il-10 increases the number of cfu–gm generatedby ex vivo expansion of unmanipulated human mncsand...

IL-10 INCREASES CFU–GM GENERATION

Volume 41, May 2001 TRANSFUSION 659www.transfusion.org

The transplantation of HPCs from various sourcesincluding bone marrow (BM), peripheral blood(PB), and cord blood (CB) has become a standardstrategy for protecting against the hematologic

toxicity of myelosuppressive or myeloablative anticancerchemotherapy.1,2 HPCs mobilized into PB by growth factorswith or without chemotherapy have been found to haveseveral advantages over BM progenitors, and it is predictedthat PB progenitor cells (PBPCs) will ultimately replace BMprogenitor cells.3,4 In the autologous transplant setting, how-ever, it is often not possible to collect an adequate numberof PBPCs from patients with impaired hematopoiesis, be-cause of extensive previous chemotherapy.5,6 Allogeneic pro-genitor cell sources are limited by MHC considerations andthe shortage of donors.7,8 Banking of CB has begun recently,but it is still not known if CB samples contain enough pro-genitor cells for engraftment in an average-sized adult. More-over, large numbers of progenitor cells are required for repeti-tive clinical use after high-dose chemotherapy9 andtherapeutic gene transfer.10 Because of these considerations,strategies that can increase the number of progenitor cells areclearly desirable and could be of major clinical benefit.11-13

IL-10 increases the number of CFU–GM generatedby ex vivo expansion of unmanipulated human MNCs

and selected CD34+ cells

Thomas Wagner, Gerhard Fritsch, Renate Thalhammer, Paul Höcker, Gerhard Lanzer,

Klaus Lechner, and Klaus Geissler

BACKGROUND: Ex vivo expansion strategies with dif-ferent cytokine combinations are currently used by sev-eral groups as a means of increasing the number ofHPCs for a variety of special clinical applications. Be-cause there is little information on the potential role ofIL-10 in such ex vivo expansion models, the effect of thiscytokine on the generation of myeloid progenitor cells insuspension cultures was investigated.STUDY DESIGN AND METHODS: On the basis of datafrom the literature and from new experiments, the com-bination of SCF and IL-3 at concentrations of 100 ng permL and 100 U per mL, respectively, was chosen as thestandard cocktail. The addition of IL-10 to such culturesresulted in a marked and dose-dependent potentiationof myeloid progenitor cell production.RESULTS: Using unmanipulated leukapheresis compo-nents from 13 individuals (including lymphoma and can-cer patients and normal donors), the expansion multipleof CFU–GM after 14 days as compared with pre-expan-sion values was 9.54 ± 2.31 times by SCF/IL-3 and46.38 ± 7.37 times by the combination of SCF/IL-3 and100 ng per mL of IL-10 (p<0.001). IL-10 also potentiatedCFU–GM generation from selected CD34 PBMNCs (n =9) with an expansion of 17.22 ± 7.04 times versus 45.67± 16.78 times using the SCF/IL-3 and SCF/IL-3/IL-10combination, respectively (p<0.05). Moreover, expan-sion-promoting effects of IL-10 were observed in liquidcultures containing MNCs from bone marrow (n = 4) andcord blood (n = 3), but did not reach statistical signifi-cance because of the small number of samples.CONCLUSION: These results suggest IL-10 as a usefulcytokine to optimize progenitor cell-expansion strategiesfor clinical application.

ABBREVIATIONS: BD = Becton Dickinson; BM = bone marrow;

CB = cord blood; FSC = forward scatter; IGF1 = insulin-like

growth factor 1; IMDM = Iscove’s modified Dulbecco’s medium;

LT-CIC(s) = long-term culture-imitating cell(s); PB = peripheral

blood; PBPC(s) = PB progenitor cell(s); SSC = side scatter.

From the Department of Blood Group Serology and Transfusion

Medicine, University Clinics of Graz, Graz, Austria; the Division

of Hematology and Hemostaseology, Department of Internal

Medicine I, and the Departments of Laboratory Medicine and of

Transfusion Medicine, University of Vienna; and the Children’s

Cancer Research Institute, St. Anna Children’s Hospital, Vienna,

Austria.

Address reprint requests to: Klaus Geissler, MD, Department

of Division of Hematology and Hemostaseology, Internal Medi-

cine I, University of Vienna, Waehringerguertel 18-20, A-1090

Vienna, Austria; e-mail: [email protected].

Received for publication March 13, 2000; revision received

July 10, 2000, and accepted July 18, 2000.

TRANSFUSION 2001;41:659-666.

T R A N S P L A N T A T I O N A N D C E L L U L A R E N G I N E E R I N G

WAGNER ET AL.

660 TRANSFUSION Volume 41, May 2001 www.transfusion.org

Several groups have shown that ex vivo culture systemsusing appropriate cytokine combinations may be helpfulin expanding the number of human progenitor cells.14,15 Exvivo-generated progenitor cells have been shown to pro-duce or at least enhance hematopoietic engraftment aftermyeloablative cancer therapy in animal models and pa-tients.16,17 Traditionally, selected CD34+ cells are used for exvivo expansion of progenitor and postprogenitor cells bymost investigators,18-20 but a substantial loss of HPCs oftenoccurs because of the selection process.21 Significant CFU–GM production can be obtained with unmanipulatedPBPCs, but some inhibition of ex vivo expansion by CD34-cells has been reported.22

IL-10 is a 35-kD, protein, originally identified by virtueof its ability to inhibit cytokine synthesis in helper 1 T-cellclones.23,24 It is primarily produced by MNCs, and it pos-sesses a wide range of activities on different hematopoieticcells.25 The main feature of this cytokine is a suppressiveeffect on cytokine expression.26 The effect of IL-10 on theex vivo expansion of HPCs has not been investigated so far.Theoretically, IL-10 may affect in vitro progenitor cell gen-eration by the potential suppression of cytokines releasedfrom accessory cells in unmanipulated PBPCs. On the otherhand, IL-10 has been shown to restore viability of bcl-2antisense-treated primary human CD34+ cells in overnightliquid cultures, which suggests an antiapoptotic effect of IL-10 on HPCs.22 In this study, therefore, we investigated theeffect of IL-10 on the generation of progenitor cells in sus-pension cultures. We show that the addition of IL-10 to liq-uid cultures markedly increases the number of CFU–GMgenerated by ex vivo expansion of both unmanipulatedhuman PBPCs and selected CD34+ cells.

MATERIALS AND METHODSPBPCsAfter informed consent was given we collected PBPCs byapheresis either from patients with various malignant dis-eases (Table 1) during hematopoietic recovery after chemo-therapy or from healthy donors. Apheresis was performedwith a cell separator (COBE Spectra, Cobe BCT, Lakewood,CO).

In part, apheresis samples were processed immedi-ately for CD34+ selection (Isolex 300i, Baxter HealthcareCorp, Irvine, CA) to enrich CD34+ PBPCs, as published.27

After their separation through the CD34 magnetic column,the purity of CD34+ cells in the progenitor cell fraction wasmore than 92 percent (range, 92-99.5%). Cell viability was>90 percent in all cases.

BMMNCsAfter the subjects gave informed consent, BM samples wereobtained by aspiration into sterile tubes containing heparinwith no preservative. BMMNCs were harvested after a

ficoll-hypaque gradient centrifugation (400 × g, 30 min,1.007 g/mL). The low-density cells were collected from theinterface between density solution and plasma, washedtwice, and resuspended in Iscove’s modified Dulbecco’smedium (IMDM, GIBCO, Paisley, UK).

Human umbilical CBCells were obtained from normal human umbilical CB thatwas scheduled for discard after delivery of the infant andafter the collection of samples necessary for routine test-ing. CB was collected as described previously,28 and MNCswere harvested as described above.

Assessment of CD34+ cellsCD34+ cells in apheresis components were assayed as pre-viously described. Samples were labeled with FITC-conju-gated CD45 MoAb29 and PE-conjugated CD34 antibody orisotype control antibodies (Becton Dickinson [BD] San Jose,CA) for 30 minutes at 4°C. Twenty thousand cells were ana-lyzed for each sample. Measurement on a flow cytometer(FACSCalibur, BD) was evaluated by using acquisition andanalysis software (CELLQUEST, BD) for data acquisitionand exploratory multidimensional software (PAINT-A-GATE PRO, BD) for data evaluation.

ReagentsRecombinant human IL-10 (rHuIL-10; specific activity, 1-2× 106 U/mg) was provided by Schering-Plough Corp.(Kenilworth, NJ). The rHu-GM–CSF and rHu-IL-3 were pro-vided by Sandoz (Basel, Switzerland). The rHu-SCF and in-sulin-like growth factor 1 (IGF1) were obtained fromPharma Biotechnologie Hannover (Hannover, Germany).The rHu-IL-6 was purchased from Serotec (Oxford, UK),rHu-IL-1β from Endogen (Woburn, MA), and rHuFlt3-Lfrom ImmunoKontact (Frankfurt, Germany).

TABLE 1. Cell sources for progenitor cell expansionSource Samples

Unmanipulated PBPCsNormal donors 01, 3Multiple myeloma 02, 7, 11, 12Breast cancer 05, 6, 9, 13Hodgkin’s disease 10Non-Hodgkin’s lymphoma 08Ovarian cancer 04

CD34+ cellsNormal donors 15, 19Breast cancer 14, 17, 20Hodgkin’s disease 21Non-Hodgkin’s lymphoma 16, 18Multiple myeloma 22

BMMNCsNormal donors 23, 24, 25, 26

CBNormal donors 27, 28, 29



Progenitor cell assayCFU–GM were assayed as described previously.30,31 Cellswere cultured in 0.8-percent methylcellulose, 30-percentFCS (INLIFE, Wiener Neudorf, Austria), 10-percent BSA(Behring, Marburg, Germany), α-thioglycerol (10–4 mol/L),and IMDM (GIBCO). Cultures were stimulated with 100 Uper mL of rHu-GM-CSF and 10 U per mL of rHu-IL-3.CD34+ cells and BMMNC or CB or PBPCs depending on thetype of cells collected from the donor were plated in dupli-cate at 5 × 103 and 30 × 103 per mL, respectively. After a cul-ture period of 14 days (37°C, 5% CO2, full humidity), cultureswere examined under an inverted microscope. Aggregateswith more than 40 translucent, dispersed cells werecounted as CFU–GM. Colonies with unclear morphologywere picked, transferred to a glass slide, and stained to con-firm the cell composition of the colonies by conventionallight microscopy.

Ex vivo expansionExpansion cultures were incubated at 37°C in 5-percent CO2

and full humidity with IMDM as growth medium, supple-mented by 10-percent FCS. Ex vivo expansion under vari-ous cytokine combinations was analyzed in either 24-wellor 48-well plates (Falcon, Heidelberg, Germany) with a cul-ture volume of 1 mL. Cultures were performed in the pres-ence of 100 ng per mL of SCF and 100 U per mL of rHu-IL-3. The rHu-IL-10 was added at a concentration of 0.1 ng permL to 100 ng per mL. BMMNCs or CB or PBPCs, depend-ing on type of cells collected from the donor and CD34+cells were seeded at 1 × 106 per mL and 1 × 105 per mL, re-spectively. Cultures were incubated for 14 days. No addi-tional feeding was performed during the culture period. Inall cultures, cell viability was determined by trypan blueexclusion and found to be above 96 percent. On Day 14, cellswere counted with an electronic counting device (CoulterElectronics, Luton, UK) and subsequently plated on culturedishes in duplicate using between 3 × 103 and 12 × 103 cellsper mL.

Cytospin preparationsCytospin preparations of the ex vivo-expanded cells wereconducted (Shandon Cytospin III, Southern Product,Astmoor, UK) at 30 × g for 10 minutes. Slides were stainedaccording to a modified Wright technique.

Four-color flow cytometric analysisEx vivo-expanded cells were processed as previously de-scribed32 using a conventional lyse-and-wash proce-dure.33,34 Predominantly employed conjugated MoAbs wereCD3 (UCHT1), CD4 (MT310), CD8 (DK25), CD14 (TÜK4),CD15 (C3D1), CD19 (HD37), and CD45 (T29/33) (all fromDako, Glostrup, Denmark); CD33 (P67.6), CD34 (HPCA-2),CD38 (HB-7), CD45RA (2D1), and CD56 (NCAM 16.2) (allfrom BD). The parameters acquired per cell were forward

light scatter (FSC) and 90° side light scatter (SSC), and fourfluorescence signals (FL1, FL2, FL3, FL4). The compensa-tion was set as determined for the respective MoAb com-binations. Data acquisition and evaluation was performedas described above.

Data analysisThe absolute number of CFU–GM per culture was calcu-lated by multiplying their incidence (/cell seeded in meth-ylcellulose) by the number of nucleated cells present ineach culture. Expansion was calculated as the final CFU–GM number divided by the initial numbers of CFU–GM.The paired t test was used to determine the significance ofdifferences. A p value <0.05 was considered significant.

RESULTSEffect of IL-10 on the CFU–GM expansion fromunmanipulated leukapheresis componentsOf the cytokine combinations that have been used in thelast years by different groups for expansion of myeloid pro-genitors, IL-3 and SCF were the only cytokines that werepresent in each cocktail. Therefore, we hypothesized thatboth growth factors may also be of critical importance inexpansion experiments using unmanipulated PBPCs. Infact, none of the cytokines IL-1, IL-6, GM–CSF, Flt3-L, orIGF1 was able to greatly improve CFU–GM expansion (datanot shown). Therefore, IL-3 and SCF at concentrations of100 U per mL and 100 ng per mL, respectively, were cho-sen as the standard cocktail for further experiments. Theaddition of IL-10 to such cultures resulted in a marked anddose-dependent potentiation of myeloid progenitor cellgeneration (Fig. 1). To investigate the reproducibility of our

Fig. 1. Dose-dependent potentiating effect of IL-10 on the gen-

eration of CFU–GM from an ex vivo-expanded unmanipulated

leukapheresis component (Sample 5). Unmanipulated PBPCs

were expanded in the presence of SCF and IL-3 and of increas-

ing concentrations of IL-10 (0.1-100 ng/mL) for 14 days. The

absolute number of CFU–GM per culture was calculated by

multiplying their incidence (/cells seeded in methylcellulose)

by the number of nucleated cells present in each culture.

WAGNER ET AL.


initial observation in a larger number of patients, we per-formed ex vivo expansion experiments using leukapheresiscomponents from 13 individuals. Not unexpectedly, weobserved a high patient-to-patient variability regardingCFU–GM generation by IL-3/SCF, which is probably due todifferences in disease, mobilization regimen, and priortherapy. Despite this marked variation, the addition of IL-10 potentiated CFU–GM generation in all cases tested(Table 2). In 13 PBPC samples, the expansion multiple ofCFU–GM after 14 days was 9.54 ± 2.31 times by SCF/IL-3and 46.38 ± 7.37 times by the combination of SCF/IL-3 andIL-10 at 100 ng per mL (p<0.001). It is interesting that therewas one sample with no CFU–GM expansion by SCF/IL-3at all but a 65-times expansion by the addition of IL-10 tothe culture system (Sample 7).

Effect of IL-10 on the CFU–GM expansion fromselected CD34+ cellsTo investigate the potential usefulness of IL-10 as an ex vivoexpansion-promoting cytokine in highly purified progeni-tor cells, selected CD34+ cells from patients as well ashealthy donors were also used for expansion experiments.Again, we observed a high variability of CFU–GM genera-

tion, which was a mean 17.22 ± 7.04-times with IL-3/SCF(Table 3). The addition of IL-10 increased CFU–GM num-bers in all nine experiments over the values with the stan-dard IL-3/SCF combination, leading to a mean 45.67 ±16.78-times expansion (p<0.05).

Effect of accessory cells on progenitor cellexpansionHaving observed CFU–GM expansion by IL-3/SCF and itspotentiation by IL-10 in unmanipulated as well as selectedCD34+ cells, we were interested in further elucidating thepossible role of accessory cells (CD34–) in our culture sys-tem. Therefore, we used the Isolex 300i to separate leuka-pheresis components from patients into CD34+ and CD34–cell populations and performed expansion experimentsusing the cell populations separately or together and withor without IL-10. To exclude any potential CFU–GM genera-tion derived from CD34– cells, the cells were irradiated with30 Gy. As shown in Table 4, accessory cells (CD34– cells) didnot show any inhibitory effect on the ex vivo-expansionpotential of CD34+ cells, but rather increased the CFU–GMexpansion, which was three times higher than with selectedCD34+ cells alone. As expected from our previous experi-

ments, IL-10 potentiated CFU–GM ex-pansion from CD34+ cells alone and inthe presence of accessory cells.

Effect of IL-10 on expansion ofCFU–GM from other cell sourcesAlthough CD34+ cells selected fromunmanipulated PBPCs are currently themost commonly used cell componentfor ex vivo strategies, the generation ofCFU–GM from other cell sources such asBMMNCs and CB has been reported.35,36

In our hands both BMMNCs and CBwere suitable for myeloid progenitor cellexpansion by IL-3/SCF with or withoutIL-10. As shown in Table 5, BMMNCsfrom healthy donors were expanded10.75 ± 2.95-times and 25.5 ± 6.36-timesby SCF/IL-3 and SCF/IL-3/IL-10, respec-tively. Using CB, drawn after the deliveryof healthy newborns, CFU–GM genera-tion was 23.67 ± 10.73-times by IL-3/SCFand 86.33 ± 54.35-times by IL-3/SCF/IL-10 (Table 6).

Effect of IL-10 on total cell number,morphology, and imm unophenotypeof expanded cellsThe expansion of total cells in suspen-sion cultures containing unmanipulatedPBPCs was significantly decreased in the

TABLE 2. Effect of IL-10 on CFU–GM expansion from unmanipulated PBPCsCFU–GM (numbers/well)

Sample Before After 14-day suspension culturenumber expansion SCF/IL-3 Expansion (×) SCF/IL-3/IL-10 Expansion (×)

01 265 2,106 8 5,550 2102 270 2,376 8 6,750 2503 125 1,184 9 9,000 7204 130 1,216 9 9,912 7705 120 1,600 13 11,032 9206 200 5,568 28 12,240 6207 610 0 0 39,600 6508 300 4,380 14 8,316 2809 570 910 2 36,018 6310 1,330 1,400 1 29,250 2211 980 5,000 5 41,478 4212 1,120 4,750 4 8,838 813 1,060 24,620 23 27,412 26

Mean ± SEM 545 ± 120 4,239 ± 1,770 9.54 ± 2.31 18,876 ± 3,785 46.38 ± 7.37

TABLE 3. Effect of IL-10 on CFU–GM expansion from CD34+ selected cellsCFU–GM (numbers/well)


14 70 2,175 31 2,805 4015 218 1,750 8 3,750 1716 694 1,635 2 3,880 617 926 2,880 3 7,300 818 570 3,744 7 8,008 1419 500 6,500 13 26,350 5320 200 4,494 22 23,310 11721 189 3,144 2 25,344 1422 170 11,380 67 24,081 142Mean ± SEM 393 ± 97 4,189 ± 1,031 17.22 ± 7.04 13,870 ± 3,502 45.67 ± 16.78



presence of IL-10 from that with controls (2.41 ± 0.28-timesvs. 3.42 ± 0.62-times; p<0.05), but it was similar in CD34+cell cultures (13.67 ± 2.77-times vs. 14.78 ± 2.82-times). Withrespect to morphology, we observed an increased matura-tion of normal monocytic cells into macrophage-like cellsby IL-10 as we have previously reported in chronicmyelomonocytic leukemia cells.37 No other changes in cellmorphology were seen under IL-10. With respect to CD34cell measurement by flow cytometry, no significant effectwas observed from the addition of IL-10 after ex vivo expan-sion. Regarding the immunophenotype of cultured cells, apanel of MoAbs including CD3, CD4, CD8, CD14, CD15,CD19, CD33, CD38, CD45, CD45RA, and CD56 was testedin a four-color flow cytometric analysis, but again no sig-nificant changes were found in cell cultures with or with-out IL-10 (data not shown).

DISCUSSIONIncubation of selected CD34+ cells with different cytokineshas been the most commonly applied strategy for ex vivoexpansion of HPCs. However, each currently available tech-nique used for purification of CD34+ cells leads to an ap-proximately median 50-percent loss (range, 14-83%) of

CD34+ cells.21,38,39 To achieve maximumnumbers of progenitor cells for trans-plantation, therefore, an ex vivo expan-sion system suitable for unmanipulatedHPCs collected by apheresis would cer-tainly be of interest. Using IL-10 in addi-tion to SCF and IL-3, we demonstratedthat substantial numbers of CFU–GMcan be generated in vitro fromunmanipulated PBPCs. The median in-crease in myeloid progenitors was 42-times on Day 14. Thus, the expansionpotential of our culture system usingunmanipulated PBPCs is comparable tothat of many other recently reportedprotocols that used selected CD34+cells.40-44 Considering the substantial cellloss by the CD34+ cell-selection proce-dure, therefore, our strategy may be atleast as efficient or even more efficient

with respect to the total amount of HPCs available for clini-cal use.17

The effect of IL-10 on long-term culture-initiating cells(LT-CICs) has not been investigated in this study and re-mains to be shown. LT-CICs are generally considered asHPCs relatively close to the pluripotent stem cell with thepotential for long-term reconstitution. In ex vivo expansionsystems previously reported that the yield of LT-CICs rangedfrom a moderate loss of 50 percent45 up to a certain degree(1.23-times46) of expansion.40 The significance of LT-CICrecovery in such cultures with respect to clinical effects,however, seems to be limited. In a recently reported PhaseI study,45 all patients showed adequate hematopoietic re-constitution up to a year after transplant, despite the lossof early progenitors during 12-day expansion of BMMNCsin controlled perfusion bioreactors.

Ex vivo expansion of unmanipulated PBPCs has beenreported to reduce the possibility of malignant cell con-tamination in disseminated cancer.15 There are only limiteddata regarding the expression of IL-10 receptors on humantumor cells. B-cell chronic lymphocytic leukemia cells havebeen shown to express IL-10 receptors, and their prolifera-tion seems to be inhibited by the addition of IL-10.47 Wehave reported that leukemic cells from patients with

TABLE 4. Effect of accessory cells on progenitor cell expansionCFU–GM (numbers/well)

After 14-day suspension culture

Sample number Before expansion AC* alone SCF/IL-3 SCF/IL-3/IL-10 AC/SCF/IL-3 AC/SCF/IL-3/IL-10

16 694 0 1,635 3,880 4,900 06,86417 926 0 2,880 7,300 7,975 09,28018 570 0 3,744 8,008 8,442 13,806

Mean ± SEM 730 ± 104 0 2,753 ± 612 6,396 ± 1,274 7,105 ± 1,111 9,983 ± 2,035

* Irradiated accessory cells.

TABLE 5. Effect of IL-10 on CFU–GM expansion from BMMNCsCFU–GM (numbers/well)


23 100 1,680 17 02,867 2924 400 1,300 03 16,940 4225 700 9,525 13 10,373 1526 820 8,325 10 13,200 16Mean ± SEM 505 ± 161 5,207 ± 2,161 10.75 ± 2.95 10,845 ± 2,980 25.5 ± 6.36

TABLE 6. Effect of IL-10 on CFU–GM expansion from CBCFU–GM (numbers/well)


27 870 13,475 15 29,445 3428 48 2,170 45 9,333 19529 1,740 19,370 11 51,968 30

Mean ± SEM 886 ± 488 11,671 ± 5,046 23.67 ± 10.73 30,249 ± 12,314 86.33 ± 54.35

WAGNER ET AL.


chronic myelomonocytic leukemia also express IL-10 re-ceptors and that cell surface binding of IL-10 inhibits cellgrowth through the suppression of endogenous GM–CSFrelease.37 Because of its immunosuppressive effects, IL-10seems to be at least partly responsible for tumor escape invivo,48 but this effect is unlikely to play a significant role inan ex vivo system.

Apart from PB and BM, CD34+ cells can be obtainedfrom umbilical CB.49-51 Although the proliferation potentialof CB-derived progenitors seems to be more pronouncedthan that of progenitors from adult sources,28,52 it is still notknown if CB samples contain enough progenitor cells toachieve engraftment in an average-sized adult. Here wedemonstrate that IL-10 is effective in increasing the expan-sion of progenitor cells derived from CB and may help makethis progenitor cell source suitable for a larger number ofpatients.

There are few data to substantiate the view that CD34+cells should be enriched to high purity to avoid the possibleinhibitory effects of CD34– cells. In fact, contaminatingautologous CD34– cells have been reported to inhibit nucle-ated cell production.22 In another study, the total cell expan-sion was about 10 times greater in CD34+ cell cultures, butCFU–GM expansion, was shown to be similar for bothunseparated MNCs and CD34+ cell cultures.40 In our study,the addition of irradiated accessory cells (CD34– cells) didnot show any inhibitory effect on the ex vivo expansionpotential of CD34+ cells, but rather it increased CFU–GMexpansion, which was three times higher than that withselected CD34+ cells alone.

Despite the marked amplification of progenitor cells byIL-10, the total number of blood cells was unchanged orlower in the presence of IL-10 than in controls. The reasonfor this is not completely clear, but it could be due to IL-10-mediated suppression of the release of endogenous growthfactor by accessory cells. Because lineage-restricted growthfactors such as GM–CSF and G–CSF have not been addedto our culture system, the endogenous release of thesemolecules within such cultures may play a significant rolein the generation of mature cells. IL-10 has been shown toinhibit the synthesis of growth-stimulatory cytokines in avariety of hematopoietic cells and thus may lead to a de-crease in the total number of cells in liquid cultures. In semi-solid cultures, we showed that IL-10 inhibits the autono-mous formation of myeloid colonies by reducingendogenous GM–CSF.53

In conclusion, our results demonstrate that IL-10 in-creases the number of HPCs generated by ex vivo expan-sion of both unmanipulated human PBMNCs and selectedCD34+ cells.54,55 Current ex vivo expansion strategies aregreatly effective in patients with a stable progenitor cellpool but may fail in patients in whom hematopoiesis wasperturbed by prior chemotherapy. Our study included pa-tients who had small progenitor yields that may be insuffi-

cient for HPC transplantation. IL-10 was markedly effectivein expanding the number of HPCs, even in these patients.Such patients are likely to have the greatest clinical benefitfrom ex vivo strategies if this procedure helps to generateadequate amounts of HPCs to make them suitable formyeloablative therapy.56

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il-10 increases the number of cfu–gm generatedby ex vivo expansion of unmanipulated human mncsand...

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