1

Theevolvingparadigmofhematopoieticstemcellsandtheirderivedlineages1

2

ElisaLaurenti1,#,BertholdGöttgens1,#3

1:DepartmentofHaematologyandWellcomeTrustandMRCCambridgeStemCell4

Institute,UniversityofCambridge,CambridgeUK5

#:correspondingauthors:EL:el422@cam.ac.uk;BG:bg200@cam.ac.uk6

7

Preface8

Thedevelopmentofmaturebloodcellsfromhaematopoieticstemcells(HSCs)haslongservedasa9

paradigmforstemcellresearch,withthehaematopoieticdifferentiationtreeusedwidelyasamodel10

for maintenance of hierarchically organised tissues. Recent results and new technologies have11

challenged the demarcations between stem and progenitor populations, the timing of cell fate12

choices and the contribution of stem and multipotent progenitor cells to steady-state blood13

maintenance. These evolving views of haematopoiesis have broad implications for our14

understanding of adult stem cell function, as well as the development of new therapies for15

malignantandnon-malignanthaematopoieticdiseases.16

17

Introduction18

WhenErnstHaeckelfirstusedthewordstemcell(Stammzelle)in1868,asaDarwinistheuseditto19

refertotheprimordialunicellularorganismfromwhichallmulticellular lifedescended1.Thisstem20

cell thereforesatat the rootofabranching family tree, incidentally calleda stemtree inGerman21

(Stammbaum,e.g.a treethatshowswherethingsstemfrom).Haeckel’sbiogenetic law(ontogeny22

recapitulatesphylogeny)shortlythereafterpromptedhimtousethestemcelltermalsotodescribe23

the fertilised egg. Histopathologists subsequently applied this stem cell concept to normal and24

leukaemichaematopoiesis,putting forwardtheconceptofacommonprogenitorofredandwhite25

blood cells 2 aswell as a commonprecursorofmyeloidand lymphoid leukaemic cells 3. From the26

very beginning, the stem cell concept has thus been framed into a tree-like model, where27

multipotentstemcellsgiverisetotheirprogenythroughanorderedseriesofbranchingsteps.28

The first invivoassay for stemcell functionwasbasedon the rescueof lethal irradiation through29

bonemarrow transplantation 4, followedby the firstestimationof stemcellnumbersbycounting30

haematopoieticcoloniesinthespleensoftransplantedmice(spleencolonyformingunitassay,CFU-31

S).ThisnotonlyprovidedanestimateofCFU-S frequencyat1 in10,000bonemarrowcells 5,but32

also delivered the first definitive proof for in vivo multipotent progenitor cell function based on33

2

tracking cytogenetic abnormalities within individual CFU-S colonies 6. Fluorescence activated cell34

sorting (FACS) subsequently facilitated the purification of transplantable hematopoietic stem cells35

(HSCs), with a landmark 1988 publication7 demonstrating the utility of positive and negative36

selection.HSCshavehistoricallybeendefinedonthebasisoftwoessentialproperties:self-renewal37

and multipotency. Operationally this is tested via transplantation experiments. In contrast,38

progenitors are defined by the absence of extended self-renewal and a restricted lineage39

differentiationcapacity(mostoftenbi-orunilineage),sothattheyareusuallylostwithinthefirst2-340

weeksaftertransplantation8.41

CharacterisationofprogenitorpopulationsdownstreamoftheHSCresultedaroundtheyear2000in42

amodelofthehaematopoieticdifferentiationtreestillshowninmanytextbookstoday(Fig.1A).In43

thismodel, the first branch point segregates lymphoid potential from all other lineages (myeloid,44

erythroidandmegakaryocytic), followedbyanumberof furtherbranching stepsoneither sideof45

the tree progressing from multi- to bi- and finally unipotent progenitor cells. The subsequent46

introduction of additional surface markers suggested several modifications of this classical tree,47

including lymphoid andmyeloid fates remaining associated until further down the tree 8–10, early48

megakaryocytebranching11,12aswellassubdivisionofthemultipotentprogenitorcompartmentinto49

distinctsubpopulations13,14(Fig.1B).Moreover,thepictureisfurthercomplicatedbecausetheHSC50

poolitselfisfunctionallyandmolecularlyheterogeneous11,12,15–20.Thesestudiesaremostadvanced51

inthemurinesystem,wherewenowhavewhatatfirstglanceappearstobeabewilderingnumber52

ofdifferentstructuresforthehaematopoietictree.Whileitislikelythatallthesestructurescapture53

trueaspectsofHSCdifferentiation,collectivelytheywouldbedifficulttosqueezeintoasingle,rigid54

branchingtree.Newwaysofnotonlythinkingabout,butalsographicallyrepresentingtheprocess55

of HSC differentiation are thus required. Below we will illustrate how new technologies are56

challenging the classical view of the hematopoietic hierarchy as a highly compartmentalised and57

stablestructure.Theemergingpictureisoneofacollectionofheterogeneouspopulationsorganised58

hierarchically,withgradualprogression fromonetothenext,andwhichremainshighly flexible to59

meetthechangingneedsofblooddemand.60

61

Stemcellsandprogenitorsboundaries62

Withself-renewalandmultipotencyattheheartofwhatdefinesanHSC,muchresearchhasbeen63

investedintounderstandingtheunderlyingcellularandmolecularprocesses.64

65

DefiningtheHSCstate66

3

Atthecellularlevel,switchingoffself-renewalcoincideswithturningonlineageprograms.Itthus67

seemedplausiblethatthiswouldalsobetrueatthemolecularlevel,andtheconceptofmultilineage68

primingwasproposedearlyonasapossibleunderlyingmolecularmechanismbywhichHSCs69

maintainmultipotentiality21(Box1).Howevertheadventofgenomictechnologies14,22–25coupled70

withmousegeneticsstudies,hasdemonstratedthattheHSCtranscriptionalprogrammeisdefined71

byacollectionofuniquemetabolicandcellularproperties,whicharenotintuitivelylinkeddirectly72

withmultipotency.Approximately70%ofallexpressionchangesbetweenHSCsandearly73

progenitorsoccurindependentlyoflineagechoice24,withasimilardichotomyalsoatthelevelsof74

methylation26–29andchromatinaccessibility30.Correspondingly,HSCsresideinaquiescent19,20,31,32,75

autophagy-dependent33,34andglycolytic35,36statemarkedbylowmitochondrialactivity37,38and76

tightlycontrolledlevelsofproteinsynthesis,belowthoseofmostotherhaematopoieticcelltypes39.77

Stem-cellspecificstressresponseandqualitycontrolmechanismsallowpreservationoftheintegrity78

oftheHSCcompartmentafterexposuretoDNA40,41andproteindamage42,43ormetabolicstress33,34.79

Incontrast,progenitorsarehighlyproliferativeandmetabolicallyactivecellsdependentonoxidative80

metabolismandmitochondrialfunction.81

Importantly,so-calledHSCspecificcharacteristicsarenotabsolute:HSCsoccasionallydivideduring82

homeostasis and get activated in response to stress, and therefore transiently pass through a83

proliferativestate.It isalsoworthnotingthatearlylymphoidprogenitors(mouseLMPP9orhuman84

MLP8)sharecommontranscription(TF)networkswithHSCs24,whichintheformerpushtowardsB85

cell differentiation24 while in the latter inhibit self-renewal44,45. While the induction of lineage-86

specific transcriptional programs may occur largely independently of the loss of stem cell87

characteristics,regulatorssuchasRunx1canbeinvolvedinboth46suggestingthatmuchremainsto88

belearnedabouthowlineagedecisionsarecoordinatedwithchangesofcellularstate.89

90

HSCself-renewalandcellcycleinterplay91

Whether the variability in HSC outputs is an intrinsic system property, a reflection of stochastic92

behaviour,environmentalinfluences,technicallyrelatedvariabilityoracombinationofallofthese,93

hasbeenandstillisasubjectofdebate.Nonetheless,inthepast10-15years,thisheterogeneityin94

behaviourhasbeensignificantlyformalised,anditisnowacceptedthatHSCsareheterogeneousin95

terms of durability of engraftment upon transplantation, cell cycle properties and differentiation96

(Table1).97

98

HSCsfirstofalldifferinthedegreetowhichtheyself-renew,equatingtothenumberofsymmetric99

divisions that anHSC canmakeover its lifetime.Operationally, the fieldnowwidely accepts that,100

bothinmouseandhuman,HSCthatrepopulateintransplantationassaysformorethan16weeksin101

a primary transplantation and at least in a second round of transplantation are considered Long-102

4

Term (LT-) HSCs 17,47–50. If cells can produce all differentiated cell types and engraft transiently in103

primary(andinsomecasessecondary)transplants,theyarereferredtoasIntermediate(IT-)HSCs16,104

Short-Term (ST-) HSCs or MultiPotent Progenitors (MPPs) 13,14,47 depending on the length and105

robustnessofthegraftproduced.106

107

Heterogeneity inself-renewalcapacityappearstobedirectlycorrelatedtothetimeHSCsspend in108

quiescence. Label retaining studieshavedemonstrated that themostdormantHSCs (which retain109

the label overmonths at steady-state) display themost robust and longest repopulation capacity11019,20,51–53. Two cell cycle parameters are inversely correlated with repopulation capacity: the111

frequencyofdivision(lengthof intervalbetweendivisions),butalsothetimeasingleHSCtakesto112

exitquiescenceinvitro16,54.Ofnote,thelevelofpre-existentCDK6mRNAandproteininquiescent113

HSCsdirectlydetermines thekineticsofquiescenceexit54 ,andmaythusserveasamarkerof the114

quiescentstate32,54(lessCDK6correspondingtohigherdormancy).Dormancyisalsoassociatedwith115

highlevelsofvitaminAmetabolismviaretinoicacidsignalling32,highlevelsofp5732,55,lowprotein116

synthesis32,lowMycactivity32,56,andmayexistasacontinuumofquiescentstatesbetweenthemost117

dormant HSCs and their activated counterparts 32. While dormant HSC forced into activation by118

stresssignalscanreturntodormancy19,51,Bernitzetal.estimatedthat4divisionsinadulthoodare119

sufficientforirreversiblelossofself-renewal53.120

121

Anumberof label-retainingassayshavebeendevelopedtoallow isolationofHSCsbasedon their122

divisionhistory19,20,32,51–53,57.Onemajorlimitationofalllabel-retainingstudiestodateisthattherate123

of symmetric divisions is inferred or indirectly measured at the bulk level. However, direct124

experimentalmeasurementofasymmetricversussymmetriclabel-dilutingdivisionswillberequired125

to understand the dynamics at play within the HSC compartment. Novel integrative tools, most126

likelyatthesinglecell level,willhavetobedevelopedtoaddressthischallenge.Nonetheless, it is127

alreadyapparentthatdistinctcellcyclepropertieswithintheHSCpoolareintimatelylinkedtoHSC128

function. Coupledwith a wide range in division frequencies (depending on the HSC subset, from129

onceamonthtotwiceayearinmouse19,51,53,58),thismeansthattherewillbesubstantialvariationin130

thecontributionofdistinctHSCsubsetstobloodformation.131

132

HeterogeneityinHSClineageoutput133

The capacity to give rise to all differentiated blood cell types is a fundamental aspect of what134

constitutes an HSC. It is however now accepted that there are distinct differentiation behaviours135

within theHSC andMPP13,14 compartments (Table 1, Fig. 1B). Using limiting dilution analysis and136

singlecell transplantation, theMüller-SieburgandEavesgroupsdescribedHSCs thatdiffer in their137

relative myeloid and lymphoid output 15,59. More recently the Jacobsen and Nakauchi groups138

5

identified HSCs that predominantly differentiate towards megakaryocytes and platelets (platelet-139

biased)11,12.Therearehoweverlimitationstosinglecelltransplantation,inparticularthatverylow140

contributionstocertainlineagesmaybemissed.Moreover,acellwithunilineagereadoutmayhave141

thepotentialtogiverisetootherlineagesinadurablefashioninotherconditions,andbothplatelet-142

biasedHSCandlong-livedplateletprogenitors11,60,61maycoexistandbedifficulttodistinguish.143

144

Importantly, HSC heterogeneity is not simply stochastic as it can be propagated through serial145

transplantation 15,18,62,63 , indicating intrinsic programming, themolecular basis forwhich however146

remains unclear. As discussed below, these findings have important conceptual implications,147

because theyquestionatwhatcellular stage lineagechoicesoccur.Recentevidencesuggests that148

theoverallpicturemayevenbemorecomplex,withdistinctmetabolicneeds64andclonalexpansion149

capacity of HSCs. There are examples where HSCs with high clonal expansion capacity generate150

subsetswithloweroutput65,butalsocasesinwhichHSCswithverymodestclonalexpansioninthe151

firsttransplantation,generatethemostrobustgraftsuponserialtransplantation66,67.152

153Rethinkingbloodlineagesrelationships154

A number of recent studies at the single cell level have questioned the routes by which lineage155

differentiationoccurs.156

157Singlecellassaystostudypotential158

Cell fate decisions (Box1) are executed at the level of individual single cells. To understand their159

regulation,itisthereforeimperativethatboththebiologicalassaystestingtheircellularfunctionas160

well as the biochemical assays examining their molecular profiles are performed at single cell161

resolution.Whilemostadvancedinthemouse,invivotransplantationassayshavebeenandremain162

fundamental for our understanding of HSC biology, as the only assay that can test for HSC self-163

renewal. Because of the suboptimal support of human cells from the mouse microenvironment,164

xenotransplantscannotrobustlyread-outallpossibledifferentiationroutes,particularlynotatsingle165

celllevel.Thelast10yearshavethereforeseenacollectiveeffortindefiningthelineagepotentialof166

single human progenitor cells by using highly defined in vitro models that can support the167

differentiationofmostmaturebloodcelltypes.WorkfromtheDickandVyasgroups,togetherwith168

studiesinmicefromtheJacobsengroupdescribedthatthefirstrestrictioninlineagepotentialdoes169

not segregate lymphoidandmyeloidpotential aspostulatedby theCMP-CLPmodel (Fig.1A), but170

thatthesepotentialsremaincoupledintheLMPP9,10andMLP8compartments(Fig.1B).Manyother171

subpopulations, most with either bi- or unilineage capacity, have since been found both in the172

lympho-myeloidbranch68andthemyelo-erythroid-megakaryocyticbranch69–73.Altogetheritseems173

that few single cells read-out as multipotential within the progenitor compartment. There are174

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howeversomecaveatswithsuchinterpretations,becausestronginstructivesignalsprovidedbythe175

in vitro cultures, or potentially high stress levels that HSC are exposed to during single cell176

transplantsmaypromoteunilineageoutput.Moreover,agivencellmaybebipotentialbasedonits177

molecular state, but if it makes a lineage choice before dividing, it will read out as unipotent in178

functionalassays.Nonethelesstheevidencetodatesuggeststhatlineagechoiceoccursearlierthan179

previouslythought,andasrecentlyshownfordendriticcells74,likelyalreadywithinthephenotypic180

HSCpopulations.181

182

Single-celltranscriptionallandscapes183

Single cell expression analysis of RNA was first reported over 25 years ago75, but remained low184

throughputbothinnumberofgenesandcellsuntilmicrofluidicapproacheswereintroduced.These185

quicklypromptedstudiesreportingtheexpressionofdozensofgenesinhundredsofsingleHSCsand186

progenitor cells (HSPCs)76 , and provided new insights into core regulatory circuits 77, novel187

progenitor populations 70,71 , cellular hierarchies in normal and transformed haematopoiesis 78,188

dissociationbetweenself-renewalpotentialandactivationoflineageprograms79,andthemolecular189

overlapbetweenHSCpopulationspurifiedwithfourdifferentcellsortingstrategies80.However,itis190

notpracticaltoassaymorethan200genespersinglecellwithPCRbasedmethods,andthesegenes191

need to be predefined thus limiting the scope for novel discoveries. A real breakthrough was192

thereforeprovidedbytechnicalinnovations,whichnowmakeitpossibletoperformtranscriptome-193

wideRNA-Seqinthousandsofsinglecells.194

Followingalandmarkpaperreportingthetranscriptomesformorethan2600murinesinglemyelo-195

erythroid progenitor cells 81, a subsequent report of 1600 transcriptomes ranging from true long-196

term HSCs to progenitors of all major lineages focused on generic stem cell functions such as197

metabolicandcellcyclestatus82.Anumberofalgorithmshavebeendeveloped83–87basedonthe198

idea that single cell transcriptomes represent snapshots of single cells as they traverse199

differentiation landscapes (Fig. 1C). A recent study on human bone marrow haematopoiesis200

comprehensivelysampledtheHSPCcompartment88.Computationalpredictionssuggestiveofearly201

lineagerestrictionwereunderpinnedbyinvitrosinglecellcultureassays,whichleadtheauthorsto202

propose a model whereby acquisition of lineage-specific fates is a continuous process, and203

unilineage-restricted cells emerge directly from a continuum of low-primed undifferentiated204

haematopoieticstemandprogenitorcells,withoutanymajortransitionthroughmulti-andbipotent205

stages. This is supported by other studies 69,81,85 , which highlighted the abundance of unipotent206

progenitorswithincompartmentsthat,atthepopulationlevel,aremultipotent.207

There are however caveats with this new model and its heavy reliance on scRNA-Seq. A major208

conundrumhere is thatsurfacemarkercombinationscanreadilysplit theHSPCcompartment into209

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functionallydistinctsubpopulations,includingwithinthespaceproposedtobeacontinuumoflow-210

primed cellswhen analysed by scRNA-Seq.One possible explanation is that there is a decoupling211

betweensteadystatemRNAandproteinexpressionlevels.Thecounterargumenthereisthatmulti-212

omics analysis of highly purified bulk HSPC populations has shown good concordance between213

mRNA and protein for themajority of genes14. It is possible also that functional heterogeneity of214

HSCsisprimarilydeterminedattheepigeneticlevel.Futuremeasurementsofchromatinaccessibility215

and histonemarks ideally at single cell resolutionmay reveal suchmechanisms. A third possible216

explanation is that even though purifying cells based on protein markers followed by functional217

assayssuggestscleansplitsintodistinctcelltypeswithdifferentbiologicalfunctions,thechangesin218

biological functionality are in reality much more gradual, where there is no binary biological219

differencebetweenforexampleLT-HSCsandMPPs,but insteadany individualcellsitssomewhere220

alongacontinuousspectrum.AfourthpossibleexplanationisthatcurrentscRNA-Seqmethodsare221

not effective at distinguishing between closely related cell types, because many of the shared222

biologicalprocesses(e.g.cellcycle,metabolism,motility)generatesubstantialheterogeneity,which223

may exceed the number of differentially expressed genes between closely related stages of224

haematopoietic maturation. The degree to which early haematopoiesis is characterised by a225

continuumversusdistinctpopulationsthereforeremainsaquestionrequiringfurtherinvestigation.226

227

Newrepresentationsofhaematopoiesis228

An immediate challenge for the field is how all the recent findings can be reconciled into new229

graphicalmodels describing the hierarchical organisationof haematopoiesis. It seems clear that a230

treewhereacircledepictseachsuccessiverestrictioninpotentialandeachcircleisconnectedtoa231

few others by arrows is an over-simplification. First, the circle is intuitively viewed as a232

homogeneoussetofcellswithspecificcharacteristics,avisionincompatiblewiththelargedegreeof233

heterogeneity observed experimentally. Second, these trees indicate a restricted set of possible234

transitionsbetweencircles,whichlikelyunderestimatesthepossibledifferentiationjourneysinvivo.235

Becausecellsurfacemarkerscanhighlyenrichforparticularbehaviours(differentiation,self-renewal236

orproliferativeoutput),amodelinwhichalllineagesbranchoutdirectlyfromtheHSCcompartment237

also seems somewhatunrealistic (Fig. 1C).We thusproposehereanalternative visualisation (Fig.238

2A), in which trajectories of differentiation are mapped over the areas that have long been239

representedascircles,highlightingboththediversityinpossibleroutesandtheprevalenceofearly240

lineage choice. In addition, it is important to remember that one HSC will produce a very large241

numberofprogeny,which increasesexponentiallywitheachdivision,anelementsofar ignoredin242

graphical representations of hematopoiesis (including Fig.2A). Divisional histories are difficult to243

measure, and are likely to be heterogeneous, but should nevertheless be incorporated in future244

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experimental and computational analyses,which could result in new graphical representations of245

thebloodsystem(Fig.2B).246

247

248

Makingbloodatsteadystateandunderstress249

In addition to defining the routes of lineage differentiation, another important question is to250

understand the quantitative contributions of HSCs and progenitors to daily and emergency251

haematopoiesis.252

253

Studyingunperturbedhaematopoiesis254

Whilethehaematopoieticdifferentiationtreeiswidelyusedasaparadigmforhowahierarchically-255

organizedtissueismaintained, it is importanttorememberthatthistreewaslargelyderivedfrom256

experiments thatmeasure cell potential in colony or transplantation assays, rather than cell fate257

during steady state differentiation.However, if a single cell gives rise to two lineages in a colony258

assay, this does not prove that the same cell, when left alone in an unperturbed bone marrow259

environment,wouldhavedonethesame.Tounderstandthedynamicsofbloodformation,cellscan260

be individually tagged (by retro- or lentiviral insertions, barcodes etc), and transplanted into261

recipientmice tomeasure the contribution of each clone over time. Progressivelymore sensitive262

methodshavebeenused inmouse,primatesandhumans 89–93, collectively supporting the lineage263

biasesand/orrestrictionsobservedinsinglecelltransplants. Importantly,onlyalimitednumberof264

HSCproducethevastmajorityofthedifferentiatedcellsinatransplantationsetting,consistentwith265

studiesofthedivisionalhistoryofHSC19,20,51–53,57,whichdemonstratethatonlytheveryraremost266

dormantHSCcanprovidelife-longreconstitutionaftertransplant.267

Substantial excitementwas raised by new technologies to assess the lineage output of individual268

stemandprogenitorcells inunperturbedhaematopoiesis94.Doxycycline inducedmobilizationofa269

sleepingbeautytransposon instem/progenitorcellswasemployedtogenerateunique integration270

events,whichserveasbarcodesthentrackedbysequencing.Consequently,comparingbarcodesin271

mature lineages following a pulse-chase label allows reconstruction of single cell behaviours in272

native, unperturbed haematopoiesis. In contrast to transplantation approaches, analysis of273

unperturbed haematopoiesis suggested that (i) MPPs contribute predominantly to the myeloid274

lineage during steady state, and (ii) cells functioning as HSCs in transplantation do not play a275

significantroleinsteadystatehaematopoiesis,whichinsteadseemstobedrivenalmostentirelyby276

cellswithintheMPPcompartment.277

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An alternative genetic fate mapping system based on Cre-loxP induced recombination of a278

transgenic barcode cassette recently achieved temporally-controlled barcode induction in single279

cells,anddemonstratedthatwhenHSCsarelabelledatthefetalliverstage,theirdescendantsinthe280

adultwillmostlycontributetomultiplelineages95.However,megakaryocyticfatewasnotanalysed,281

andwhentheanalysiswasrepeatedinadultbonemarrow,fewbarcodesweredetectedinHSCsas282

wellasmatureprogeny.GiventhateachfetalliverHSCdividesandthereforegiverisestomultiple283

HSCs in the adult, conclusions about the lineage contribution of individual adult HSCs therefore284

remained preliminary. Another study from the Camargo group96 addressed this issue more285

comprehensivelybycarryingouta30-weekpulse-chaseexperimentinadultmicewiththesleeping286

beauty barcode system. 133 barcodes were detected in HSCs and at least one of four mature287

lineages (megakaryocyte,erythroid,granulocyte,B-cell). Interestingly,morethanhalfof these133288

HSCbarcodeswerepresentonlyinmegakaryocytes,andonlyaminorityoftheremainingbarcodes289

were present in more than one mature lineage. Coupled with analysis at shorter pulse-chase290

intervalsandcomprehensivesinglecellRNA-Seqanalysis,thisstudythereforeconcludedthatduring291

homeostatic unperturbedhaematopoiesis, (i)megakaryocytes can arise independently fromother292

lineages, and (ii) the phenotypic LT-HSC population as defined by transplantation assays actively293

contributestomegakaryocyteoutput.294

In theHSCcompartment,a transposontagmayoftenbepresent in justoneor twocells,because295

HSCcloneswill rarelyamplifyduringunperturbedhaematopoiesis, thusmakingbarcodedetection296

lessreliable.ItisthereforenoteworthythatboththeRodewaldandReizisgroups97,98foundthatthe297

HSCcompartmentcontributedmoretomultilineagebloodproductionthanwhatwasestimatedby298

thetransposonapproach. Buschetal.alsoestimatedthekineticswithwhichcells transit through299

theirdifferentiationtrajectories.Interestingly,fluxintothelymphoidbranchis180foldlessthanin300

themyeloidbranch.Fluxintotheerythroidlineagewasnotassessed,butislikelytobeevenhigher301

than in themyeloid (Fig. 2b). Flux analysis also found substantial self-renewal capacity in the ST-302

HSC/MPP compartment, consistent with a recent report of long-term normal haematopoiesis in303

micewheretheHSPCcompartmentwasablatedby90%99.304

Futureapproachesare likelytoemploybarcodesthatareexpressedunderastrongpromoter,and305

thereforecanbedetectedreliablybyscRNA-Seq.Thiswouldaffordtruesinglecellresolutionforthe306

analysis of clonal relationships and single cell transcriptomes, offering the exciting possibility of307

defining the native hierarchy agnostic of sorting strategies that were developed using308

transplantation. Another pertinent question is towhat extent laboratorymice kept under sterile,309

pathogen-free conditions are a suitable model for human haematopoiesis, which is constantly310

challengedbyexposuretoinfectiousagents,andhastofunctionoveramuchlongerlifespan.Long-311

term follow-up of autologous transplant patients has already revealed previously unknown312

functional aspects of human haematopoiesis, such as the number, stability and dynamics of313

10

individualHSCsovermanyyears100.Backgroundsomaticmutationsrepresentuniquebarcodesthat314

canbeexploited to reconstruct clonal lineage relationships101,102 andmay thus representanother315

approachtoinvestigatethedynamicsofsinglehumanHSPCsoverextendedperiodsoftime.316

317

Hematopoiesisisflexibleinspaceandtime318

Bloodproductionneedstohavetheflexibilitytoadapttodrasticchangesofdemand,withevidence319

accumulatingthatHSCpropertiesanddifferentiationjourneyscanadapt.Asreviewedelsewhere103,320

haematopoieticdevelopment in theembryo is complex,witha seriesof transienthaematopoietic321

wavesacrossseveralorgans(Fig.3). Importantly,fetalandadultHSChavefundamentallydifferent322

regulation and behaviours (reviewed in 104). In mice, there is a switch from a proliferative to a323

quiescentstatebetween3and4weeksofage,whichcoincideswithdecreaseinself-renewal105.In324

humans, the timing of the fetal to adult switch somewhat differs.When compared to adult bone325

marrow,humanHSCsfromneonatalcordbloodhaveincreasedproliferativepotential(asexpected326

from mouse studies), but their cell cycle properties already resemble the adult configuration 54.327

Consistently, telomere length in granulocytes, a surrogate for HSC division rate, rapidly declines328

duringthefirstyearofhumanlife106.Thereisevidencealsothatterminallydifferentiatedcellsare329

produced differently during fetal and adult hematopoiesis, becausemanymore single progenitor330

cells from human fetal liver produce 2 lineages or more than from adult bone marrow 69.331

Interestingly, the relative proportion of HSC subsets also change over time with balanced HSC332

predominating in fetal liver,while lymphoid-deficient (also calledmyeloid-biased)HSCaccumulate333

duringageing107.TheeffectsofageingonHSCandmoregenerallybloodproductionarenumerous334

andhavebeenreviewedelsewhere108.ChangesinboththecompositionoftheHSCpool107,109,110as335

wellas themolecularcircuitryof individualHSCs34,111–113canbeduetoeitherextrinsicor intrinsic336

properties,includingalterationsinthemicroenvironment,theproliferativehistoryandaccumulation337

ofmutations.338

The HSC niche is a highly complex ecosystem that sustains HSC function, in particular promoting339

survival and long-termmaintenance of the HSC pool 114. If and how niche interactions shape the340

activityofdistinctHSCsubsetsandthedifferentiationjourneysofHSCsremainsunclear.Singlecell341

transplants and clonal trackinghave shown that clonesdisplay stereotypical behaviourover serial342

rounds of transplantation, arguing that their characteristic outputs are not extensively niche-343

dependent 15,59,62,63. However, it is possible that distinct HSC subsets may have different niche344

preferences.Furthermore,eventhoughthevastmajorityofHSCarelocatedinthebonemarrowin345

adults, a small percentage of HSC are released in the blood with circadian-clock controlled346

patterns115,andHSCscanbefoundalsointhespleen116andlungs117(Fig.3).Theroleoftheseextra-347

medullarynichesandwhethertheyhostspecificsubsetsofHSCorinfluencetheirdifferentiationor348

11

clonalexpansioncapacitywillhave tobeexploredat singlecell resolution. Finally,many typesof349

stress directly affect HSC function: DNA damage 40,41, inflammation 118, acute or chronic350

infection119,120, psychosocial stress 121, metabolic stress33 and obesity 122. For most of these351

processes, insightshavebeengainedon themolecularmechanismsdriving the changes inHSCor352

progenitorcell function. Interestinglythough,therearealsoexamplesshowingthat,atthecellular353

level, not all HSC or progenitor subsets equally respond to these stresses. For example, in354

emergency myelopoiesis, MPP2 and MPP3 drive enhanced production of GMPs 13,123, which355

reorganisethemselvesspatiallyandactivateaself-renewalnetwork123.Wearethusonlybeginning356

tounderstandhowstressresponsescanreshapetherelativeabundanceandpossiblydifferentiation357

trajectories of different HSPC subsets, and less still is known about the cellular and molecular358

control/feed-backmechanismsthatmaintainand/orre-establishhomeostasis.359

360

ImplicationsforHumanDisease361

Ourunderstandingofhaematopoiesis iscurrentlyundergoinganumberofshifts.Theseinclude:(i)362

demarcationsbetween stemandprogenitor cells that previouslywere considered rather rigid are363

becoming increasingly blurred, (ii) cell fate choices upstream of the classically defined bi-364

/oligopotent progenitor cellsmay bemore prevalent than previously thought, (iii) the loss of key365

stem cell characteristics may be largely decoupled from the initiation of specific lineage366

differentiation programmes, and (iv) measuring cellular fates in vivo without the need for highly367

disruptivetransplantationprocedureshashighlightedapreviouslyunderappreciatedimportanceof368

ST-HSC/MPPs in unperturbed haematopoiesis. These and other revisions of our understanding of369

haematopoiesis as a stem cell developmental system have major implications for the diagnosis,370

prognosisandtreatmentofhaematologicaldiseases.371

372

Haematologyasaclinicaldisciplinehasa long track recordofbeinganearlyadopterof the latest373

technologicaldevelopments,whichrecentlyincludedthefirstsuccessfulgenetherapytrials124,125as374

wellassomeofthefirstcomprehensivecancergenomestudies126.Withrespecttohaematopoietic375

malignancies,muchcurrentresearchfocusesonidentifyingthecelloforigin,whichacquiredthefirst376

somatic mutation within the multistep progression towards a full-blown malignancy. It is widely377

accepted now that stem and progenitor cells play a major role in the development of myeloid378

malignancies (CML,AML)andmyeloproliferativeneoplasms.More recentevidencealso implicates379

HSCsandlymphoidprogenitorsintheearlystagesofhairycell leukemia127andlymphoma128.It is380

beyond the scope of this review to list the effects of each of the driver mutations on HSC and381

progenitor cell function, but it isworthnoting that eachoneof themwill reshape thebalanceof382

12

differentiation trajectories and generate complex clonal dynamic patterns. Since cellular context383

influencesthepotentialimpactofleukaemogenicmutations,thenewlyrecognizedfluidityofcellular384

states within the HSPC compartment implies greater disease heterogeneity between patients,385

becauseevenwhentwopatientscarryidenticalfoundermutations,thelikelihoodthattheyarosein386

identical cellular states is small.Moreover, themalignant transformation is likely toopenupnew387

molecular states/trajectories. In AML for example, leukemic stem cells are defined by a chimeric388

transcriptionalstate10,129,andsinglecellproteomicapproacheshavedemonstratedtheexistenceof389

distinctdifferentiationtrajectoriesformalignantcells130.390

391

Given the increasing recognition of discrepancies between the clonal behaviour of native392

unperturbed HSPCs versus transplantation, xenotransplantation of human leukaemic cells into393

immune-compromised mice will share similar limitations, where exposing leukaemic cells to a394

transplantation assaymay induce cellular behaviour that would never occur in a human patient.395

Exciting prospects may be offered here by newmodels that may permit transplantation without396

irradiation 131 and the use of ossicles templatedwith human bonemarrow stromal cells 132,133. It397

nevertheless seems imperative to invigorate research efforts that directly track disease in human398

patients. This will require careful design of patient cohorts and new screening technologies,399

includingmulti-omics single cell technologies to define the cellular state as well as themutation400

burdenofindividualcells,asdemonstratedrecentlyforBCR-ABL134.401

402

Bonemarrowtransplantationislikelytoremainanimportantcurativetherapyformanyleukaemia403

patients, andwith progress in the cell and gene therapy field,may findmuchwider applicability.404

Sincesuitabledonormaterialforbonemarrowtransplantationremainsratelimiting,onepromising405

avenuehereistoproduceHSCsfromothercelltypesbyborrowingregulatoryprocessesknownto406

beimportantduringdevelopmentalhaematopoiesis,asillustratedbytherecentgenerationofstem407

cellswithlong-termengraftingcapabilityfromhumanpluripotentandmouseadultendothelialcells408135,136. A better understanding of the HSC state and cell fate decision making will provide new409

avenuestodevelopprotocolsforinvitroamplificationofHSCs137–139,andalsofacilitateoptimization410

of protocols for robust genome engineering of HSCs with vast potential for treating common411

diseases ranging from inherited red blood cell to autoimmune disorders. Expansion of clinical412

applicationswillgreatlybenefitfromabetterunderstandingoftheself-renewalanddifferentiation413

potential of individual HSPCs, coupledwith robust prediction algorithms frommolecular profiling414

datatoevaluatetheefficacyofcelltherapyproducts.Haematopoiesisisthereforewellpositionedto415

lead theway in the gene and cell therapy arena, andwemaynot be too far away froma future416

13

where haematopoiesiswill be firmly establishednot just as a stem cell, but also as a therapeutic417

paradigm.418

419

420

ACKNOWLEDGMENTS421

WethankDrDavidKentforcriticalreadingofthemanuscript.E.L.issupportedbyaSirHenryDale422

fellowshipfromtheWellcomeTrust(WT)/RoyalSociety.ResearchintheLaurentiandGottgens423

laboratoriesissupportedbytheWT,CRUK,Bloodwise,MRC,BBSRC,NIH-NIDDK,andcoresupport424

grantsbytheWTandMRCtotheWT-MRCCambridgeStemCellInstitute.425

426

AUTHORS’CONTRIBUTIONS427

E.L. andB.G. contributedequally to thewriting andeditingof themanuscript aswell as to figure428

preparation.429

430

14

TABLES431

Table1:summaryofphenotypicorfunctionallydefinedHSCsubsetsreportedtodate.432

ListedaresubtypesofHSCsdefinedbasedoncombinationsofcellsurfacemarkersand/orfunction.433AglobalinterpretationoftheliteratureonHSCbiologyiscomplicatedbythefactthateachstudy434usuallyusesonlyoneoftheclassificationschemesbelow,sotheextentofoverlapbetweenHSC435subsetsremainstobeclarified.N.A.:notassessed.436

Species Name Cellsurfacephenotype

Self-renewal Cellcycleproperties

Differentiation Refs

Mouse LT-HSC Lin-Sca1+cKit+CD34-CD150+

CD135-CD48-±EPCR+±Rholo

High 17,47,48,140

IT-HSC Lin-Sca1+cKit+

CD34loCD135-

RholoCD49bhi

Intermediate ShortG0exit

16

ST-HSC/MPP1

Lin-Sca1+cKit+CD135-CD150-CD48-

Low

a N.A. High N.A.Ly-deficient

15,107

b N.A. High N.A.Balanced

g N.A. Intermediate N.A.My-deficient

d N.A. Low N.A.My-deficient

MPP2 Lin-Sca1+cKit+oCD135-CD150+CD48+

Low

similar

Ly-deficient13,14

MPP3Lin-Sca1+cKit+oCD135-CD150-CD48+

LowBalanced

MPP4Lin-Sca1+cKit+oCD135+CD150-CD48+

LowLy-biased

d-HSC N.A. Higher dormant 19,20,51–53 a-HSC N.A. Lower activated Human LT-HSC Lin-CD34+

CD38-CD45RA-

CD49f+CD90+±Rholo

High LongG0exit

50,54

ST-HSC/MPP

Lin-CD34+CD38-CD45RA-

CD49f+CD90+

Low ShortG0exit

CD34-LT-HSC

Lin–CD34–

CD38–CD93hiHigh Highly

quiescent 66

15

437

FIGURES438

Fig.1:Timelineofhierarchicalmodelsofhematopoiesis.439

A. Visualisation based on state-of-the-art around the year 2000: HSCs are represented as a440

homogeneouspopulationdownstreamofwhichthefirst lineagebifurcationseparatesthemyeloid441

and lymphoid branches via the CMP and CLP populations;B. During the years 2005 to 2015 this442

visualisation incorporates new findings: theHSCpool is nowaccepted to bemoreheterogeneous443

both in terms of self-renewal (vertical axis) and differentiation properties (horizontal axis), the444

myeloid and lymphoid branches remain associated further down in the hierarchy via the LMPP445

population,theGMPcompartmentisshowntobefairlyheterogeneous141.C.From2016onwards,446

singlecelltranscriptomicssnapshotsindicateacontinuumofdifferentiation.Eachreddotrepresents447

asinglecellanditslocalisationalongadifferentiationtrajectory. 448

449

Fig.2:Trajectorybasedvisualizationsofthehematopoietichierarchy.450

A. 2D visualisation of early hematopoiesis. Continuous lines: trajectories of differentiation for451

differenttypesofsinglecellspresentinthephenotypicHSCcompartment(greyshadedarea).Along452

thesetrajectories,cellsandtheirprogenypassthroughprogenitorcompartmentscommonlydefined453

byspecificcombinationsofcellsurfacemarkers(shadedareas).Horizontallinesrepresentsnapshots454

ofthelineagepotentialofthecellspresent ineachphenotypiccompartment(singlecolourcircles:455

unilineage cells, 2 colours circles: bi-lineage cells, 3 colours circles: tri-lineage cells, black circles:456

multipotent cells). In most progenitor compartments the number of unilineage cells outnumbers457

that of bi- or tri-lineage ones. The figure illustrates differentiation trajectories reported in the458

literaturetodate,buttheirproportionsmaynotreflectthe invivosituation.B.3Dvisualisationof459

theprogenyofone singleHSC.Red,blueandgreen respectively represent theerythroid,myeloid460

andlymphoidlineage.Cellhistory,divisionandprogenitorexpansionshouldallbeconsideredwhen461

modellingthedifferentiationjourneyofoneHSCandallitsprogeny.Inanadulthuman,therearean462

estimated3000-10000HSCs,whichmostlikelydivideonlyfromonceevery3monthstoonceevery3463

years58.Humansproduceanestimated1.4x1014maturebloodcells/year142.Theamplificationfroma464

fewthousandHSCsthereforeisstaggeringandmustincludeastrongcontributionfromatransient-465

amplifying compartment. Also, because there are many more terminally differentiated erythroid466

cellsthanmyeloidcells,andevenlesslymphoidcells,allwithdifferentturn-overrates,thefluxinto467

eachcompartmentmustbehighlyregulated.468

469

16

Fig.3:ThecompositionoftheHSPCcompartmentchangesinspaceandtime.470

HSPCsare found inmanyorgans in thebodyacrossa lifetime.Cellsofdifferentcolours represent471

distinctHSPCsubsets.TodateitisunclearwhetherallHSPCsubsetsanddifferentiationtrajectories472

are present in the same proportions in each of the organs. Current evidence suggests that age-473

relatedchangesresultfromacombinationofshiftsinthecompositionoftheHSPCpool,aswellas474

phenotypicchangesinparticularcelltypesdrivenbyintrinsicgeneticandepigeneticchangesaswell475

assystemicalterationsofthemicroenvironment.AGM:AortaGonadMesonephros.476

477

BOXES478

BOX1:Mechanisticunderpinningsofcellfatechoice479

Cell fate decisions entail a choice between alternative gene expression programs, executed by480

transcriptional and epigenetic regulators. Transcription factors (TFs) bind specific sequencemotifs481

within promoter, enhancer and silencer regions. Cell type specific expression is achieved through482

combinatorial TF interactions, which form key building blocks of wider regulatory networks. TF483

complexesrecruitepigeneticregulatorstomodulatetheactivationstatusofagenelocus,whichcan484

be transmitted to subsequent cell generations as so-called “epigenetic memory”. Importantly, a485

progenitorcell canbecomeprimedbyopeningup regulatoryelementsassociatedwithgenes that486

drivedifferentiationdownaspecificmaturelineage143.487

Multipotentcellsarebelievedtoexhibitmultilineagepriming,whichentailssimultaneous,low-level488

activation of expression programs for alternate lineages. Lineage choice then constitutes one489

program “winning out”while the alternative program is extinguished. Cross-antagonism between490

pairsoflineage-determiningTFsrepresentsanattractivemechanisticmodel,whichinitiallyfocussed491

ontheerythroidregulatorGata1andthemyeloidregulatorPu.1.Morerecentsinglecelltime-lapse492

imaginghoweverquestionedwhetheranerythroid/myeloidfatechoiceis indeeddrivenprimarily493

by Gata1/Pu.1 cross-antagonism144 . Evidence for lineage priming and its resolution by TF cross-494

antagonism has been reported at single cell resolution for other TF pairs, such as the495

neutrophil/macrophagefatechoicecontrolledbyGfi1/Irf887.496

497

Bothinstructiveandstochasticmodelshavebeenproposedasmechanismstriggeringthe498

upregulationofonelineageprogramoveranother.Stochasticheregenerallyreferstorandom,499

unequaldistributionofmoleculesfollowingcelldivision.Instructivemodelspositthatlow-level500

expressionofacytokinereceptorissufficientforacelltoberesponsivetoexternalsignals,asshown501

17

foranumberofmyeloidcytokines145.Autoregulationandfeedforwardloopsalsoplayimportant502

rolesinfatechoicedecisions.Forexample,Gata1positiveautoregulationstabilizeserythroidfate146503

,Pu.1positiveautoregulationstabilizesmyeloididentity147,andthehighlyconnectedtriadofGata2,504

Tal1/SclandFli1isthoughttostabilizethestem/progenitorstate148,149.Infeedforwardloops,an505

upstreamregulatorinducesitstargetdirectlyaswellasthroughanintermediateregulator150.Feed-506

forwardmotifscanfilterouttransientsignals,andwhencoupledwithautoregulation,generate507

forwardmomentum.508

509

510

18

511

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882

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Figure 1

Megak Ery NeutroMonoEosino

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Figure 2

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Figure 3