Simply
red:
A
novel
spectrophotometric
erythroid
proliferation
assay
as
a
tool
for
erythropoiesis
and
erythrotoxicity
studies
$
Daniela
Boehm
*
,
Angus
Bell
DepartmentofMicrobiology,SchoolofGenetics&Microbiology,MoyneInstituteofPreventiveMedicine,TrinityCollegeDublin,Dublin2,Ireland
A R T I C L E I N F O
Articlehistory:
Received7April2014
Receivedinrevisedform22July2014 Accepted22July2014
Availableonline24July2014
Keywords:
Erythropoiesis Hemoglobin Cytotoxicity Anemia Malaria
A B S T R A C T
Mostmammaliancellproliferationassaysrelyonmanualorautomatedcellcountingortheassessment ofmetabolicactivityincolorimetricassays,withtheformerbeingeitherlaborandtimeintensiveor expensiveandthelatterbeingmultistepproceduresrequiringtheadditionofseveralreagents.The proliferationoferythroidcellsfromhematopoieticstemcellsandtheirdifferentiationintomaturered blood cells is characterized by the accumulation of large amounts of hemoglobin. Hemoglobin concentrationsareeasilyquantifiableusingspectrophotometricmethodsduetothespecificabsorbance peakofthemolecule’shememoietybetween400and420nm.Erythroidproliferationcanthereforebe readilyassessedusingspectrophotometricmeasurementinthisrange.Wehaveusedthisfeatureof erythroid cellstodevelopa simpleerythroid proliferationassaythatisminimallylabor/time-and reagent-intensiveandcouldeasilybeautomatedforuseinhigh-throughputscreening.Suchanassaycan beavaluabletoolforinvestigationsintohematologicaldisorderswhereerythropoiesisisdysregulated, i.e.,eitherinhibitedorenhanced,intothedevelopmentofanemiaasaside-effectofprimarydiseases suchasparasiticinfectionsandintocyto-(particularlyerythro-)toxicityofchemicalagentsordrugs.
ã2014TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/3.0/).
1.Introduction
Erythropoiesis is one of the body’s most productive cell proliferation processes, yielding an average of 21011 new
erythrocytesfromhematopoieticstemcellsofthebonemarrow everyday toreplace those lost tosenescence and destruction
[25]. A reduced erythropoietic output or the production of malfunctioning erythrocytes leads to anemia which can have severe and even fatal consequences when tissues are insuffi -ciently suppliedwith oxygen [17]. Homeostasis of erythrocyte productionisprimarilyregulatedbythehormoneerythropoietin (EPO), whose production is upregulated upon tissue oxygen depletion[9,30].However, numerousfactors –both exogenous (such as toxins) and endogenous (such as inflammatory cytokines) – can inhibit proliferation and/or differentiation of erythroid cells [27]. In addition, the requirement for large amountsofironforhemoglobinizationmakestheprocesshighly
dependent on the availability of sufficient concentrations of transferrin-boundiron[16].Indiseasesofchronicinflammation suchasrheumatoidarthritis,erythropoiesisisimpairedbothby the direct action of proinflammatory cytokines such as tumor necrosis factor (TNF)-
a
and interferon (IFN)-g
and by upregulation of the liver hormone hepcidin, the primary regulator ofironuptakeandstorage, leadingtoa reductionin the amount of bio-available iron in circulation [10]. Many chemotherapeuticagentshavealsobeenfoundtoinduceanemia eitherthroughaninhibitionoftheproductionofEPOoradirect cytotoxic/inhibitoryeffectonerythroidcells[6,31].A number of infections with parasitic agents such as
Plasmodium, Schistosoma, Leishmania or hookworms result in anemia[22,24].Inthecaseofintestinalinfections,thisanemiais believedtobecausedprimarilybyintestinalhemorrhage,reduced iron absorption or decreased bioavailability of iron [29]. Inflammatoryresponsestotheinfectionsincludingthesecretion ofproinflammatorycytokinesand/ortheresultantupregulationof hepcidinadditionallyappeartoinhibit erythropoiesis,asin the anemia of chronic disease [4,27]. In the case of malaria and leishmaniasis, evidence existsthat parasitic productsmay also directly impede erythroid proliferation and/or differentiation
[13,33].
On theother hand, erythropoiesis can become dysregulated in certain myeloproliferative disorders leading to uncontrolled proliferationoferythroidcells.Intheerythroleukemiapolycythemia $This isanopen-accessarticledistributedunderthetermsoftheCreative
Commons Attribution-NonCommercial-No Derivative Works License, which permitsnon-commercial use, distribution,and reproduction in any medium, providedtheoriginalauthorandsourcearecredited.
*Corresponding author. Current address: Dublin Institute of Technology, DepartmentofFoodScienceandEnvironmentalHealth,Dublin1,Ireland. Tel.:+35314024477;fax:+35316799294.
E-mailaddress:[email protected](D.Boehm).
http://dx.doi.org/10.1016/j.btre.2014.07.005
2215-017X/ã2014TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/3.0/). ContentslistsavailableatScienceDirect
Biotechnology
Reports
vera for example a mutation in the Janus tyrosine kinase JAK2renderserythroidproliferationindependentoferythropoietin andcausesexcessiveredcellproduction[20,23].
In vitromethods for thegeneration of erythroidcellsfrom hematopoieticstemcellsderivedfromvarioussourceshavebeen established and shown to yield both high proliferation of erythroid cells and produce functional, mature, enucleated reticulocytes or erythrocytes, thus faithfullyrecapitulating the invivoprocess[3,11,12].Ingeneral,thedifferentiationprocessof erythroidprogenitorcellsandtheirmaturationischaracterized by the acquisition of specific erythroid features including particularsurfacemarkers,an exitfrom thecell cycleandthe accumulationoflargeamountsofhemoglobinthatisresponsible forthecells’abilitytobindoxygen[35,39].Atetramerof4globin chains with a central heme molecule, hemoglobin shows a spectrophotometricabsorbancepeakbetween400and420nm, whichhasbeenexploitedforthequantificationofhemoglobinin solutionbyHarboeandothers[5,14,15].Asthischaracteristiccan beusedfor hemoglobinquantificationnot onlyinsolution but alsowhencell-bound,wehavedevelopedaspectrophotometric assayforassessingerythroidproliferationbasedonabsorbance at405nm.
2.Materialsandmethods
All chemicals were obtained from Sigma–Aldrich (Arklow, Ireland)unlessstatedotherwise.
2.1.Cellisolation
Mononuclearcells(MNC)wereisolatedfromperipheralblood buffycoats obtained from theIrish Blood Transfusion Services (Dublin, Ireland) using density gradient centrifugation with histopaque-1077. CD34+ cells were isolated from mononuclear cellsviaimmuno-magneticseparationusinganti-CD34magnetic beads according to the manufacturer’s instructions (Miltenyi Biotec,Bisley,UK).
2.2.Cellculture
Cultures were initiated from frozen or freshly isolated mononuclearcells or CD34+cells. For depletion of leukocytes, mononuclearcellswerepre-cultured for2 daysin IMDM with stabilizedglutamine(Biochrom,Berlin,Germany)containing5% (vol/vol) heparinizedhuman plasmaand3U/mlEPO (Janssen-Cilag,Dublin,Ireland).Cells werethenwashedandculturedin erythroidproliferation medium [12] consistingof IMDM with 330
m
g/ml iron-saturated human transferrin and 10m
g/ml recombinanthumaninsulin,supplementedwith5%heparinized human plasma, 100ng/ml stem cell factor (SCF) (Cambridge Biosciences, Cambridge,UK), 5ng/ml interleukin-3 (IL-3)(R&D Systems, Minneapolis, MN, USA), 3U/ml EPO and 106M hydrocortisonefor 1–2 days. CD34+ cells were plateddirectly into the appropriate conditions. Manual cell counting was performedusingthetrypanblueexclusionmethodwithtrypan bluediluted1/6inphosphatebufferedsaline(PBS)andaddedto cellsat1:1or1:4ratios.2.3.Plasmodiumfalciparumconditionedmedium
P.falciparum-conditionedmediumwas obtainedfrom blood-stage cultures of P. falciparum 3D7 cultures grown in RPMI mediumsupplemented with5%(wt/vol)Albumax1 ina candle jar according to published methods [8]. P. falciparum cultures were grown to 10–15% parasitemia, washed two times with wash medium consisting of RPMI supplemented with 0.18g/l
sodium bicarbonate and one time with IMDM and then
resuspended in IMDM. For higher protein content, cultures were concentrated 6–8 fold by resuspension in lowervolumes of IMDM after washing and cultured for 3–4h to obtain conditioned medium. Conditioned medium was obtained by centrifuging the culture supernatant for 10min at 2000rpm (823g)followedbyfiltrationthrougha0.2
m
mfilterandused at upto80% (vol/vol) in erythroidmedium.2.4.Absorbance-basedassay
Cells(CD34+cellsorpre-culturedMNCs)weresubsequently seeded in erythroidproliferation mediumcontaining5% hepa-rinized humanplasma,100ng/mlSCF, 5ng/ml IL-3 and 3U/ml EPOasstandardconditions.Theseconditionsservedasapositive control for erythroid proliferation. As a negative control, cells wereseededinpureIMDMmediumlackinggrowthfactorsand plasma.Erythropoiesisinhibitingagentswereaddedatdifferent concentrations or growth factorconcentrations werevaried to assesstheeffectonerythroidproliferation.Cellswereseededin 96-wellflat-bottomplatesat1–5105cells/mlandculturedina
humidifiedincubatorat37C and5%CO2forupto21days.All
conditionsweresetupintriplicateandforeachconditionawell containing the appropriate medium blank without cells was included.
Absorbance measurements of plates with lid were taken at 405nm using a Synergy H1 (Biotek, Potton, UK) plate reader preheated to 37C and following 2min of linear shaking at 567cyclesperminute(cpm).
2.5.Analysis
Results from manual cell counting were determinedas the mean and standard deviation of the cell concentrations of triplicatewells.
Results fromspectrophotometric measurements were deter-mined as the mean absorbance of triplicate wells and their standard deviation. The mean values were corrected by the absorbanceofthemediumblankandpresentedaspercentageof thepositivecontrol.StatisticalanalysiswasperformedusingPrism software(LaJolla,CA,USA).
3.Results
3.1.Methodprinciple
The Harboe method has been established for determining hemoglobinconcentrationsinsolutionusingspectrophotometric measurement at 415nm and has been validated for assessing hemolysisinredcellsamples[14,15].Weadaptedthismethodfor estimating erythroid cell concentrations in unlysed culture samples and determining erythroid proliferation in a non-invasive manner.Hemoglobin showsmaximumlightabsorption between400and420nmandwefoundabsorbanceat405nmand 415nm to correlate linearly (R2=0.9999) allowing the use of 405nmabsorbancefilterscommonlyavailableonstandardplate readers(Fig. 1a).Weestablishedthatthelysisoferythrocytesisnot necessary for reliable hemoglobin quantification and that cell suspensions could be directly subjected to spectrophotometric measurement (Fig. 1b, R2=0.9905). Initial assay set-up was
performed using samples of native erythrocytes isolated from donorbloodsuspendedinPBSandabsorbancemeasurementsat 405nmwerefoundtocorrelatelinearly(R2=0.998)withmanual
their absorbance (erythrocyte equivalents/ml=(5,413,000
91,210)A405(154,70080,730)).
Absorbance measurements were obtained from in vitro erythroidculturesatvarioustimepointsofcultureusingaplate readerpre-heatedto37C,andplateswereagitatedtodisperse cells evenly in the microwells before measurement. The absorbance values werecorrected using the absorbance of the mediumofeach conditionand normalizedtoapositivecontrol cultureonthesameplatetodeterminethehemoglobinizationas percentageofthepositivecontrolthatinturncorrelatedwiththe cellexpansion(Fig.2).
3.2.Stability
Hemoglobinization begins at the proerythroblast stage and two thirds of a cell’s total hemoglobin are produced by the erythroblast while the remaining third is synthesized at thereticulocytestage[35].Inculture,cellscontaineddetectable amountsofhemoglobinfromday8afterseedingintoerythroid medium,showedstrongincreaseinhemoglobinizationoverthe next 7 days and reached a plateau thereafter. Absorbance measurements based on hemoglobin remained stable over extended periods of time showing only slight decreases in
Fig.2.Outlineofset-upandmeasurementprincipleofspectrophotometricerythroidproliferationassay.MNC=mononuclearcell.
Fig.1.(a–c)Hemoglobinmeasurementat405nmcanbeusedtodeterminethecellconcentrationsofunlysedsamplesoferythrocytes:(a)hemoglobinabsorbanceof erythrocytelysatesat415and405nmcorrelatedlinearlywitheachother.(b)Theabsorbancesat405nmofunlysederythrocytesamplesshowedlinearcorrelationwiththose oferythrocyteslysedosmoticallyindistilledwater.(c)Theconcentrationsoferythrocytesamplesasdeterminedbymanualcountingusingtrypanbluecorrelatedlinearly withabsorbancemeasurementsat405nm.DilutionsofnativeerythrocytesinPBSwereusedbetween1105
and1107
absorbance after further 10 days (Fig. 3), indicating that this moleculeisnotreadilydegradedevenwhenitisreleasedintothe culturesupernatantuponcelldeathandrupture.
3.3.Optimizationofcultureconditions
Cell concentrations and absorbance measurements for ery-throidculturescorrelatedlinearlyandwhilestandarddeviations werelargerthanfornativeredbloodcellsamples,thesevaried comparably for both measurement principles due to higher biologicalvariationbetweentriplicatewells.Thelinearfunction obtained for a culture at day15 of erythroid culture (Fig. 4b) indicatedalowerabsorbanceforagivencellconcentrationthan obtainedfortheerythrocytestandardcurveastheseerythroidcells had not yet fully hemoglobinized. While the ex vivo culture methoddoes not yield fullymature erythrocytes but produces predominantly reticulocytes, a linear correlation between cell concentrationsandabsorbancecouldbedemonstratednotonlyfor
Fig.4.(aandb)Optimizationofcultivationconditions:(a)theeffectofdifferentgrowthfactor(EPO,SCF)andplasma(PP)concentrationsonerythroidproliferationcouldbe determinedusingthespectrophotometricerythroidproliferationassay.Thepositivecontrolwasgrowninstandardconditionsof5%plasma,100ng/mlSCF,5ng/mlIL-3and 3U/mlEPO.Concentrationsofplasma,SCForEPOwerevariedindividuallywhiletheotherfactorsweremaintainedatstandardconcentrations.Resultsarethemean andstandarddeviationoftriplicatesexpressedaspercentageofpositivecontrol.(b)Theassaycoulddistinguishbetweenconditionspromotingorinhibitingerythroid proliferationequallywellasmanualcellcounting,showinglinearcorrelation(R2
=0.9313)betweenthecellconcentrationandtheabsorbanceofrespectivesamples.Results areshownasmeanandstandarddeviationoftriplicatedeterminationsonday15ofculture.
mature erythrocytes (Fig. 1c) but also for ex vivo generated erythroidcellswhichrepresentamixedpopulationoferythroid cellsofdifferentmaturities.Comparisonwiththeinternalpositive control(standardgrowthconditions)thusallowedfora determi-nationofreducedorenhancederythroidproliferation.Usingthis method,cultureconditionswhicharelessfavorabletoerythroid expansion,e.g.,reducedconcentrationsofplasmaorofthegrowth factorsstemcellfactor(SCF)orEPOintheerythroidmedium,could bedetermined(Fig.4aandb).
3.4.ErythroidinhibitionbyP.falciparum
Using this method, we were furthermore able to detect erythropoiesis inhibiting activity in medium conditioned by blood-stage cultures of themalarial parasiteP. falciparum. The assaywas abletodistinguishbetweenerythropoiesis-inhibiting and -promoting conditions to the same extent as manual cell countingusingtrypan blueexclusion (Fig.5a) and was able to detect differential responses to different concentrations of the inhibitorymedium(Fig.5b).
3.5.Cytotoxicagents
Dimethylsulfoxide (DMSO) is a commonly used solvent for drugs that show limited solubility in aqueous solutions but it
effects a range of biological functionsand cancause toxicside effectsinvivo[37].DMSOisalsousedastheprimary cryoprotec-tive agentfor hematopoieticstem cellsfor transplantationasit reducescelldamageduetocrystalformationand protectscells fromdehydration.Ithas,however,beenshowntobetoxictothese cellsatelevatedconcentrationsresultinginaround25%ofviable celllossat5%(vol/vol) DMSOandupto50%at 10%DMSO[1]. DMSO therefore presents a useful candidate molecule for evaluating the potential of this assay for the assessment of chemicalcytotoxicity.Highconcentrations(20%and10%) abrogat-edallcellgrowthand verylowlevelsofhemoglobinformation weredetectedat5%and2%DMSOwhereasconcentrationsbelow 1%showednoinhibition(Fig.6a).Theapplicabilityoftheassayfor toxicologicalstudieswasfurtherdemonstratedbytheuseofthe antibioticchloramphenicolwhichhasbeenfoundtocausebone marrowsuppressionandaplasticanemiainvivo[34].Usingourin vitro system, concentration-dependent inhibition of erythroid growthwasobserved,witha1mg/mlconcentrationofthedrug almostabolishingerythropoiesiswhereas12.5
m
g chlorampheni-col/ml still caused about 50% growth reduction (Fig. 6b). The suitability of an assay for high-throughput screening (HTS) dependsonitsperformanceandsensitivitywhichcanbeassessed usinga number ofstatisticalparameters includingtheZ0 factorcommonlyusedtoquantifythequalityofanassay[19].Inatotalof 10differentassaysthat werevalidated,ourspectrophotometric erythroidproliferationassayperformedwellwithintheacceptable limitsandshowedanaverageZ0of0.67(Table1).
4.Discussion
Erythropoiesis is one of the body’s most proliferative cell production processes and dysregulation of this process can have life-threatening consequences. In the absorbance based erythroid proliferation assay presented here, we exploit the featuresoferythroidcultures–highcellexpansioninvitroand accumulation of large amounts of spectrophotometrically quantifiablehemoglobin–todevelopanovelresearchtool.
Research continues into the development of suitable drug treatmentsforerythroleukemiassuchaspolycythemiavera(PV). Thesedrugscurrentlyincludehydroxycarbamide(hydroxyurea), pipobromanorinterferon,butthesetherapiescanincreasetherisk of transformation to myelofibrosis or leukemia [21,26]. An erythroid proliferation assay based on PV hematopoietic stem cellscouldthereforesignificantlyfacilitatethescreeningfornovel compoundsthatreduceerythroidproliferationtonormallevelsin this type of myeloproliferative disorder. As venesection in an attempt to lower hematocrit levels is one of the primary treatmentsforPV,mononuclearcellsfromPVpatientswouldbe readilyavailablefromthesephlebotomiesandapatient’sowncells could even be used totest for responsivenessto specific drug treatments.
Ontheotherhand,suchscreeningmayenableidentificationof erythropoiesisstimulatingagentsinconditionswheretheprocess is inhibited such as those of Diamond Blackfan anemia, a congenital hypoplastic anemia characterized by mutations in genesencodingribosomalproteinsleadingtoreducedproduction oferythrocytes[7].
Anemicconditionswhereerythroidinhibitionmaybeadirect result of the action of inhibitory pathogen-derived factors as suspectedinmalaria[2]orLeishmaniainfectionscouldalsobenefit fromascreeningtoolfortheidentificationofthecausativefactors andmethodsoftheirinactivation.
Finally,drugcytotoxicitystudies–anderythrotoxicityofcancer chemotherapeuticsinparticular–maybesignificantlyfacilitated byahigh-throughputassay,reducingtheneedforanimalmodels and cutting both time and cost requirements. A number of
Fig.5.(aandb)ErythroidinhibitionbyproductsofP.falciparum:(a)absorbance measurementandmanualcellcountingcoulddistinguishbetweenconditions promotingerythroidproliferation(containingoptimalconcentrationsofgrowth factors[100ng/mlSCF,5ng/mlIL-3,3U/mlEPO]andmedium[5%plasma])or reducing erythroid proliferation (medium conditioned with products of P.
falciparum).Resultsarepresentedasmeanandstandarddeviationoftriplicate
cytotoxicityassaysarecommerciallyavailableandhavebeenused forhigh-throughputscreening.Mostofthesearecolorimetricor
fluorescentassaysthatrelyoneitherthemeasurementofenzyme activitiesinviable cellsordetectenzymesreleasedintoculture supernatantsuponcelldeathusingestablishedcelllines[28,40].
Among the most common assays are those based on the
measurementof activitiesof ATP,lactate dehydrogenase(LDH),
aminopeptidase,orproteases,ordeterminationofredoxpotential viaresazurinortetrazoliumbasedcompounds(MTT,MTS,XTT). Whilemanyoftheaforementionedassayshaveestablishedtrack records for cytotoxicity studies, their limitation lies in the requirementfortheadditionofreagentsmakingthembothmore costandlabor-intensiveandpreventingcontinuousmeasurement ofaculture.Recentadvancesindrugcytotoxicitytestinginclude
Table1
Assayperformance:evaluationofstandardparametersusingthemean(m)andstandarddeviation(s)ofthepositive(max=standarderythroidgrowthconditions)and negativecontrol(min=nogrowthfactors)showsgoodassayperformanceandsensitivityforthespectrophotometricassaywhenusedtoanalyzetheeffectofDMSO, chloramphenicolandinatotalof10differentassays.Resultsarecalculatedusingthegivenequationsandtheaverageassayperformanceisshownasmeanandstandard deviationoftherespectiveparametersfor10assaysperformedbetweendays7and12oferythroidcultures;valuerangesaregiveninparentheses.
Parameter DMSO
day10
Cm day7
Averageofn=10assays day101.5(7–12)
Acceptablelimits
Coefficientofvariation(max) %CV=smax/mmax100
7.6% 5.4% 6.02.6%(2.5–9.9%) <15%
Coefficientofvariation(min) %CV=smin/mmin100
11.6% 11.3% 10.55.4%(0–17.2%) <15%
Signaltonoiseratio S:N=(mmaxmmin)/smin
17.8 49.1 34.116.5(14.3–63.8)
Signaltobackgroundratio S:B=mmax/mmin
3.1 6.5 4.61.1(3.1–6.5) >2-fold
Signalwindow
SW=(mmaxmmin3(smin+smin))/smax
4.4 11.7 9.94.6(4–17.4) >2-fold
Z0factor
Z0=(3smax+3smin)/|mmaxmmin|
0.49 0.75 0.670.11(0.49–0.86) >0.5
the development of microfluidic cell culture systems or
‘lab-on-a-chip’devices[36,38].Thesedeviceshavetheadvantage ofallowingnon-invasiveandcontinuousmonitoringbutaremore complexandcostlyintermsofequipment.
Automation of thespectrophotometric assaydescribed here shouldbe easilyachievable through anadaptation tocommon microplate-handlingroboticset-ups. While an internal positive control to which results are normalized reduces the need for plate-to-plate standardization, this could nonetheless be facilitated, e.g., for cytotoxicity studies of candidate drugs, by establishinglargeMNCpoolsorusingerythroleukemiacelllines. A limitingfactor totheuseof ahemoglobin-basedassayfor cytotoxicitystudiesishoweveritsinabilitytodistinguishbetween liveanddeadcells,asitcanonlydetermineeffectsonthegrowth/ hemoglobinizationoferythroidcellsbutcannotdetectthedeathof alreadyfullyhemoglobinizedcells.Hemoglobinizationcontinues pastthestagewhereerythroidcellsbecomecellcyclearrestedand cease proliferating and large amounts of hemoglobin are still synthesizedatthereticulocytestage[35].Theassayisthusableto detectcytotoxiceffectsonerythroidcellsduringgrowthphase,as hemoglobinization would cease prematurely, but unable to differentiatebetweenintacthighlyhemoglobinizedreticulocytes andhemoglobinwhichmayhavebeenreleasedintosolutionby lysedcellsinlatestagesofculture.
Thespectrophotometric assayhasbeensuccessfullyusedfor thedetectionoferythropoiesisinhibitingactivityinmediumfrom
P.falciparumculturesandtodeterminepreferentialgrowthfactor concentrations for erythroid expansion. It can further detect cytotoxic components and react in a concentration-responsive manner. Overall, this method provides the means for rapid assessment of erythroid proliferation – either enhanced or inhibited–comparedtoastandardcontrolandcanthusbehighly beneficialininitialscreeningstagestoselectpotentialconditions orcandidatemoleculesofinterest.Designofexperiment(DoE)has beengrowingin significancefor processoptimizationanddrug designapplications[18,32].Coupling DoEforerythroidsystems with an automatable assay such as this one to obtain the experimentalresultsonwhichtobuildthedesign andverifyits predictioncouldallowfortheacquisitionoflargeamountsofdata inshortperiodsoftime.Foranassaytobeimplementablein high-throughputscreeningithastomeetanumberofcriteria:itshould be simple, automatable, show sensitive and reproducible performanceandlastbutnotleastbeeconomicallyfeasible.We believethisassayfulfillsallofthesecriteriaandpresentsagood candidateforHTS.
Few cells in the human body lend themselves to the establishmentofa colorimetricproliferation assayas readilyas erythroid cells which simply produce the red read-out dye themselves–thenextstepisdevelopingtheapplications.
Acknowledgement
ThisworkwassupportedbyfundingfromtheIrishResearch Council(IRC).
References
[1]F.AbrahamsenJenny,M.BakkenAnne,Ø.Bruserud,Cryopreservinghuman peripheralbloodprogenitorcellswith5-percentratherthan10-percentDMSO resultsinlessapoptosisandnecrosisinCD34+cells,Transfusion42(2002) 1573–1580.
[2]A.Bell,D.Boehm,Anti-diseasetherapyformalaria–‘resistanceproof’?Curr. Pharm.Des.19(2013)300–306.
[3]D.Boehm,W.G.Murphy,M.Al-Rubeai,Thepotentialofhumanperipheral bloodderivedCD34+cellsforexvivoredbloodcellproduction,J.Biotechnol. 144(2009)127–134.
[4]F. Burte, B.J. Brown, A.E. Orimadegun, W.A. Ajetunmobi, N.K. Afolabi,F. Akinkunmi,O. Kowobari,S.Omokhodion,K.Osinusi,F.O.Akinbami,W.A.
Shokunbi,O.Sodeinde,D.Fernandez-Reyes,Circulatoryhepcidinisassociated withtheanti-inflammatoryresponsebutnotwithironoranemicstatusin childhoodmalaria,Blood121(2013)3016–3022.
[5]P.Cookson,J.Sutherland,R.Cardigan,Asimplespectrophotometricmethodfor thequantificationofresidualhaemoglobininplateletconcentrates,VoxSang. 87(2004)264–271.
[6]M.Dicato,L.Plawny,M.Diederich,Anemiaincancer,Ann.Oncol.21(2010) vii167–vii172.
[7]S.R.Ellis,J.M.Lipton,DiamondBlackfananemia:adisorderofredbloodcell development,in:J.B.James(Ed.),CurrentTopicsinDevelopmentalBiology, AcademicPress, 2008,pp.217–241(Chapter8).
[8]B.J. Fennell, Z.A. Al-shatr, A. Bell, Isotype expression, post-translational modificationand stage-dependent productionoftubulinsin erythrocytic Plasmodiumfalciparum,Int.J.Parasitol.38(2008)527–539.
[9]J.W.Fisher,Erythropoietin:physiologyandpharmacologyupdate,Exp.Biol. Med.228(2003)1–14.
[10]T.Ganz,E.Nemeth,Hepcidinandironhomeostasis,Biochim.Biophys.Acta (BBA)–Mol.CellRes.1823(2012)1434–1443.
[11]M.C.Giarratana,L.Kobari,H.Lapillonne,D.Chalmers,L.Kiger,T.Cynober, M.C.Marden,H.Wajcman,L. Douay,Exvivogenerationoffullymature humanredbloodcellsfromhematopoieticstemcells,Nat.Biotechnol.23 (2005)69–74.
[12]M.C.Giarratana,H.Rouard,A.Dumont,L.Kiger,I.Safeukui,P.Y.LePennec, S.Francois,G.Trugnan,T.Peyrard,T.Marie,S.Jolly,N.Hebert,C.Mazurier, N.Mario,L.Harmand,H.Lapillonne,J.Y.Devaux,L.Douay,Proofofprinciple for transfusion of in vitro-generatedred blood cells, Blood 118 (2011) 5071–5079.
[13]K.Haldar,N.Mohandas,Malaria,erythrocyticinfection,andanemia,Hematol. Am.Soc.Hematol.Educ.Program(2009)87–93.
[14]V.Han,K.Serrano,D.V.Devine,Acomparativestudyofcommontechniques usedtomeasurehaemolysisinstoredredcellconcentrates,VoxSang.98 (2010)116–123.
[15]M.Harboe,Amethodfordeterminationofhemoglobininplasmaby near-ultravioletspectrophotometry,Scand.J.Clin.Lab.Investig.11(1959)66–70. [16]M.W.Hentze,M.U.Muckenthaler,N.C.Andrews,Balancingacts:molecular
controlofmammalianironmetabolism,Cell117(2004)285–297. [17]V.M.Hodges,S.Rainey,T.R.Lappin,A.P.Maxwell,Pathophysiologyofanemia
anderythrocytosis,Crit.Rev.Oncol./Hematol.64(2007)139–158. [18]M.M. Hunt, G. Meng, D.E. Rancourt, I.D. Gates, M.S. Kallos, Factorial
experimental design forthe culture ofhuman embryonic stemcells as aggregates in stirred suspension bioreactors reveals the potential for interactioneffectsbetweenbioprocessparameters,TissueEng.PartCMethods 20(2014)76–89.
[19]J.Inglese,R.L.Johnson,A.Simeonov,M.Xia,W.Zheng,C.P.Austin,D.S.Auld, High-throughputscreeningassaysfortheidentificationofchemicalprobes, Nat.Chem.Biol.3(2007)466–479.
[20]C.James,V.Ugo,J.-P.LeCouedic,J.Staerk,F.Delhommeau,C.Lacout,L.Garcon, H.Raslova,R.Berger,A.Bennaceur-Griscelli,J.L.Villeval,S.N.Constantinescu, N. Casadevall, W. Vainchenker, A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera, Nature 434 (2005) 1144–1148.
[21]J.-J.Kiladjian,S.Chevret,C.Dosquet,C.Chomienne,J.-D.Rain,Treatmentof polycythemiaverawith hydroxyurea andpipobroman: final resultsofa randomizedtrialinitiatedin1980,J.Clin.Oncol.29(2011)3907–3913. [22]C.H.King,K.Dickman,D.J.Tisch,Reassessmentofthecostofchronichelmintic
infection: a meta-analysis of disability-related outcomes in endemic schistosomiasis,Lancet365(2005)1561–1569.
[23]R.Kralovics,F.Passamonti,A.S.Buser, S.-S.Teo,R.Tiedt,J.R.Passweg,A. Tichelli,M. Cazzola, R.C.Skoda, Again-of-function mutation of JAK2 in myeloproliferativedisorders,N.Engl.J.Med.352(2005)1779–1790. [24]W.P.Lafuse,R.Story,J.Mahylis,G.Gupta,S.Varikuti,H.Steinkamp,S.Oghumu,
A.R.Satoskar,Leishmaniadonovaniinfectioninducesanemiainhamstersby differentiallyalteringerythropoiesisinbonemarrowandspleen,PLoSOne8 (2013)e59509.
[25]C.Leberbauer,F.Boulmé,G.Unfried,J.Huber,H.Beug,E.W.Müllner,Different steroidsco-regulatelong-termexpansionversusterminaldifferentiationin primaryhumanerythroidprogenitors,Blood105(2005)85–94.
[26]M.F.McMullin,Areviewofthetherapeuticagentsusedinthemanagementof polycythaemiavera,Hematol.Oncol.25(2007)58–65.
[27]F.Morceau, M.Dicato,M.Diederich,Pro-inflammatorycytokine-mediated anemia:regardingmolecularmechanismsoferythropoiesis,Mediat.Inflamm. 2009(2009)405016.
[28]A.L.Niles,R.A.Moravec,T.L.Riss,Invitroviabilityandcytotoxicitytestingand same-wellmulti-parametriccombinations forhigh throughputscreening, Curr.Chem.Genomics3(2009)33–41.
[29]M.V.Periago,J.M.Bethony,Hookwormvirulencefactors:makingthemostof thehost,MicrobesInfect.14(2012)1451–1464.
[30]T.D.Richmond,M.Chohan,D.L.Barber,Turningcellsred:signaltransduction mediatedbyerythropoietin,TrendsCellBiol.15(2005)146–155.
[31]J.L.Spivak,Theanaemiaofcancer:deathbyathousandcuts,Nat.Rev.Cancer5 (2005)543–555.
[32]S.E.Swalley,J.R.Fulghum,S.P.Chambers,Screeningfactorseffectingaresponse insolubleproteinexpression:formalizedapproachusingdesignof experi-ments,Anal.Biochem.351(2006)122–127.
inhibiting erythropoietin-induced proliferation of erythroid precursors, J.Infect.Dis.(2013).
[34]J.A. Turton, C.M. Andrews, A.C. Havard, T.C. Williams, Studies on the haemotoxicityofchloramphenicolsuccinateintheDunkinHartleyguinea pig,Int.J.Exp.Pathol.83(2002)225–238.
[35]M.J.Weiss,C.O.dosSantos,Chaperoningerythropoiesis,Blood113(2009) 2136–2144.
[36]E.WitkowskaNery,E.Jastrze˛bska,K.Zukowski,_ W.Wróblewski,M.Chudy, P.Ciosek,Flow-throughsensorarrayappliedtocytotoxicityassessmentincell culturesfordrug-testingpurposes,Biosens.Bioelectron.51(2014)55–61.
[37]D.C. Wood, J. Wood, Pharmacologic and biochemical considerations of dimethylsulfoxide,Ann.N.Y.Acad.Sci.243(1975)7–19.
[38]M.-H.Wu,S.-B.Huang,G.-B.Lee,Microfluidiccellculturesystemsfordrug research,LabChip10(2010)939–956.
[39]A.Zeuner,F.Martelli,S.Vaglio,G.Federici,C.Whitsett,A.R.Migliaccio,Concise review: stem cell-derived erythrocytes as upcoming players in blood transfusion,StemCells30(2012)1587–1596.
