C De Bruyn, A Delforge, L Lagneaux and D Bron. Summary:

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1 () 25, Mcmilln Pulishers Ltd All rights reserved / $15. Chrcteriztion of CD34 susets derived from one mrrow, umilicl cord lood nd moilized peripherl lood fter stem cell fctor nd interleukin 3 stimultion C De Bruyn, A Delforge, L Lgneux nd D Bron Service de Médecine Interne et Lortoire de Recherche et d Investigtion Clinique, Institut J Bordet, Brussels, Belgium Summry: We chrcterized CD34 + cells purified from one mrrow (BM), moilized peripherl lood (PB) nd cord lood (CB) nd we tried to estlish correltions etween the cell cycle kinetics of the CD34 + CD38 nd CD34 + CD38 + supopultions, their sensitivity to SCF nd IL-3 nd their expression of receptors for these two CSFs. At dy, significntly fewer immture CD34 + CD38 cells from CB nd moilized PB re in S + G2M phses of the cell cycle (respectively 2. ±.4 nd.9 ±.3%) thn their BM counterprt (5.6 ± 1.2%). A 48-h incution with SCF + IL-3 llows significnt increse in the percentge of cycling CD34 + CD38 cells in CB (19.2 ± 2.2%, P.2) nd PB (14.1 ± 5.5%, P.5) while the prolifertive potentil of BM CD34 + CD38 progenitors remins constnt (8.6 ± 1.%, NS). CD123 (IL-3 receptor) expression is similr in the three sources of hemtopoietic cells t dy nd fter 48-h culture. CD117 (SCF receptor) expression, lthough very heterogeneous ccording to the supopultions nd the sources of progenitors evluted, seems not to correlte with the difference of progenitor cell sensitivity to SCF nor with their prolifertive cpcity. Considering the importnce of the c-kit/scf complex in the dhesion of stem cells to the microenvironment, severl oservtions re relevnt. The density of CD117 ntigen expression (expressed in terms of men equivlent solule fluorescence, ) is significntly lower on fresh PB cells thn on their BM (P.17) nd CB (P.4) counterprts, prticulrly in the immture CD34 + CD38 popultion (56 ± 131, 2121 ± 416 nd 1192 ± 129 respectively); moreover, when PB nd BM CD34 + CD38 cells re stimulted for 48 h with SCF + IL-3, the CD117 expression decreses y 1.5- nd 1.66-fold, respectively. This reduction could modify the functionl cpcities of ex vivo PB nd BM mnipulted immture progenitor cells. () 25, Keywords: c-kit; cell cycle; cord lood; one mrrow; peripherl lood Correspondence: Dr C De Bruyn, Institut Jules Bordet, Rue Héger-Bordet, 1, Brussels, Belgium Received 4 My 1999; ccepted 14 Octoer 1999 Umilicl cord lood (CB), moilized peripherl lood (PB) nd one mrrow (BM) re currently used s sources of hemtopoietic stem cells. 1,2 However, severl phenotypic nd functionl differences etween hemtopoietic stem cells from these three tissues hve een descried during the lst few yers. 3 6 The CD34 + CD38 phenotype corresponds to primitive supopultion of progenitor cells in BM s well s in CB nd in moilized PB, tht cn e distinguished functionlly from the CD34 + CD38 + popultion y severl in vitro ssys. 7 Although for the three sources, the CD34 + CD38 phenotype is consistent in defining the most primitive progenitors, 8,9 functionl differences etween CD34 + CD38 cells from the three tissues hve een descried. CD34 + CD38 cord lood cells hve higher cloning efficiency, proliferte more rpidly in response to cytokine stimultion nd generte more progenitors thn their counterprt in one mrrow. 1 In this study, we chrcterized CD34 + cells purified from the three sources y compring their cell cycle sttus nd their cpcity to form lst colonies in contct with BM strom. Moreover, we evluted the expression of receptors to SCF (CD117) nd IL-3 (CD123) on the cell surfce of ech supopultion t dy nd 48 h fter stimultion y SCF + IL-3. The rtionle for utilizing this cytokine comintion is its ility to promote in vitro expnsion of CB CD34 + nd CD34 + CD38 cells. 11 The ims of our study were to estlish reltions etween the cell cycle kinetic of CD34 + supopultions, their sensitivity to colony-stimulting fctors nd their expression of SCF nd IL-3 receptors. Mterils nd methods Cells Umilicl cord lood cells were collected fter full-term deliveries. Immeditely fter delivery, the cord ws clmped, the umilicl vein ws ctheterized septiclly nd the smple collected y grvity into 4 ml lood unit pck contining 35 ml citrte-phosphte-dextrose denine-1 solution (CPD-A) (Bxter Fenwl, L Châtre, Frnce). Bone mrrow cells were otined y spirtion from the sternum of norml donors. The smples were nticogu-

2 378 lted with 1 IU/ml heprin (Rhône-Poulenc Rorer, Brussels, Belgium). Peripherl lood cells were otined fter cytpheresis of norml donors moilized y 1 g/kg of G-CSF (Neupogen, Amgen, Thousnd Oks, CA, USA). Informed consent ws otined from norml donors efore ny smple collection. CD34 positive cell selection on Midimcs immunomgnetic selection systems (Miltenyi Biotec, Bergisch Gldch, Germny) According to the mnufcturer s instructions, 5 to mononucler cells suspended in uffer contining PBS nd.5% ovine serum lumin (BSA), were incuted for 15 min t 4 C with locking regent (humn IgG) nd simultneously with nti-humn CD34 ntiody (QBEND1). After wshing, the cells were incuted for 15 min t 4 C with superprmgnetic MACS microeds recognizing QBEND1. The cells were then wshed nd loded on to high grdient mgnetic seprtion column (LS + column) wshed with 3 ml uffer. The unlelled cells were wshed five times through the column using 3 ml uffer. After removing the column from the mgnetic seprtion unit, the lelled cells re collected y wshing LS + column with 5 ml uffer. Selected cells re wshed once in PBS efore use. Suspension culture CD34 + cells re plted in 24 well pltes t 1 4 to 1 5 cells/ml in serum-free medium (CellGRO, BioWhittker, Wlkersville, MD, USA) with SCF (1 ng/ml) nd IL-3 (2 ng/ml). Cells were mintined t 37 C under 5% CO 2 in 1% humidity for 2 dys. Vile cell numer, phenotype nd cell-cycle sttus nlysis were performed t dy nd fter 2 dys of culture. Phenotype nlysis Two-fluorescence nlysis: Cell suspensions were incuted for 3 min t room temperture in the drk with fluorescein isothiocynte (FITC) conjugted nti-cd38 (Immunotech, Mrseille, Frnce) ssocited with phycoerythrin (PE) conjugted nti-cd34 (HPCA-2, Becton Dickinson, Sn Jose, CA, USA), PE conjugted nti- CD117 (Immunotech) or PE conjugted nti-cd123 (Phrmingen, Sn Diego, CA, USA). After wshing, the cells were nlyzed using n EPICS-XL flow cytometer (Coulter, Hileh, FL, USA). The percentge of positive cells ws determined y reference to nonspecific stining with ntiodies of the sme isotype (IgG1/IgG1, Becton Dickinson). The density of CD117 nd CD123 ws lso expressed s men equivlent solule fluorescence () evluted y reference to clirtion eds (Dko Fluorospheres; Dko, Glostrup, Denmrk). Triple-fluorescence nlysis: In ddition, for severl smples, cells were triple-stined using FITC-conjugted nti-cd38 nd phycoerythrin-cynin 5.1 (PE-Cy5) conjugted nti-cd34 (Immunotech) ssocited with either PEconjugted nti-cd117 or PE-conjugted nti-cd123. The percentges nd the of CD117 positive cells mong CD38 + nd CD38 supopultions were evluted fter gting on the CD34 + popultion. Non-specific stining ws performed using the Opticlone (IgG1-FITC/IgG1-PE/IgG1- PE-Cy5) from Immunotech. Doule lelling nlysis of DNA content nd of CD38 expression 1 5 cells were incuted for 3 min t room temperture with 2 l FITC conjugted nti-cd38 (Immunotech). After incution, cells were wshed nd resuspended in 1 l of PBS. 2 l of permeilizing regent (Coulter DNA prep regent) were dded nd the suspension ws mixed vigorously for t lest 45 s. After mixing, 38 l of propidium iodide (PI) solution (Coulter) were dded nd the cells were incuted for 24 h t 4 C in the drk. The cell cycle sttus of the cell suspension ws nlyzed y flow cytometry. Doulets were excluded y pproprite gting in the width vs re dot plot. Dt were nlyzed using Elite 4. Softwre (Coulter). Blst-colony forming cell ssy (Bl-CFC) This ssy is dependent on the ility of the stem cell popultion to recognize nd ttch to preformed mrrow derived stroml lyer nd to grow in this environment without ddition of exogenous growth fctors. The feeder lyer is otined y culturing during 4 to 6 weeks one mrrow mononucleted cells in the presence of methylprednisolone nd is composed of firolsts, mcrophges nd ft cells. Stroml lyers were prepred y plting one mrrow mononucler cells in 1 ml -MEM (Gico) contining 2% FCS supplemented with methylprednisolone (Solumedrol) (Upjohn, Klmzoo, MI, USA) in 35-mm Petri dishes. The stroml cultures were fed weekly y complete replcement of the culture medium. After 4 to 6 weeks, stroml lyers re confluent. At this time, cells from the strting CD34 + cell suspension nd from cells recovered fter 48-h culture were incuted for 2 h t 37 C nd 5% CO 2 on confluent mrrow-derived stroml lyers to llow dherence to the strom. The stroml lyers were then wshed three times with lph-mem to remove ll cells which hd not dhered. The cultures were then overlid with 1 ml of.3% gr in lph-mem supplemented with 2% FCS nd were incuted for 7 dys t 37 C nd 5% CO 2. All colonies closely ssocited to the stroml lyer nd contining more thn 2 cells were counted. 12 Sttisticl nlysis The comprisons etween either phenotype or cell cycle sttus t dy nd fter 48-h culture or etween phenotypes of CD38 nd CD38 + popultions, in the sme type of smple, re performed using the Student s pired t-test. Comprisons etween phenotype from different types of smple re performed using the Student s t-test.

3 Tle 1 Cell phenotype t dy nd fter 48 h stimultion using SCF + IL-3 dy 48 h % % % % CD34 + CD34 + CD38 CD34 + CD34 + CD38 CB (n = 9) 85.9 ± ± ± ± 5.3 BM (n = 5) 96.6 ± ± ± ± 3.3 PB (n = 6) 87.9 ± ± ± ± 14.4 Results CD34CD38 expression of selected cells t dy nd fter 2 dy stimultion y SCF + IL-3 As shown in Tle 1, the purity of CD34 + cells, otined using the Midimcs system, ws greter thn 85%. However, to exclude contmintion y CD34 cells, the phenotypes of the progenitor cells were studied y dul color fluorescence, nd lso y triple color fluorescence, nlysis fter gting on the CD34 positive cell frction. After 2 dys of culture, the numer of cells remins constnt for the three sources of hemtopoietic stem cells evluted, the expression of CD34 nd CD38 ntigens is not rdiclly modified. Cell cycle sttus of CD34 + cells from CB, BM nd moilized PB t dy nd fter 48-h stimultion y SCF + IL-3 In the first set of experiments, the cell cycle sttus of CD34 + CD38 nd CD34 + CD38 + cells is evluted y flow cytometry. As shown in Tle 2, cycle nlysis of the two supopultions indictes tht t dy, the lrge mjority of the whole CD34 + cell popultion from CB s from BM nd moilized PB were in G/G1 phses of the cell cycle. Significntly more CD34 + CD38 + cells were in prolifertive phses of the cell cycle (S+G2M) s compred with CD34 + CD38 cells, in CB, BM nd PBSC. On the other hnd, significntly more BM CD34 + CD38 cells were in S+G2M s compred with PB CD34 + CD38 cells (P =.28). No significnt differences etween cell cycle sttus of CD34 + CD38 cells re oserved etween CB nd BM, nor etween PB nd CB. After 48 h incution of CD34 + cells in the presence of SCF nd IL-3, the percentge of BM CD34 + CD38 cells in S+G2M phse of the cell cycle does not significntly increse ut in contrst, it increses y 7.7- nd 1.2-fold for CB nd PB, respectively. Our dt suggest tht the CD34 + CD38 cells from BM nlyzed t dy re more ctively cycling thn their PB nd CB counterprt, however fter 48-h incution with SCF + IL-3 this percentge does not increse. Clonogenic cpcities of cells efore nd 48 h fter stimultion with SCF + IL-3 To support our oservtions tht CB nd PB CD34 + CD38 cells re more prolifertive fter stimultion using SCF + IL-3 thn t dy, we hve plted these cells in culture system llowing the growth of strom-dependent progenitors (Bl-CFC). These cells re minly present in the CD34 + CD38 frction 13 nd we cn thus expect tht this ssy reflects primrily the clonogenic cpcities of the CD34 + CD38 frction. For CB nd PBSC, we hve otined respectively 5.2 ± 1 (P.9) (n = 5) nd 6.1 ± 1.2 (P.22) (n = 4) fold more Bl-CFC with CD34 + cells stimulted during 48 h y SCF + IL-3 thn using fresh CD34 + selected cells. For BM, the numer of Bl-CFC ws not significntly different efore nd fter incution with CSFs (n = 5). These results confirm the incresed prolifertive cpcities of immture PB nd CB CD34 + CD38 cells fter incution with SCF + IL- 3 while BM immture progenitors mintin similr cpcities. CD117 expression on CD34 + supopultions from CB, BM nd moilized PB t dy nd fter 48-h culture The fct tht CB nd moilized PB CD34 + CD38 cells seem to e more responsive to SCF nd IL-3 thn their BM counterprt, suggesting different sensitivity of progenitors to these growth fctors, my result from different receptor expression on the cell surfce. Therefore, we exmined the expression of SCF nd IL-3 receptors on CD34 + CD38 + nd CD34 + CD38 cells purified from CB, BM nd PB. This expression is evluted in terms of receptor density () (Figure 1) nd of percentge of positive cells (Tle 3). Due to the reltively high purity of CD34 + cells otined using the Midimcs seprtion system, the results oserved fter two or triple fluorescence nlysis were not different nd were thus pooled. 379 Tle 2 SCF + IL-3 Cell cycle nlysis of CD34 + cells nd of the two supopultions CD34 + CD38 nd CD34 + CD38 + t dy nd fter 48 h stimultion y CD34 + CD34 + CD38 + CD34 + CD38 dy 48 h P dy 48 h P dy 48 h P CB (n = 8) 5.9 ± ± ± ± 5.5 NS 2. ± ± BM (n = 8) 11.8 ± ± ± ± ± ± 1. NS PB (n = 6) 2. ± ± ± ± 9.9 NS.9 ± ± Percentge of CD34 +, CD34 + CD38 + nd CD34 + CD38 cells in S + G2M phses of the cell cycle, expressed s men ± s.e.m. P represents significnt difference etween percentges oserved t dy nd fter 48 h culture. It ws clculted using the Student s pired t-test.

4 * c 35 c Figure 1 CD117 expression on () CB(n = 9), () BM(n = 5) nd (c) PB (n = 6) CD34 + supopultions t dy, ; nd fter 48 h culture with SCF + IL-3,. Results re expressed s men ± s.e.m. of men equivlent solule fluorescence (). *Represents significnt difference etween CD117 expression t dy nd fter 48 h culture. When we compred the density of receptors on the cell surfce (Figure 1), CD117 expression is significntly lower on CD34 + CD38 cells thn on CD34 + CD38 + cells, in CB (P.4) s well s in PB (P.11) nd in BM (P.2). This difference etween the two susets remins significnt fter 48-h culture (respectively P.1, P.5 nd P.1 for CB, PB nd BM). On the other hnd, it is cler tht t dy, on the immture CD34 + CD38 popultion, CD117 expression is significntly lower on moilized PB cells thn on their CB (P.1) nd BM (P.1) counterprts. When the Figure 2 CD123 expression on () CB(n = 9), () BM(n = 5) nd (c) PB (n = 6) CD34 + supopultions t dy, ; nd fter 48 h culture with SCF + IL-3,. Results re expressed s men ± s.e.m. of men equivlent solule fluorescence (). CD34 + cells re incuted for 48 h with SCF + IL-3, the CD117 expression on totl CD34 + cells increses in CB nd PB while it decreses in BM. This effect is proly due to the increse of CD117 expression on CD34 + CD38 + cells from CB nd PB nd the decrese on BM cells. Surprisingly, CD117 expression decreses on PB nd BM CD34 + CD38 cells while it remins stle in CB. The regultion of CD117 expression fter short-term incution with SCF + IL-3 seems very heterogeneous ccording to the type of smples nd the supopultion evluted. Nevertheless, it could e noted tht, fter 48 h culture, CD117 expression

5 Tle 3 CD117 expression on CD34 +, CD34 + CD38 + nd CD34 + CD38 cells t dy nd fter 48 h culture with SCF + IL Dy 48 h CD34 + CD34 + CD38 + CD34 + CD38 P CD34 + P CD34 + CD38 + P CD34 + CD38 P P CB (n = 9) 57.5 ± ± ± ± 2.6 NS 64.2 ± 5.8 NS 31.4 ± 7.6 NS.8 BM (n = 5) 67.2 ± ± ± 9. NS 48.2 ± ± 4.6 NS 25.7 ± PB (n = 6) 43.1 ± ± ± ± 8.1 NS 47.3 ± 8.9 NS 14.1 ± 6.3 NS.2 Results re expressed s the men percentge ± s.e.m. of CD117 positive cells in ech supopultion. P represents significnt difference etween CD117 expression on CD34 + CD38 + nd CD34 + CD38 cells nd re clculted using the Student s pired t-test. P represents significnt difference etween CD117 expression t dy nd fter 48 h culture in the three supopultions nd re clculted using the Student s pired t-test. on PB progenitor cells is significntly lower thn on their CB nd BM counterprts (respectively P.6,.2,.4 nd P.1,.1,.1 for the CD34 +, CD34 + CD38 + nd CD34 + CD38 cells). When we compred the percentge of CD117 positive cells, the sme differences etween PB vs CB nd BM progenitor cells, lthough less spectculr, re lso oserved. At dy s fter 48 h, we did not oserve significnt difference etween CD117 expression on CB nd BM cells. CD123 expression on CD34 + supopultions from CB, BM nd moilized PB t dy nd fter 48 h culture (Figure 2) (Tle 4) CD123 expression is not significntly different in the three sources of hemtopoietic cells. After 48 h of culture the density of IL-3 receptors increses in ll supopultions while the percentges of positive cells remin constnt. We did not oserve significnt differences etween the three tissues. Discussion Moilized peripherl lood (PB) nd umilicl cord lood cells (CB) re two lterntive sources of llogeneic stem cells to one mrrow (BM) for trnsplnttion. 1 There re incresing reports demonstrting differences in phenotype nd functionl properties etween hemtopoietic progenitors from PB, CB nd BM. 5,1,14 16 The most striking difference etween BM, PB nd CB is in the hemtopoietic recovery following trnsplnttion which is fster fter PB nd delyed fter CB trnsplnttion when compred to BM. 1,2 The difference of engrftment kinetics etween the three sources of progenitor cells re not yet fully understood. The impct of cell dose is proly importnt ut remins controversil. 17 On the other hnd, it seems resonle to hypothesize tht qulittive difference etween CD34 + popultions of the three sources could lso ply role in engrftment kinetics. Comprtive studies of the CD34 + supopultions in the three sources of stem cells my contriute to understnding the mechnisms involved in hemtologic recovery fter trnsplnttion. In this study, we hve first identified the cell cycle sttus of CD34 + CD38 nd CD34 + CD38 + supopultions in stedy stte BM, PB nd CB smples. In ccordnce with previous studies, it ppers tht the gret mjority of CD34 + cells from CB nd prticulrly PB, re in G/G1 phses of the cell cycle wheres more thn 1% of the BM CD34 + cells re in S + G2M phses. We hve lso oserved this difference etween on the one hnd, CB nd PB nd on the other hnd, BM, for the immture CD34 + CD38 frction ( 2% in S+G2M for PB nd CB nd 5% for BM). Short-term incution of these cells with SCF + IL- 3 llows significnt increse in the percentge of cycling CD34 + CD38 cells in CB nd PB while the prolifertive potentil of BM CD34 + CD38 cells is not significntly modified. It seems thus possile to trigger during shortterm culture, the cycling sttus of PB nd CB progenitor cells without rdiclly modifying their numer or their phenotype. Tking into ccount this prticulr spect, it ppers tht the CB CD34 + progenitors seem to hve more Tle 4 CD123 expression on CD34 +, CD34 + CD38 + nd CD34 + CD38 cells t dy nd fter 48 h culture with SCF + IL-3 Dy 48 h CD34 + CD34 + CD38 + CD34 + CD38 P CD34 + P CD34 + CD38 + P CD34 + CD38 P P CB (n = 9) 6.5 ± ± ± ± 7.8 NS 67.9 ± 8.5 NS 4.5 ± 9.4 NS.4 BM (n = 5) 65.9 ± ± ± ± 3.2 NS 55.6 ± 5.8 NS 36.1 ± 7.3 NS NS PB (n = 6) 68.1 ± ± ± 12.1 NS 64.8 ± 6.1 NS 69.1 ± 6.3 NS 48. ± 6.7 NS.9 Results re expressed s the men percentge ± s.e.m. of CD123 positive cells in ech supopultion. P represents significnt difference etween CD123 expression on CD34 + CD38 + nd CD34 + CD38 cells nd re clculted using the Student s pired t-test. P represents significnt difference etween CD123 expression t dy nd fter 48 h culture in the three supopultions nd re clculted using the Student s pired t-test.

6 382 similrities with PB thn with BM CD34 + cells. These surprising results cnnot explin the different ehvior etween CB nd PB cells fter trnsplnt. Therefore, cell cycle differences re unlikely to explin the mrked difference in post-trnsplnt recovery etween PB nd CB. Our results, demonstrting tht PB CD34 + cells nd prticulrly CD34 + CD38 cells respond rpidly to SCF + IL-3, seem to confirm the oservtion of Lemoli et l 23 which hve shown tht the mjority of moilized peripherl lood CD34 + cells, including LTC-IC, re not quiescent (G phse), ut re in G1 phse of the cell cycle nd re thus redy to progress into S-phse under CSF stimultion. The ility of CB nd PB immture progenitors to exit G/G1 phses more rpidly thn their BM counterprt could derive from difference in receptor expression for the two cytokines used (SCF nd IL-3). We hve thus investigted the expression of SCF nd IL-3 receptors on the different CD34 + supopultions nd the short term in vitro modultion of this expression y cytokines. It is well documented tht the SCF/c-kit interction pys crucil role in the erly stge of hemtopoiesis. However, conflicting reports hve descried c-kit expression nd little is known concerning the modultion of this expression on the immture nd more committed progenitor cells. Therefore, it will e importnt to elucidte c-kit expression on different supopultions of progenitors. Our results, in ccord with those from severl others groups, 7,27,28 show tht the c-kit expression on PB CD34 + cells is significntly lower thn tht of BM nd CB CD34 + cells which express high level of c-kit receptors. This difference, lso oserved in CD34 + CD38 + cells, is prticulrly mrked on immture CD34 + CD38 cells. Moreover, in PB, when we stimulted the CD34 + cells y SCF + IL-3, CD117 expression decreses on CD34 + CD38 cells while it increses on CD34 + CD38 + cells. After 48 h culture, CD117 expression on PB CD34 + CD38 cells is thus very low. These results seem to e in contrdiction with the pprent greter sensitivity of immture PB progenitors to SCF. In cord lood, CD117 expression remins constnt in the CD34 + CD38 supopultion nd increses in CD34 + CD38 + cells. This is contrry to BM progenitors in which CD117 expression decreses on ll supopultions studied. Our results indicte tht CD117 expression lthough different etween the three tissues, seems not to correlte with the difference in sensitivity of progenitor cells to SCF nor with the prolifertive cpcity of progenitors induced y SCF stimultion. The low expression of c-kit on PB cells tht we hve oserved in this study ws previously relted to the moiliztion of CD34 + cells into the circultion fter G-CSF dministrtion. 29,3 This oservtion gives new importnce to c-kit expression in moiliztion nd in dhesion of hemtopoietic stem cells to one mrrow microenvironment. In fct, it is well known tht c-kit is not only cytokine receptor ut lso n dhesion molecule. 31,32 The SCF/c-kit complex ws previously shown to ply n importnt role in the homing of hemtopoietic stem cells to the one mrrow microenvironment. 33 As we postulted tht the hemtopoietic recovery fter trnsplnttion is dependent in vivo to homing of cells to one mrrow stroml cells nd to prolifertion of hemtopoietic stem cells in this microenvironement, we cn suppose tht in vitro down-regultion of the CD117 expression, s oserved for the PB immture progenitors cells, my impired engrftment. This hypothesis is confirmed y dt from Gothot et l 34 who showed tht 36 h stimultion y cytokines ctivtes cell cycle nd decresed the ility of humn PB cells to repopulte SCID/NOD mice nd from Szilvssy et l 35 who recently oserved the sme decline in the homing cpcity of murine progenitors fter ex vivo culture. In the sme wy, severl uthors hve descried tht trnsplnttions with ex vivo expnded CD34 + cells do not permit reconstitution of stle long-term hemtopoiesis, 36,37 indicting loss of repopulting cpcities of immture progenitor cells during ex vivo culture. These oservtions, lthough controversil, 38 suggest tht functionl modifictions could occur during the ex vivo mnipultion nd hve n impct on the engrftment cpcities of stem cells. In conclusion, these experiments provide evidence tht short term ex vivo culture cn modify stem cells t different levels (their cell cycle sttus nd the expression of dhesion molecules) nd cn influence the engrftment cpcity of these cells. Moreover these modifictions re very heterogeneous depending on the source of stem cells nd the supopultions studied. On the other hnd, the in vitro prolifertive, s well s dhesive, cpcities of progenitor cells, lthough very importnt, do not seem to e the only limiting fctors for the in vivo cell engrftment. Further studies will e needed to elucidte the reltionship etween cell ehvior, cell cycle chnges, expression of dhesion molecules, cytokine receptor expression nd the cpcity of cells to dhere to the one mrrow microenvironment. Acknowledgements We thnk Amgen for providing us with SCF. C De Bruyn is supported y grnt FNRS-Télévie No References 1 Arcese W, Avers F, Bndini G et l. Clinicl use of llogeneic hemtopoietic stem cells from sources other thn one mrrow. Hemtologic 1998; 83: Ruinstein P, Crrier C, Scrdvoou A et l. Outcomes mong 562 recipients of plcentl-lood trnsplnts from unrelted donors. New Engl J Med 1998; 22: Trycoff CM, Aoud MR, Lver J et l. Rpid exit from G/G1 phses of cell cycle in response to stem cell fctor confers on umilicl cord lood CD34 + cells n enhnced ex vivo expnsion potentil. Exp Hemtol 1994; 22: Fritsch G, Stimpfl M, Kurz M et l. The composition of CD34 supopultions differs etween one mrrow, lood nd cord lood. Bone Mrrow Trnsplnt 1996, 17: Wng JCY, Doedens M, Dick JE. Primitive humn hemtopoietic cells re enriched in cord lood compred with dult one mrrow or moilized peripherl lood s mesured y the quntittive in vivo SCID-repopulting cell ssy. Blood 1997; 89: Almici C, Crlo-Stell C, Wgner JE et l. Biologic nd phenotypic nlysis of erly hemtopoietic progenitor cells in umilicl cord lood. Leukemi 1997; 11:

7 7 Reems JA, Torok-Sor B. Cell cycle nd functionl differences etween CD34 + /CD38 hi nd CD34 + /CD38 lo humn mrrow cells fter in vitro cytokine exposure. Blood 1995; 85: Terstppen LWMM, Hung S, Sfford M et l. Sequentil genertions of hemtopoietic colonies derived from single nonlinege committed progenitor cells. Blood 1991; 77: Civin CI, Almeid-pord G, Lee MJ et l. Sustined, retrnsplntle, multilinege engrftment of highly purified dult humn one mrrow stem cells in vivo. Blood 1996; 88: Ho Q-L, Shh AJ, Thiemnn FT et l. A functionl comprison of CD34 + CD38 cells in cord lood nd one mrrow. Blood 1995, 86: De Bruyn C, Delforge A, Bron D et l. Ex vivo expnsion of CD34 + CD38 cord lood cells. J Hemtother 1997; 6: Gordon MY, Dowding CR, Riley GP, Greves MF. Chrcteristion of strom-dependent lst colony forming cells in humn mrrow. J Cell Physiol 1987; 13: Gordon MY, Lewis JL, Grnd FH et l. Phenotype nd progeny of primitive dherent humn hemtopoietic progenitors. Leukemi 1996; 1: De Bruyn C, Delforge A, Bron D et l. Comprison of the coexpression of CD38, CD33 nd HLA-DR ntigens on CD34 + purified cells from humn cord lood nd one mrrow. Stem Cells 1995; 13: Srour EF, Bregni M, Trycoff CM et l. Long term hemtopoietic culture-inititing cells re more undnt in moilized peripherl lood grft thn in one mrrow ut hve more limited ex vivo expnsion potentil. Blood Cells Mol Dis 1996; 22: Weekx SFA, Vn Bockstele DR, Plum J et l. CD34 ++ CD38 nd CD34 + CD38 + humn hemtopoietic progenitors from fetl liver, cord lood nd dult one mrrow respond differently to hemtopoietic cytokines depending on the ontogenic source. Exp Hemtol 1998; 26: Mielcrek M, Torok-Stor B. Phenotype nd engrftment potentil of cytokine-moilized peripherl lood mononucler cells. Curr Opin Hemtol 1997; 4: Donhue RE, Kiry MR, Metzger ME et l. Peripherl lood CD34 + cells differ from one mrrow CD34 + cells in thy-1 expression nd cell cycle sttus in nonhumn primtes moilized or not moilized with grnulocyte colony-stimulting fctor nd/or stem cell fctor. Blood 1996; 87: Leitner A. Lck of DNA synthesis mong CD34 + cells in cord lood nd in cytokine-moilized lood. Br J Hemtol 1996; 92: Uchid N, He D, Frier AM et l. The unexpected G/G1 cell cycle sttus of moilized hemtopoietic stem cells from peripherl lood. Blood 1997; 89: Ymguchi M, Ikeuchi K, Hirym F et l. Different dhesive chrcteristics nd VLA-4 expression of CD34 + progenitors in G/G1 versus S+G2/M phses of the cell cycle. Blood 1998; 92: Croockewit AJ, Rymkers RA, Smeets ME et l. The low cycling sttus of moilized peripherl lood CD34 + cells is not restricted to the more primitive sufrction. Leukemi 1998; 12: Lemoli RM, Tfuri A et l. Cycling sttus of CD34 + cells moilized into peripherl lood of helthy donors y recominnt humn grnulocyte colony-stimulting fctor. Blood 1997; 89: Rtjczk MZ, Pletcher CH, Mrlicz W et l. CD34 +, kit +, rhodmine 123 low phenotype identifies mrrow cell popultion highly enriched for humn hemtopoietic stem cells. Leukemi 1998; 12: Doi H, In M, Ymmoto Y et l. Pluripotent hemtopoietic stem cells re c-kit low. Proc Ntl Acd Sci USA 1997; 94: Sogo S, In M, Ogt H et l. Induction of c-kit molecules on humn CD34 + /c-kit low cells: evidence for CD34 + /c-kit low cells s primitive hemtopoietic stem cells. Stem Cells 1997; 15: Ske H, Ohmizono Y, Tnimuki S et l. Functionl differences etween supopultions of moilized peripherl loodderived CD34 + cells expressing different levels of HLA-DR, CD33, CD38 nd c-kit ntigens. Stem Cells 1997; 15: Ske H, Yht N, Kimur T et l. Humn cord loodderived primitive progenitors re enriched in CD34 + c-kit cells: correltion etween long-term culture-inititing cells nd telomerse expression. Leukemi 1998; 12: Steen R, Tjonnfjord GE, Groseth GLA et l. Efflux of CD34 + cells from one mrrow to peripherl lood is selective in stedy-stte hemtopoiesis nd during G-CSF dministrtion. J Hemtother 1997; 6: Kroger N, Zeller W, Hssn HT et l. Difference etween expression of dhesion molecules on CD34 + cells from one mrrow nd G-CSF-stimulted peripherl lood. Stem Cells 1998; 16: Broudy VC. Stem cell fctor nd hemtopoiesis. Blood 1997; 9: D Aren G. Thy-1 (CDw9) nd c-kit receptor (CD117) expression on CD34 + hemtopoietic progenitor cells: five dimensionl flow cytometry study. Hemtologic 1998; 83: Kodm H, Nose M, Niid S et l. Involvement of the c-kit receptor in the dhesion of hemtopoietic stem cells to stroml cells. Exp Hemtol 1994; 22: Gothot A, vn der Loo JCM, Clpp DW, Srour EF. Cell cycle relted chnges in repopulting cpcity of humn moilized peripherl lood CD34 + cells in non-oese dietic/severe comined immune-deficient mice. Blood 1998; 92: Szilvssy SJ, Bss MJ, Vn Znt G, Grimes B. Orgn-selective homing defines engrftment kinetics of murine hemtopoietic stem cells nd is compromised y ex vivo expnsion. Blood 1999; 93: Alcorn MJ, Holyoke TL, Richmond L et l. CD34 positive cells isolted from cryopreserved peripherl lood progenitor cells cn e expnded ex vivo nd used for trnsplnttion with little or no toxicity. J Clin Oncol 1996; 14: Holyoke TL, Alcorn MJ, Richmond L et l. CD34 positive PBPC expnded ex vivo my not provide durle engrftment following myeloltive chemordiotherpy regimens. Bone Mrrow Trnsplnt 1997; 19: Stiff P, Oldenerg D, Hsi E et l. Trnsplnttion of ex vivo expnded cells grown in n Astrom stroml-sed perfusion iorector from smll mrrow liquots (4 ml) produces durle hemtopoietic reconstitution fter ltive chemotherpy. Blood 1997; 9 (Suppl. 1):