J.Cell.Mol.Med. Vol 6, No 4, 2002 pp. 609-620

A major goal of experimental and clinical hematol-ogy is the identification of mechanisms and condi-

tions that support the expansion of transplantablehematopoietic progenitor cells. The lympho-hematopoietic system is composed of cells withshort lifespan. In contrast, their cellular sourceadult hematopoietic stem cells (HSCs) are long-lived population that is not depleted during a life-time of normal functioning. The importance of

Ex vivo differentiation of umbilical cord blood progenitorcells in the presence of placental conditioned medium

Mihaela Chivu a, Carmen C. Diaconu a, Lorelei Brasoveanu b, Irina Alexiu a, Coralia Bleotu a, G. Banceanu c, D. Miscalencu d, C. Cernescu a, *

a "St. S. Nicolau" Institute of Virology, Bucharest, Romaniab Center of Immunology, Bucharest, Romania

c Department of Obstetrics and Gynecology, Polizu Hospital, Bucharest, Romaniad Faculty of Biology, University of Bucharest, Romania

Received: August 1, 2002; Accepted: November 20, 2002

AbstractHematopoetic stem cells (HSC) are the progenitors for the lympho-hematopoietic system, with long lifespan and high prolifera-tion potential. Transplantation of HSC from bone marrow or peripheral blood represents a standard therapy in severe hematolog-ical conditions. A possible alternative source of HSC is the umbilical cord blood, prepared by various separation procedures fol-lowed by expansion in cultures supplemented with hematopoietic growth factors. In order to check the effects of placental con-ditioned medium (PCM) from placental cells culture upon viability of HSC, we added plasma, PCM, dimetil sulfoxyde or heminin HSC cultures. Flow cytometry or direct scoring of solid cultures using CD45+, CD34+, CD71+ and CD14+ fluorescent-labeledmonoclonal antibodies evaluated the effects upon cell proliferation and colony forming ability of HSC cultures, versus controls.PCM produced the highest proliferation, followed by plasma, DMSO and hemin. PCM improved the survival time and maintaineda higher proportion of immature cells. PCM stimulates the differentiation towards myeloid lineage progenitor cells (>90% beingCD45+), increasing the percentage of CD14+, granulocites /monocytes precursors. It is highly suggestive that PCM containsgrowth factors or cytokines, which regulate the development of HSC. Characterization of these factors is in progress.

Keywords: placental conditioned medium • hematopoietic progenitor cells • umbilical cord blood and differentiation

* Correspondence to: Prof. Dr. C. CERNESCU,"St. S. Nicolau" Institute of Virology, 285, Sos. Mihai Bravu,Bucharest 79650, Romania, Tel/Fax: +4021.324.25.90, E-mail: cernescu@valhalla.racai.ro

Introduction

HSC is due to the potential of this population toeither give rise to differentiated progeny or to selfrenew producing more HSCs [1]. During the lastdecade it has become routine to isolate and identi-fy human HSCs based mainly on characteristic cellsurface proteins [2].

Transplantation of HSCs from bone marrowand mobilized peripheral blood turn out to be astandard therapy in malignant and in non-malig-nant conditions [3]. The lack of suitable donors isan important constraint. The discovery that umbil-ical cord blood (UCB) contains high numbers ofHSCs and can be used as an alternative source forallogeneic stem cells (SC) transplantation led tothe establishment of several UCB cell bank inmany hematology clinics [4]. Obstetric factors, theprocedure followed and the equipment used toperform the collection influence the final yield.The nucleated cell count is constant in sequentialfractions but a significant increase in CD34+ cellcontent in the last versus first fraction wasobserved and procedures of enrichment have beenapplied [5-8].

The progress that has been made in obtainingenriched HSC from different sources allows com-parative studies on their regenerative capacity.When a primitive subpopulation of HSC wasobtained either from fetal liver, UCB or adult bonemorrow the number of cells that generate differen-tiated progeny, inversely correlated with develop-mental age [9]. There are also qualitative differ-ences. Only the embryonic stem cells are trulypluripotent cells that can give rise to all cell typespresent in the organism. Adult stem cells have tis-sue or organ specific potential, which can give riseto the differentiated cells types. However, in someinstances, SCs from UCB or adult bone morrowhave been shown to express developmental poten-tial matching the breadth of that SCs obtainedfrom embryos.

Until now there are few studies proving that spe-cialized cells derived from cultured UCB progeni-tors can function after transplantation. It is perhapsnot surprising that cells generated in vitro are notequivalent to those arising in vivo given the exten-sive cellular interactions and “microenvironmenttraining” that take place during development [6].

Ex vivo expanded progenitor cells have beenproposed as a source of cells to support high-dosechemotherapy and to decrease or eliminate the peri-

od of neutropenia and trombocytopenia followingtransplantation [10].

During the process of differentiation, pluripotenthematopoietic stem cells become committed tomyeloid or lymphoid precursor cells and eventuallydifferentiate into functional, morphologically dis-tinct end-stage cells loosing the proliferative poten-tial. Proliferation and differentiation depends ofgrowth factors, cytokines, hormones, and mitogens[11]. Differentiation of promyelocytic cells intomonocytes/macrophages or into granulocytes can beinduced in vitro by treating the cells with differentagents like IFN-gamma or phorbol 12-myris-tate-13-acetate (PMA) for monocytes/macrophages,dimethyl sulfoxide (DMSO) for granulocytes [12]and hemin for erythroblasts [13]. Using conditionedmedia has become a new way for stimulate cellulargrowth [14, 15]. We hypothesized that conditionedmedia harvested from culture of placental cellscould increase proliferation or induce differentiationamong hematopoietic progenitor cells. This paperpresents the data according to this hypothesis.

Materials and methods

Umbilical cord blood collection

Human UCB was obtained from normal full-term deliver-ies from Department of Obstetrics and Gynecology, PolizuHospital, Bucharest. The blood was collected according toinstitutional guidelines, using citrate phosphate dextroseadenine as anticoagulant. Informed consent was obtainedin all cases. Infectious diseases screening in mothers wascarried out for syphilis, hepatitis B and C, HIV 1 and 2,cytomegalovirus. The positive donations were discarded.The procedure for mononuclear cells separation was start-ed no later than six hours after delivery

Cell processing

Low-density mononuclear cells were isolated by UCBcentrifugation (30 min at 500 g) using Histopaque(Sigma; density = 1.007 g/mL). The cells present at theinterface with the gradient medium were collected,washed twice in phosphate buffer saline and resuspend-ed in Iscove’s modified Dulbecco’s medium (IMDM,Sigma) supplemented with 2% newborn calf serum

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(NCS, Sigma). Total numbers of nucleated and viablecells were evaluated by Trypan-Blue dye exclusion test.

Placental conditioned medium (PCM)

The major goal of this study was to see if the con-ditioned medium harvested from placental cells cul-ture can sustain in vitro the viability and prolifera-tion potential of freshly isolated HPC cells. Cell cul-tures were established from 4 full-term placental tissuesby tripsin digestion. 1.5×106 cells were plated in 75 cm2

flasks in Dulbecco MEM: F12 medium (Sigma) with10% NCS, 400 UI/mL penicillin, 200 µg/mL strepto-mycin. Regular passages were done twice a month witha 1:2 split ratio and the supernatant was collected duringone month, centrifuged at 500 g, filtrated through 0.22µm membrane, aliquoted and frozen at -200°C.

Effects of PCM on survival andproliferation of HPC

Mononuclear cells were seeded at a density of 1.5×106/mLin IMDM with 10% NCS. In several samples, medium con-ditioned by placental cells was added (from 10% to 100%vol.). Cultures were incubated at 37°C, 5% CO2, in ahumidified atmosphere. After 48 hours, the number of liv-ing cells was evaluated by the Trypan-Blue exclusion test.

Generation of progenitor cell subsets

The induction of differentiation was done as previouslydescribed [16, 17]. Briefly, 1.5×106 mononuclear cells permL were plated in IMDM with 10% NCS, 400 UI/mLpenicillin, 200 µg/mL streptomycin, 2 µmol L-glutamine(Gibco-BRL) and 1% autologus plasma. The cultures weresupplemented with PCM (final concentration 20% v./v.) orwith 1% DMSO (Sigma), 0.1 mM hemin (Sigma), as con-trol differentiation agents. After 6 days cells were analyzedby determining cell growth and use in functional assays.

Effects of PCM on human embryonic cells

Human embryonic cells were seeded at a density of0.4×105/mL in DMEM: F12 (Sigma) with 400 UI/mLpenicillin, 200 µg/mL streptomycin. In culture mediumwas added: 10% NCS or 10% NCS + 20% PCM or 20%

PCM. Viability was monitored after 3 days by theTrypan-Blue exclusion test.

Flow cytometric analysis

Acquisition of cells was performed using a FACScanflow cytometer (Becton Dickinson – BD) equipment.Control and induced cells were collected, washed twicein PBS, 0.1% BSA, 0.1% sodium azide at 4°C andlabeled for 30 min at 4°C with either fluorescein (FITC)-or phycoerythrin (PE)-conjugated monoclonal antibodiesdirected against CD34, CD71, CD45, CD14, HLA-DRand isotypic IgG control antibodies (all from BectonDickinson). After two washes with phosphate bufferedsaline, 10,000 cells were subsequently analyzed. Datawere evaluated using Win MDI 2.8 research software.

Hematopoietic colony assays

Progenitor cells were evaluated in agar solid cultures. Theculture medium consisted of 0.3% agar (Gibco BRL),20% NCS, 1% bovine serum albumin (Merck), 5×10-4 M2-mercaptoethanol, 1% non-essential aminoacids (GibcoLife-Sciences) in IMDM. The medium was supplement-ed with placental conditioned medium (10 and 20%), 2%autologous plasma to support the clonal growth ofhematopoietic progenitor cells. DMSO 1% and hemin 0.1mM were used as control differentiation agents. MNCfrom UCB were plated at a final concentration of 5×105

cells in 35 mm Petri dish in triplicate and incubated 14days at 37°C and 5% CO2. After 14 days of culture,colonies were scored in the same dish using an invertedmicroscope. Hematopoietic colonies were classifiedaccording to “Atlas of Human Hematopoietic Colonies”[18] as: BFU-E, erythroid colonies of more than 50hemoglobinized cells grouped in one or several tight clus-ters CFU-GM – more dispersed colonies containing bothgranulocytes and monocytes – and CFU-GEMM, thatappear as colonies with a central mass of erythroid cellssurrounded by larger individual cells from granulo-cyte/macrophage/megakaryocyte lineages.

Statistical Analysis

Statistical analysis was performed by using the Student’st-test. Differences with p < 0.05 were considered to besignificant.

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Results

Effects of PCM on survival andproliferation of HPC

Four cultures of placental cells were initiated andculture media were tested with similar results (datanot shown). For determining the appropriate vol-ume of PCM that can be used in supplementing cul-ture conditions, we assessed first the effect of PCMon cell survival. The best results were obtainedwhen 20% vol. (Fig. 1) of PCM were added toIMDM. The kinetic analysis of UCB cells showedin this case a 1.25 fold increase of nucleated cellsduring the first 2 days in culture with PCM with theviability above 98%.

Generation of progenitor cell subsets

We found that conditioned media harvested fromplacental cells slightly stimulated the proliferation offreshly isolated HPC in the first 3 days in culture andimproved their survival (Fig. 2). The results showthat the viability of cells cultured for 6 days in PCMmedium decreased by only 19.6% compared with thecells that were suspended in IMDM not-conditioned,which decreased by 80%. Thus, these data demon-strate that placental cells secrete factors that mayimprove survival of lineage-specific progenitor cells.

Following treatment with DMSO, cell prolifera-tion was strongly reduced after 4 days of culture.Also, the hemin-treated cells ceased to proliferateafter 4 days. The cells were more than 80% viableat the end of the culture period, except for theDMSO-treated cells, in which viability at 6 dayswas about 28.6%.

Effects of PCM on human embryonic cells

To evaluate the specificity of PCM stimulatoryeffect we tested another type of cell. Human embry-onic cells were culture in presence of PCM. Resultsshowed that the combination between PCM andNCS did not succeeded to improve cells growth ascompared to NCS only. A difference was obviouswhen cells were grown in PCM; an improving via-bility was obtained compared to control mediumonly, from 25 to 50% (Fig. 3).

Flow cytometric analysis

Flow cytometric analysis of control and inducedcells was performed after 3 days in culture (Fig. 4)and the results are shown as the percentage of eventsregistered in the particular gates. When cells werecultured in the absence of inducers, percentage ofcells in granulocytic gate was as low as

Fig. 1 Effect of PCM on survival of freshly isolated HPC cells (1.5×106/mL). Cells were cultured for 48 hours inconditioned medium or in a medium with 10% NCS. Cell viability was evaluated by Trypan-Blue exclusion test. Barsare the mean ± SD of the results of 3 experiments.

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0.74 ± 0.09% (Fig. 4 A4) while, when cells werestimulated with PCM (final concentration 20% v/v)and autologous plasma, percentage was significant-ly increased to 24.39 ± 0.56% (Fig. 4 D4).

The characteristics of cells in these conditionswere further assessed on the basis of antigen sur-face expression. Fig. 5 shows the flow cytometricanalysis of a representative 3-day time-courseexperiment. The vast majority of cells obtainedafter 3 days of culture maintained CD45+ pheno-type. However, during days 1 to 3, the percentage ofCD34+ and CD71+ cells increased, suggesting anactivation of proliferation process. Taking into con-sideration that, majority of these cells is slightly dif-ferentiated (> 90% being CD45 positive), and thatthe percentage of CD14+ cells increased we thinkthere is a proliferation of granulocytes/monocytesprecursors.

Hematopoietic progenitor cells content

In order to evaluate clonogenicity in solid cultures,PCM was added in various proportions with orwithout autologous plasma. HPC content in differ-ent plating conditions was determined by colony

assays (Fig. 6). When cells were cultured in theabsence of inducers factors, they produced7.6 ± 1.5 colonies per 5×105 plated cells.Introducing in culture 2% of autologous plasmaincreased this growth to 9 ± 0 colonies. The high-est number of colonies (27.6 ± 1.5) was obtainedwhen cells were stimulated with PCM (final con-centration 20% v/v) and autologous plasma.16.66% of them (4.6/27.6) corresponded to ery-throid progenitors; 51.81% (14.3/27.6) weremyeloid progenitors, and 31.15% corresponded tomultipotent progenitors (Table 1). The total num-ber of colonies stimulated by each of these factorsalone was lower than using both of them. WhenDMSO (a well known myeloid differentiationagent) was added in culture the number of myeloidcolonies increased with 152.4% from 6.3 ± 0.5 to9.6 ± 1.1 (p>0.05) but still remained low comparedto culture stimulated with 20% PCM and plasma14.3 ± 1.5 (p<0.05). Besides, a higher proportionof immature, multipotent progenitors was observedwhen using PCM and plasma (p<0.05). In contrast,the number of erytroid colonies does not show asignificant increase. Only hemin was able to stim-ulate the number of BFU-E, 9 ± 1, as compared to4.3 ± 1 for PCM.

Fig. 2 Cell growth curves of UCB mononuclear cells inpresence of various agents. Cells were seeded at an ini-tial density of 1.5×106 cells/mL without (λ) or with 20%PCM (∆), 1% DMSO (υ), 0.1 mM hemin (ν) and werecultured for 6 days without any change of media. Themean±SD of the results of 3 experiments

Fig. 3 Cell growth curves of human embryonic cellsgrown with/without PCM. Cells were seeded at a densi-ty of 0.4×105/mL in DMEM (ν); 10% NCS (λ), 10%NCS + 20% PCM (∆) or 20% PCM (υ) were added.Viability was monitored after 3 days by the 0.5%Trypan-Blue exclusion test. The mean±SD of the resultsof 3 experiments.

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Discussion

Development of ex vivo culture conditions thatfacilitate in vitro maintenance and expansion oftransplantable HSCs represent a major challenge instem cell research. This is a necessary first steptoward a better understanding of the regulatory pro-cess governing the development of all hematopoiet-

ic lineages from HSCs. Furthermore, establishmentof such culture conditions is a prerequisite forpotential ex vivo manipulation and expansion oftransplantable HSCs in several clinical applicationssuch as gene therapy, tumor cell purging, and stemcell transplantation [19].

The aim of the present study was to explore thehypothesis that conditioned media harvested from

Fig. 4 Representative dot plots (n = 3) of UCB cells induced with DMSO, hemin and PCM. Dot-plot of cells thatwere cultured in medium without inducer (A – first panel); presence of DMSO (B – second panel); presence of hemin(C – third panel); presence of PCM (D – forth panel). Index 1 – total UCB cells; Index 2 – cells from lymphocytegate; Index 3 – cells from monocyte gate; Index 4 – cells from granulocyte gate.

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placental cells culture would increase proliferationor induced differentiation among early hematopoi-etic cells in vitro. In humans, placenta has two lay-ers: the fetal placenta and the junctional zone calledbasal plate. The layer closest to the fetus contains anetwork of mingled fetal and maternal blood ves-sels separated by trophoblast giant cells. Next to thejunctional zone are maternal decidual cells that linethe uterus. This architecture leaves little doubt thatthe majority of placental dispersed cells used fortissue cultures initiation are from vascular endothe-lium. We speculate that, having extensive prolifera-tion and differentiation potential, endothelial cellscan provide growth factors in response to local trig-gers required by the organ they are recruited to. The

Roman statesman Cato the Elder left a recipe for apopular pie called placenta from which the modernname “pizza” comes. The anatomists give this nameto the organ conceived to exchange the nutrients,gases and waste between maternal and fetal blood.Here we demonstrated other virtues of this enig-matic organ.

We treated cells with PCM, hemin, and DMSO(as control differentiation agents) and determinedtheir differentiation stage by three types of assays:cell proliferation, colony-forming ability and flowcytometric analysis. We found that conditionedmedium harvested from placental cells slightlystimulated the proliferation of freshly isolated HPCcultured for 6 days and improved their survival with

Fig. 4 Surface antigen phenotype characterization of 3 days PCM-treated mononuclear cell cultures. Isotype-matched control is indicated by solid area. Phenotypic analysis demonstrated that cells predominantly developedalong a myeloid differentiation pathway. 92.86% from entire population expressed CD45 – leucocytic antigen,31.39% were CD14 positive and 37.38 % of them presented other myeloid marker HLA-DR. In addition, a signifi-cant increase in CD34 and CD71 percentage of positive cells was obvious.

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60%. When we evaluate the possible effect of PCMon human embryonic cells, results showed that thecombination between PCM and NCS did not suc-ceeded to improve cells growth as compared toNCS only. Thus, we suppose there is either a lowerlevel of growth factors directed towards fibroblasticcells (such as fibroblast growth factor – FGF) inPCM, or a lack of hematopoietic cytokine receptorson human embryonic cells.

HPC content in different plating conditions wasdetermined by colony assays. Results showed thatcells from umbilical cord blood undergo clono-genic maturation even in the absence of exogenousgrowth factors. Schibler et al. analyzing thegrowth-factor-independent colonies have foundtranscripts for GM-CSF and IL-3, suggesting thatcord blood progenitors are able to produce somehematopoietic cytokines [20]. Other authors haveshown that normal human CD34+ cells expressmRNA for several hematopoietic regulators: Kitligand, Epo, IL-1, IL-6, TGF-β1 [21-24]. Majka et

al. reported that the β-chemokines RANTES, MIP-1α, and MIP-1β are endogenously secreted byCD34+ cells and megakaryoblasts, and auto protectthem from infection by R5 HIV [23]. They alsoreported that human CD34+ cells secrete IL-16,which could possibly protect them from infectionby X4 HIV [25]. Therefore, therapeutic agents thatmay stimulate the endogenous synthesis of thesechemokines in human hematopoietic cells couldprotect them from HIV infection [11]. The secre-tion of various chemokines by normal humanCD34+ cells is intriguing and has important conse-quences for cell biology.

Taking into account the numerous growth fac-tors, cytokines and chemokines secreted by severalnormal human cells, using conditioned media hasbecome a new way for stimulate cellular growth.

We found in agar culture experiments that moremyeloid lineage-specific progenitor cells, such asCFU-GM, and CFU-GEMM, retained their differ-entiation potential if they were cultured in medium

Fig. 6 Multiple lineage differentiation of human pro-genitor cells (a) CFU-GM – one disperse colonie con-taining both granulocytes and monocytes (x30);(b) BFU-E – hemoglobinized cells grouped in one tightcluster (x40); (c) CFU-GEMM, appear as one coloniewith a central mass of erythroid cells surrounded by larg-er individual cells (x40).

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conditioned by placental cells. To evaluate its rolein differentiation and maturation of hematopoieticprogenitors, PCM was added in various proportionswith or without autologous plasma. The highestnumber of colonies (27.6 ± 1.5) was obtained whencells were stimulated with PCM (final concentra-tion 20% v/v) and autologous plasma. Probably,PCM contains several growth factors, especiallythose involved in proliferation and differentiation tomyeloid lineage. The number of colonies detectedwith PCM reflects additive or synergistic actionsbetween the growth factors.

When DMSO was added in culture the numberof myeloid colonies increases but still remained sig-nificantly low compared to culture that stimulatedby 20% PCM and plasma. Enhanced detection ofCFU-GM, after DMSO treatment, was directly cor-related with decreased detection of CFU-GEMM. Ahigher proportion of immature, bipotent progenitorswas observed when using PCM and plasma. In con-trast, number of erytroid colonies does not show asignificant increase. Only hemin was able to stimu-late BFU-E formation.

Differentiation of the cells was also determinedby flow cytometric analysis. Exposure of the cells toPCM for 3 days results in an increase of the percent-age of cells in granulocyte gates. In contrast, per-centage of cells in granulocyte gate was lower upontreatment with DMSO. Phenotypic analysis demon-strated that cells predominantly express surface anti-gens such as CD45, HLA-DR and developed along

to myeloid differentiation pathway. In addition, a sig-nificant increase in CD34 and CD71 percentage ofpositive cells was obvious. Taking into considerationthat, majority of these cells is slightly differentiated(> 90% being CD45 positive) and that the percentageof CD14+ cells increased we think there is a prolifer-ation of granulocytes/monocytes precursors. Theseobservations are correlated with the increase of thepercentage of cells in granulocyte gates and withresults obtained in clonogenic assay. We suggest thatplacental conditioned medium induces a differentiat-ed proliferation, process that might be sustained byplacental cells shedding factors.

The endogenously secreted factors presumablypresents in PCM may play a key role in the biolo-gy of early hematopoietic cells. In vitro, whenrecombinant molecules are used, the effect is dif-ferent. It is considerate that endogenously secretedfactors even if they are secreted at very low levelsmay be more biologically effective than recombi-nant proteins employed at similar doses [26]. Forexample, autocrine TGF-b1 secretion in humanhematopoietic progenitors up-regulates bcl-2expression and regulates the survival and differen-tiation of HSCs by cell cycle-independent mecha-nisms [27] whereas exogenously added TGF-b1affects the cell cycle and inhibits proliferation ofthese cells [28]. A full understanding of the preciserole in normal hematopoiesis of the numerousendogenously secreted regulators will require moreinvestigation.

ADDITION OFCOLONIES / 5 × 105 PLATED CELLS

BFU-E CFU-GM CFU-GEMM TOTAL

control 2.6 ± 1.1 2.6 ± 0.5 2.3 ± 0.5 7.6 ± 1.52

2 % plasma 3 ± 0 3 ± 0 3 ± 0 9 ± 0

20 % PCM 3.3 ± 1.1 6 ± 1 4 ± 1 13.3 ± 1.54

plasma + 10% PCM 4.3 ± 1.1 6.3 ± 0.5 5 ± 2 15.6 ± 2.08

plasma + 20% PCM 4.6 ± 1.5 14.3 ± 1.5 8.6 ± 1.5 27.6 ± 1.52

plasma + 10% PCM + DMSO 4.6 ± 0.5 9.6 ± 1.1 5.3 ± 1.1 19.6 ± 2.8

plasma + 10% PCM + hemin 9 ± 1 4.3 ± 1.1 5 ± 1.7 18.3 ± 3.05

Table 1. Clonogenic maturation of umbilical cord blood hematopoietic progenitors. Responsiveness inCFU to stimulation with various agents. Results show that treatment with PCM is superior. The data rep-resents mean ± SD of 3 experiments.

In conclusion, we propose that PCM induce pro-liferation and differentiation of myeloid lineage-progenitor cells. It is clear that these processes weresustained by several growth factors contained byPCM. Therefore, the future study project of ourteam is to characterize the PCM by detecting thecytokine composition in an attempt to understandhow PCM can interfere and regulate various stagesof hematopoiesis. Better understanding of mecha-nisms that regulate synthesis of these endogenousfactors may allow developing of new therapeuticstrategies, improving antileukemia treatments, con-tribute to AIDS-free status or provide an answer forefficient ex vivo stem cell expansion.

Acknowledgements

The authors would like to thank Dr. Gratiela Tardei forexpertise and consultation in FACS analysis. We alsothank Mihaela Rusu for coordinating the collection ofthe human umbilical cord blood samples.

This work was supported by grants from CNCSIS (no. 591/2002), with MEC support, andNational-Research-Development Program VIASAN(no. 051/2001-2004), Romania.

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