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CD34+ cells from placental/umbilical cord bloodBrazilian Journal of Medical and Biological Research (2005) 38: 1775-1789ISSN 0100-879X

I mmunophenotype of hematopoieticstem cells from placental/umbilical cordblood after culture

1Laboratório de Hematologia, Faculdade de Farmácia, 2Departamento de Genética,Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil3Faculdade de Farmácia, Pontifícia Universidade Católica do Rio Grande do Sul,Porto Alegre, RS, Brasil4Stem Cell Biology, 5Immunogenetics, New York Blood Center, New York, NY, USA

P. Pranke1,3,4, J. Hendrikx4,G. Debnath4, G. Alespeiti4,

P. Rubinstein5, N. Nardi2and J. Visser4

Abstract

Identification and enumeration of human hematopoietic stem cellsremain problematic, since in vitro and in vivo stem cell assays havedifferent outcomes. We determined if the altered expression of adhe-sion molecules during stem cell expansion could be a reason for thediscrepancy. CD34+CD38- and CD34+CD38+ cells from umbilicalcord blood were analyzed before and after culture with thrombopoietin(TPO), FLT-3 ligand (FL) and kit ligand (KL; or stem cell factor) indifferent combinations: TPO + FL + KL, TPO + FL and TPO, atconcentrations of 50 ng/mL each. Cells were immunophenotyped byfour-color fluorescence using antibodies against CD11c, CD31, CD49e,CD61, CD62L, CD117, and HLA-DR. Low-density cord blood con-tained 1.4 ± 0.9% CD34+ cells, 2.6 ± 2.1% of which were CD38-negative. CD34+ cells were isolated using immuno-magnetic beadsand cultured for up to 7 days. The TPO + FL + KL combinationpresented the best condition for maintenance of stem cells. The totalcell number increased 4.3 ± 1.8-fold, but the number of viable CD34+

cells decreased by 46 ± 25%. On the other hand, the fraction ofCD34+CD38- cells became 52.0 ± 29% of all CD34+ cells. Theabsolute number of CD34+CD38- cells was expanded on average 15 ±12-fold when CD34+ cells were cultured with TPO + FL + KL for 7days. The expression of CD62L, HLA-DR and CD117 was modulatedafter culture, particularly with TPO + FL + KL, explaining differencesbetween the adhesion and engraftment of primary and cultured candi-date stem cells. We conclude that culture of CD34+ cells with TPO +FL + KL results in a significant increase in the number of candidatestem cells with the CD34+CD38- phenotype.

CorrespondenceP. Pranke

Laboratório de Hematologia

Faculdade de Farmácia, UFRGS

Av. Ipiranga, 2752

90160-000 Porto Alegre, RS

Brasil

Fax: +55-51-3316-5437

E-mail: ppranke@adufrgs.ufrgs.br

Research supported by New York

Blood Center, New York, USA, and

CAPES.

Received January 21, 2005

Accepted August 15, 2005

Key words• Hematopoietic stem cells• CD34+CD38- cells• Human umbilical cord

blood• Ex vivo expansion• Adhesion molecules

Introduction

A small population of primitive hemato-poietic stem cells (HSCs) is present in thebone marrow. These cells are defined bytheir ability to self-renew as well as to differ-entiate into committed progenitors of the

different myeloid and lymphoid compart-ments generating all of the blood cell lin-eages (1). The complexity of this system isenormous, since as many as 1010 erythro-cytes and 108-1010 white blood cells are pro-duced each hour each day during the lifetimeof the individual. Over the past 15 years,

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human umbilical cord blood (HUCB) hasbeen clinically investigated as an alternativesource of HSCs for allogeneic transplanta-tion of patients lacking a human leukocyteantigen-matched marrow donor. However,the number of HSCs in HUCB samples islimited. Identification of conditions that sup-port the self-renewal and expansion of hu-man HSCs remains a major goal of experi-mental and clinical hematology. Expansionof human stem cells in ex vivo culture willlikely have important applications in trans-plantation, stem cell marking, and gene thera-py (2). The CD34+ protein is a surface glyco-protein expressed on HSCs and progenitorcells in early developmental stages in HUCBand bone marrow, as well as on endothelialcells. The CD34+CD38- immunophenotypedefines a primitive subpopulation of pro-genitor cells in fetal liver and fetal or adultbone marrow (3-5). About 1% of bone mar-row cells express CD34, and generally lessthan 1% of these cells are CD38-negative.Hence, the frequency of the CD34+CD38-

population is about 1 in 10,000, or evenlower. Phenotypic analyses of several cellsurface markers reveal that even this rarepopulation is heterogeneous (6). Ex vivoculture is a crucial component of severalclinical applications of stem/progenitor cells.A single stem cell has been proposed to becapable of more than 50 cell divisions ordoublings in vivo and as such has the capac-ity to generate up to 1015 cells, or sufficientcells for up to 60 years. The proliferation anddifferentiation of cells is controlled by agroup of hematopoietic growth factors. Rep-lication of this enormous cell amplificationwith hematopoietic growth factors in vitrowould allow the generation of large numbersof cells that could be used for a variety ofclinical applications (7).

Several culture systems have been devel-oped to expand HSCs (5). Piacibello et al. (1)reported the differential ability of FLT-3 ligand(FL), thrombopoietin (TPO), kit ligand (KL),and interleukin-3 (IL-3), alone or combined,

to support different stages of hematopoiesis inlong-term stroma-free suspension cultures ofHUCB CD34+ cells. Several studies have de-scribed the effects of TPO alone in culture,which can stimulate early proliferation, sur-vival or differentiation of progenitor cells incord blood or bone marrow (8).

The proliferation and differentiation ofHSCs are controlled not only by solublegrowth factors, but also by adhesion to stro-mal cells and matrix molecules. The expres-sion of adhesion molecules has attractedspecial attention, as their expression on HSCsand on endothelial and stromal cells plays apivotal role in the process (9). These mol-ecules permit the interaction with variousregulatory elements present in the microen-vironment, which includes stromal cells,extracellular matrix molecules and solubleregulatory factors such as cytokines andgrowth/differentiation factors (10).

Adhesion molecules include integrins,selectins and molecules from the immuno-globulin superfamily.

The objective of the present study was toinvestigate the behavior of umbilical cordblood CD34+CD38+ and CD34+CD38- cellscultured with different combinations ofgrowth factors, with respect to their viabil-ity, immunophenotype and self-renewal anddifferentiation capacities. Adhesion mol-ecules representing the integrins (CD11c orintegrin α-chain, CD49e or α-5 chain andCD61 or ß-3 chain), selectins (CD62L orLECAM-1) and the immunoglobulin super-family (CD31 or PECAM-1) were analyzed.The expression of HLA-DR and CD117 (c-kit or stem cell factor receptor), which repre-sent differentiation markers for CD34+ cells,was also investigated.

Material and Methods

Human umbilical cord blood cells

A total of 27 cord blood samples wereused in this study. Umbilical cord blood

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samples obtained after deliveries (≥29 weeks)were collected in sterile bags containing ci-trate-phosphate-dextrose. Samples were ob-tained at the Umbilical Cord Blood Bank ofthe New York Blood Center (New York,NY, USA). Blood was collected accordingto an Institutional Review Board-approvedprotocol. Units that are not used in the Pla-cental Blood Program are destined to re-search. Since collection is performed on de-livered placentas, the blood is considereddiscarded tissue. Blood not used for clinicaltransplants is not identified and is used with-out informed consent.

Isolation of CD34+ cells

Low-density mononuclear cells (MNCs)were isolated using density gradient cen-trifugation on Ficoll-Paque 1.077 g/cm2

(Amersham Pharmacia, Piscataway, NJ,USA), modified by the addition of 1 Mphosphate buffer, pH 7.6, to the Dulbecco’sphosphate-buffered saline (GibcoBRL, Gai-thersburg, MD, USA) used to dilute the blood.This modification improved the isolation ofthe mononuclear fraction, since the harvestedcell population contained 50% less reticulo-cytes and less than 50 to 60% erythrocytes.CD34+ cells were harvested from the MNCsusing automated magnetic cell sorting(MACS) High Gradient Magnetic Separa-tion Columns for positive selection (MiltenyiBiotec, Bergisch Gladbach, North Rhine-Westphalia, Germany). The magneticallylabeled cells were enriched by passing themtwice through positive selection columns.

Antibodies

For the analysis of CD34+ cells, mono-clonal antibodies (PharMingen/Becton Dick-inson, San Jose, CA, USA) specific for thefollowing human antigens were used: CD34/FITC (clone 34374X lot MO46959), CD38/APC (clone HIT2), CD11c/PE (clone B-IY6), CD31/PE (clone WM59), CD49e/PE

(clone IIA1), CD61/PE (clone VI-PL2),CD62L/PE (clone Dreg 56), CD117/PE(clone YB5.B8), and HLA-DR/PE (cloneG46-6), as well as isotype control antibodies(clones MOPC-21): mouse IgG1,k/FITC,IgG1,k/PE, and IgG1,k/APC.

Flow cytometry analyses

Processing for four-color fluorescenceflow cytometry was done within 36 h ofcollection using at least 10,000 CD34+ cells,before culture and after 4 and 7 days ofculture. Cells were incubated with anti-CD34/FITC and anti-CD38/APC antibodies com-bined with PE-conjugated antibodies specif-ic for CD11c, CD31, CD49e, CD61, CD62L,CD117, or HLA-DR. All incubations weredone for 30 min at 4ºC, and cells were washedwith phosphate-bufered saline. 7-Aminoac-tinomycin D (Molecular Probes, Inc., Eu-gene, OR, USA) at a final concentration of 1µg/mL was used to identify dead cells. Flowcytometry was performed on a FACScaliburinstrument (Becton Dickinson) equipped withan argon-ion laser tuned at 488 nm. TheCELLQuest software (Becton Dickinson)was used for data analysis. Between 5,000and 50,000 events were collected for eachanalysis. The gating strategy used can besummarized as follows. First, viable cellswere gated, followed by a gating of the cellcluster in forward and side scatter, and usingthe FITC channel of CD34+ cells. Amongthe CD34+ cells, CD38-negative and CD38-positive cells were gated and the frequencyof cells positive for the third antibody wasanalyzed.

Analysis of the cell frequency among thedifferent populations was done using theHendrikx and Pranke Plot program (data notshown) (11), a novel method to facilitatevisualization of complex flow cytometry datasets across four dimensions in just a fewgraphs. In short, samples are divided intoclusters, and the mean fluorescence of thecluster versus the frequency of the cluster is

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plotted per cluster. In addition, data and cellsuspension are shown as third and fourthdimensions, identified as symbol shape andsymbol color.

Ex-vivo expansion cultures

MACS-isolated CD34+ cells were cul-tured in 24-well plates (Multiwell™ TissueCulture Plate, Becton Dickinson) in 2 mLIscove’s modified Dulbecco’s medium withL-glutamine and 25 mM HEPES buffer(GibcoBRL), supplemented with hydro-cortisone (10-5 M, Sigma, St. Louis, MO,USA), 2-mercaptoethanol (5.5 x 10-5 M,GibcoBRL), penicillin G (100 units/mL,GibcoBRL), streptomycin (0.1 mg/mL,

Figure 1. Effect of different com-binations of growth factors oncell proliferation after 4 and 7days in culture. Sixteen differentunits of cord blood were used,and samples 2, 3, 4 and 11 weresplit into two different cultures (aand b), using 2 combinations ofgrowth factors. TPO = thrombo-poietin; FL = FLT-3 ligand; KL =kit ligand.

Table 1. Volume of the umbilical cord blood samples and frequency of mononuclearand CD34+ cells obtained after the isolation on Ficoll-Paque and automated magneticcell sorting columns, respectively.

Mean ± SD Range

Blood volume (mL) 45.2 ± 5.6 32-57MNC (x 107) 10.4 ± 5.7 2.5-23.0MNC (/mm3 blood) 2287 ± 1132 532-4510CD34+ cells (x 106) 1.3 ± 0.8 0.3-4.0CD34+ cells (/mm3 blood) 28.0 ± 16.7 6.4-85.0% CD34+ cells among MNCs 1.4 ± 0.9 0.4-4.9

Data are reported as mean ± SD for N = 27. MNC = mononuclear cells.

GibcoBRL) and 1% bovine serum albumin(Sigma). Cell concentrations varied between2.5 and 5.5 x 105/well. Human growth fac-tors used were: TPO (Kirin Brewery, KirinBrewery, Chuo-ku, Tokyo, Japan), FL(Amgen Inc., Thousand Oaks, CA, USA)and KL (Amgen Inc.), also called steel factoror stem cell factor, at concentrations of 50ng/mL each. The combination of hemato-poietic growth factors used were: TPO + FL+ KL; TPO + FL and TPO. The cultures weremaintained at 37ºC and 5% CO2 in a humidi-fied atmosphere and analyzed on days 4and 7.

Results

CD34+ cells were isolated from a total of27 cord blood samples, and the number ofMNCs as well as CD34+ cells was analyzedin a Neubauer chamber. As shown in Table1, the concentration of CD34+ cells was 28cells/mm3 of cord blood (yield after Ficolland MACS procedure). The purity of thiscell fraction was 93.2 ± 3.6% (87-99%; N =10). Since 22.2 ± 11.3% (12.5-50.6%) of theCD34+ cells were dead, the average fre-quency of viable CD34+ among total cellswas 71.0 ± 13.1% (39.4-86.5%).

CD34+ cells were cultured with three

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combinations of growth factors (Figure 1).With TPO + FL + KL, the average increasein cell number in 9 samples studied was 3.55± 1.6-fold after 7 days of culture. Only 1sample (3.a) showed a 0.56-fold decrease incell number on day 4, but by day 7 thenumber of the cells in this sample had in-creased. The observation of individual cul-tures (Figure 1) showed some heterogeneityamong samples, particularly when TPO +FL was used. With this combination ofgrowth factors, the total number of cellschanged very little until day 4. From day 4until day 7 of culture, however, in one samplethe cell number decreased 0.65-fold, in 2samples it showed no major variation, and inthe remaining 2 a considerable increase (2.57± 0.5-fold) in total cell number was ob-served. Considering the total culture period,in 2 samples the cell number decreased 0.59± 0.11-fold and in 3 of them it increased 1.92± 0.56-fold. In all 6 samples cultured withTPO, the total cell number decreased duringthe first 4 days. After that, in 4 of them therewas an increase, not enough, however, toreach the original number. Taken as a whole,cell numbers decreased 0.55 ± 0.26-fold withTPO only.

Further analyses were done in 10 of thesamples. Three of them were separated intotwo cultures submitted to different treat-

ments. The analysis of cell viability duringthe period of culture (Table 2) showed that,for cultures in the presence of TPO + FL +KL, the absolute number of viable cells in-creased 4.27 ± 1.82-fold in the 4 samplesstudied. In one sample (3.a) the number ofviable cells decreased 0.5-fold from day 0 today 4, but increased 3.4-fold from the 4th tothe 7th day. In cultures with TPO + FL, thenumber of viable cells increased 1.94 ± 0.56-fold in 3 samples, and decreased 0.76-fold inone sample. In the 5 samples cultured withTPO, the number of viable cells decreased0.35 ± 0.28-fold.

The viability of CD34+ cells was increasedafter culture with TPO + FL + KL and withTPO + FL, and maintained in the presence ofTPO, but the frequency of viable CD34+

cells among total viable cells decreased(Table 2). From day 0 to day 7 of culturewith TPO + FL + KL, the number of viableCD34+ cells decreased 0.46 ± 0.25-fold inthe 4 samples studied, whereas with TPO +FL and TPO alone this decrease was 0.30 ±0.12- and 0.13 ± 0.08-fold, respectively.

Of particular interest are the results re-garding the frequency of CD34+CD38- cellsduring the culture period. As shown in Table2, the number of viable CD34+CD38- cellsincreased in some of the culture conditions.With TPO + FL + KL, this increase was

Table 2. Total and CD34+ cell viability and frequency of CD34+ and CD34+CD38- cells on day 0 and afterculture with three different combinations of growth factors.

TPO + FL + KL (N = 4) TPO + FL (N = 4) TPO (N = 5)

Day 0 Day 4 Day 7 Day 4 Day 7 Day 4 Day 7

Total cell viability 76.1±13.0 81.7±6.0 78.6±5.8 68.9±17.6 72.2±8.1 54.2±14.9 52.9±13.0 (N = 10)

CD34+ cell viability 76.0±12.7 90.2±4.0 92.3±5.9 83.2±7.5 87.7±7.0 78.6±11.0 79.8±14.0 (N = 10)

Viable CD34+ cells 93.3±2.1 35.0±23.3 10.9±5.0 63.9±16.9 23.9±22.0 80.7±7.0 41.8±21.1among viable cells (N = 10)

Viable CD38- cells 2.6±2.1 16.0±19.9 52.0±28.8 1.6±0.9 9.1±8.6 1.8±1.6 2.5±2.4among CD34+ cells (N = 8)

Data are reported as % ± SD. TPO = thrombopoietin; FL = FLT-3 ligand; KL = kit ligand.

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14.59 ± 11.81-fold in 3 samples. In onesample, cell numbers were not determinedon day 0, but from day 4 to day 7 the numberof viable CD34+CD38- cells increased 4.27-fold. In one sample (3.a), the number ofCD34+CD38- cells decreased 0.23-fold fromday 0 to day 4, but increased 23-fold untilday 7. For cultures with TPO + FL, thesecells increased 2.79 ± 2.29-fold in the 3samples studied from day 0 to day 7 and inone sample for which the analysis was notdone on day 0, they increased 1.22-fold fromday 4 to day 7. In the presence of TPO,however, the number of viable CD34+CD38-

cells decreased 0.26 ± 0.31-fold in 5 samples.The expansion and proliferation of

CD34+CD38- cells were significantly in-creased in the presence of TPO + FL + KL ascompared to the other two combinations ofgrowth factors. An interesting correlationcan be seen between this effect and totalincrease in cell numbers, which is directlyproportional to the increase or decrease inCD34+CD38- cells (Figures 1 and 2). WhenTPO + FL + KL were used, in 4 samples,total cell numbers as well as CD34+CD38-

cells were gradually increased during cul-ture. In Figure 1, it can be seen that total cell

numbers increased from the first to the fourthday in 8 of 9 samples cultured in the pres-ence of TPO + FL + KL. Only one sample(3.a) showed a 0.6-fold decrease in cell num-ber until the fourth day in culture, but thisnumber increased around 3.4-fold from day4 to day 7. The same pattern was observedfor CD34+CD38- cells (Figure 2), as well asin cultures where TPO + FL or TPO alonewere used as growth factors. On the otherhand, no correlation was observed betweenthe initial number of CD34+CD38- cells andtotal cell growth or with the expansion of theCD34+CD38- cells. Similarly, no correlationwas detected between the initial number ofviable CD34+ cells and cell growth or num-ber of CD34+CD38- cells (results not shown).

The immunophenotypic profile of freshlyisolated CD34+CD38+ and CD34+CD38- cellswas also investigated. In 7 of 8 samplesanalyzed, the number of CD34+CD38+ cellspositive for CD11c was low (less than 20%)and the fluorescence was dim or, alterna-tively, all cells were negative. Only onesample showed around 45% of positive cellswith dim fluorescence. The CD34+CD38-

population could be analyzed in only 5samples due to low event numbers. The

Figure 2. Number of viableCD38- cells among viableCD34+ cells in culture. Note thelogarithmic scale on the ordi-nate. ND = not determined; TPO= thrombopoietin; FL = FLT-3ligand; KL = kit ligand.

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samples presented the same predominantpattern as observed for CD34+CD38+ cells.

All 8 samples showed 100% of the cellspositive for CD31, with a bright fluores-cence, and about 70-100% of the cells withregular fluorescence for CD49e. For CD61,however, among CD34+CD38+ cells eitherthe number of positive cells was low (lessthan 15%), with regular fluorescence, or100% of the cells were negative. In one ofthe samples, the positive population con-tained a small (around 3%) subpopulation ofbright cells. Because the number of the eventswas low in CD34+CD38- cells, we couldanalyze only 4 of 8 samples.

Cells were heterogeneous for CD62Lexpression. About 43 ± 17 and 27 ± 17% ofCD34+CD38+ and CD34+CD38- cells, re-spectively, were positive with a regular meanfluorescence intensity. In one sample, a sec-ond population of around 20%, amongCD34+CD38+ cells, presented bright fluo-rescence for CD62L. In 2 samples the num-ber of events among CD34+CD38- cells wastoo low to be analyzed.

The pattern for HLA-DR was very het-erogeneous among samples. This marker waspresent in about 54 ± 28 and 34 ± 31% ofCD34+CD38+ and CD34+CD38- cells, re-spectively, with a regular mean fluorescenceintensity. In one of the samples, a smallpopulation of the bright cells was also seenamong the CD34+CD38+ population, whereasin another two populations, very few posi-tive cells with dim and bright cells wereobserved.

The fluorescence pattern observed forCD117 was complex. Among CD34+CD38+

cells, two clusters could be observed, onewith a high frequency of cells (80 ± 10%,range: 59-91%) with regular fluorescenceand another composed of few cells (6 ± 5%)with bright or very bright fluorescence.Among the CD34+CD38- cells, 56 ± 24%presented regular mean fluorescence inten-sity, with no bright cells observed.

The immunophenotypic profile of um-

bilical cord blood CD34+ cells was analyzedafter 4 and 7 days of culture with TPO + FL+ KL, TPO + FL and TPO. In some of thesamples, particularly among CD34+CD38-

cells, the analysis was not possible due tovery small numbers of events. Culture withTPO + FL or TPO alone was also a factorwhich decreased the cell number to a levelbelow meaningful analysis in some cases.

The patterns for CD11c, CD31, CD49e,and CD61 between CD34+CD38+ andCD34+CD38- cells were not modified afterthe culture in all combinations of growthfactors (data not shown). For CD62L, how-ever, the number of positive cells and thefluorescence intensity increased from day 0to day 4 and again from day 4 to day 7 in allculture conditions (Figure 3). In only onesample, cultured with TPO alone, did thenumber of positive cells increase from day 0to day 4 but it decreased a little from day 4 today 7 (data not shown).

In cultures with TPO + FL + KL, thenumber of cells positive for HLA-DR andthe intensity of fluorescence increased fromthe day 0 to day 4, but decreased a little fromday 4 to day 7 between CD34+CD38+ andCD34+CD38- cells (Figure 3). In cultureswith TPO + FL, the reactivity pattern did notchange among CD34+CD38+ cells, exceptfor one sample in which the number of posi-tive cells increased from day 0 to day 4 butremained unaltered until the end of the cul-ture. Similar patterns were observed inCD34+CD38- cells, but the number of eventswas very low. The results were heteroge-neous in cells cultured with TPO alone (datanot shown). Among CD34+CD38+ cells, thenumber of positive cells increased signifi-cantly in 3 samples, whereas in the remain-ing 2 no changes were observed. Two of the4 samples analyzed for CD34+CD38- cellsremained the same and in 2 the number ofHLA-positive cells increased.

Culture with TPO + FL or TPO alonereduced the number of cells brightly positivefor CD117 among CD34+CD38+ cells in

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comparison with day 0. The presence ofTPO + FL + KL, however, induced a de-crease in CD117-positive cells (Figure 3).Among CD34+CD38- cells, the results weremore heterogeneous. After culture with TPO+ FL + KL, a cluster of few cells with dim orregular fluorescence could be observed, butno bright cells. The number of positive cellsincreased after culture with TPO + FL but nobright cells were seen. When TPO alone wasused, two clusters were observed: a highnumber of cells with mean fluorescencearound 100 and another one with few cells ofmean fluorescence around 1000.

In some samples, after culture with TPO+ FL (samples 5 and 6) or TPO alone (samples9 and 3.b) the number of CD34+CD38- cellswas too low to analyze for CD117 reactivity.On day 0, the average frequency of CD117-negative cells was 44.2 ± 26.3% (N = 5).After culture with TPO + FL + KL, 84.5 ±11.8% of the cells were negative for CD117on day 4 (N = 3) and 96.0 ± 2.7% (N = 4) onday 7. After culture with TPO + FL, 11.3 ±

5.1 and 7.5 ± 9.2% of the CD34+CD38- cellswere CD117-negative on day 4 (N = 3) andday 7 (N = 2) respectively, whereas culturewith TPO resulted in 7.3 ± 2.1 and 7.5 ±6.4% CD117-negative cells on days 4 (N =3) and 7 (N = 2), respectively.

Discussion

Lack of CD38, HLA-DR and lineage-committed antigens, as well as the co-ex-pression of Thy-1 (CDw90) and c-kit recep-tor (CD117), have been shown to identifythe candidate hemopoietic stem cells (12).However, a better knowledge and standard-ization of the phenotype of umbilical cordblood CD34+ cells is critical since HUCBvolume is limited (13). The present studyaimed to contribute to the characterizationof CD34+ cells from umbilical cord blood,analyzing their phenotype and behavior be-fore and after culture with different combi-nations of growth factors.

The frequency of CD34+ cells among

Figure 3. Frequency of cellspositive for CD62L/PE, CD117/PE and HLA-DR/PE amongCD34+CD38+ and CD34+CD38-

cells on day 0 and after 7 days ofculture with TPO + FL + KL. TPO= thrombopoietin; FL = FLT-3ligand; KL = kit ligand. PE = fluo-rochrome phycoerythrin. M1 =isotype control; M2 = cells withregular fluorescence; M3 = cellswith bright fluorescence.

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HUCB MNCs in our study was 1.4% (0.4-4.9%), in agreement with other studies. Thisfrequency is similar to that in harvested pel-vic bone marrow (1.0 ± 0.3 vs 0.8 ± 0.4%)(14). About 1% of bone marrow cells ex-press CD34, and generally less than 1% ofthese cells are CD38-negative (6,11,15). Inother studies, Campagnoli et al. (16) showedthat the concentration of CD34+ cells in wholeblood samples in term fetal blood was 0.4 ±0.03% of total CD45+ cells, and Hao et al. (3)showed that the frequency of CD34+ cellsamong total MNCs in cord blood was 0.36 ±0.33% with a large variation among samples(range 0.02 to 1.43%; N = 30).

In the present study, we found a largevariation in the frequency of CD34+ cellsamong HUCB MNCs, from 0.4 to 4.9%.Although correlation between total nucle-ated cell and CD34+ cells in HUCB has beenreported, within groups of samples with simi-lar total nucleated cell counts a high degreeof variation (at times exceeding 10-fold) inCD34+ cells is observed. CD34 counts inHUCB can be as low as 0.1% of total nucle-ated cell as reported by Yap et al. (17), andD’Arena et al. (18) observed 0.01-1.71%CD34+ cells among HUCB cells. Differentexplanations have been given for the varia-bility found in the frequency of CD34+ cellsin HUCB. There is evidence that, althoughthe CD34 population frequency is a reliableindicator of the progenitor potential ofHUCB, it is nevertheless heterogeneous innature. On the other hand, these heteroge-neous results can reflect differences in thesensitivity of the methods employed by thedifferent groups. CD34+ HSCs have alsobeen shown to vary with gestational age,mode of delivery and positioning of the de-livered neonate after delivery. Yap et al. (17)found that CD34+ cells accounted for 5.1 ±1.0% of CD45+ cells in first trimester blood,significantly more than in term cord blood(0.4 ± 0.03%).

Controversial results have been publishedregarding the frequency of CD38- cells

among cord blood CD34+ cells. We foundthat 2.6 ± 2.1% (range 0.55-5.57) of theCD34+ cells were CD38-negative on day 0,which agrees with reports showing that mostCD34+ cells present the CD38 antigen inHUCB (4) and in mobilized peripheral bloodcells (19). Campagnoli et al. (16) reportedthat the percentage of CD34+ cells which areCD38- was 3.9 ± 0.9% (N = 5), whereasCD34+CD38- cells have been reported tocomprise 0.05 ± 0.08% of the MNCs presentin cord blood (3). Timeus et al. (9) observedthat the number of CD34+CD38- cells wassignificantly higher in cord blood than inbone marrow (16 ± 8.8 and 4.7 ± 3% of totalCD34+ cells, respectively). However, thenumber of CD38- cells among HUCB CD34+

cells was reported as 11% (18), or 34.9 ±3.4% (20).

Studies comparing the three differentcompartments have shown that the propor-tion of CD34+CD38- cells is greater in HUCBas compared to peripheral blood (9,21,22).This might explain the successful clinicaluse of HUCB even when a small number ofcells is used, making the presence of theseantigens candidate predictive parameters forclinical use of HUCB samples (13).

The frequency of viable CD34+ cells af-ter isolation was lower than that found, forinstance, in samples processed in cord bloodcenters. It is possible that the extensive ma-nipulation involved in the immunomagneticprocedure, not performed in cord blood cen-ters, decreases cell viability. It is known thatdifferent factors involved in the processingof hematopoietic cells, such as a 48-h delayin their analysis or freezing and thawinghave a negative impact on their biology (23).

Although cell numbers were higher afterculture, particularly in the presence of TPO+ FL + KL, and cell viability was increasedor did not show a difference, the number ofCD34+ cells showed a marked decrease.These results indicate that, during culture, aproportion of stem cells differentiate andlose the CD34 surface marker. In our study

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we used three different combination of he-matopoietic growth factors. TPO is a pri-mary regulator of megakaryocyte and plate-let production and might also play an impor-tant role in early hematopoiesis (24). It is animportant cytokine in the early proliferationof human primitive as well as committedhematopoietic progenitors, and in the ex vivomanipulation of human hematopoietic pro-genitors (9). TPO has also been observed tosuppress apoptosis of CD34+CD38- cells inculture, showing a potential role in main-taining quiescent primitive human progeni-tor cells viable (25). Liu et al. (26) used acombination of growth factors with and with-out TPO and showed a significant expansionof CD34+ cells from HUCB and neonatalblood to early and committed progenitors, inthe presence of this factor. This potentialrole of TPO in the early hematopoietic dif-ferentiation was explored in the present study,in which TPO was used in all combinationsof growth factors.

FL, a class III tyrosine kinase receptorexpressed on primitive human and murinehematopoietic progenitor cells, is able toinduce proliferation of CD34+CD38- cellsthat are non-responsive to other early actingcytokines and to improve the maintenanceof progenitors in vitro (5). The expansion ofnonadherent cells from umbilical cord blood,for instance, has been shown to be greaterwith TPO + FL + KL than TPO + FL, andgreater in this combination than with TPOalone. Similarly, the expansion of CD34+

CD38- was greater with TPO + FL than withTPO alone, and the percentage of CD34+

cells was greater with TPO + FL than withTPO + FL + KL (1). Our data agree withthese results, since the absolute number ofCD34+CD38- cells increased considerablywhen TPO, FL and KL were used as growthfactors. The number of those cells increasedin a few samples when we used TPO and FLand decreased when we used just TPO. It hasalready been shown that, although TPO alonecan stimulate limited clonal growth, it

synergizes with the KL, FL, or IL-3 to po-tently enhance clonogenic growth. Ramsfjellet al. (24) showed that whereas KL and FL incombination stimulate the clonal growth ofonly 3% of CD34+CD38- cells, 40% of thosecells are recruited by TPO + FL + KL, dem-onstrating that TPO promotes the growth ofa large fraction of CD34+CD38- progenitorcells.

An interesting correlation can be madebetween the number of CD34+CD38- cellsand total increase in cell numbers, whichwas directly proportional to the increase ordecrease in CD34+CD38- cells. When TPO+ FL + KL were used total cell numbers aswell as CD34+CD38- cells presented a gradualincrease during culture. These results indi-cate that self-renewal and differentiation ofCD34+CD38- cells were significantly in-creased in the presence of TPO + FL + KL ascompared to the other two combinations ofgrowth factors.

Finally, it is interesting to observe that,although an increase in total cell counts andin CD34+CD38- cell number was induced bythe growth factors, particularly in the TPO +FL + KL combination, this increase wassmaller than reported in other studies (1). Inthose studies, as well as in several otherones, however, cells were cultured with fetalcalf serum or pooled human serum, whereaswe employed serum-free media. We believethat serum-free medium allows a better con-trol of the role that individual cytokines andtheir combination have on cell growth anddifferentiation. Furthermore, many of thesestudies analyze the behavior of the cells inlong-term culture, whereas in the presentstudy cells were cultured for only one week.Our aim was to expand CD34+CD38- cellswithin a short period of time to use theexpanded population for transplants.

We investigated the expression of sev-eral cell adhesion molecules and other pro-teins among CD34+CD38+ and CD34+CD38-

cells in umbilical cord blood, before andafter culture with TPO + FL + KL, TPO + FL

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or TPO. Adhesion molecules play a role inthe migration of hematopoietic progenitorcells and in the regulation of hematopoiesis.Cell adhesion molecules are highly expressedin both HUCB and bone marrow CD34+

CD38- cells. It has been shown that mol-ecules such as CD11a and CD62L are moreexpressed in HUCB than in the bone marrowCD34+CD38- subset, suggesting a possibleadvantage in homing and engraftment ofmore undifferentiated HUCB as opposed tobone marrow HSCs (27).

The expression of CD11c on HUCBCD34+ cells in fresh samples was rare, asalready reported for bone marrow (28) andHUCB (6) CD34+ cells. This adhesion mol-ecule has a role in the linkage to receptors onstimulated endothelium (Nancy Hogg,www.ncbi.nlm.nih.gov/prow). PECAM-1expression was observed on all CD34+ cells,with high fluorescence, in all samples. Otherreports have also shown high expression ofCD31 on bone marrow (28) and cord blood(6) CD34+ cells. This molecule is involvedin the adhesion between cells such as endo-thelial cells and leukocytes (Muller WA,www.ncbi.nlm.nih.gov/prow), as well as inthe interaction between hematopoietic cellsand extracellular matrix components in bonemarrow. CD11c and CD31 were homoge-neously expressed, presenting the same pat-tern among CD34+ CD38+ or CD34+CD38-

before and after culture.We found a large number of CD34+

CD38+ and CD34+CD38- cells positive forCD49e in all samples both before and afterculture. This molecule corresponds to the α-chain of the VLA-5 integrin, and is stronglyinvolved in the binding of bone marrowprogenitor cells to extracellular matrix com-ponents (29). It is interesting, however, thatdifferent studies report conflicting results.Asosingh et al. (28) showed that all CD34+

cells in normal bone marrow expressedCD49e, while cord blood and mobilizedCD34+ cells had a lower expression of thismolecule than bone marrow CD34+ cells.

On the other hand, Timeus et al. (9) showedgreater expression of this molecule on CD34+

of cord blood than bone marrow. In otherstudies, cord blood CD34+ cells have beenreported to express VLA-5 in a pattern re-markably similar to that of bone marrowCD34+ cells. Denning-Kendall et al. (30)showed that the expression of VLA-5 onCD34+ and CD34+CD38- cells increased af-ter 7 days of culture with KL, FL, TPO, andG-CSF.

CD61 has been observed in small levelson HUCB CD34+ cells: less than 20% (13) or20.2 ± 16.1% (4). In our study, the expres-sion of this antigen on HUCB CD34+ cellswas also rare in CD34+CD38+ or CD34+

CD38- cells before and after culture. L-se-lectin is involved in the homing of CD34+

cells after peripheral blood MNC transplan-tation. The majority of the CD34+ cells hadCD62L on the membrane surface. HUCBand mobilized blood CD34+ have been shownto present a higher expression of CD62Lthan bone marrow CD34+ cells (28). CD62Lwas also more frequently expressed in theHUCB than in the bone marrow CD34+CD38-

subset, suggesting a possible advantage inhoming and engraftment (9,27). In the pres-ent study, CD62L expression was heteroge-neous, and the CD34+CD38+ population pre-sented a slightly higher frequency of posi-tive cells. Surbek et al. (31) showed thatCD62L on CD34+ stem and progenitor cellsin umbilical cord blood change during gesta-tion. This could explain the great variabilityin the frequency of CD62L-positive cellsobserved in different samples, since in thepresent study the gestational age presented arange from 29 weeks to term.

An interesting effect was observed forthe expression of CD62L after culture. Thenumber of CD62L-positive CD34+CD38-

cells and the CD62L expression on thesecells increased during culture in all condi-tions. Denning-Kendall et al. (30) have alsofound increased expression of L-selectin (orCD62L) on CD34+ and CD34+CD38- cells

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after 7 days of culture with KL, FL, TPO,and G-CSF.

Timeus et al. (9) have shown that a shortexposure to cytokines increases L-selectinexpression in the more differentiated he-matopoietic progenitors, CD34+CD38+ cells,which could improve their homing in a trans-plant setting. After transplantation of HSCs,adhesion molecules play a major role in themultistep process of engraftment in whichL-selectin is suggested to be of relevance.Gigant et al. (32) showed a higher frequencyof CD62L-positive cells in peripheral bloodthan in bone marrow or cord blood, andYoung et al. (33) reported increased expres-sion of CD62L expression on mobilized pe-ripheral blood CD34+ cells cultured withTPO + FL + KL. The present study showeda significant increase of L-selectin-positivecells, suggesting improved homing ability inHUCB cultured with growth factors in allcombinations.

There is evidence that cord blood, bonemarrow and peripheral blood-derived HSCsare highly heterogeneous for a number ofantigens useful for HSC enumeration byflow cytometry (6). HLA-DR is expressed inthe majority of HUCB (4,13) and peripheralblood (13,19) CD34+ cells. While De Bruynet al. (21) showed that the co-expression ofCD34 with HLA-DR was not significantlydifferent in HUCB and bone marrow (86.3 ±2.7 and 92.7 ± 5.1%, respectively), Cho et al.(22) showed that CD34+HLA-DR+ cell fre-quencies did not differ significantly betweenthose two compartments and MNCs. Veryheterogeneous results were found for HLA-DR in the present study which, due to thesmall number of cells in some of the experi-mental conditions, made their interpretationdifficult. The great heterogeneity of positivecells in fresh samples, as well as small differ-ences after culture, could be explained bydifferent gestational ages. Fetal liver cells,for instance, have been shown to presentlower proportions of CD34+HLA-DR+ thanHUCB, showing that the composition of

fetal leukocytes changes during develop-ment and with gestational age (34). Thefrequency of HLA-DR-positive cells was alittle higher among CD34+CD38+ thanCD34+CD38- cells. This result supports theconclusion that these molecules are moreexpressed in more differentiated cells.

The expression of c-kit (CD117) onCD34+CD38+ cells separated the cells intotwo populations in all samples, with morethan 60% of the cells showing regular fluo-rescence, and a small population of bright orvery bright cells. In this way, three fractionswere described: negative cells, cells withregular fluorescence, and cells with highfluorescence. Among CD34+CD38- cells,however, the frequency of c-kit-positive cellswas slightly lower and bright cells were notobserved. Although the number of samplesin the present study was relatively small, theresults show that, after culture with TPO +FL + KL, the number of CD34+CD38-CD117-

cells increased. This number decreased whenwe used TPO + FL or TPO alone. Culture ofCD34+ cells with TPO + FL + KL thus signifi-cantly increases the number of candidate stemcells with the CD34+CD38- (c-kit-) pheno-type. On the other hand, the down-regula-tion of c-kit may be due to the presence ofKL in the growth factor combination, sincethis factor was essential to expand CD34+

CD38- cells.The most primitive human hematopoi-

etic progenitor cells have demonstrated co-expression of c-kit, FLT-3 and Thy-1, beingnegative for HLA-DR, CD38 and lineagemarkers (12,24). CD117 expression has thusbeen reported to characterize true stem cells(35). The c-kit receptor, a member of the Igsuperfamily of adhesion molecules, is in-volved in the interactions of CD34+ cellswith other cells and stroma in bone marrow,mobilized peripheral blood and HUCB. Thec-kit receptor was detected on the majorityof CD34+ HSCs, particularly on HUCB inthe studies of D’Arena et al. (12). On theother hand, Sakabe et al. (19) showed that

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the expression of the c-kit receptor on mobi-lized peripheral blood CD34+ cells was ap-proximately 20% of that on bone marrow- orHUCB-derived CD34+ cells which expresshigh levels of c-kit receptor.

Studies about the expression of c-kit onmobilized peripheral blood CD34+ cellsshowed three fractions, namely CD34+c-kithigh, CD34+c-kitlow and CD34+c-kit- cells(19). Different levels of CD117 antigen werealso shown in HUCB. While Gunji et al. (36)demonstrated that myeloid progenitors areenriched in CD34+c-kithigh cells and eryth-roid progenitors are more enriched in CD34+

c-kitlow cells, Sakabe et al. (19) showed thaterythroid progenitors are highly enriched inmobilized peripheral blood CD34+c-kithigh

cells, and that CFU-GM is enriched in mobi-lized peripheral blood CD34+c-kit- cells.Primitive progenitors with self-renewal po-tential may be present in the mobilized pe-ripheral blood CD34+c-kit- or CD34+c-kit low

cell population. Laver et al. (37) reportedthat the HUCB-derived CD34+c-kitlow cellpopulation contains the majority of quies-cent progenitors and blast cell colony form-ing cells. Thus, the CD34+CD38- or CD34+c-kit- or low immunophenotype defines primi-tive progenitor cells in fetal liver, fetal bonemarrow, adult bone marrow, mobilized pe-ripheral blood, and HUCB (19). The expres-sion of c-kit may therefore be useful in iden-tifying HUCB progenitors with long-termengraftment capability (37).

HUCB has recently been explored as analternative HSC source for allogeneic trans-plantation in both adults and pediatric pa-tients with hematological malignancies andmarrow failure syndromes. HUCB transplan-tation is particularly important in patientswho lack HLA-matched marrow donors,permitting the use of HLA-mismatched graftsat 1-2 loci (or antigens) without higher riskfor severe graft-versus-host disease relativeto bone marrow transplantation from a full-matched unrelated donor (38). Since the firstcase reported in 1988, more than 3700 pa-

tients have received HUCB transplants for avariety of malignant and non-malignant dis-eases. Due to the relatively low number ofstem cells in HUCB, limited by the bloodvolume which can be collected, the vastmajority of recipients (2/3) were childrenwith an average weight of 20 kg(www.netcord.org). The ex vivo expansionof HSCs thus represents an attractive ap-proach to overcoming the current limitationsregarding adult HUCB transplantation.

CD34 selection and ex vivo expansion ofHUCB prior to transplantation are feasible.Serum-free media, in some cases with com-plementation of growth factors such as TPO,KL and FL (39,40), have been shown toallow the expansion and transplantation ofHSCs. The ex vivo expansion of HUCB he-matopoietic stem and progenitor cells hasbeen shown, for instance, to increase celldose and reduce the severity and duration ofneutropenia and thrombocytopenia aftertransplantation. Additional accrual, however,will be required to assess the clinical effi-cacy of expanded HUCB progenitors. Somestudies have shown that the ex vivo expan-sion of cord blood CD34+ cells results in thegeneration of increased mature cells andprogenitors that are capable of more rapidengraftment in fetal sheep compared tounexpanded HUCB CD34+ cells (39). Enu-meration of CD34+CD38- cells is correlatedwith the number of committed progenitorsand the capacity of generating CD34+ cells.This is an important parameter if expansionprotocols are to be used in clinical transplan-tation, since CD34+CD38- cells are a goodpredictive marker of cloning ability and ex-pansion potential of CD34+ cord blood cells(40).

In conclusion, in this study the resultsindicating that culture of HUCB CD34+ cellswith the combination TPO + FL + KL in-duced an increase in total cell counts as wellas in CD34+CD38- cell number suggest thatthis growth factor combination induces anexpansion of very primitive stem cells. The

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use of allogeneic cord blood products as asource of cellular support for patients re-ceiving high-dose chemotherapy has beenlimited primarily by the low numbers ofcells in a HUCB unit. The results of thepresent study and others, however, showthat the true stem cell can be expanded invitro. Furthermore, our data show that the

discrepancy between current in vitro and invivo read-out systems to assess candidatestem cells may be affected by changes inadhesion molecules. Further studies shoulddetermine what culture conditions and cellpopulations are needed for a range of clini-cal applications, improving the use of cordblood for transplantation in adults.

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