cd34-positive cells: biology and clinical … cd34-positive cell: deﬁnition and ... in ad d i ti...

The CD34-positive cell: definition andmorphology

Cellular expression of the CD34 antigen iden-tifies a morph o l ogi c a lly and immu n o l ogi c a llyheterogeneous cell population that is function-a lly ch a racteri zed by the in vi tro c a p a bi l i ty togen era te cl onal aggrega tes derived from earlyand late progenitors and the in vivo capacity toreconstitute the myelo-lymphopoietic system ina supralethally irradiated host.1-3

Im mu n o h i s toch emical studies have dem on-s tra ted that the CD34 anti gen is stage but notl i n e a ge specific. In fact, indepen den t ly of thed i f feren ti a tive lineage, it is ex pre s s ed on ly byon togen eti c a lly early cell s .4 For ye a rs a majorob s t acle to the morph o l ogical iden ti fic a ti on ofp ut a tive hem a topoi etic stem cell has been thed i f f i c u l ty in sep a ra ting them from their directprogeny. The use of CD34 and other su i t a bl ecell surface markers (i.e. CD33, CD38, HLA-DRa n ti gens) in flu ore s cen ce - activa ted cell - s orti n gtechniques or other cell separation methods hasallowed considerable progress in this field.

Positively selected, lineage committed CD34+

cells and more immatu re, lineage nega tiveC D 3 4+ C D 3 3– H LA- D R- cells are shown inF i g u re 1 and Figure 2, re s pectively. On May -Grünwald-Giemsa stained preparations, CD34+

cells are medium sized cells having large nuclei,eccen tri c a lly su rro u n ded by narrow rims ofdeep blue cytoplasm occ a s i on a lly con t a i n i n gcytoplasmic granules. Some normal CD34+ cellnu cl ei show one or more pale blue nu cl eo l i .

Ta ken toget h er, these findings ref l ect the het-erogeneous proliferative status and protein syn-thesis of this cell population. Conversely, earlierhematopoietic progenitors, identified as CD34+

C D 3 3– H LA- D R–, seem to be more hom oge-neous in size (small lymphocyte-like cells) andl ack cytoplasmic gra nules and prom i n en tnucleoli. Again, the morphology of this cellularpop u l a ti on appe a rs to ref l ect the functi on a lch a racteri s tics of these cells (e.g. low pro tei nsynthesis, very low pro l i fera tive activi ty wi t hpredominantly G0 phase).

Several mon ocl onal anti bodies (MY10, 12.8,B1-3C5, 115.2, ICH3, TUK3, etc.) ra i s edagainst the leukemic cell lines KG1 or KG1a andan anti - en do t h elial cell anti body (QBEND10)assigned to the CD34 cluster have been shownto identify a transmembrane glycoproteic anti-gen of 105-120 kD ex pre s s ed on 1-3% of nor-mal bone marrow cells, 0.01-0.1% peri ph era lbl ood cells and 0.1-0.4% cord bl ood cell s .5

Different antibodies recognize distinct epitopesof the same antigen. CD34 antigen expression isassociated with concomitant expression of sev-eral other markers that can be classified as lin-eage non-specific markers (Thy1, CD38, HLA-DR, CD45RA, CD71) and lineage spec i f i cm a rkers, including T- lym ph oid (T d T, CD10,CD7, CD5, CD2), B-lym ph oid (T d T, CD10,CD19), myeloid (CD33, CD13) and megakary-oc ytic (CD61, CD41, CD42b) markers .5 Th eex pre s s i on of lineage non - s pecific markersallows the heterogeneous CD34+ population to

Correspondence: Prof. Sante Tura, Istituto di Ematologia L. e A. Seràgnoli, Policlinico S. Orsola, via Massarenti 9, 40138 Bologna, Italy.Acknowledgments: the preparation of this manuscript was supported by grants from Dompé Biotec SpA, Milano, and Amgen S.p.A., Milano, Italy.Received October 18, 1994; accepted May 2, 1995.

CD34-POSITIVE CELLS: BIOLOGY AND CLINICAL RELEVANCECarmelo Carlo Stella, Mario Cazzola,* Paolo De Fabritiis,° Armando De Vincentiis,# AlessandroMassimo Gianni,@ Francesco Lanza,^ Francesco Lauria,** Roberto M. Lemoli,°° Corrado Tarella,##

Paola Zanon,@@ Sante Tura°°

Cattedra di Ematologia, Università di Parma, Parma; *Dipartimento di Medicina Interna e Terapia Medica, Sezione diMedicina Interna e Oncologia Medica, Università di Pavia e IRCCS Policlinico S. Matteo, Pavia; °Dipartimento di BiopatologiaUmana, Sezione di Ematologia, Università “La Sapienza”, Roma; #Dompé Biotec SpA, Milano; @Centro Trapianto MidolloOsseo “Cristina Gandini”, Divisione di Oncologia Medica C, Istituto Nazionale Tumori, Milano; ^Istituto di Ematologia eFisiopatologia dell’Emostasi, Università degli Studi di Ferrara, Ferrara; **Istituto di Scienze Mediche, Università degli Studi diMilano, Milano; °°Istituto di Ematologia “Lorenzo e Ariosto Seragnoli”, Università degli Studi di Bologna, Bologna; ##Cattedradi Ematologia, Università di Torino, Torino; @@Amgen S.p.A., Milano, Italy

reviewHaematologica 1995; 80:367-387

368

be divi ded into two disti n ct su bpop u l a ti on sch a racteri zed, re s pectively, by low or highexpression of Thy1, CD38, HLA-DR, CD45RA,CD71. These two cell su bpop u l a ti ons con t a i ne a rly and late hem a topoi etic progen i tor cell s ,respectively.6-8

In ad d i ti on to conven ti onal immu n o l ogi c a lm a rkers cl a s s i fied on the basis of their assign a-ti on to specific clu s ters of differen ti a ti on ,CD34 cells ex press receptors for a nu m ber ofgrowth factors. Two disti n ct families of rel a tedreceptors have been iden ti f i ed: (i) tyro s i n ekinase receptors, including the stem cell factorreceptor (SCF-R, CD117) and the mac roph a geco l ony - s ti mu l a ting factor receptor (M-CSF- R ,CD115); (ii) hem a topoi etic receptors not con-taining a tyrosine kinase domain, su ch as thegra nu l oc yte - m ac roph a ge co l ony - s ti mu l a ti n gf actor receptor (GM-CSF-R, CDw116).5 , 9

The iden ti fic a ti on of new markers sel ectivelyex pre s s ed on pri m i tive lym ph o h em a topoi eti ccells (CD34+ C D 3 8–) repre s ents a sti mu l a ti n gre s e a rch field. In this con text, stem cell tyro s i n ekinase receptors (STK), su ch as STK-1, a hu m a nh om o l ogue of the mu rine Flk-2/Flt-3, are ofp a rticular rel eva n ce .1 0 - 1 2 The ligands for thesereceptors might repre s ent new factors able tos el ectively con trol stem cell sel f - ren ewal, pro l i f-era ti on and differen ti a ti on .1 3 - 1 5

Clonogenic and biologic activityThe structural and functional integrity of the

h em a topoi etic sys tem is maintained by a rel a-

tively small pop u l a ti on of stem cells, loc a tedmainly in the bone marrow, that can (i) under-go sel f - ren ewal to produ ce more stem cells or(ii) differentiate to produce progeny which areprogre s s ively unable to sel f - ren ew, irrevers i blycom m i t ted to one or another of the va ri o u sh em a topoi etic lineages, and able to gen era tecl ones of up to 105 l i n e a ge - re s tri cted cells thatmature into specialized cells.16-18

The dec i s i on of a stem cell to ei t h er sel f -ren ew or differen ti a te and the sel ecti on of aspecific differentiation lineage by a multipotentprogen i tor du ring com m i tm ent are intri n s i cproperties of stem cell progenitor cells and arereg u l a ted by stoch a s tic mech a n i s m s .1 9 Su rviva land amplification of hematopoietic progenitorsare controlled by a number of regulatory mole-cules (hem a topoi etic growth factors) interact-ing according to com p l ex mod a l i ties (syner-gism, rec ru i tm ent, antagon i s m ) .1 9 A furt h erl evel of hem a topoi etic con trol is exerted bynu clear tra n s c ri pti on factors that activa te lin-e a ge - s pecific genes reg u l a ting growth factorresponsiveness and/or the proliferative capacityof hematopoietic cells.20

Detection of the most primitive hematopoiet-ic cell types is now po s s i ble due to the tech-nique of long-term bone marrow culture. In thecase of human bone marrow, a 5- to 8-wee ktime peri od bet ween initi a ting cultu res andassessing clonogenic progenitor numbers allowsqu a n ti f i c a ti on of a very pri m i tive cell in thestarting population, the so-called long-term cul-ture-initiating cell (LTC-IC).21 Committed prog-

C. Carlo Stella et al.

Figure 1. May-Grünwald-Giemsa (MGG) staining of cytospin preparation ofenriched CD34+ cells, highly purified by avidin-biotin immunoaffinity.

Figure 2. MGG staining of cytospin preparation of enriched CD34+ CD33–

HLA-DR– cells. The CD34+ cell fraction was further depleted of CD33+ HLA-DR+ cells by immunomagnetic separation.

369CD34-positive cells

enitors of the various hematopoietic cell classescan be qu a n ti t a ted by a nu m ber of short - termc u l tu re cl on ogenic assays .1 9 CD34 anti genex pre s s i on assoc i a ted with low CD38 andC D 4 5 RA ex pre s s i on and va ri a ble HLA- D Rexpression is a typical feature of LTC-IC, CFU-Blast, CFU-T, CFU-B. In con trast, CD34 anti-gen expression associated with CD38 and HLA-DR ex pre s s i on is a typical fe a tu re of mu l ti po-tent (CFU-GEMM) and lineage - re s tri cted(CFU-GM, CFU-G, CFU-M, BFU-E, CFU-E,B F U - Meg, CFU-Meg) hem a topoi etic progen i-tor cells5 (Figure 3). Recently reported data haves h own that low or absent ex pre s s i on of theThy1 or SCF receptor can be efficiently used toen ri ch pri m i tive hem a topoi etic progen i torsf rom the heterogen eous CD34+ cell pop u l a-ti on .7 , 9 Al t h o u gh the CD34 anti gen is vi rtu a llyexpressed by all progenitor cells, the percentageof CD34+ cells with assaya ble in vi tro cl on o-genic activity ranges from 10 to 30%. The prob-lem of non-clonogenic CD34+ cells is still openand not adequately explained by the presence of

lym ph oid progen i tors wh i ch are not assaya bl ewith current in vitro systems. Non-proliferatingC D 3 4+ cells might repre s ent a su bpop u l a ti onthat is not re s pon s ive to conven ti onal myel oi dh em a topoi etic growth factors. The non - pro l i f-era ting CD34+ su b s et might requ i re the pre s-ence of co-factors, such as the ligand of STK-1or the hepatocyte growth factor, able to activates tem cell - s pecific genes whose ex pre s s i on is aprerequ i s i te for acqu i ring re s pon s iveness toconventional growth factors.14,22,23

In let h a lly irrad i a ted non - human pri m a te s ,both auto l ogous and all ogen eic CD34+ cells havebeen shown to have the capac i ty to recon s ti tutethe myel o - lym ph opoi etic sys tem, thus su gge s t-ing that the stem cell re s pon s i ble for hem a topoi-etic recon s ti tuti on is CD34+.2 4 , 2 5 It has also beens h own that human CD34+ H LA- D R– cells tra n s-p l a n ted in utero in the fetus of sheep initi a teand sustain a ch i m eric hem a topoiesis produ c i n ghuman progen i tor cells of all differen ti a tive lin-e a ge s .2 6 Auto l ogous CD34+ cells en ri ch ed byavi d i n - bi o tin co lumns have been shown to bea ble to recon s ti tute myel o - lym ph opoiesis inp a ti ents receiving high - dose ch em orad i o-t h era py.2 7 The re sults of studies using CD34+

bone marrow cells in the all ogen eic set ting inp a ti ents receiving both rel a ted as well as unrel a t-ed all ogen eic marrow transplants wi ll soon beava i l a bl e .2 7 In ad d i ti on, trials are planned thatwi ll use all ogen eic peri ph eral bl ood CD34+ cell sei t h er alone or with marrow.2 7

Characterization and function of the CD34cell surface molecule

The CD34 cell su rf ace molecule has been bi o-ch em i c a lly ch a racteri zed and both the hu m a nc D NA and gene have been cl on ed and sequ en c-ed in the last few ye a rs .2 8 , 2 9

CD34 is a one-pass type I tra n s m em brane gly-copro tein with a molecular wei ght of 105-120kDs in ei t h er the redu ced or unredu ced form2 8

( F i g u re 4). CD34 pro tein is not hom o l ogous toa ny other known pro tein. The minimum size ofthe CD34 pro tein is 354-amino acids and con-tains nine sites for N-glyco s yl a ti on and a severa lfor O-glyco s yl a ti on that are essen tial con s ti-tu ents of the three ep i topes of the molecule; thisFigure 3. Cellular organization of the hematopoietic system.

370 C. Carlo Stella et al.

m o l ecule is also ri ch in sialic acid. Its bi och em i-cal com po s i ti on su ggests a mu c i n - l i ke stru ctu reand in some re s pects re s em bles leu co s i a l i n(CD43). Sequ en ce com p a ri s ons bet ween hu m a nand mouse CD34 show a very low level of iden-ti ty in the glyco s yl a ted regi on, 70% iden ti ty inthe gl obular domain, and 92% in the tra n s-m em brane and cytoplasmic regi ons.

Using a KG1 cell line libra ry, it has been

shown that the human CD34 gene is located onchromosome 1, and recent studies with in situhybri d i z a ti on have assign ed its loc a l i z a ti on toband 1q32, in close prox i m i ty to other gen e sthat en code growth factors or functi on mole-cules su ch as CD1, CD45, TGF2, laminin,LAM/GMP, etc.28

Seven CD34 mon ocl onal anti bodies (Mo Ab s )were clu s tered at the 4th Work s h op on Leu ko-c yte Di f feren ti a ti on An ti gens (Vi enna, 1988),3 0 , 3 1

and another 15 Mo Abs were veri fied as recog-nizing the CD34 molecule du ring the 5thIn tern a ti onal Work s h op on Leu koc yte Di f feren-ti a ti on An ti gens (Bo s ton, 1983), the most directevi den ce being re activi ty with cells tra n s fectedwith CD34 cDNA and binding to CD34pro tei n .3 2 , 3 3 The ep i tope spec i fic i ty of the CD34a n ti bodies was cl a s s i f i ed into three disti n ctgroups according to the sen s i tivi ty of the ep i-topes to en z ym a tic cl e ava ge (wh i ch was per-form ed using neu raminidase, chym opapain andglycopro tease from Pa s teu rella haem olyti c a) ,re activi ty with fibroblasts and high en do t h el i a lvenules, and cross bl ocking ex peri m ents (Ta bl e1 ) .3 4 , 3 5 We know, in fact, that glycopro tease fromPa s teu rella haem olyti c a s pec i f i c a lly cl e aves on lypro teins containing sialyl a ted O-linked gly-c a n s .3 4 Ba s ed on these data, it can be furt h erpo s tu l a ted that class III ep i topes are more prox i-mal to the ex tracellular side of the cell mem-brane than class I and class III ep i topes.

Fu rt h erm ore, for most CD34 Mo Abs (wi t hfew excepti ons) cross bl ocking ex peri m ents are

Fig. 4

GPI anchor

Lipid bilayer

N-glycosylation site

O-glycosylation site

Figure 4. Schematic representation of the CD34 cell surface molecule.

Epitope class Clones CD34 reactivity

paraffin frozen westernsection section blotting

I. Sensitive to neuraminidase (from Vibriocholerae), chymopapain, glycoprotease (fromPasteurella haemolytica)

14G3, BI3C5, My10,* 12.8,*ICH3,* Immu-133, Immu-409

positive negative positive

II. Resistant to neuraminidase. Glycoprotease,and chymopapain sensitive

43A1, MD34.3,MD34.1, MD34.2, QBend10,4A1, 9044, 9049

positive positive positive

III. Resistant to neuraminidase, chymopapainand glycoprotease

CD34 9F2,HPCA2,581, 553.563, Tuk 3, 115.2

negative positive negativeTable 1. Epitope specificity of CD34MoAbs as assessed by their differentialsensitivity.

*Incomplete digestion by neuroaminidase.

in agreem ent with the cl a s s i f i c a ti on based onen z ym a tic cl e ava ge of the CD34 pro tein. Ino t h er words, using a cocktail of CD34 Mo Ab s ,CD34 re activi ty is bl ocked on ly in the case thatMo Abs bel on ging to the same CD34 ep i tope ares i mu l t a n eo u s ly em p l oyed. On the con tra ry, thecom bi n ed use of Mo Abs recognizing class II andIII or class I and II or class III ep i topes does nota f fect cell re activi ty. Moreover, CD34 Mo Ab sdefining class III ep i topes are unable to re actwith CD34 glycopro tein in We s tern bl o t sbecause this ep i tope is sen s i tive to den a tu ra ti on.

The pattern of expression of CD34 antibodiesexhibited by CD34+ acute leukemias is partiallyin accord a n ce with that derived from ep i topemapping based on the differential sensitivity ofCD34 to enzymatic treatment. However, aboutone third of CD34 Mo Abs do not seem tobel ong to any of these su b groups and for thispeculiar pattern of expression are referred to asatypical CD34 reagents. The widest variation inCD34 Mo Ab re activi ty has been dem on s tra tedin ac ute myel oid leu kemia (AML) samples,allowing us to postulate the occurrence of aber-rant anti gens or of disti n ct ep i topes in su b-groups of leu kemias. Al tern a tively, it can behypo t h e s i zed that the ex pre s s i on of differen ta n ti bodies could ref l ect the degree of matu ra-tion of leukemic cells. The presence of new, dis-ti n ct non overl a pping ep i topes could be pro-posed on the basis of the data published so farin the litera tu re. As far as the ex pre s s i on ofCD34 in normal and leu kemic cells is con-cerned, it has been calculated by flow cytometrythat the nu m ber of molecular equ iva l ents ofs o lu ble flu oroch rome (MESF) ex pre s s ed byl eu kemic and normal progen i tors ra n ges from18,200 to 322,000 and from 8,000 to 124,000,respectively.

Recent data co ll ected by Lanza et al.3 6 s eem toi n d i c a te that class I-type Mo Abs are more sen s i-tive to freezing procedu res than class II and IIIMo Abs, since the ep i tope is not iden ti fia ble fo l-l owing a frozen / t h awed met h odo l ogy.

The functi on of CD34 su rf ace glycopro tein inh em a topoi etic stem and progen i tor cells is sti llthe obj ect of deb a te. In light of recent fin d i n gs ,it would seem to play a rel evant role in modu-l a ting cell ad h e s i on .3 6 Fu rt h erm ore, it has been

dem on s tra ted that CD34 prob a bly acts as anad h e s ive ligand for L- s el ectin. It has been fur-t h er po s tu l a ted that the CD34 molecule co u l dp l ay a pro tective role against pro teo lyti cen z ym e - m ed i a ted damage due to its high nu m-ber of O-glyco s yl a ti on sites. The cytop l a s m i cdomain contains two sites for pro tein kinase Cph o s ph oryl a ti on and one for tyrosine ph o s ph o-ryl a ti on .2 8

Given the discordant reactivity of these mole-cules, the choice of the CD34 MoAb to employmay be important when analyzing cell positivi-ty for the CD34 molecule in both leukemic andnormal samples, and may be responsible for thed i f feren ces reported by va rious aut h ors in theliterature concerning the prognostic role playedby this anti gen in ac ute myel oid leu kem i a s .3 7

The type of CD34 Mo Ab used to enu m era teprogen i tor cells is prob a bly rel evant in theperi ph eral bl ood stem cell autograft set ting aswell, since both early and late engraftment fol-l owing tra n s p l a n t a ti on are, to some ex ten t ,related to the number of hemopoietic stem cellscollected at the time of blood or bone marrowh a rvests, and to the degree of progen i tor cellmaturation related to the expression of lineagemarkers such as HLA-DR, CD71, CD38, CD33,and myeloperoxidase.

Techniques for CD34+ cell separationA nu m ber of different tech n i ques have been

proposed for separating CD34+ cells. The com-m on aim is to produ ce a cell pop u l a ti on wi t hoptimal purity and viability by means of a lowcost, rapid and simple sep a ra ti on tech n i qu e .The first separation techniques exploited para-m eters su ch as size and cell den s i ty and wererepresented by Ficoll-Hypaque and Percoll den-s i ty grad i ents. In the last two dec ades, thedevel opm ent of mon ocl onal anti bodies hasa ll owed a more specific and careful cell sel ec-tion by identifying surface antigens used as tar-gets for cell separation (Table 2).

FACS (Fluorescence Activated Cell Sorter)F l ow cytom etry is able to phys i c a lly sep a ra te

d i f ferent pop u l a ti ons after incubati on of cell swith flu oroch rom e - con ju ga ted mon ocl on a l



antibodies. This cell sorting technique can yielda high ly puri f i ed (> 98%) cell pop u l a ti on. Inad d i ti on, the use of el ectronic ga tes all owss el ecti on and recovery of several su bpop u l a-tions according to antigenic expression and dif-ferent characteristics such as size and cytoplas-mic gra nu l a ri ty. This tech n i que has been veryuseful for studying CD34+ sub-populations butcannot be employed to select large numbers ofcells due to its com p l ex i ty and low recovery.The recent development of high-speed cell sort-ing, however, might allow clinical utilization ofthis technique.

PanningAn ti-CD34 mon ocl onal anti bodies bound to

one of the su rf aces of cell cultu re flasks wererecently used to select CD34+ cells. When a cellsuspension is introduced into the flask, the pos-i tive pop u l a ti on is bl ocked on the plastic su r-f ace, while CD34 nega tive cells remain in su s-pension and can be easily eliminated. Adherentcells should contain the CD34+ population witha viability >90%.38

Immunomagnetic systemsIm mu n om a gn etic be ads are uniform, su per-

p a ra m a gn etic, po lys tyrene be ads with affinitypurified anti-mouse Ig covalently bound to thesu rf ace. Th ey are equ a lly su i ted for nega tiveand positive cell separation; the rosetted targetcell can easily be isolated by applying a magneton the outer wall of the test tube for 1-2 min-utes. Im mu n om a gn etic be ads co u p l ed wi t hCD34 mon ocl onal anti bodies can be uti l i zedfor positive selection of CD34+ cells to obtain apop u l a ti on with > 80% vi a ble CD34+ cell s .3 9

Si m i l a rly, immu n om a gn etic be ads can beem p l oyed for nega tive dep l eti on with mon o-cl onal anti bodies binding lineage - s pec i fic anti-gens.

High-affinity chromatography based on biotin-avidin interaction

This met h od is based on an immu n oad s orp-ti on tech n i que that relies on the high affinityi n teracti on bet ween the pro tein avidin and thevitamin bi o tin. Avi d i n - bi o tin immu n oad s orp-ti on has been em p l oyed for both po s i tive sel ec-

ti on and dep l eti on of spec i fic cell pop u l a ti on s .4 0

The instru m ent inclu des a set of non - reu s a bl eprodu cts including bi o ti nyl a ted anti CD34 anti-body, plastic bags, filters and a co lumn ofavi d i n - bi o tin be ads. An autom a ted vers i on con-tro ll ed by a com p uter wh i ch guara n tees repro-du c i bi l i ty of opera ti on and redu ces risks ofopera tor errors has been devel oped for cl i n i c a luse. Its capac i ty has recen t ly been incre a s ed sothat a single co lumn can process more than5 031 01 0 bone marrow or peri ph eral bl ood cell sin 1-2 hours and sustain bone marrow en gra f t-m ent in pati ents su bm i t ted to autogra f t .4 1

CD34-positive subpopulations: phenotypicand functional analysis

The normal CD34+ cell population likely con-tains progen i tors of all human lym ph o -h em a topoi etic lineages, including stem cell sc a p a ble of hem a topoi etic recon s ti tuti on afterbone marrow tra n s p l a n t a ti on .1 Levels of CD34ex pre s s i on decline with differen ti a ti on; con s e-qu en t ly, the earliest cl on ogenic cells (CFU-blast, LTC-IC, etc.) express the highest levels ofCD34, while the most differen ti a ted (CFU-G,CFU-M, CFU-E, CFU-Meg) ex press on ly lowl evels of CD34 (Figure 5). The CD34 anti genhas been used to identify, enumerate and isolatecells from different lym ph o - h em a topoi etic lin-

Table 2. Recovery of CD34-positive cells after different separation tech-niques.

Enrichment Recovery Large-scaleseparation

(% CD34+ (% CD34+

in the recovered of the original population) sample)

Negative depletionby immunomagnetic 20-60 30-60 nobeads

Positive selection byimmunomagnetic 60-80 30-60 yesbeads

Fluorescence activatedcell sorter (FACS) > 95 30 - 50 time consuming

Panning 50 - 80 30 - 60 yes

Ceprate SC® 50 - 80 40 - 70 yes


eages, as well as develop in vitro tests for indi-rect evaluation of cells with different functionaland clonogenic capacity.42

Pre-clinical studiesSeveral animal-human systems have been cre-

a ted to uti l i ze ch i m eras for the stu dy of lym-ph o - h em a topoiesis in vivo. The first ex peri-m ents dem on s tra ted the fe a s i bi l i ty of tra n s-planting human fetal stem cells to sheep fetuses;the po s tnatal pre s en ce of human cells in thes h eep was doc u m en ted at several points inti m e .4 3 Fu rt h erm ore, some early CD34+ su b-populations were able to repopulate sheep bonem a rrow; animals were tra n s p l a n ted in uterowith CD34+/ D R– cells and the pre s en ce of ach i m eric pop u l a ti on with the functi onal ch a r-acteri s tics of hem opoi etic progen i tors wasdem on s tra ted in the marrow and peri ph era lblood in a percentage of cases.44 Berenson et al.45

also showed how hem a topoi etic progen i tors(positive for the Ia antigen and subsequently forthe CD34 antigen) could reconstitute the mar-row of let h a lly irrad i a ted dogs. Of the seven

animals tre a ted, all showed com p l ete marrowengraftment after reinfusion of Ia-positive cells;on ly one dog died from infecti on .4 5 Si m i l a rly,m a rrow cells from 5 pri m a tes (baboons) weretre a ted in vi tro with a bi o ti nyl a ted anti - C D 3 4antibody and then passed through a column ofavidin; after autograft, all the animals showedmarrow engraftment followed by hematologicalrecon s ti tuti on com p a ra ble to that of con tro lanimals.46 Furthermore, the demonstration thata ll ogen eic CD34+ cells can recon s ti tute the he-m a topoi etic sys tem in let h a l ly irrad i a tedb a boons con f i rm ed that this cell pop u l a ti onincludes pluripotent stem cells.25

Lymphoid precursor cellsThe CD34+ cell com p a rtm ent contains all the

cells ex pressing terminal deox y nu cl eo ti dyl tra n s-ferase (T d T), wh i ch is an intra nu clear en z ym eex pre s s ed in early lym ph oid cells under goi n gi m mu n ogl obulin or T- cell receptor gen ere a rra n gem ent. Flow cytom etry has shown thatthe great majori ty of TdT+ cells in the marrowcoex press CD34, CD19 and CD10 (B-cell pre-

Figure 5. Functional differentiation and antigenic expression of hematopoietic cells.

c u rs ors), as well as T- cell differen ti a ti on anti-gens su ch as CD7, CD5 and CD2. A small pro-porti on of CD34+/ T d T+ cells coex press CD10,wh i ch might repre s ent a com m on lym ph oi dprogen i tor for both B and T lineage s .4 7 Recen t ly,Mi ll er et al. reported that CD34+ cells may alsogen era te NK cells in vi tro.4 8

Granulocyte-macrophage precursorsMa rrow eryt h roid progen i tors lack spec i f i c

m a rkers and therefore are difficult to iden ti f y.G lycoph orin A- d i rected mon ocl onal anti bod i e srecogn i ze all hem ogl obi n i zed cells, but this mol-ecule is ex pre s s ed in on ly a small proporti on ofC D 3 4+ cells and is absent in cl on ogenic cell s .4 9

Hi gh levels of CD45 are pre s ent on BFU-E,but this anti gen is lost by the CFU-E stage ;5 0

h owever, the CD45RO isoform is well repre-s en ted on earl i er eryt h roid progen i tors, wh i l ethe CD45RA isoform is pre s ent on com m i t tedmyel oid progen i tors. The ex pre s s i on of CD71(transferrin receptor) is currently considered tobe the specific antigen for the CD34+ erythroidpop u l a ti on. CD71 is pre s ent at high levels onerythroid progenitor cells and at very low levelsin all the other progenitor cells.51 Expression ofCD71 increases from stem cells to BFU-E, thendeclines during erythroid maturation. In addi-ti on, marrow CD34+ eryt h roid cells migh tex press IL-3, GM-CSF (CD116) and eryt h ro-poi etin receptors, based on the acti on of thesegrowth factors on CFU-erythroid cells.52

Myel oid prec u rs or cl on ogenic cells (CFU-GM, CFU-G, CFU-M, CFU-MK) coex pre s sCD34, HLA-DR, CD117 (c - k i t), CD45RA ,CD33 and CD13; CD15 is present at low levelson CFU-G, while CFU-M spec i f i c a lly ex pre s sCD115 (M-CSF receptor). CFU-MK are theonly CD34+ cells which express the platelet gly-copro teins iden ti f i ed by the CD61, CD42 andCD41 mon ocl onal anti bod i e s .5 Den d ri tic cell salso originate from bone marrow, but the con-ditions that direct their growth and differentia-ti on are sti ll poorly ch a racteri zed. GM-CSFs ti mu l a tes the growth of den d ri tic cells frommouse peri ph eral bl ood; however, it wasrecently reported that CD34+ cells may give riseto den d ri ti c / L a n gh erans cells after sti mu l a ti onwith GM-CSF and tumor necrosis factor-a.53

Multilineage progenitors and stem cellsCFU-GEMM contain prec u rs or cl on ogen i c

cells of both myel oid and eryt h roid lineage sand ex press CD34, HLA-DR, CD38, CD117and CD45RA. Th ey also ex press low amountsof CD33, but not CD13. In a hypothetical dif-ferentiation scheme involving pluripotent stemcells, the lympho-hemopoietic compartment isthe next cell type and can be identified in vitrowith CFU-Blast and LTC-IC.

The lack of ex pre s s i on of CD38 is the mostimportant characteristic of these early progeny,wh i ch repre s ent 1% of CD34 and less than0.01% of mononuclear cells. The lack of CD38a ll ows sep a ra ti on of com m i t ted progen i tors( C D 3 4+/ C D 3 8+) from earl i er com p a rtm en t s( C D 3 4+/ C D 3 8–) by a single marker com bi n a-tion.54

Fu rt h erm ore, the earliest CD34+ cells coex-press low levels of CD45RO6 and are nega tivefor staining with the fluorescent dye rhodamine123. The role of HLA-DR in defining earlier celltypes is sti ll con troversial; a series of evi den cei n d i c a tes that the stem cell com p a rtm ent hasthe CD34+/ C D 3 8–/ H LA- D R+ ph en o type .8 , 5 4

Recent works, however, have not found HLA-DR in the earliest cells.55 These data were con-firmed by the possibility that HLA-DR expres-sion may discriminate the Ph-positive leukemiccom p a rtm ent (HLA- D R+) from normal re s i d-ual cells (HLA- D R–) in ch ronic myel oi dleukemia.56

Adhesion molecules and cytokine receptorsA nu m ber of molecules within the integri n

family have been shown to mediate interactionsbet ween early CD34-po s i tive cells and bon em a rrow stromal cells. These inclu de VLA-4 / VCAM-1, VLA- 5 / f i bron ectin, LFA-1 orICAM, and several others. Each adhesion mole-cule appe a rs to med i a te a spec i fic cell interac-ti on. Hem a topoi etic growth factors may beactive in a solu ble form or in a mem bra n e -a n ch ored form; ad h e s i on molecules may becrucial for allowing anchored growth factors tobind the target cell. Table 3 lists the main adhe-s i on molecule and growth factor receptorsexpressed on CD34-positive cells.


CD34 expression on normal and neopl a s tic cell sIt has been known that CD34 mon ocl on a l

a n ti bodies bind spec i f i c a lly to vascular en do-t h el ium ever since a 110 kd pro tein ex tractedfrom freshly isolated umbilical vessel endotheli-um was iden ti f i ed with CD34 anti bodies inWestern blots and in Northern blot analysis.57,58

CD34 molecules have a striking ultrastructurall oc a l i z a ti on on en do t h elial cells: they are con-centrated primarily on the luminal side, in par-ticular on membrane processes, many of whichi n terd i gi t a te bet ween ad jacent en do t h el i a lcell s .5 7 , 5 8 Si n ce this regi on is an important sitefor leu koc yte ad h e s i on and tra n s en do t h el i a ltra f fic, in con trast to previous ex peri en ce s ,3 3 i thas been hypo t h e s i zed that CD34 may beantagonistic or inhibitory to the adhesive func-ti ons of vascular en do t h el ium. This was su p-ported by the dem on s tra ti on that CD34 gen eex pre s s i on at the mRNA level is rec i proc a llydown - reg u l a ted wh en ad h e s i on molec u l e sICAM-1 and ELAM-1 are up-regulated by IL-1du ring inflammatory skin lesions assoc i a tedwith leu koc yte infiltra ti on .3 3 , 5 9 Fu rt h erm ore ,ad d i ti onal studies con du cted on both para f f i nem bed ded and cryopre s erved secti ons dem on-s tra ted that fibroblasts also re act with anti -CD34 Mo Ab s .6 0 However, it should be notedthat while CD34 MoAbs reacted with all classes

of ep i topes on cryopre s erved secti ons, class IIIep i topes were not recogn i zed by specific anti -CD34 Mo Abs on paraffin em bed ded secti on s .Levels of CD34 expression, highest in immatureh em a topoi etic prec u rs or cells, dec rease pro-gressively with cell maturation.

Rega rding hem a to l ogic malignancies, CD34 isex pre s s ed in a large percen t a ge of ac ute leu ke-m i a s .6 1 The flu ore s cen ce inten s i ty of CD34ex pre s s i on is va ri a ble and high er in ac ute lym-ph obl a s tic (ALL) than in ac ute myel obl a s ti cl eu kemia (AML). In these latter pati ents, theCD34 anti gen is found on 40-60% of leu kem i cblasts and is most frequ en t ly assoc i a ted wi t hM0, M1 and M4 FAB cyto type, secon d a ryl eu kemias, karyo typic abn orm a l i ties invo lvi n gch rom o s ome 5 or 7, P170 ex pre s s i on and poorprogn o s i s .6 1 - 6 4 Thus, CD34 ex pre s s i on may becon s i dered the most pred i ctive nega tive factor inAML pati ents stri ct ly correl a ted with inten s i tyof ex pre s s i on .6 5 , 6 6 In ALL, CD34 is ex pre s s ed in70% of pati ents, parti c u l a rly in those with a B-l i n e a ge ph en o type. In these pati ents, unlikeAML cases, the clinical rel eva n ce of CD34ex pre s s i on is con troversial; however, accord i n gto the fin d i n gs of a Ped i a tric Onco l ogy Gro u p,6 7

its ex pre s s i on in B-lineage cases was assoc i a tedwith hyperd i p l oi dy, lower frequ ency of initi a lcen tral nervous sys tem (CNS) leu kemia and af avora ble prognosis. In T- cell ALL cases, on theo t h er hand, CD34 ex pre s s i on showed a po s i tivecorrel a ti on with initial CNS leu kemia and CD10n ega tivi ty, but not with any pre s en ting favor-a bl e - risk ch a racteri s ti c s .6 7 , 6 8

Lastly, CD34 and HLA-DR expression may bevery useful in discri m i n a ting bet ween the veryfew benign primitive hematopoietic progenitorsand their malignant co u n terp a rts in pati en t swith chronic myeloid leukemia (CML). In fact,it has been demonstrated that normal progeni-tor cells are CD34+ and HLA-DR–, while malig-nant progen i tor cells, wh i ch ex h i bit Ph andbc r / a bl re a rra n gem ent, ex press HLA-DR anti-gens.56

Positive and negative regulators of hematopoi-etic progenitor cells

The hematopoietic stem cell is defined by its


Table 3. Adhesion molecule and growth factor receptors expressed onCD34-positive cells.

Antigen name CD Ligand

GlyCAM-L/selectin none adhesion molecule

ICAM1,2,3 CD11a/CD18 adhesion molecule

H-CAM CD44 adhesion molecule

VLA-4 CD49d/CD29 adhesion molecule

VLA-5 CD49d/CD29 adhesion molecule

FGF-R none growth factor

M-CSF CD115 growth factor

GM-CSF-R CD116 growth factor

SCF (c-kit) CD117 growth factor

Interferon g-R CD119 growth factor

IL 7-R CD127 growth factor

ex ten s ive sel f - ren ewal capac i ty, mu l ti l i n e a ged i f feren ti a ti on po ten tial and capac i ty for ofl on g - term recon s ti tuti on of normal marrowf u n cti on in let h a lly irrad i a ted animals. 6 8

Tra n s p l a n t a ti on of retrovi ra lly marked mu ri n es tem cells has shown that on ly a few mu l ti l i n-eage progenitor cells induce the repopulation ofen gra f ted hem a topoi etic ti s su e ,6 9 su gge s ti n gthat hematopoiesis may be supported by a suc-cession of short-lived clones. Moreover, experi-mental evidence indicates that the processes ofs el f - ren ewal, differen ti a ti on and sel ecti on oflineage potentials are intrinsic properties of thes tem cells and occur according to a stoch a s ti c( ra n dom) model .7 0 In the ste ady state, most ofthe stem cells are qu i e s cent (G0 phase) andbegin active cycling ra n dom ly.7 1 Convers ely,su rvival and pro l i fera ti on of hem a topoi eti cprogen i tors are reg u l a ted by cytokines, wh i chalso act in preventing apoptosis.72 According tothis model, the induction of differentiation by acytokine may be considered as the consequenceof the pro l i fera ti on of a specific pop u l a ti ons ti mu l a ted by that factor. The broad termcytokines includes growth factors such as fibrob-last growth factor, col o ny sti mu l a ting facto rs

(CSFs) (e.g. gra nu l oc yte - C S F, gra nu l oc yte -m ac roph a ge-CSF), and interl eukins (ILs).Depending on their target cells and differen tproliferative potential, cytokines can be dividedi n to three categori e s :7 1 1) late - acti n g, lineage -specific factors; 2) intermediate-acting, lineage-nonspecific factors; 3) early-acting growth fac-tors inducing the recruitment of dormant earlyprogenitors in the cell cycle (Figure 6).71

The majori ty of late - acting factors su pport thepro l i fera ti on and matu ra ti on of lineage - com-m i t ted progen i tors and the functi onal proper-ties of term i n a lly differen ti a ted cells. Eryt h ro-poi etin (Epo) is the phys i o l ogic reg u l a tor of ery-t h ropoi e s i s7 3 and throm bopoi etin has the samerole in throm bopoi e s i s ,7 4 while M-CSF and IL- 5a re con s i dered specific for mac roph a ges andeo s i n ophils, re s pectively. G-CSF exerts its activi-ty on com m i t ted neutrophil prec u rs ors ,a l t h o u gh it has been shown to be a syner gi s ti cf actor for pri m i tive hem a topoi etic cell s .7 5

In term ed i a te - acti n g, lineage - n on s pecific fac-tors inclu de IL-3, IL-4 and GM-CSF. Th ei ractivi ty is mainly directed tow a rd progen i torcells in the intermediate stages of hematopoieticdevelopment. However, they interact with later-


Figure 6. Cytokines exert their activity at different levels of the hematopoietic differentiation pathway. Each progenitor cell is concurrently affected by multipleregulators.


acting growth factors for the produ cti on ofm ore matu re cell s ,7 6 as wel l as with thec y tokines capable of tr i ggering stem cellcycling. On their own, they act as survival fac-tors for stem cells and appear to stimulate earlyh em a topoi etic prec u rs ors on ly after their ex i tfrom G0.77

Several cytokines have recen t ly been iden ti-fied for their capacity to stimulate the prolifera-ti on of the earliest hem a topoi etic cells. Ma pp-ing studies of normal blast cell co l ony form a-ti on from single progen i tors have shown thatIL-6, G-CSF, IL-11, stem cell factor (SCF), IL-12and l eu kemia inhibi to ry facto r (LIF) recruit dor-mant stem cells into the cell cycle that are thena ble to re s pond to ad d i ti onal growth factors .7 7

Wh ereas the perm i s s ive factors retain limitedpro l i fera tive po ten tial by them s elves, theystrongly enhance the stimulatory activity of IL-3, GM-CSF, G-CSF and EPO on CD34+ a n dmore immature CD34+ lineage-progenitors.78 Inaddition to positive interaction with intermedi-a te-and late - acting growth factors, SCF syner-gi s ti c a lly or ad d i tively augm ents the co l ony -forming abi l i ty of other early - acting growt hf actors, su ch as IL-11, IL-12 and IL-6, onmyel oid and bi l i n e a ge (i.e. lym ph omyel oi d )pri m i tive cell s .7 9 Recen t ly, the ligands for theSTK-1 or FLT3 receptor, and the hep a toc ytegrowth factor were shown to be able to stimu-l a te very pri m i tive hem a topoi etic progen i torcells. However, their bi o l ogical activi ty is sti llunder investigation.

The main sources of both positive and nega-tive reg u l a tory pro teins are acce s s ory myel oi dcells and the stromal com pon ent of the bon emarrow. In general, microenvironment cells donot con s ti tutively produ ce cytokines, ra t h ertra n s c ri pti on and/or tra n s l a ti on processes arerapidly induced by cytokines such as IL-1, TNF.The extracellular matrix also participates in theregulation of hematopoiesis by binding growthf actors and pre s en ting them in a bi o l ogi c a llyactive form to bone marrow progenitor cells.80

Most data su ggest that stem cells ex press lowl evels of growth factor receptors and requ i remu l tiple pro l i fera tive sti muli to en ter into thecell cycle, while com m i t ted progen i tor cells canbe ef fectively sti mu l a ted by indivi dual cyto-

k i n e s .2 3 Com bi n a ti ons of two or more growt hf actors8 1 can sti mu l a te hem a topoi etic cells ei t h erby amplifying the progeny cell produ cti on ofs i n gle prec u rs ors (syner gy) or by inducing ad d i-ti onal cl on ogenic cells to pro l i fera te (rec ru i t-m ent). Examples of these two types of en h a n ce-m ent are given in Figures 7 and 8, wh ere CD34+

C D 3 3– D R– cells are simu l t a n eo u s ly sti mu l a tedby two or three growth factors. The molec u l a rbasis reg u l a ting the com p l ex interp l ay bet weenc ytokines is sti ll largely unknown. However, on epropo s ed mechanism for growth factor syner-gism is the indu cti on of CSF receptors on earlyh em a topoi etic progen i tor cell s .8 2 This appe a rs tobe a coord i n a te cascade tra n s activa ti on via up-m odu l a ti on of growth factor receptors that lead sto pro l i fera ti on and differen ti a ti on of hu m a nm a rrow cell s .8 3 Convers ely, the stru ctu ra lh om o l ogy bet ween some growth factors ,8 4 t h epre s en ce of shared receptor su bunits on the cellm em bra n e8 5 and com m on sign a l - tra n s du cti on

Figure 7. Human CD34+ CD33- DR- cells were stimulated by IL-9 (A) or IL-9and SCF (B) in the presence of EPO. The addition of SCF induced thegrowth of large multicentered BFU-E colonies containing > 10,000 cells.The different size of the colonies indicates amplification of stem cell prog-eny (synergy).

A

B

pro tei n s8 6 provi de a po ten tial ex p l a n a ti on fort h eir functi onal similari ties and the app a ren tredundancy of their activi ty.

The growth of hem a topoi etic progen i tor cell sis also reg u l a ted by solu ble nega tive reg u l a torssu ch as mac roph a ge inflammatory pro tei n - 1a( M I P- 1a), tu m or nec rosis factor-a (T N F-a) ,i n terferons (IFNs), pro s t a glandins and tra n s-forming growth factor-b (TG F-b). The TG F-bf a m i ly of pro teins inclu des at least five isoform s(TG F-b1-5) wh i ch are en coded by differen tgen e s8 7 and produ ced by stromal cells, platel et sand bone cells. Moreover, a su b s et of very pri m-i tive mu rine hem a topoi etic cells has been shownto sec rete active TG F-b1 by an autoc rine mech-a n i s m .8 8 TG F-b1 and 2 isoforms are bi m od a lreg u l a tors of mu rine and human hem a topoi eti cprogen i tor cells, and their activi ty is based uponthe differen ti a ti on state of the target cells andthe pre s en ce of growth factors .8 9 In the hu m a ns ys tem, for instance, CFU-GEMM derived fromp u ri f i ed CD34+ C D 3 3– cells are inhibi ted byTG F-b1, wh ereas more com m i t ted progen i torssu ch as CFU-G or CFU-GM are not affected. Inad d i ti on, high pro l i fera tive po ten ti a l - C F C( H P P-CFC) re s pon s ive to a com bi n a ti on ofCSFs are markedly inhibi ted by TG F-b1, wh i l em ore matu re CFU-GM are actu a lly en h a n cedwh en GM-CSF is used as co l ony forming fac-tor.7 8 TG F-b1 and 2-indu ced myel o su ppre s s i onis parti a lly co u n teracted by early - acting growt hf actors su ch as G-CSF, IL-6 and fibrobl a s tgrowth factor.9 0 On the other hand, TG F-b3 hasbeen shown to be a more po tent su ppre s s or of

human BM prec u rs ors and its activi ty onh em a topoiesis is on ly inhibi tory,8 9 a l t h o u gh thes y n er gi s tic growth factors IL-11 and IL-9 seemto be able to oppose the nega tive reg u l a ti on ofTG F-b3 on human CD34+ cell s .9 1 Several po ten-tial modes of acti on of the TG F-b f a m i ly havebeen su gge s ted, including down - m odu l a ti on ofc ytokine receptors ,9 2 i n teracti on with the under-ph o s ph oryl a ted form of the reti n obl a s tom agene produ ct in late G1 ph a s e ,9 3 and altera ti on ofgene ex pre s s i on .9 4 E a rly studies have shown thatTG F-b1 and 3 exert their activi ty on norm a land leu kemic cells by len g t h ening or arre s ti n gthe G1 phase of the cell cycl e ,9 5 and this ef fect isf u n cti onal to pro tect normal CD34 po s i tive cell sf rom the tox i c i ty of alkyl a ting agents in vi tro.9 6

More recent inve s ti ga ti ons have dem on s tra tedthat TG F-b reg u l a tes the re s pon s iveness of miceh em a topoi etic cells to SCF, wh i ch is known tobe the main syner gi s tic factor for both mu ri n eand human stem cells, thro u gh a dec rease in c -kit m R NA stabi l i ty that leads to dec re a s ed cell -su rf ace ex pre s s i on .9 7

Similarly to TGF-b, TNF-a has been reportedto have both inhibitory and stimulatory effectson hem a topoi etic progen i tor cells. TNF-apo ten ti a ted the IL-3 and GM-CSF- m ed i a tedgrowth of human CD34+ cells in short-term liq-uid cultu re assay. However, it inhibi ted thegrowth prom o ting activi ty of G-CSF.9 8 TwoTNF receptors with molecular wei ghts of 55and 75 kd, respectively, have recently been iden-ti f i ed .9 9 Wh ereas the p55 receptor med i a te sT N F-a ef fects on com m i t ted progen i tor cell s ,the p75 receptor is invo lved in signaling theinhibition of murine primitive cells.

Fu rt h erm ore, it was recen t ly shown thatT N F-a is capable of inhibi ting the mu l ti -growth factor (GM-CSF, IL-3, G-CSF, SCF, IL-1 ) - depen dent growth of human HPP- C F Cderived from CD34+ cells thro u gh interacti onwith both p55 and p75 receptors, while the p55receptor exclu s ively med i a tes the bi f u n cti on a lactivity of TNF-a on more mature BM precur-sors responsive to single cytokines.100

M I P- 1a is a pepti de of 69-amino acids with am o l ecular wei ght of 7.8 kd produ ced by activa t-ed mac roph a ges, T- cells and fibrobl a s t s .1 0 1 Itbel on gs to a large family of put a tive cyto k i n e s


Figure 8. The addition of SCF to IL-11, in the presence of EPO, results in amuch higher number of colony-forming cells derived from CD34+ CD33–

DR– progenitors (right), as compared to the IL-11 and EPO combination(left) (recruitment).

that inclu des MIP- 1b, MIP-2 and IL-8 (ch em o-kines). Bi o l ogic ch a racteri z a ti on has shown thatM I P- 1a en h a n ces the M-CSF- and GM-CSF-depen dent growth of CFU-GM, while it inhibi t sthe co l ony - forming abi l i ty of hem a topoi eti cprec u rs ors pre s ent in a cell pop u l a ti on en ri ch edfor CD34+ + D R+ cells sti mu l a ted with eryt h ro-poi etin, IL-3 and GM-CSF.1 0 2

Ta ken toget h er, these re sults indicate that acom p l ex interp l ay bet ween po s i tive and nega tivereg u l a tory pro teins determines the pro l i fera ti onor inhibi ti on of early hem a topoi etic progen i torcells (Figure 9). In gen eral, the activi ty ofi n h i bi tors of hem opoiesis appe a rs to berevers i ble, lineage - n on s pec i fic and directed at thee a rly stages of differen ti a ti on. In ad d i ti on, TG F-bhas shown some degree of differen tial activi tybet ween normal and neop l a s tic lym ph oid cell s .9 6

Thus, nega tive reg u l a tors may be cl i n i c a lly rel e-vant to the pro tecti on of the hem a topoi etic stemcell com p a rtm ent from the do s e - l i m i ting tox i c i tyof neop l a s tic disease thera py.9 6 , 1 0 3 , 1 0 4

Coll e ction of CD34+ cell sBone marrow and peri ph eral bl ood are the

on ly sources of immatu re hem opoi etic prec u r-s ors iden ti fied as CD34+ cells. A diagn o s tic mar-row sample contains on ly a few CD34+ cell s ,while even fewer of them are pre s ent in peri ph er-

al bl ood samples taken under ste ady state con d i-ti ons. Large qu a n ti ties of CD34+ cells can be co l-l ected with massive marrow harvests, su ch as fortra n s p l a n t a ti on purposes. Nevert h eless, marrowC D 3 4+ cells are som ewhat elu s ive due to thei rs c a t tering among the predominant CD34+

h em a topoi etic pop u l a ti on .1 0 5 Recen t ly devel opedcell sep a ra ti on procedu res all ow co ll ecti on ofh i gh ly en ri ch ed CD34+ cell pop u l a ti on s .However, this is gen era lly accom p l i s h ed thro u gha s pec i fic and of ten unaccept a ble cell loss.1 0 6 So farthe limited nu m ber of immatu re prec u rs orscom m on ly obt a i n ed from both bone marrowand peri ph eral bl ood has been the major ob s t acl eto a simple iden ti fic a ti on and analysis of marrowC D 3 4+ cells. In deed, the growing interest inC D 3 4+ cells is pri m a ri ly the re sult of the devel op-m ent of new thera peutic mod a l i ties that all oweasy access to large qu a n ti ties of hem opoi eti cprec u rs ors thro u gh the peri ph eral bl ood. The keyrole in these innova tive approaches is repre s en tedby the introdu cti on of hem opoi etic growth fac-tors for clinical use.1 0 7

At pre s ent GM-CSF and G-CSF are the mostex ten s ively em p l oyed and stu d i ed hem opoi eti cgrowth factors in the clinical set ti n g. From thevery begi n n i n g, it was ob s erved that GM-CSF orG-CSF ad m i n i s tra ti on was assoc i a ted with ani n c rease in circ u l a ting hem opoi etic progen i-tors .1 0 8 , 1 0 9


Differentiation

Self renewal

Negative Positive Survival

IFN a, b, gTGF-b3

TGF-b1-2

TNF-aMIP-1a

SCFIL-11IL-12IL-6G-CSFIL-1IL-9(?)

IL-11GM-CSF

Figure 9. Interplay between positive andnegative regulatory proteins acting onhematopoietic stem cells.

L a ter on, it was dem on s tra ted that this ph e-n om en on could be ex ten s ively and reprodu c i blya m p l i fied by com bining growth factor ad m i n i s-tra ti on with high - dose ch em o t h era py.1 1 0 - 1 1 2

In deed hem opoi etic progen i tors are massively,t h o u gh tra n s i en t ly, mobi l i zed into peri ph era lbl ood du ring hem opoi etic recovery fo ll owi n gh i gh - dose ch em o t h era py given with growth fac-tor su pport. Su ch an abu n d a n ce of immatu reh em opoi etic cells makes them easily recogn i z-a ble by cell sorting tech n i ques that em p l oy anti -CD34 Mo Ab s .1 1 3 Un der optimal con d i ti ons, theproporti on of CD34+ cells may re ach va lues ash i gh as 20-30% of the total leu koc yte count. Ins te ady state con d i ti ons CD34+ cells do notexceed 4% of the total marrow pop u l a ti on ,while they are undetect a ble by cell sorting tech-n i ques in the peri ph eral bl ood. Ch em o t h era pyand growth factors do not merely indu ce a rel a-tive increase of immatu re hem opoi etic cell s ;t h eir absolute nu m ber is amplified several ti m e sover basal con d i ti ons. This all ows co ll ecti on ofsu f fic i ent amounts of hem opoi etic progen i torsfor autogra f ting purposes by means of a fewl eu k a ph eresis procedu re s .1 1 0 , 1 1 3 , 1 1 4

Va lues of 10-2031 04 CFU-GM/kg repre s en tthe minimal required dose for marrow engraft-ment with peripheral blood progenitors.115-117 Inf act, mu ch high er qu a n ti ties of circ u l a ti n gprogen i tors can be co ll ected using appropri a temobilization procedures. Under optimal condi-ti ons, circ u l a ting CD34+ cell peak va lues mayrange between 150 and 700/µL on days of maxi-mal mobi l i z a ti on. As a rule, at least 831 06

C D 3 4+ cells/kg or more can thus be co ll ectedwith 1 to 3 leu k a ph eresis procedu re s .1 1 4 Th e s ehu ge qu a n ti ties, approx i m a tely corre s pon d i n gto 503104 CFU-GM/kg, must be considered thei deal threshold dose of peri ph eral bl ood prog-enitors for autografting purposes. Indeed valuesof 831 06 C D 3 4+ cells/kg or more guara n tee arapid and du ra ble hem opoi etic recovery wh enc i rc u l a ting progen i tors are used as the soles o u rce for marrow recon s ti tuti on fo ll owi n gsubmyeloablative treatment.118-121

Thus far, massive CD34+ cell mobi l i z a ti on hasbeen most com m on ly ob s erved wh en growt hf actor is ad m i n i s tered fo ll owing high - do s ec ycl oph o s ph a m i de, given at 7 g/sqm. In deed

ch em o t h era py that indu ces profound leu koc y-topenia seems to be crucial for optimal mobi-l i z a ti on; for instance, cycl oph o s ph a m i de atdoses lower than 7 g/sqm produ ces a redu cedm obilizing sti mu lu s .1 1 1 , 1 2 2 Several other ch em o-t h era py sch edules have also been su cce s s f u llyem p l oyed for mobi l i z a ti on purposes. The pri n-cipal ch em o t h era py pro tocols reported to beh i gh ly ef fective in inducing CD34+ cell mobi l i z a-ti on are su m m a ri zed in Ta ble 4.110, 111, 115, 117, 122-127

As stre s s ed earl i er, hem opoi etic growth factorsp l ay a key role in mobi l i z a ti on. This has beencl e a rly doc u m en ted with cycl oph o s ph a m i de. Am edian peak va lue of 75 CD34+ cells/µL hasbeen recorded fo ll owing high - dose cycl oph o s-ph a m i de alone, wh ereas 420 and 500/µL are them edian va lues recorded wh en GM-CSF or G-C S F, re s pectively, are ad ded to cycl oph o s-ph a m i de .1 1 2 , 1 1 3 , 1 2 8 , 1 2 9 Ex ten s ive growth factor-i n du ced mobi l i z a ti on is furt h er su b s t a n ti a ted bythe po s s i bi l i ty of co ll ecting su f f i c i ent CD34+

cells using growth factor alon e .1 3 0 , 1 3 1 In this set-ting the most promising ex peri en ces have been


Table 4. Main chemotherapy protocols employed in hematopoietic progeni-tor mobilization.

Authors Protocol Drug(s) Dosagecharacteristics employed

Gianni et al.110 single agent cyclophosphamide high

Tarella et al.123 single agent etoposide highGianni et al.124

Kotasek et al.121 single agent cyclophosphamide intermediateTarella et al.122

Dreyfus et al.125 single agent cytarabine high

Schimazaki et al.126 multiple drugs etoposide highcytarabine

Kawano et al.115 multiple drugs daunorubicin intermediatecyclophosphamideetoposide

Pettengell et al.127 multiple drugs doxorubicin intermediatecyclophosphamideetoposide

Hass et al.117 multiple drugs cytarabine highmitoxantrone

produ ced with G-CSF. The re sults reportedi n d i c a te new opportu n i ties for the uti l i z a ti on ofm obi l i zed progen i tors in all ogen eic tra n s p l a n t a-ti on procedu re s .1 3 2 , 1 3 3 Com bi n ed ch em o t h era pyand growth factors remain the most ef f i c i en tm obi l i z a ti on procedu re at this time. However,on going studies are directed at improving mobi-l i z a ti on ef fic i ency with growth factors alone. Forexample, G-CSF at doses high er than 5µ g / k g / d ay up to 20 µg/kg/day may have high erm obi l i z a ti on capac i ty.1 3 4 Fu rt h er improvem en tm i ght derive from the clinical ava i l a bi l i ty of newc ytokines, su ch as IL-3, SCF and others, to beem p l oyed alone or in com bi n a ti on with G- orG M - C S F.1 3 5 - 1 3 7 However, the po ten ti a lly highm obilizing activi ty of su ch new cytokine com bi-n a ti ons must be accom p a n i ed by no or very fews i de ef fects in order to be con s i dered for a wi declinical app l i c a bi l i ty.

Mobilizing pro tocols gen era lly inclu de dailydel ivery of growth factors, starting 1 to 3 daysa f ter ch em o t h era py ad m i n i s tra ti on and con-ti nuing until harve s ting procedu res are com-p l eted. Growth factor is usu a lly ad m i n i s teredfor a total of 7-12 con s ec utive days. The mostconven i ent ro ute of del ivery is su bc ut a n eo u s ,with 1 to 2 doses per day. Progen i tor cell har-vests are perform ed du ring hem opoi etic recov-ery, provi ded that progen i tor cell mobi l i z a ti onis doc u m en ted. In deed va rious para m etersh ave been con s i dered as an indirect indicati onof progen i tor mobi l i z a ti on, including ani n c rease in WBC or, altern a tively, in mon o-c ytes, basophils or platel et s .1 3 8 - 1 4 0 However,C D 3 4+ cell eva lu a ti on remains mandatory foran acc u ra te defin i ti on of the ex tent of progen i-tor mobi l i z a ti on .1 4 1 , 1 4 2

C D 3 4+ cells should be mon i tored daily fromthe early stages of hem opoi etic recovery.Detecti on of circ u l a ting CD34+ cells is not su f fi-c i ent re a s on for starting leu k a ph eresis proce-du res; an adequ a te nu m ber of CD34+ cells (>20-50/µL) and WBC > 1 . 031 09/L and platel et co u n t> 3 031 09/L are requ i red for safe and ef fectiveprogen i tor cell harve s ti n g.1 1 4 , 1 4 2 Leu k a ph ere s e sa re perform ed using con ti nu o u s - flow bl ood cells ep a ra tors. A com p l ete leu k a ph eresis procedu regen era lly takes 2-3 hours and the total bl oodvo lume proce s s ed ra n ges from 6 to 10 li-

ters .1 1 4 , 1 4 0 , 1 4 3 , 1 4 4 The harve s ted cells are re su s pen dedin freezing med ium and then cryopre s erved forsu b s equ ent tra n s p l a n t a ti on. One to 3 leu k a-ph ereses repe a ted on con s ec utive days usu a llyprovi de large amounts of progen i tor cells capa-ble of rapid en gra f tm ent after su bmyel oa bl a tivetre a tm en t s .

Factors and procedu res favoring CD34+ cellm obi l i z a ti on have been well establ i s h ed .However, other con d i ti ons advers ely influ en c i n gthe mobi l i z a ti on ph en om en on should be con-s i dered. A major limitati on is repre s en ted byi m p a i red marrow functi on, as can occur in pre-vi o u s ly tre a ted pati en t s .1 1 1 In fact, pati ents atfirst relapse fo ll owing a single tre a tm ent pro to-col maintain an adequ a te mobi l i z a ti onc a p ac i ty;1 4 5 h owever, few if any mobi l i zed CD34+

cells can be obt a i n ed from heavi ly tre a tedp a ti ents previ o u s ly ex po s ed to mu l tiple ch em o-t h era py co u rses. Mobi l i z a ti on is also profo u n dlyredu ced and of ten to t a lly abo l i s h ed in pati en t sprevi o u s ly ex po s ed to rad i o t h era py, espec i a lly ifit was del ivered to the pelvis or to ex ten ded ver-tebral are a s .1 1 7 , 1 2 3 L a s t ly, marrow inva s i on bytu m or cells may nega tively affect mobi l i z a ti onc a p ac i ty. This is typ i c a lly reported in myel om ap a ti ents in wh om the ex tent of CD34+ cells of tencorrel a tes with the degree of marrow invo lve-m ent by tu m oral plasma cell s .1 2 2 In con clu s i on ,s everal new fin d i n gs have dra m a ti c a lly improv-ed the procedu res for co ll ecti on of largeamounts of CD34+ cells. However, optimal mar-row functi on is a prerequ i s i te for ex p l oi ting fullythe activi ty of all those factors known for thei rm obi l i z a ti on - i n ducing capac i ty.

CD34+ positive cells in umbilical cord bloodSignificant numbers of human hematopoietic

stem cells can be found in umbilical cord bloodand can be used for all ogen eic bone marrowtra n s p l a n t a ti on. Mayani et al.1 4 6 found that 1-2% of the total nu m ber of cord bl ood - derivedl ow - den s i ty cells ex press high levels of theCD34 antigen and low or undetectable levels ofthe antigens CD45RA and CD71. These popu-lations were highly enriched in clonogenic cells(34%), in particular in multipotent progenitors(12%). By cultu ring these cells at low con cen-


tra ti on for 8-10 days in high ly defin ed seru m -free liquid cultures supplemented with varioush em a topoi etic cytokines, it was po s s i ble toach i eve a significant ex p a n s i on (abo ut 100-fold) of the CD34+ cell population.

These last results were recently confirmed byTraycott et al.1 4 7 in an el egant stu dy showi n gthat umbilical cord bl ood CD34+ cells ra p i dlyexit G0-G1 phases and start to cycle in responseto stem cell factor. This fe a tu re would makecord bl ood CD34+ cells more su i t a ble candi-dates than bone marrow cells for ex vivo expan-sion.

It is clear that in vitro expansion and matura-tion of hematopoietic progenitor cells might beof particular rel eva n ce for tra n s p l a n t a ti on ofcord blood hematopoietic cells; however, infor-m a ti on abo ut the ef fects of su ch an ex p a n s i onon the cells required for long-term hematopoi-etic reconstitution is highly desirable.

The dual role of peri p h eral bl ood hem a topo i eti cprogen i tor cells in oncoh em a tol ogy

C D 3 4+ progen i tor cells circ u l a ting in theperipheral blood represent an enriched and eas-ily accessible source of two distinct cell popula-tions: committed progenitor cells and hemato-poietic stem cells.

Al t h o u gh circ u l a ting progen i tors are com-monly called stem cells, the presence of circulat-ing stem cells has been formally proved only inmice.148 In humans the presence of hematopoi-etic stem cells in the peripheral blood is almostcertain (see below), but to call reinfusion of cir-culating progenitors ‘transplantation’ of periph-eral bl ood stem cells (PBSC or similaracronyms) is hardly appropriate. This terminol-ogy overl ooks the fact that the trem en do u sinterest in peripheral blood autografting is not(at least so far) a con s equ en ce of its being asimple su rroga te for auto l ogous bone marrowtra n s p l a n t a ti on (as the term PBSC would su g-gest), but rather derives from the unique prop-erty of this procedure to reduce the duration ofthe severe pancytopenia that fo ll ows su bmye-l oa bl a tive tre a tm ents from two - t h ree wee k s( wh en bone marrow cells are used) to a fewdays only.110,149 The reason for the rapid recovery

which occurs after circulating progenitor auto-tra n s f u s i on (CPAT) has not been form a llyproved, but it is most likely a con s equ en ce ofthe mu ch larger (10- to 100-fold high er )amount of com m i t ted progen i tors rei n f u s edwh en a pati ent is autogra f ted with bl ood -derived (as oppo s ed to marrow - derived) cell s .The most likely hypothesis is that these lateprogen i tors (po s t - progen i tors) of gra nu l oc yte sand platel ets are capable of giving rise tomature progeny within a few days, thus allow-ing submyeloablated patients to survive the ini-tial aplastic phase.

The fundamental role of com m i t ted progen i-tor cells was el ega n t ly proved in mice by Jon e set al.1 5 0 None of the let h a lly irrad i a ted animalstra n s p l a n ted with a pure pop u l a ti on of stemcells free of more matu re progen i tors (CFU-GM, CFU-S) su rvived the initial aplasia. Am ore recent paper1 5 1 ch a ll en ged this ‘co nven-tional wi sd o m’ m odel, and maintained thatperi ph eral bl ood stem cells per se a re capable ofra p i dly matu ring in vivo.

Ot h er aut h ors, using a very similar approach ,re ach ed an opposing con clu s i on .1 5 2 In hu m a n sthe most convi n c i n g, albeit indirect, evi den cein favor of the role of com m i t ted progen i tors inaccel era ting po s t - transplant recovery was pro-vi ded by Robert s on et al. ,1 5 3 who doc u m en ted as i gn i fic a n t ly pro l on ged hem a topoi etic recoveryfor pati ents under going auto l ogous bone mar-row tra n s p l a n t a ti on purged ex vivo with anti -CD33 mon ocl onal anti bodies. This re su l t ,wh i ch occ u rred after a tre a tm ent that sel ective-ly kills the most matu re progen i tor cell s( ex pressing the CD33 su rf ace anti gen), repre-s ents convincing indirect proof of the role ofcom m i t ted progen i tors in early en gra f tm en t .

The clinical role of com m i t ted progen i tors inreducing the morbi d i ty and mort a l i ty of high -dose thera py has ex p a n ded since hem opoi eti cgrowth factors have become cl i n i c a lly ava i l a bl e .In fact, infusion of rhGM-CSF or rh G - C S F, inp a rticular after ad m i n i s tra ti on of myel o tox i cdoses of certain stem cell - s p a ring agents, all owseasy co ll ecti on of an amount of CFU-GM/kgbody wei ght 10 to 100 times high er than thatcon t a i n ed in a bone marrow harve s t .1 1 0

As alre ady doc u m en ted by a large nu m ber of


383

p a pers, the use of circ u l a ting progen i tors hasbro u ght abo ut a dra m a tic ch a n ge in the per-s pectives of high - dose su bmyel oa bl a tive regi-m ens. In fact, these on ce spec i a l i zed, ex pen s iveand high ly toxic tre a tm ents are now well to l er-a ted, easy to ad m i n i s ter, and cl i n i c a lly usef u l( cost ef fective). As an example, it is worth men-ti oning the initial Milan Ca n cer In s ti tute ex pe-ri en ce in a group of over 50 poor- risk bre a s tc a n cer pati ents who received high - dose mel-phalan plus an optimal amount of circ u l a ti n gprogen i tors ( � 531 04 C F U - G M / k g bodywei ght). These pati ents requ i red a median of10.5, 11 and 12.5 days to score > 0.5, 1.0 and2 . 031 09 /L neutrophils/µL, re s pectively.Moreover, more than half of them did notrequ i re platel et tra n s f u s i ons, while the rem a i n-ing ones needed on ly one or two tra n s f u s i on sdu ring the first week after autogra f ti n g. Su chmild to modera te tox i c i ty was never de s c ri bedbefore the clinical ava i l a bi l i ty of com m i t tedprogen i tor cells.

In con clu s i on, born as a ‘co m pa s s i o n a te’ su r-roga te of bone marrow autogra f ti n g, tod ayC PAT is ra p i dly rep l acing ABMT. In fact, tod ayit is the latter that should be con s i dered a ‘co m-pa s s i o n a te need ’ procedu re, useful in those fewp a ti ents unable to mobi l i ze a su f fic i ent nu m berof circ u l a ting progen i tor cell s .

The second, disti n ct role of circ u l a ting prog-en i tors is rel a ted to the pre s en ce of stem cell s ,i .e. to ti po tent prec u rs ors re s pon s i ble fordu ra ble recon s ti tuti on of all lym ph o - h em a to-poi etic lineages fo ll owing marrow abl a tivet h era py. Si n ce vi rtu a lly no high - dose tre a tm en tthat can be safely ad m i n i s tered to humans isgenu i n ely myel oa bl a tive, formal proof of theex i s ten ce of circ u l a ting stem cells must aw a i tei t h er stable tra n s du cti on of DNA markers intoa utogra f ted cells or the use of this cell pop u l a-ti on for all ogra f ti n g.

These experiments are presently underway ins everal labora tori e s ,1 5 4 , 1 5 5 and prel i m i n a ry datado con f i rm the pre s en ce of stem cells in theperipheral blood of humans. Their utilization isex pected to revo luti on i ze fields like all ogen ei cbone marrow transplantation and somatic genetherapy, whenever the target of genetic manipu-lations is the hematopoietic cell.156

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