macrophages (CFU-GM) (Figures 3 & 4). Themost immature (multipotent) CFC measurable containsgranulocytes, erythrocytes, macrophages, and oftenmegakaryocytes and are usually measured at day 12after culture initiation. This CFC is usually termed asCFU-mix as it may not always contain megakaryocytesbut does contain erythroid and granulocyte/macrophagecells. B and T lymphocytes in vitro requires special-ized co-culture systems, thus are more difficult toassess20,21.

CFU-GM is the most attractive progenitor for invitro hematotoxicology studies because of its role indrug-induced neutropenia, its predictive value andtechnical simplicity22. CFU-GM assay has been vali-dated for testing drug-associated hematotoxicity involv-ing mouse bone marrow and human cord blood cellsas well as for predicting in vivo human Maximal Tole-

rated Dose (MTD) values by extrapolating in vivo dataon mouse toxicity. Amongst murine models, for invivo toxicological studies rats are the most frequentlyused animals. The rat CFU-GM assay is widely usedfor its capability to evaluate in vitro hematotoxicity.Pessina et al., reports a refined and optimized standardoperating procedure (SOP) based on the use of cryo-preserved progenitors for murine CFU-GM assayapplicable for in vitro toxicity testing of compounds.The researchers also presented a prevalidation studyfor intra-laboratory and inter-laboratory variability ofSOP for CFU-GM assay and its performance for thein vitro determination of the inhibitory concentration(IC) values of drugs on rat myeloid progenitors. ThisSOP proposes to reduce the number of rats used inexperimental procedures and increases the homogenei-ty of the data obtained from CFU-GM assay23,24.

A second type of assay measures human stem cellsindirectly via their production of CFU-GM progeni-tors. Hematopoietic cells added to preformed culturesof bone marrow stroma produces long-term bone mar-row cultures (LT-BMC). Cultures are maintained witha regimen of depopulation and feeding for five weeks.Then the presence of stem cell activity is quantifiedfrom the number of CFU-GM colonies produced. Enu-meration of the stem cells (LTC-IC for long-term cul-ture initiating cells) requires limiting dilution analyses

In-Vitro Hematological Toxicity Prediction by Colony-Forming Cell Assays 171

Figure 2. Hematopoietic Colony Assay Procedure.

because the number of CFU-GM produced per stemcell varies under stressful stimulus25.

Yu-xiao Liu et al. successfully generated CFU-GM,CFU-Mix, and CFU-E colonies from co-culture system.In this method Human embryoid bodies (hEBs) obtain-ed from human stem cells were treated with cell extractfrom human fetal liver tissue and co-cultured withhuman fetal liver stromal cells feeder to induce hema-topoiesis26 (Figure 5).

A third assay called the P-delta assay, is simpler butstill requires limiting dilution analysis. In this assay,primitive cells are separated from progenitors by ex-ploiting their ability to adhere to tissue culture plastic.Stem cells are quantified from their ability to produceCFU-GM over a period of one week2,3.

Stromal Cell Assay or Non-hematopoieticProgenitor Assays

Bone marrow mesenchymal stem cells (BMMSCs)are a heterogeneous population of postnatalprecursorcells with the capacity of self-renewal and differentia-tion into osteoblasts, chondrocytes, adipocytes, and

neural cells. BMMSCs are thought to be derived fromthe bone marrow stromal compartment. They havecapacity of adhering to culture dishes generating col-ony-forming unit-fibroblasts (CFU-F)27. The mannerin which chemicals affect the proliferation capacity ofstromal progenitor cells can be investigated in vitroby determining their effects upon the capacity of stro-mal progenitors to form colonies of adherent fibrob-last-like cells that is colony forming units of fibroblast(CFU-F)10. Several chemicals and drugs are capableof producing hemopoietic dysfunction by disturbingthe functional activity of the stroma28-30. In this respectthe hemopoietic re-feeding of chemically treated stromais expected to facilitate an understanding of the roleof stroma in hematotoxicity31,32.

Application, Limitation andFuture Prospectives

In vitro tests for hematotoxicity have been appliedin three principal domains of toxicology, i.e., food,


Figure 3. Human Colony Forming Cell (CFC) Assay (“Image courtesy: R&D Systems, Inc., Minneapolis, MN, USA”).

CFU-E (Colony forming unit-erythroid): Clo-nogenic progenitors that produce only one ortwo clusters with each cluster containing from8 to approximately 100 hemoglobinized erythro-blasts. It represents the more mature erythroidprogenitors that have less proliferative capacity.

BFU-E (Burst forming unit-erythroid): Thesize of the colony can be described as small (3to 8 clusters), intermediate (9 to 16 clusters), orlarge (more than 16 clusters) according to thenumber of clusters present. These are primitiveerythroid progenitors that have high prolifera-tive capacity.

CFU-G (Colony forming unit-granulocyte):Clonogenic progenitors of granulocytes that giverise to a homogeneous population of eosinophils,basophils or neutrophils.

CFU-M (Colony forming unit-macrophage):Clonogenic progenitors of macrophages thatgive rise to a homogenous population of macro-phages.

CFU-GM (Colony forming unit-granulocyte,macrophage): Progenitors that give rise to col-onies containing a heterogeneous population ofmacrophages and granulocytes. The morphologyis similar to the CFU-M and CFU-G descriptions.

CFU-GEMM (Colony forming unit-granulo-cyte, erythrocyte, macrophage, megakaryo-cyte): Multi-lineage progenitors that give rise toerythroid, granulocyte, macrophage and mega-karyocyte lineages, as the name indicates.

environment related and the other one associated withchemicals and drugs.

In food toxicology, clonogenic assays have beenused to identify the origin of blood disorders inducedby food contaminants such as mycotoxins, heavy me-tals etc.33. In environmental related aspects, toxicity ofpesticides, natural toxins and chemicals has been de-monstrated with Hematopoietic clonogenic assays34-36.

The hematopoietic colony-forming-cell (CFC) assayis a valuable tool for toxicity screening in therapeuticdrug development and assessment of maximum tolerat-ed dose (MTD) in human before phase 1 trials37,38. Invitro tests determines the safety margins by reducing

toxicological uncertainties due to animal/human extr-apolation and provide a more rational basis for calculat-ing clinical dosages for setting human exposure limits.An in vitro assay could highlight the potency differ-ence between humans and the preclinical test species,so that the starting dose in phase I clinical trials couldbe considerably closer to the MTD without compromis-ing safety. Thus, not only would phase I clinical trialswould be completed more quickly, but fewer patientswould be treated with ineffective doses. In this respectpredictivity of the data obtained from animal studiescould be increased by in vitro tests, and the level ofuncertainty concerning human safety could be decreas-


(a) BFU-E

(c) CFU-M (left) and CFU-G (right)

(b) CFU-GM

(d) CFU-GEMM

Figure 4. Mouse/Rat Colony Forming Cell (CFC) Assays. (a) BFU-E (Burst forming unit-erythroid); (b) CFU-GM (Colony form-ing unit-granulocyte, macrophage); (c) CFU-G (colony forming unit-granulocyte) & CFU-M (colony forming unit-macrophage);(d) CFU-GEMM (Colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte) (“Image courtesy: R&D Systems,Inc., Minneapolis, MN, USA”).

Figure 5. Typical CFU-GM, CFU-Mix, and CFU-E colonies derived from co-culture system (Image courtesy: Liu et al., 2010).

GM Mix E

ed39. This approach is most accurate when the dose-limiting toxicity in the experimental species is bonemarrow suppression. However, the potential of com-parative in vitro hematotoxicology studies to identifydifferences in drug tolerance even for non-myelosup-pressive compounds has justified its use as a ‘sentineltissue’ in the National Cancer Institute’s toxicologyprogram since 198622.

The data produced by CFC assays are often morepredictive in clinical situation compared to those withusing cell lines. Therefore, in vitro assays play an im-portant role in identification of toxic compounds andexclude them way before reaching costly drug develop-ment and clinical trials process. It has been estimatedthat the average drug takes 10-15 years to get to marketat a cost of approximately $800 million. The use ofsensitive and high throughput screening in vitro assaysduring the drug screening process can help to elimi-nate late drug failure due to hematotoxicity. It saves alot of the time and cost which is currently associatedwith drug development process40.

In in-vitro models for testing toxicity there is a par-ticular challenge to determine the effect of hepaticmetabolism on toxins. A co-culture model has beendeveloped enabling the effect of any metabolisedagent on another cell type to be assessed. The modelutilises HepG2 liver spheroids as a source of metabo-lic enzymes, which have been shown to more closelyresemble human liver than traditional monolayer cul-tures. The developed model was subsequently validat-ed using several chemotherapeutic agents, both pro-drugs and active drugs, with resulting mesenchymalstem cell (MSC) damage closely resembling effectsseen in patients following chemotherapy41.

However, despite the obvious value and utility ofCFC assays in the hematological toxicity, the assayshave certain drawbacks as they are very much time-consuming and also face certain technical challenges.Manually determining the number of colonies is high-ly subjective, requires technical expertise and lacksstandardization. Although side-by-side cell populationsand species comparisons can be performed but due tothe low-throughput capability of the system this canonly be done on a small scale. This almost precludesthe use of the assay during the drug development pro-cess, except where a drug is known to interfere withthe differentiation process. These are relevant in smallscale experiments, since these assays are limited bythe manual colony counting process42,43. Clonogenicassay miniaturisation in order to reduce cost and in-creasing the number of compounds tested in lessertime for colony scoring has been proposed in 2004 byMalerbaetal. Cells were cultured in 96-well platesaccording to ECVAM Standard Operating Procedures.

Only 100μL/well of methyl cellulose suspension wasseeded into 96-well plates. BFU-E and CFU-GM col-onies were scored using an inverted microscope at 25Xmagnitude. Rich and Hall in 2005 redesigned the col-ony-forming assay methodology into ATP-based bio-luminescence proliferation assay readout. The newassay has been named HALO® (Hemotoxicity Assaysvia Luminescence Output). HALO® does not sufferfrom the drawbacks of the manual clonogenic assays.It is rapid, completed in half the time of the manualclonogenic assay, highly sensitive, non subjective andstandardized. It is based on the use of a highly sensi-tive bioluminescence signal with high-throughput capa-bility to measure the proliferative capacity of differentcell populations from different sources and speciessimultaneously42. The proliferation status of cell isproportional to the intra-cellular ATP concentration(iATP). The 96-well plate format provides high-throu-ghput capability. When different cells are stimulatedwith various growth factors and/or cytokines to detectlympho-hematopoietic population (including CFU-GEMM, CFU-GM, CFU-MK, BFU-E), the iATP canbe released by lysis after incubation and act as a limit-ed substrate for highly sensitive luciferin/luciferasereaction to produce bioluminescence in the form oflight. The light is measured in a plate of Luminometreusing 96 or 384-well plate format. In addition, the sys-tem provides multifunctional capability so that up to14 different lympho-hematopoietic populations fromdifferent hematopoietic sources (peripheral blood andbone marrow and cord blood from humans) from fivedifferent species i.e. human, non-human primate, dog,rat, and mouse can be detected and measured simulta-neously. The HALO Platform has been validatedagainst the manual clonogenic assay. To be used forscreening compounds, HALO® has been Compared toclassical clonogenic assays that had been used to studya number of varied compound43.

Conclusions

An inherent and persistent problem of using in vitroassays during the early stages of drug development asa alternative method for in vivo hematotoxicology stud-ies is that the complex behaviour of a compound invivo system is very different to that of in vitro models.

Improvement in the existing technology, preciselyrefined and optimized standard operating procedure(SOP) makes researcher capable of Recreating an invivo-like environment in vitro. Use of more sensitiveand specific toxicity screening tools are very help fullto overcome the drawbacks of in-vitro manual assaysas they reduce costs, increase number of compounds


tested and time for colony scoring. These tests havethe potential to reduce the number of animals requiredfor hematotoxicity testing and to permit the refinementof those animal procedures that have to be conducted.Identification of new precursor stem cells and develop-ment of more sensitive assays to define these cells arein progress.

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in-vitro hematological toxicity prediction by colony-forming cell assays

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