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Methods in Molecular Biology 2567 Louis M. Pelus Jonathan Hoggatt Editors Hematopoietic Stem Cells Methods and Protocols M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Hematopoietic Stem Cells Methods and Protocols Edited by Louis M. Pelus Department of Microbiology & Immunology and Department of Medicine/Hematology Oncology, Indiana University School of Medicine, Indianapolis, IN, USA Jonathan Hoggatt Moderna Therapeutics, Cambridge, MA, USA Editors LouisM.Pelus JonathanHoggatt DepartmentofMicrobiology& ModernaTherapeutics ImmunologyandDepartment Cambridge,MA,USA ofMedicine/Hematology Oncology,IndianaUniversity SchoolofMedicine Indianapolis,IN,USA ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2678-8 ISBN978-1-0716-2679-5 (eBook) https://doi.org/10.1007/978-1-0716-2679-5 ©SpringerScience+BusinessMedia,LLC,partofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. Cover Illustration Caption: The cover photo shows trilineage differentiation of expanded human bone marrow MesenchymalStem/StromalCells(MSC)toOsteoblasts(left),Adipocytes(Middle)andChondrocytes(right). MSC areintegralcomponentsofthehematopoieticnichethatnurtureandeducateHematopoieticStemCells.Osteogenic differentiationwasdeterminedbystainingformineralizationofextracellularmatrixandcalciumdepositsusingAlizarin Red S. Adipocytes were identified by production of lipid droplets using Oil Red O staining. Alcian blue staining of glycosaminoglycansdeterminedchondrocytedifferentiation.ThecoverphotoisprovidedbyDr.PratibhaSingh(Indiana UniversitySchoolofMedicine,Indianapolis,IN,USA). ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface Thefieldofexperimentalhematology,orhematopoiesisorhematopoieticstemcellbiology aswemostlyrefertoittoday,hasgrowntremendouslyandcontinuestogrowspurredonby ever-increasing technology and sophistication. Having entered the field in the early 1970s (LMPelus),Iwasamazedbytheabilityoftechniquestovisualizethegrowthofindividual bone marrow cells into colonies of myeloid and erythroid cells when given magical organ- conditioned medium and the utility to evaluate responses to exogenously added entities. Lethally irradiated mice could be salvaged by reintroduction of cells that formed spleen nodules.Thepoorman’ssortingexperimentswherebylineagespecificitycouldbeobserved and altered by separating cloned daughter cells, and the long-term culture systems that allowedforevaluationofhematopoieticdifferentiationprovidedsomebasicunderstanding ofstemandprogenitorcellfunction. We have come so far from then. The hematopoietic hormones were cloned and made readily available. FACS machines were developed for analysis and selection of single cells. Monoclonalantibodiesanduniquefluorochromesandmasscytometryweredevelopedthat allow us to use these machines to quantify stem and progenitor cell populations and signaling pathways and transcription factors in these cells. Genetic tools to turn on, turn off, or delete genes in living organisms were developed, as were single cell genomics and proteomics.Insitumethodsletuspeerintothelivingbonemarrow.Newgeneticorganisms and more relevant animal models continue to emerge to evaluate true in vivo function of stemandprogenitorcellsandvalidatethefindingsuncoveredwithallthesetools.Withthese tools,wehaveagreaterunderstandingofhematopoiesisandcellfunction,andnewtechno- logicaladvancesoffer thepromisetolearnmore. Previouseditionsofthisseriescoveringhematopoieticstemcellprotocolshaveprovided thorough coverage of many developments in the field, focused on user friendlyand detail- oriented format, to allow those inexperienced or new to the areas apply these methods to their research and take advantage of the exceptional guidance offered by expert faculty. In this new edition, our aims were to update technical advances in areas covered in previous editions and incorporate new techniques focused on the molecular/genetic, cellular, and whole organism levels. We were also keen to incorporate methods that apply stress to hematopoiesis,whichwebelieveisamajorpathwaytounderstandhematopoieticstemcell biology and drive research in many unexpected and exciting ways in the future. While not exhaustivebyanymeans,thiseditionnowcontainschaptersfocusedonbetter understand- ingtheroleofhematopoieticnichesandtheircellularcomponents.Lastly,wehaveincluded chaptersdedicatedtoinvivomodelsthattestandquantitatestemcellfunctionandarekey tofurtherdevelopmentoftherapeuticapplications. Thisbookwouldnotbepossiblewithoutthetimeandeffortofallthecontributingauthors. Theirdedicationtotheintentofthisseriesisoutstanding,andwethankthemfortheirsupport. Wehopethatthisresourcewillbeavaluedadditiontoalllaboratoriesfocusedonunderstanding hematopoieticstemcellbiologyandthetherapeuticadvancesthatcanbederived. Indianapolis,IN,USA LouisM.Pelus Cambridge,MA,USA JonathanHoggatt v Contents Preface ..................................................................... v Contributors................................................................. ix PART I OVERVIEW 1 TheFactoryofBloodProduction:HematopoieticStemCells ........ ........ 3 JonathanHoggattandLouisM.Pelus PART II MOLECULAR MODIFICATION AND EVALUATION 2 DetectionofDNADamageinHematopoieticStemCells..... ....... ........ 11 SaipriyaAyyarandIsabelBeerman 3 EvaluatingHistoneAcetylationinMouseHematopoieticStem andProgenitorCellsUsingChromatinImmunoprecipitation ........ ........ 29 LiqiongLiu 4 CRISPRGeneEditingofHematopoieticStemandProgenitorCells.......... 39 RezaShahbazi,PatriciaLipson,KarthikeyaS.V.Gottimukkala, DanielD.Lane,andJenniferE.Adair 5 LentiviralTransductionofNonhumanPrimateHematopoieticStem andProgenitorCells ....... ........ ....... ....... ........ ....... ...... .. 63 ChuanfengWu,SoGunHong,AylinBonifacino, andCynthiaE.Dunbar PART III CELLULAR ISOLATION AND CHARACTERIZATION 6 IdentificationofNonhumanPrimateHematopoieticStemandProgenitor Cells....... ........ ....... ........ ....... ....... ........ ....... ........ 87 StefanRadtkeandHans-PeterKiem 7 IsolationandCharacterizationofFetalLiverHematopoieticStemCells ....... 99 DiegoA.L(cid:1)opezandAnnaE.Beaudin 8 UtilizingCyTOFtoExamineHematopoieticStemandProgenitor Phenotype ......... ....... ........ ....... ... .... .... .... ....... ........ 113 SafaF.MohamadandMaeganL.Capitano 9 HematopoieticStemCellIdentificationPostirradiation....... ....... ........ 127 AndreaM.Patterson,ChristieM.Orschell,andLouisM.Pelus PART IV THE HEMATOPOIETIC NICHE AND ACCESSORY CELLS 10 IntravitalMicroscopyforHematopoieticStudies..... ........ ....... ........ 143 MyriamL.R.HaltalliandCristinaLoCelso vii viii Contents 11 LaserMicromachiningofBoneasaToolforStudyingBone MarrowBiology .... ....... ........ ....... ....... ........ ....... ........ 163 ChristaHaase,DmitryRichter,andCharlesP.Lin 12 MSCandHSPCCoculture:MimickingExVivoBoneMarrowNiche......... 181 PratibhaSingh 13 IsolationofThymusStromalCellsfromHumanandMurineTissue .......... 191 KarinGustafssonandDavidT.Scadden PART V TRANSPLANTATION AND IN VIVO MODELS 14 ExperimentalModelsofMouseandHumanHematopoieticStemCell Transplantation..... ....... ........ ....... ....... .... .... ..... .. ........ 205 ScottH.Cooper,MaeganL.Capitano,andHalE.Broxmeyer 15 HematopoieticStemandProgenitorCellIdentification andTransplantationinZebrafish..... ....... ....... ........ ....... ........ 233 EllenFraint,PengLv,FengLiu,TeresaV.Bowman, andOwenJ.Tamplin 16 EstablishingaMurineModeloftheHematopoieticAcuteRadiation Syndrome.......... ....... ........ ....... ....... ........ ....... ....... . 251 P.ArturPlett,LouisM.Pelus,andChristieM.Orschell 17 PurinergicSignalingandItsRoleinMobilizationofBoneMarrow StemCells ......... ....... ........ ....... ....... ........ ....... ........ 263 MalwinaSuszynska,MateuszAdamiak,ArjunThapa, MonikaCymer,JaninaRatajczak,MagdalenaKucia, andMariuszZ.Ratajczak Index ...................................................................... 281 Contributors JENNIFERE.ADAIR • FredHutchinsonCancerCenter,Seattle,WA,USA;Universityof Washington,Seattle,WA,USA MATEUSZADAMIAK • DepartmentofRegenerativeMedicine,WarsawMedicalUniversity, Warsaw,Poland SAIPRIYAAYYAR • EpigeneticsandStemCellUnit,TranslationalGerontologyBranch, NationalInstituteonAging,Baltimore,MD,USA ANNAE.BEAUDIN • DepartmentofInternalMedicine,DivisionofHematologyand HematologicMalignancies,UniversityofUtahSchoolofMedicine,SaltLakeCity,UT, USA ISABEL BEERMAN • EpigeneticsandStemCellUnit,TranslationalGerontologyBranch, NationalInstituteonAging,Baltimore,MD,USA AYLINBONIFACINO • TranslationalStemCellBiologyBranch,NationalHeart,Lung,and BloodInstitute,NationalInstitutesofHealth,Bethesda,MD,USA TERESAV.BOWMAN • GottesmanInstituteforStemCellBiologyandRegenerativeMedicine, DepartmentofDevelopmentalandMolecularBiology,andDepartmentofMedicine (Oncology),AlbertEinsteinCollegeofMedicineandMontefioreMedicalCenter,Bronx, NY,USA HALE.BROXMEYER • DepartmentofMicrobiology&Immunology,IndianaUniversity SchoolofMedicine,Indianapolis,IN,USA MAEGANL.CAPITANO • DepartmentofMicrobiologyandImmunology,IndianaUniversity SchoolofMedicine,Indianapolis,IN,USA CRISTINALOCELSO • ImperialCollegeLondon,London,UK;TheFrancisCrickInstitute, London,UK SCOTTH.COOPER • DepartmentofMicrobiology&Immunology,IndianaUniversitySchool ofMedicine,Indianapolis,IN,USA MONIKACYMER • DepartmentofRegenerativeMedicine,WarsawMedicalUniversity, Warsaw,Poland CYNTHIAE.DUNBAR • TranslationalStemCellBiologyBranch,NationalHeart,Lung,and BloodInstitute,NationalInstitutesofHealth,Bethesda,MD,USA ELLENFRAINT • DepartmentofPediatrics(PediatricHematology/OncologyandCellular Therapy)andDepartmentofDevelopmentalandMolecularBiology,AlbertEinstein CollegeofMedicineandMontefioreMedicalCenter,Bronx,NY,USA KARTHIKEYAS.V.GOTTIMUKKALA • FredHutchinsonCancerCenter,Seattle,WA,USA KARINGUSTAFSSON • Center forRegenerativeMedicine,MassachusettsGeneralHospital, Boston,MA,USA;HarvardStemCellInstitute,Cambridge,MA,USA;Departmentof StemCellandRegenerativeBiology,HarvardUniversity,Cambridge,MA,USA CHRISTAHAASE • WellmanCenter forPhotomedicine,MassachusettsGeneralHospitaland HarvardMedicalSchool,Boston,MA,USA;Center forSystemsBiology,Massachusetts GeneralHospitalandHarvardMedicalSchool,Boston,MA,USA MYRIAML.R.HALTALLI • ImperialCollegeLondon,London,UK;TheFrancisCrick Institute,London,UK;Wellcome–MedicalResearchCouncilCambridgeStemCell Institute,UniversityofCambridge,Cambridge,UK JONATHANHOGGATT • ModernaTherapeutics,Cambridge,MA,USA ix x Contributors SOGUNHONG • TranslationalStemCellBiologyBranch,NationalHeart,Lung,andBlood Institute,NationalInstitutesofHealth,Bethesda,MD,USA HANS-PETERKIEM • StemCellandGeneTherapyProgram,FredHutchinsonCancer ResearchCenter,Seattle,WA,USA;DepartmentofMedicine,UniversityofWashington SchoolofMedicine,Seattle,WA,USA;DepartmentofPathology,UniversityofWashington SchoolofMedicine,Seattle,WA,USA MAGDALENAKUCIA • StemCellInstitute,JamesGrahamBrownCancerCenter,Universityof Louisville,Louisville,KY,USA;DepartmentofRegenerativeMedicine,WarsawMedical University,Warsaw,Poland DANIELD.LANE • FredHutchinsonCancerCenter,Seattle,WA,USA CHARLESP.LIN • WellmanCenter forPhotomedicine,MassachusettsGeneralHospitaland HarvardMedicalSchool,Boston,MA,USA;Center forSystemsBiology,Massachusetts GeneralHospitalandHarvardMedicalSchool,Boston,MA,USA;HarvardStemCell Institute,Cambridge,MA,USA PATRICIALIPSON • FredHutchinsonCancerCenter,Seattle,WA,USA FENGLIU • StateKeyLaboratoryofMembraneBiology,InstituteofZoology,Chinese AcademyofSciences,Beijing,China;InstituteforStemCellandRegeneration,Chinese AcademyofSciences,Beijing,China;UniversityofChineseAcademyofScience,Beijing, China LIQIONGLIU • DepartmentofMicrobiologyandImmunology,IndianaUniversitySchoolof Medicine,Indianapolis,IN,USA;TriArmTherapeutics,Shanghai,PRChina DIEGOA.LO´PEZ • DepartmentofPathology,UniversityofUtahSchoolofMedicine,SaltLake City,UT,USA PENGLV • StateKeyLaboratoryofMembraneBiology,InstituteofZoology,ChineseAcademy ofSciences,Beijing,China;InstituteforStemCellandRegeneration,ChineseAcademyof Sciences,Beijing,China;UniversityofChineseAcademyofScience,Beijing,China SAFAF.MOHAMAD • DepartmentofHematologyandOncology,BostonChildren’sHospital/ HarvardSchoolofMedicine,Boston,MA,USA CHRISTIEM.ORSCHELL • DepartmentofMedicine/HematologyOncology,Indiana UniversitySchoolofMedicine,Indianapolis,IN,USA ANDREA M.PATTERSON • DepartmentofMedicine/HematologyOncology,Indiana UniversitySchoolofMedicine,Indianapolis,IN,USA LOUISM.PELUS • DepartmentofMicrobiology&ImmunologyandDepartmentof Medicine/HematologyOncology,IndianaUniversitySchoolofMedicine,Indianapolis,IN, USA P.ARTURPLETT • DepartmentofMedicine/HematologyOncology,IndianaUniversitySchool ofMedicine,Indianapolis,IN,USA STEFANRADTKE • StemCellandGeneTherapyProgram,FredHutchinsonCancerResearch Center,Seattle,WA,USA JANINARATAJCZAK • StemCellInstitute,JamesGrahamBrownCancerCenter,Universityof Louisville,Louisville,KY,USA MARIUSZZ.RATAJCZAK • StemCellInstitute,JamesGrahamBrownCancerCenter, UniversityofLouisville,Louisville,KY,USA;DepartmentofRegenerativeMedicine, WarsawMedicalUniversity,Warsaw,Poland